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Birkhäuser Advances in Infectious Diseases
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10117 Berlin
Germany
Community-Acquired
Pneumonia
Edited by N. Suttorp, T. Welte and R. Marre
Birkhäuser Verlag
· ·
Basel Boston Berlin
Editors
Norbert Suttorp Reinhard Marre

Charité – University Medicine Berlin University of Ulm
Dpt. of Internal Medicine Medical Microbiology and Hygiene
Augustenburger Platz 1 Robert-Koch-Str. 8
D-13353 Berlin D-89081 Ulm
Germany Germany
Tobias Welte
Medizinische Hochschule Hannover
Carl-Neuberg-Str. 1
D-30625 Hannover
Germany
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Printed on acid-free paper produced from chlorine-free pulp. TFC '
Cover illustration: Colored transmission electron micrograph of Mycoplasma pneumoniae
(green) demonstrating flask-shaped morphology. The background shows a chest x-ray from
a patient. With the friendly permission of Kristen L. Hoek, Vanderbilt University Medical
Center, Nashville, and Matthias Krüll.
Printed in Germany
ISBN-10: 3-7643-7562-0 e-ISBN-10: 3-7643-7563-9
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987654321 www. birkhauser.ch
Contents
List of contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface ................................................................... ix
Tobias Welte
Diagnosis and treatment of community acquired pneumonia –
the German perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Reinhard Marre
Detection of respiratory bacterial pathogens ........................... 15
Walter Hampl and Thomas Mertens

Viral pathogens and epidemiology, detection, therapy
and resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Mathias W.R. Pletz, Lesley McGee and Tobias Welte

Resistance in Streptococcus pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Hans-Dieter Klenk
Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Matthias Krüll and Norbert Suttorp

Pathogenesis of Chlamydophila pneumoniae infections –
epidemiology, immunity, cell biology, virulence factors . . . . . . . . . . . . . . . . . 83
Dina M. Bitar, Marina Santic, Yousef Abu Kwaik

and Maëlle Molmeret
Legionnaires’ disease and its agent Legionella pneumophila . . . . . . . . . . . 111
Sven Hammerschmidt, Gavin K. Paterson, Simone Bergmann

and Timothy J. Mitchell
Pathogenesis of Streptococcus pneumoniae infections:
adaptive immunity, innate immunity, cell biology, virulence factors . . . . 139
vi Contents
Ken B. Waites, Jerry W. Simecka, Deborah F. Talkington

and T. Prescott Atkinson
Pathogenesis of Mycoplasma pneumoniae infections:
adaptive immunity, innate immunity, cell biology,
and virulence factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Pablo D. Becker and Carlos A. Guzmán

Community-acquired pneumonia: paving the way towards
new vaccination concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
List of contributors
Yousef Abu Kwaik, Department of Microbiology and Immunology, Room

MS-410, University of Louisville, Louisville, KY 40209, USA
T. Prescott Atkinson, Department of Pediatrics, University of Alabama at
Birmingham, Birmingham, AL 35249, USA
Pablo D. Becker, Department of Vaccinology, Helmholtz Centre
for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig,
Germany; e-mail: pablo-daniel.becker@helmholtz-hzi.de
Simone Bergmann, Research Center for Infectious Diseases, University of
Würzburg, Röntgenring 11, D-97070 Würzburg, Germany
Dina M. Bitar, Department of Microbiology and Department of Medical
Microbiology and Immunology, Faculty of Medicine, Al-Quds University,
Jerusalem, 19356, Israel; e-mail: dbitar@med.alquds.edu
Carlos A. Guzmán, Department of Vaccinology, Helmholtz Cen-
tre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig,
Germany; e-mail: carlos.guzman@helmholtz-hzi.de
Sven Hammerschmidt, Research Center for Infectious Diseases, University
of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany;
e-mail: s.hammerschmidt@mail.uni-wuerzburg.de
Walter Hampl, Institute for Virology, University Clinic of Ulm, Albert-Ein-
stein-Allee 11, 89081 Ulm, Germany;
e-mail: walter.hampl@uniklinik-ulm.de
Hans-Dieter Klenk, Institut für Virologie, Philipps-Universität Marburg,
Hans-Meerwein-Str. 3, 35043 Marburg, Germany;
e-mail: klenk@staff.uni-marburg.de
Matthias Krüll, Dept. Internal Medicine/Infectious Diseases and Pulmonary
Medicine, Charité, Universitätsmedizin Berlin, Augustenburgerplatz 1,
13353 Berlin, Germany; e-mail: Matthias.kruell@charite.de
Reinhard Marre, University of Ulm, Medical Microbiology & Hygiene, Rob-
ert-Koch-Str. 8, 89081 Ulm, Germany;
e-mail: reinhard.marre@uniklinik-ulm.de
Lesley McGee, Hubert Department of Global Health, Emory University,
1518 Clifton Road, Atlanta, GA, 30322, USA;
e-mail: lmcgee@sph.emory.edu
Thomas Mertens, Institute for Virology, University Clinic of Ulm, Albert-
Einstein-Allee 11, 89081 Ulm, Germany;
e-mail: thomas.mertens@uniklinik-ulm.de
viii List of contributors
Timothy J. Mitchell, Division of Infection and Immunity, Institute of Bio-

medical and Life Science, Joseph Black Building, University of Glasgow
G12–8QQ, UK
Maëlle Molmeret, Department of Microbiology and Immunology, Room
MS-410, University of Louisville, Louisville, KY 40209, USA
Gavin K. Paterson, Division of Infection and Immunity, Institute of Bio-
medical and Life Science, Joseph Black Building, University of Glasgow
G12–8QQ, UK
Mathias W.R. Pletz, Department of Respiratory Medicine, Hannover Medi-
cal School, Carl-Neuberg-Str.1, Hannover, 30625, Germany;
e-mail: pletz.mathias@mh-hannover.de
Marina Santic, Department of Microbiology and Parasitology, Medical Fac-
ulty, University of Rijeka, Croatia
Jerry W. Simecka, Department of Molecular Biology and Immunology, Uni-
versity of North Texas Health Science Center, Fort Worth, TX 76107,
USA
Deborah F. Talkington, Division of Foodborne, Bacterial and Mycotic Dis-
eases, Centers for Disease Control and Prevention, Atlanta, GA 30333,
USA
Ken B. Waites, Department of Pathology, WP 230, University of Alabama at
Birmingham, 619 19th St. South, Birmingham, AL 35249, USA;
e-mail: waites@path.uab.edu
Tobias Welte, Department of Pulmonary Medicine, Carl-Neuberg-Str. 1,
30625 Hannover, Germany; e-mail: welte.tobias@mh-hannover.de
Preface
Community-acquired pneumonia is a disease of high morbidity and mor-

tality. Demographic changes in industrialised countries with a growing
population of elderly persons will add to its significance. In the last years
much progress in the field of community-acquired pneumonia has been
achieved. Vaccination programs against influenza and Streptococcus pneu-
moniae have been established. Risk-adjusted management of patients with
community-acquired pneumonia allows to identify patients in need of hos-
pitalisation and intensive care and helps to choose an effective antibiotic
therapy. “New” pathogens such as C. pneumoniae, Legionella pneumophila,
Chlamydia-like organisms, the human coronavirus or the avian influenza
virus have been detected. In spite of all progress, clinical diagnosis of com-
munity-acquired pneumonia is by no means trivial; detection of respiratory
pathogens often fails or gives inconclusive results and duration and choice
of antibiotics still is a matter of debate.
Moreover, many patients progress from uncomplicated pneumonia to
severe pneumonia and even to pneumonia-related septic shock despite
adequate antibiotic therapy. Therefore, besides new antibiotics we definitely
need a non-antibiotic approach and a better understanding of what deter-
mines individual immune responses to pneumonia is crucial. Fundamental
molecular and cellular pathologic characteristics of disease must be linked
with clinical aspects of infection.
The present book is intended to bridge the gap between basic science,
clinical research and patient management and to crosslink patient care with
biology and microbiology. It gives a state of the art information on differ-
ent aspects of community-acquired pneumonia and allows the reader to
get data on recent developments in community-acquired pneumonia. The
editors Norbert Suttorp, Tobias Welte and Reinhard Marre themselves,
representing clinical medicine, clinical research, microbiology as well as cell
biology, hope that this book will help to manage patients with community-
acquired pneumonia and to identify promising areas of research.
Berlin/Hannover/Ulm, August 2006 Norbert Suttorp

Tobias Welte
Reinhard Marre
Community-Acquired Pneumonia 1
ed. by N. Suttorp, T. Welte and R. Marre
© 2007 Birkhäuser Verlag Basel/Switzerland
Diagnosis and treatment of community acquired

pneumonia – the German perspective
Tobias Welte
Department of Pulmonary Medicine, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1,

30625 Hannover, Germany
Abstract
Current concepts of diagnosis and treatment of CAP are risk stratified and adapted to
the national resistances of important pathogens. Thorough surveillance systems have to
be implemented in all countries.
The risk of patients can be assessed reliably with a limited number of clinical data
(CRB-65 score). Extended microbiological and laboratory diagnosis is recommended for
hospitaliszed patients only. Outpatient treatment can be performed with classic antibiot-
ics like amoxicillin or doxycyclin. Macrolides are only an alternative in these patients. In
the hospital, treatment has to be adapted to the severity of the disease. Further studies
concerning the duration of treatment and advantageous combinations are necessary.
Recommendations for treatment of CAP have to be adapted to the quickly changing
epidemiology and have to be updated every 2 to 3 years.
Introduction
Pneumonia is a worldwide, serious threat to health, and an enormous

socio-economic burden for healthcare systems. According to recent WHO
data, each year three to four million patients die from pneumonia, a large
proportion of whom are children or elderly people. Pneumonia is the third
most common cause of death among infectious diseases in the world [1].
Detailed epidemiological data is available from the USA, where two
to three million cases of CAP occur each year, leading to around 10 mil-
lion doctor-patient contacts [2]. If an estimated proportion of 20 % (half a
million) of these patients were hospitalised, the incidence is 258 hospital
admissions per 100,000 inhabitants. The requirement for hospitalization
depends on age, with the highest rates observed for patients over age 65,
among whom the necessity for hospital admissions rises by a factor of four
to around 1000 per 100,000 inhabitants [3]. In total, it is estimated that the
costs for pneumonia treatment reach 8 billion dollars annually in the USA.
2 Tobias Welte
The largest proportion of this amount is spent on elderly and hospitalized

patients.
Community Acquired Pneumonia (CAP) results in high mortality (mean
about 8%). In the US, CAP is the sixth most frequent reason for dying, and
there is an increase of 0.5 to 1% per year [2]. The increase is caused by the
growing life expectancy, by aging of the population, and by a better treat-
ment of chronic diseases. Elderly people with concomitant diseases are
more susceptible to infectious diseases [4], and have typically a spectrum
of pathogens (gram negative enterobacteriaceae, staphylococci, legionella,
bacteriaemic pneumococci), which is associated with higher mortality [5, 6].
While the mortality of CAP is low in outpatients (1%), it can rise to up to
12% in hospitalized patients [2].
Definition
Pneumonia is an infection of the alveolar space, with accumulation of

inflammatory cells and secretions in the alveoli, resulting in impaired gas
exchange [8]. Each pneumonia acquired outside of a hospital is defined
as community acquired pneumonia [4], while nosocomial pneumonia is
caused during a stay in the hospital and up to one week after discharge.
A subgroup of CAP is the ”healthcare associated pneumonia” of patients
with frequent contact with the healthcare system (haemodialysis patients,
patients in nursing homes) [9]. Although it is community acquired, this form
is treated similar to a nosocomial infection.
The approach of scientific communities to CAP is very different. A
proof of the disease is the pathologic result, combined with a positive
microbiologic specimen of the tissue. In the clinical routine, biopsies of the
lungs cannot be obtained. The typical signs on the chest radiograph can be
delayed, even with the best technical equipment. Initial diagnostics often
shows no pulmonary infiltration [10]. All other signs which are typical for
pneumonia, such as the typical sounds on auscultation, fever, cough and
sputum expectoration, dispnoea, chest pain and serological markers of
inflammation are not pathognomic, and are nearly not always present in
all patients. In the English-speaking countries, the appearance of a new or
progressive infiltration is absolutely necessary for a diagnosis of pneumo-
nia. All other criteria are of minor importance for the diagnosis [11]. The
European Respiratory Society (ERS) defines pneumonia not via a chest
radiograph finding, since many outpatients do not receive this diagnostic
and therefore chest x-ray cannot be the major criterion. The ERS defines
a “lower respiratory tract infection”, according to the clinical presenta-
tion. This includes tracheo-bronchitis, influenza infection, exacerbation of
COPD, and pneumonia [12]. The recommendations of the ERS are much
broader and cannot been compared with the narrowly focused American
recommendations for CAP.
Diagnosis and treatment of community acquired pneumonia – the German perspective 3
Aetiology
The spectrum of pathogens and resistances varies widely between conti-

nents and countries. Universal guidelines for diagnosing and treatment are
for rough orientation only; the treatment must be adapted to the specific
local situation.
Even under optimal diagnostic conditions, sufficient sputum specimen
can be obtained in only 50% of patients [13]. In the early phase of the
infection, sputum production may still be normal. In about one-third of
all cases, the specimen do not meet international quality standards, which
require a high proportion of leukocytes and a low proportion of squamous
cells (Bartlett-criteria [4]). Depending on the patient group (all patients,
all patients with positive results in the specimen, all patients who were
able to expectorate sputum, all patients who produced purulent sputum),
very different distributions of pathogens has been reported. According
to results from the German competence network for CAP (CAPNETZ
[14]), a reliable microbiologic diagnosis can be established in only 20% of
all cases [15]. Worldwide, the most important pathogen is Streptococcus
pneumoniae, followed by Haemophilus influenzae and Mycoplasma pneu-
moniae. Legionella is rare with a frequency of 4%, but associated with an
excessive mortality. This underlines the importance of the very sensitive
urinary-antigen testing in cases with clinically suspected infection with
legionella (Tab. 1). Infections with enterobacteriaceae are most common
in patients from nursing homes, elderly patients and multi-morbid patients
(cardiac and kidney diseases, neurologic disorders and chronic obstruc-
tive pulmonary disease, CODP). The mortality of these patients is much
higher than in patients who are living in the normal community [16]. In the
USA, Pseudomonas aeruginosa is also a typical pathogen in CAP [6], but
Pseudomonas is not important in middle and northern Europe.
Some studies from Italy and Spain [17] report a high prevalence of
Chlamydia pneumoniae (> 10%). These results come from serologic test-
Table 1. Clinical findings in patients with legionella infection
Fever 100%
Chills 73%
Cough 83%
Purulent sputum 50%
Chest pain 30%
Abdominal symptoms 30%
Polymyositis 78%
Neurological symptoms 23%
4 Tobias Welte
Figure 1. Association of the consumption of penicillins and the prevalence of penicillin resist-
ant pneumococci in Europe (modified according to [22]).
AT, Austria; BE, Belgium; HR, Croatia; CZ, Czech Republic; DK, Denmark; FI, Finland;
FR, France; DE, Germany; HU, Hungary; IE, Ireland; IT, Italy; LU, Luxembourg; NL, The
Netherlands; PL, Poland; PT, Portugal; SI, Slovenia; ES, Spain; UK, England only.
ing. Studies using polymerase chain reaction (PCR) show in less than 3% a
positive finding [15]. The titres of IgA and IgM may remain elevated, even
after previous or oligosymptomatic infections. The use of serologic testing
seem not to be sensible in an acute infection. Chlamydia pneumoniae might
be very prevalent is special outbreaks, but presently, the importance of C.
pneumoniae for CAP seems to be low.
Viruses had been found in a number of studies (with or without PCR)
in 10 to 1 % of all CAP cases [5, 15, 18]. The question of whether viruses
are the responsible pathogen for CAP, or if the virus induced damage of
the bronchial epithelia is the precursor of bacterial infection, is still open.
In winter, influenza viruses are most important (70% of all viruses), under-
lining the importance of influenza vaccination in the elderly because of the
prevalence and severity of pneumonia. Large trials revealed that vaccina-
tion results in lower rates of pneumonia [19]. Similar results could not be
obtained for the vaccination against S. pneumoniae, since the vaccinate did
not cover all serotypes of this pathogen. Bacteriaemic infections could be
prevented, but there is no local protection [14]. American studies revealed
that vaccination of children reduced the incidence of severe pneumonia in

adults, albeit different serotypes play the dominant role [20].
Resistances
Problems with resistances of the most important pathogens, especially

pneumococci, vary widely between different countries [21]. The main
reasons are differences in the consumption of antibiotics. There is a direct
correlation between the use of antibiotics and resistances in many countries
(Fig. 1) [22].
The significance of pathogen resistances for the outcome of a patient
is controversially discussed. Increasing resistances of pneumococci against
penicillin did not affect the mortality, even when treatment with penicillin
was continued. On the other hand, resistances against Cefuroxim had been
found to worsen mortality of pneumonia patients [23].
Pneumococci resistant against makrolides seem to be associated with
higher rates of bacteriaemia [24], but the impact on mortality in unclear.
If peumococci are resistant against fluorochinolones (there has been one
epidemic in Hong Kong), treatment with fluorochinolons had worsened the
prognosis of some individual patients [25].
An improvement of resistances can be achieved – as documented in
Scandinavia – by temporary reduction of the use of the respective classes
of antibiotics [26].
Risk stratification
The risk factors for an increased CAP mortality are age, the number of
concomitant diseases, and the place of residence before admission to the
hospital (patients from nursing homes had an eight-fold higher risk for
dying than patients coming from “regular” homes) [7].
Different scores for the estimation of the prognosis of patients with
CAP had been evaluated to substantiate the decision for hospital admission
and the decision of where to treat the patient (regular ward, intermediate
care unit, intensive care unit). Many scores designed for hospital patients
(Pneumonia Severity Index, PSI [27], CURB Score [28]) have the disad-
vantage that they need laboratory testing, what is not available in the out-
patient setting. Recent data revealed that a simple clinical score (CRB-65;
C: Confusion, R: respiratory rate > 30/min, B: blood pressure < 90 mmHg,
65: age > 65 years) allows to stratify patients into a low, moderate, or high
mortality risk group [29, 30].
The risk within the hospital can be best assessed with the modified
ATS Score [31]. If one major criterion (septic shock requiring vasopressor
therapy or mechanical ventilation) or two minor criteria (acute respiratory
6 Tobias Welte
insufficiency (PaO2/FiO2 < 250), multilobular infiltrates in the chest radio-

graph, systolic blood pressure < 90 mmHg) are present, then intensive care
treatment is necessary.
Diagnostic procedures
A chest radiograph (pa (posterior/anterior) and lateral view) is highly rec-

ommended to establish the diagnosis. However, the chest radiograph does
not have 100% sensitivity. High-resolution computed tomography (hrCT)
might show infiltrates which had not been detected in a previous chest x-ray.
If there is a clinical suspicion of pneumonia, the chest radiograph is normal,
and there is no clinical improvement of the patient, a new chest radiograph
should be performed 24 to 48 h later, or a chest computed tomography
should be performed.
Not all infiltrates in the lungs are caused by pneumonia. Congestive heart
failure, tumour, pulmonary infarction, and infiltrates due to interstitial pul-
monary diseases or systemic diseases (Wegener’s granulomatosis, collageno-
ses, and others) are important differential diagnoses for acute pneumonia.
Elevated levels of C-reactive protein (CrP) or procalcitonin III (very
expensive and not currently a routine procedure) are important parameters
[32], even if they do not prove infection. In bacterial pneumonia, leucocy-
tosis and an overrepresentation of young granulocytes can be expected.
Leucopenia might be a sign of sepsis and might therefore be a bad prog-
nostic factor.
In patients with a CRB-65 score of 0, no further laboratory diagnostic
is necessary. In hospitalized patients, the diagnostics should be focused on
inflammation, and further laboratory diagnostics should be performed,
depending on the severity of the disease and on concomitant problems.
Microbiologic testing is recommended, depending on the severity of
the pneumonia and on other risk factors. Patients with a low risk (CRB-
65 = 0) receive no benefit. However, this may change, if in addition to the
urine legionella antigen testing other quick reacting tests become available.
Preliminary results from the pneumococci antigen test do not justify a rou-
tine use of this expensive test.
Microbiologic specimen from the lower airways (sputum, bronchoalveo-
lar lavage and biopsies), pleural effusions, and blood cultures can be better
and faster collected in the hospital than in the outpatient setting. Such diag-
nostics can be recommended for all hospitalized patients in severe cases it
is mandatory.
Blood cultures show positive results in 10 to 20% only and that is a
proof. In severe infections, blood cultures must be obtained.
If legionella infection is suspected (for clinical signs see Table 1), an
antigen testing from urine specimen on Legionella pneumophilia antigen
is recommended.
Serological testing (to screen for Mycoplasma, Chlamydia and viruses)

is not generally recommended since results are difficult to interpret (see
above).
Patients who do not improve within 72 h, or who worsen in clinical state,
must be seen as non-responders [34]. In this situation, invasive diagnostic
procedures (bronchoalveolar lavage, transbronchial lung biopsy), and
– depending on the clinical course – further radiologic diagnostic (pleural
ultrasound, CT) might become necessary.
Treatment
All guidelines distinguish patients with low risk of dying (CRB-65 ) 1,

CURB ) 1, PSI I und II), who might be treated as outpatients, and patients
with high risk, who need hospital treatment. Further prognostic factors are
concomitant diseases and the type of pathogen.
Important demographic, epidemiologic, and clinical risk factors for a
specific spectrum of pathogens are:
– Previous antibiotic treatment increases the rate of resistant pathogens.

The previous antibiotic should not be continued [22].
– Recent visits to countries with high prevalence of legionellosis (e.g.
Spain, Italy) and/or contact to contaminated water has to be explored.
– Patients aged 65 and above have a higher prevalence of gram-negative
pathogens, especially if they have co-morbidities (heart, lung, kidney, liver),
previous treatment with antibiotics, or previous hospitalisation [35].
– Patients from nursing homes or with previous hospitalization are at
increased risk for infections with enterobacteriaceae and Staphylococcus
aureus, and have a higher risk for aspiration pneumonia [16].
– Chronic lung diseases are associated with Haemophilus influenzae [36].
In advanced stages of COPD, in patients with cystic fibrosis or bron-
chiectasis, there is a frequent finding of S. aureus and Pseudomonas
aeruginosa [6].
– Contact to animals: Birds: C. psittaci, sheep: C. burnetii.
– Previous treatment with steroids of at least 10 mg/day prednisolone-
aequivalent over at least 4 weeks, or structural lung diseases (COPD,
bronchiectasis, cystic fibrosis) and a hospitalization within the last 30
days (longer than a two-day stay) are associated with P. aeruginosa and
Legionella spp. [9].
The recommendations for treatment distinguish whether there are risk fac-
tors for an infection with Pseudomoas aeruginosa [6]:
– Pulmonary concomitant diseases (structural defects in COPD stadium

GOLD IV, bronchiectasis, cystic fibrosis).
8 Tobias Welte
Table 2. Treatment recommendations for patients with low risk in the outpatient setting.
Substance Dosage (per day) Duration of therapy

First choice
Aminopenicillines
– Amoxicillin * 70 kg: 1 g t.i.d p.o. 7–10 days
< 70 kg: 0.75 g t.i.d. p.o.
Or
Tetracycline
– Doxycycline 200 mg once initially, 7–10 days
* 70 kg: 200 mg once daily
< 70 kg: 100 mg once daily
Alternative
Macrolidea
– Azithromycin 500 mg once daily p.o. 3 days
– Clarithromycin 2 × 500 mg p.o. 3 days, 7–10 days
followed by 250 mg b.i.d
– Roxithromycin 1 × 300 mg oral 7–10 days
aonly in countries with low macrolide resistance
– Hospitalization within the last 30 days (longer than two days stay).
– Risk for aspiration (e.g. neurological diseases).
– Treatment with a broad spectrum antibiotic over more than 7 days
within the last month.
Depending on the number of risk factors, the probability of Pseudomonas

aeruginosa increases exponentially (to more than 50 % in three or more
risk factors).
The guidelines show wide difference in the recommended antibiotics.
The reasons are different local resistances. The recently published recom-
mendations of the ERS are presented here [12]. Updates of the recommen-
dations of the ATS and ISDA are under preparation, but not finalized yet.
Table 2 shows the recommended treatment of CAP patients with a low
risk and treatment in the outpatient setting. A follow-up to assess the suc-
cess of treatment should be performed after 72 h. Patients or persons in
their environment should be advised to contact their doctor again if fever
exceeds 4 days, dyspnoea gets worse, patients stop drinking, or conscious-
ness is decreasing.
Macrolides have been used to treat pathogens such as Chlamydia
and Mycoplasma which did not respond to beta-lactam antibiotics. Some
recently published studies [37, 38] demonstrate that patients with a low
risk of dying have no worse outcome with penicillin derivatives compared
with macrolides. Infections with Chlamydia seem to be rare, and infections
Table 3. Treatment recommendations for the calculated initial therapy of hospitalized CAP
patients (no severe pneumonia) without a risk of Pseudomonas infection
Substances Dosage Duration of therapy

Preferred in regions with low pneumococcal
resistance rate
– Penicillin G 1 Mill. IU t.i.d 7–10 days
– Ampicillin 1.5 g t.i.d i.v. 7–10 days
– Amoxicillin/Clavulansäure# 2.2 g t.i.d i.v. 7–10 days
– Ampicillin/Sulbactam 3.0 g t.i.d i.v. 7–10 days
– Cefuroxim 1.5 g t.i.d i.v. 7–10 days
– Ceftriaxon 2.0 g once daily i.v. 7–10 days
– Cefotaxim 2.0 g t.i.d i.v. 7–10 days
± Macrolides (s. Tab. 2)* 7–10 days
Alternatives
(in regions with increased pneumococcal
resistance or intolerance to preferred drugs)
Fluorquinolone§
– Levofloxacin 500 mg once daily i.v. 7–10 days
– Moxifloxacin 400 mg once daily i.v. 7–10 days
# Can be applied as sequential treatment using the same drug

* new macrolides preferred to erythromycin
§ within the fluoroquinolones, moxifloxacin has the highest antipneumococcal activity.
Experience with ketolides is limited but they may offer an alternative when oral treatment is
adequate
with Mycoplasma seem to be self-limiting in low-risk patients. In these sit-

uations, patients might be treated without intracellular active antibiotics.
Doxycyclin had been accepted as first-line treatment despite insufficient
clinical data, since there is no information about treatment failures due to
resistance.
In the outpatient setting, oral treatment is preferred. Oral cephalospo-
rins have a low bioavailability, and there are doubts about their pharmaco-
kinetics and pharmacodynamics in elderly people. Cephalosporins are an
alternative in exceptional cases only.
The recommendations for hospitalized patients are shown in Table 3.
Hospitalized patients should receive parenteral treatment, due to unreli-
able oral pharmacokinetics in elderly and severely affected patients. If the
clinical response is satisfactory, an early switch (after 72 h) to similar oral
substances is possible. Treatment success is assumed if the respiratory rate
is lower than 25/min, and oxygen saturation is above 90%, body tempera-
ture fell by at least 1°C, and the haemodynamic and neurological status are
satisfactory, and if oral feeding is possible [39].
A number of retrospective, and one prospective study [40] show that in
bacteriaemia with S. pneumoniae a combination therapy with a beta-lactam
10 Tobias Welte
Table 4. Criteria for severe community acquired pneumonia (modified according to [46])*
Baseline (minor) criteria assessed at admission

Respiratory rate > 30 min–1
Severe respiratory failure (Pa,O2/Fi,O2 ratio > 250)
Bilateral involvement in chest radiograph
Involvement of more than two lobes in chest radiograph (multilobar involvement)
Systolic blood pressure < 90 mmHg
Diastolic blood pressure < 60 mmHg
Major criteria assessed at admission or during clinical course

Requirement for mechanical ventilation
Increase in the size of infiltrates by * 50% in the presence of clinical nonresponse to
treatment or deterioration (progressive infiltrates)
Requirement of vasopressors > 4 h (septic shock)
Serum-creatinine * 2 mg·dl–1 or increase of * 2 mg·dl–1 in a patient with previous renal
disease or acute renal failure requiring dialysis (acute renal failure)
Pa,O2, arterial oxygen tension; Fi,O2, inspiratory oxygen fraction;

*, the presence of at least two minor criteria or one major criterion defines severe pneumonia,
i.e., pneumonia requiring admission at the ICU.
antibiotic and a macrolide offers the best treatment success. Despite severe
weakness in the design of these studies, combination therapy is recommend-
ed at least for severe infections. Mono therapy with a new fluoroquinolone
(moxifloxacin or levofloxacin) seems to be as effective as combination
therapy, but reliable data is missing [41].
Table 4 shows criteria for severe community acquired pneumonia
(sCAP). In this form of pneumonia, broad initial treatment effective against
all possible pathogens is necessary. The choice of antibiotics is guided by the
probability of Pseudomonas infection (Tab. 5).
In patients with sCAP, the combination therapy between a beta-lactam
and an aminoglycoside or fluorochinolone is also under discussion. Two
Meta analyses [42, 43] could not reveal an additional effect of aminogly-
cosides in infections with gram-negative pathogens. However, the data on
infections with pseudomonas are not sufficient. The recommended combi-
nation of beta-lactams with pseudomonas-active fluoroquinolones seems
to be of advantage, but this combination has not been tested systematically
until today. One abstract, published from the ECCMID meeting 2006 [44]
suggested that mono therapy with moxifloxacin, which penetrates tissue
well, could be sufficient.
Treatment failure is defined as lack of clinical improvement after 72 h of
treatment or worsening of the clinical situation. Besides extended diagnos-
tic procedures, which also allow the recognition of important complications
of CAP (pleural empyema, lung abscess), treatment must be de-escalated
target intracellular pathogens (combinations of macrolides and newer fluo-
Table 5. Treatment recommendations for the calculated initial therapy of patients with severe
CAP.
Substances Dosage Duration of therapya

No risk factors for Pseudomonas
– Ceftriaxon 2,0 g once daily i.v. 7–10 days
– + Makrolides (s. Tab. 2)
– or + Fluorquinolone (s. Tab. 3)
Risk factors for Pseudomonasa
– Piperacillin/Inhibitor 4,5 g t.i.d i.v. 7–14 days
– Cefepime 2,0 g t.i.d i.v. 7–14 days
– Carbapenem 1,0 g t.i.d i.v. 7–14 days
– + Ciprofloxacin 400 mg t.i.d i.v. 7–14 days
aminimum treatment is 7 days, maximum 14 days if there is a proof of pseudomonas or L.
pneumophilia.
roquinolones). Gram-negative pathogens and Pseudomonas must be cov-

ered (Tab. 5).
The problem of an optimized duration of treatment is controversially
discussed. First results of short-term treatment (5 days) in patients with
low risk are encouraging [45]. Hospitalized patients who were treated on a
regular ward were nearly completely free of symptoms after five days [41].
The current evidence does not allow a general recommendation of such
shor-t term therapy. Treatment duration of 10 days should only be exceeded
in rare, exceptional cases.
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1730–1754
Detection of respiratory bacterial pathogens
Reinhard Marre
University of Ulm, Institute of Medical Microbiology & Hygiene, Robert-Koch-Str. 8,

89081 Ulm, Germany
Abstract
Microbiology of community-acquired pneumonia often is based on indirect or precari-
ous evidence. Time and effort to detect a respiratory pathogen often is not sufficiently
related to its usefulness in guiding therapy. If sputa are accepted by the laboratory for
microbiological studies (Gram stain, culture), they should fulfill quality criteria such as
high number of leukocytes and low number of squamous epithelial cells. Complementary
tests such as antigen detection assays are useful adjuncts for diagnosing pneumococcal
and Legionella pneumonia. Nucleic amplification tests help to overcome the problems in
detecting Chlamydia pneumoniae, Legionella and Mycoplasma pneumoniae.
Introduction
Community acquired pneumonia is caused by a wide variety of differ-

ent bacterial species. Streptococcus pneumoniae is the leading pathogen,
followed by Haemophilus influenzae, Staphylococcus aureus, Chlamydia
pneumoniae, Mycoplasma pneumoniae and a broad spectrum of enterobac-
terial species. While it is generally agreed upon that S. pneumoniae is the
predominant pathogen in CAP, reported incidences of other species vary
considerably depending on patient population, specific epidemiological
situations and microbiological aspects such as type of specimens studied,
detection methods used and pathogen definition. Even in clinical trial con-
ditions detection of a respiratory pathogen often is based on precarious evi-
dence, namely microscopical or cultural detection of a facultative pathogen
in respiratory secretions which may be heavily contaminated by bacteria
of the oropharyngeal tract. Unequivocal, definitive evidence, i.e. growth in
blood or other usually sterile specimens is rare and selects for severe bacte-
remic pneumonia and may thus not be representative. Nucleic acid ampli-
fication techniques (NAT) such as PCR to detect microbial pathogens are
– in the absence of a solid reliable gold standard – not a reliable indicator
16 Reinhard Marre
of a causative agent. In addition, clinical patterns often do not give a clear

clue about the causative micro-organism [1].
Beyond clinical trials the situation is even worse and quality of micro-
biology of respiratory secretions often is regarded as unsatisfactory. This
resulted in the statement of Bartlett that the culture of lower respiratory
specimens may result in more unnecessary microbiologic efforts than any
other type of specimen [2]. Clinical relevance of microscopy of respira-
tory specimens too is regarded as very controversial [3] and guidelines of
the ATS and the IDSA differ with respect to the evaluation of the clinical
impact of a gram stain result of a sputum sample [4].
Because of these limitations and critical appraisal, microbiology of the
CAP may not be useful unless performed by expert microbiologists and
well trained technicians using standardized approaches for specimen work-
up, external and internal quality assurance with an active communication
network between microbiologists and clinicians. The effects of quality on
reported incidence has been illustrated by Bartlett [5], who described an
apparent decrease in the yield of S. pneumoniae from Gram staining and
culture from 80 to < 18% over the last 30 years. In evaluating 15 pub-
lished reports from North America, it was found that S. pneumoniae inci-
dence ranged from 20 to 60% [6], which is in agreement with the German
Multicenter Network CAPNETZ in which the incidence of S. pneumoniae
ranged from 28 to 48% depending of the patients population studied.
Microscopy of respiratory secretions
Microscopy of respiratory sample may help in guiding initial therapy in

CAP if a properly collected specimen is tested. Non-invasive samples such
as sputa and upper respiratory swabs give less conclusive evidence than
invasive samples (Bronchoalveolar lavages (BAL), protected specimen
brushings), but are more easily available. An essential problem is that
sputum may be contaminated by saliva. Several approaches have been
developed to assess the quality of a respiratory sample. These have been
recently described and evaluated by Sharp et al [3]. The bottom line is
that the number of leukocytes related or not to the number of squamous
epithelial cells are a criteria of quality (Fig. 1). Based on this criterion, the
number of CAP patients with a good-quality sputum has been shown by
Rosón et al to be only 40% of all patients and 60% of the patients with a
sputum sample [7]. In 35% of the patients no sputum sample was available.
In a study performed by Kalin et al 76% of the sputa were deemed puru-
lent [8]. In CAPNETZ, a Germany-wide network on CAP with more than
3,000 patients sputum was available in 45 to 70% of patients. In about half
of the patients with sputum, the sample was purulent. Non-purulent sputum
should not be used for culture and therefore rejected. If non-purulent spu-
tum is used for culture non or little pathogenic microorganisms (Candida
Detection of respiratory bacterial pathogens 17
Figure 1: Gram stain of a high-quality (purulent) (below) and a non purulent sputum (above).
18
Figure 2: Frequency of isolates in dependence of the quality of sputum. Filled columns: < 10 leukocytes per 10x field, empty columns 10–25 leukocytes per
Reinhard Marre
10x field, shaddowed columns: > 25 leukocytes per 10x field.

albicans, Escherichia coli and other enterobacteria) can be detected in high

numbers and in a high proportion of patients, thus perhaps leading to flawed
therapy decisions (Fig. 2). Although these species cannot be regarded as
pathogens in CAP, they may indicate altered colonization. Limiting identifi-
cation and work-up of the sample may lead to improved outcome as shown
convincingly by Barenfanger in the case of detection of yeasts in respiratory
specimens [9]. However, Chlamydia pneumoniae, Mycoplasma pneumoniae,
and Legionella can be detected in non-purulent sputum samples by NAT
techniques.
Many studies state that sputum can also safely be used for microscopic
detection of bacterial pathogens [3]. Positive and negative predictive value
were 90% and 62%, respectively, if patients with a definitive diagnosis of
pneumococcal pneumonia (detection of S. pneumoniae in sterile body flu-
ids or detection of the pneumococcal antigen) were evaluated, and 75 and
98% if patients with a Haemophilus influenzae pneumonia were evaluated
[7]. The corresponding sensitivity values were 35% (S. pneumoniae) and
42% (H. influenzae). Including only patients with a bacteremic pneumo-
coccal pneumonia, Musher reported a sensitivity of 57% [10]. It should,
however, be kept in mind that these studies select for patients with gen-
eralized bacterial infection and that therefore microscopy may be of less
value if patients with a less severe infection are included. In a meta-analy-
sis, Reed et al. reported a range of sensitivity values from 15 to 100%, and
of specificity from 11% to 100% in pneumococcal pneumonia [11], which
illustrates that a Gram stain in the hands of inexperienced may yield mis-
leading results.
Detection of pathogens by culture
Cultural detection of respiratory pathogens in sterile body fluids is usually

specific but limited to CAP with generalized infection. If secretions are
obtained from deeper sites of infections such as bronchoalveolar lavages or
fiber optic brush technique (FB) and if a cut-off level of 103 to 104 cfu/ml is
defined, cultural detection has a sensitivity between 42 and 95% and a speci-
ficity between 45 and 100% (reviewed by Sharp [3]). However, BAL and
FB and sterile body fluids are not available on a routine basis and therefore
microbiology has also to rely on culture of sputum. On the other hand, the
usefulness of sputum cultures is a matter of controversial debate. Heineman
et al stated that cultural detection of pathogens in sputum is meaningless
if not guided by microscopy [12]. Recent publications report either limited
value or nonvalue of microbiological studies to detect a CAP pathogen [13,
14]. Other reports indicate however that sputum cultures may reveal thera-
peutically relevant information in individual cases and that the relevance
can be increased if the sputum sample is purulent, obtained before the start
of antibiotic chemotherapy and if cultured without delay.
20 Reinhard Marre
Streptococcus pneumoniae
S. pneumoniae is the most predominant species in CAP. The incidence

ranges from 20 to 60%. About 20% of these pneumonias are bacteremic.
Pneumococci can be detected out of respiratory samples by microscopy and
culture, in addition by blood cultures and by detection of urinary antigen.
As described above, Gram stain of a sputum sample has a sensitivity of 60%
and specificity of near 100% to detect pneumococci in bacteremic patients
[7, 10]. The examination of a sputum Gram stain, however, is not as trivial as
generally believed and requires expertise of the lab personnel and an active
quality management programs.
The diagnostic value of a sputum culture in bacteremic patients has
been shown to be high if only bacteremic patients with purulent sputum
are included. In these patients it may help to guide therapy and to narrow
the spectrum of the antibiotic. The cultural isolates also allow recognizing
trends in resistance development that have implications on the improve-
ment of guidelines.
Urinary antigen assays for the detection of pneumococci (Binax NOW)
may overcome the limitations of sputum microscopy and culture, since it
is a rapid (15 min) bedside diagnostic, not limited by the availability of
purulent sputum, and does not require experienced lab personnel. The
assay detects the c polysaccharide antigen that is not capsular serotype spe-
cific. Sensitivity is reported to be 70 to 75%, specificity 90 to 95% [15-17].
Patients with a positive sputum culture may be antigen positive in about
only 50% [17], indicating either the low specificity of the sputum culture
or the low sensitivity of the antigen assay in non-bacteremic patients. In
contrast to adults, the test does not discriminate between children with and
without pneumococcal pneumonia [18].
Nucleic acid amplification assays are not useful in diagnosing a pneu-
mococcal pneumonia. When using plasma, buffy coat or urine as specimens,
Murdoch et al. studied the performance of a nested PCR targeting the
pneumolysin gene [19]. Plasma and buffy coat were only positive in 30%
of patients with a positive blood culture, urine was positive in 2% while
the antigen assay was positive in 29% of the patients. Throat swabs were
positive in 55 to 56 % by PCR, irrespective of the status as a control or as
patient.
Haemophilus influenzae
Although H. influenzae is the second or third most common species

in CAP, little attention has being paid to this species as a respiratory
pathogen. Patients with H. influenzae pneumonia often are elderly with
a history of chronic obstructive lung diseases, tumor pathology or other
causes of impaired immunity. Non-encapsulated strains of H. influenzae
predominate. Lethality of an invasive H. influenzae infection of this patient

population is high [20]. As reported by Roson et al [7] sputum microscopy
has a high sensitivity and specificity for the diagnosis of a H. influenzae
pneumonia of hospitalized patients. Detection of H. influenzae by sputum
culture may overestimate the incidence of this species in CAP since it is a
normal inhabitant of the oropharyngeal tract. Assays to diagnose H. influ-
enzae infection by antigen detection are to the best of my knowledge not
available.
Legionella
Legionella comprises a genus with about 50 different species most of which

are never or rarely encountered in the human host. The overwhelming
majority of Legionella infections are attributed to Legionella pneumophila
serotype 1. Australia, however, may be an exemption where Legionella long-
beachae is more predominant. Legionella occur in aquatic systems where
it can thrive within amoebae. When inhaled by a susceptible person (more
than 60 years of age, smoker, male, immunocompromised), it can cause a
potentially lethal pneumonia. Legionella infections occur with an incidence
of 5 to 10% in CAP in an endemic situation. Morbidity is about 20 cases of
Legionella infection per one million inhabitants.
At the time being there is no one-for-all easy and rapid test to detect
Legionella. If a Legionella infection is suspected by the clinician, differ-
ent assays should be combined. Although Legionella can be cultured from
respiratory secretion when using special media containing cysteine and iron
as essential growth factors, the detection rates are rather low and sensitivity
may not exceed 60%. The advantage of a culture is that a Legionella isolate
for further investigations (source identification) is available. Sputum Gram
stain is not helpful since the Gram negative rod Legionella is stained only
faintly by fuchsin, unless the stain is allowed to act for a prolonged time.
Direct immunofluorescence test using polyclonal or monoclonal antibodies
directed against the lipopolysaccharide can be useful in principal. Cross-
reaction of the polyclonal antiserum with Pseudomonas species [21], the low
sensitivity, the long hands-on time and the need for experienced personnel
have limited the use of this test.
The introduction of the antigen detection assay (Binax or Biotest) has
significantly contributed to a rapid and specific diagnosis of Legionella sero-
type 1 infections. Legionella infections other than Legionella pneumophila
serotype 1, however, are not covered reliably. The antigen detection assay
may also select for severe infections as shown by Yzerman [22]. For patients
with mild Legionella infection, the test sensitivities ranged only from 40
to 53%, whereas for patients with severe Legionnaires Disease (LD) who
needed immediate special medical care, the sensitivities reached 88 to
100%. Guerrero et al. reported that the Bartels EIA and the Biotest EIA
22 Reinhard Marre
had significantly higher sensitivity levels than the Binax NOW immuno-
chromatographic test (65 to 70% vs. 37%) [23].
Nucleic acid amplification assays using respiratory secretions can com-
plement the Legionella test assay system. Real-time PCR is a rapid as well
as reliable method with a sensitivity of 100% when culture proven cases
of Legionella are studied [24, 25]. The 16S rDNA, 5S rDNA or the macro-
phage infectivity potentiator (mip) gene of Legionella commonly are used
as target genes.
Antibody assay is available for the diagnosis of Legionnaire’s Disease
(LD), but its value is limited since some LD patients do not react with a
significant rise of antibodies and others have a persistant IgM antibody titer.
With the advent of molecular tests the indication of serological testing in
LD in acute care is reduced to near null.
Chlamydia pneumoniae
Chlamydia pneumoniae was identified as pulmonary pathogen only 15

years ago. Because of its debated association with coronary heart disease C.
pneumoniae has attracted considerable attention. The reported incidence
ranges from 2 to 40% depending on the epidemiological situation, site and
time of the investigation, detection methods used and patients included.
In recent years reports with low incidences (less than 2%) seem to have
increased [25–27]. The CAPNETZ trial confirmed the low incidences of C.
pneumoniae infections [28].
Most assays for the detection of C. pneumoniae are not applicable for
routine microbiology. Cell culture techniques to grow C. pneumoniae are
restricted to specialized laboratories. Even under optimal conditions (no
delay until culture, high quality specimens) cultural detection is much more
futile than, for example, cultural detection of C. trachomatis. If grown in a
first passage, many isolates do not outlive further passages. The C. pneu-
moniae-specific microimmuno-fluorescence (MIF) test has been introduced
as matter of choice to prove a C. pneumoniae infection and many clinical
CAP studies rely on this assay. In clinical practice, however, serodiagnosis
must often rely on a single serum sample and, therefore, seroconversion and/
or rise in antibody titers are missed in many patients. In addition, about 60%
of adults have antibodies against C. pneumoniae which makes it difficult
to differentiate between an acute or past infection. During the last decade,
molecular methods including conventional and real-time PCR assays have
been developed for sensitive and specific detection of C. pneumoniae DNA
in respiratory tract specimens [29]. However, there is no consensus on the
most suitable specimen, the optimal DNA isolation method and target gene.
Results of multicenter PCR comparison trials have been contradictory and
difficult to interpret [30]. Therefore, further efforts to optimize and stan-
dardize diagnostics in C. pneumoniae are urgently needed.
Mycoplasma pneumoniae
Detection of Mycoplasma pneumoniae in CAP is dealt with in the chapter

by Waites et al.
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Viral pathogens and epidemiology, detection, therapy

and resistance
Walter Hampl and Thomas Mertens
Institute for Virology, University Clinic of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany
Abstract
Worldwide community-acquired pneumonia (CAP) is one of the most frequent infectious
diseases and a leading cause of death. Several studies have shown that a pathogen could
be identified only in 50 to 60% of all patients, although in children < 6 month infectious
agents can be detected in about 90%. Viral infections are most frequent in children < 2
years (80%), whereas bacterial infections increase with age.
RSV, influenzaviruses, rhinoviruses, parainfluenzaviruses and adenoviruses are the
most common viruses associated with CAP in children. Among adenoviruses a pre-
dominance of adenovirus 7 has been reported in several countries with emergence of
highly pathogenic variants with significant lethality in young children. Many childhood
respiratory infections are caused by more than one pathogen and up to 30% mixed viral
/ bacterial infections can be observed. CAP in immunocompetent adults is rare, whereas
persons with underlaying diseases have an increased incidence of CAP. In the elderly,
RSV, influenzaviruses, parainfluenzaviruses and less frequent adenoviruses are predomi-
nant viruses causing pneumonia. Less frequently associated with CAP are the newly dis-
covered human metapneumovirus and the coronaviruses NL63 and HKU1. Hantaviruses,
involved in the hantavirus pulmonary syndrome, belong to the emerging pathogens to
date in North, Middle and South America.
For optimum diagnosis the whole spectrum of potential respiratory viral agents
should be included and multiple diagnostic techniques have to be used.
In view of the high relevance of influenzavirus for CAP influenza vaccination is highly
advisable for prevention of CAP, especially in high-risk groups.
Introduction
Viral infections are involved in 10–25% of CAP. Frequently mixed infec-

tions, viral/viral or viral/bacterial, are detected. CAP in infants and young
children is most commonly due to viral infections, with the predominance
of respiratory syncytial virus (RSV). In developing countries the incidence
of viral pneumonia is higher and is a relevant reason for death of young
children [1, 2].
28 Walter Hampl and Thomas Mertens
Viral pathogens and epidemiology
The major viral pathogens are summarized in Table 1. Usually they cause
mild self-limited illness, mainly restricted to the upper respiratory tract. The
leading viral pathogens for severe disease are RSV and influenzaviruses.
In the past two decades some new, mostly zoonotic viral pathogens have
emerged like SARS-coronavirus, avian influenzaviruses and hantaviruses
associated with hantavirus pulmonary syndrome (HPS), which has crossed
species barriers. Numerous other viruses occasionally can cause severe
respiratory disease in the lower respiratory tract, but they are better known
for other clinical manifestations (Tab. 2).
Respiratory virus infections are more common in winter and early
spring, but some cause respiratory illness without a clear seasonal pattern.
The frequency of detection of respiratory viruses varies in different stud-
ies due to methodological problems. Many studies did not include all known
respiratory viruses. Additionally, the prevalence of viruses may vary over
time and between different geographical areas. In immunocompromised
patients exogenous respiratory viruses may persist with prolonged shed-
ding.
Individuals with a subclinical infection may be an undetectable source
of transmission. Only a few studies have investigated respiratory viruses in
asymptomatic humans with very variable results. An age-dependent occur-
rence of asymptomatic respiratory infections has been reported with the
highest frequency of 68% in young children newborn to 4 years.
Factors influencing occurrence of community-acquired pneumonia

(CAP)
Virus-associated CAP occurs mainly in infants and young children, in

elderly and frail adults, in persons with underlying diseases and in immu-
nocompromised patients. Even “harmless” agents like rhino- or coronavi-
ruses frequently induce acute disorders or exacerbations in people with
chronic cardiopulmonary diseases or asthma. The main host and environ-
ment related risk factors have been investigated, but the influence of vari-
ous other factors, which can predispose CAP is controversial.
In general, the viral-associated CAP decreases with age. In several
recent studies in developed countries a pathogen was detected in 79% to
85% in immunocompetent children with CAP; 25% to 62% of the patients
had evidence of a single viral infection, 8% to 11% for viral/viral infection
and 23% to 43% a viral/bacterial infection. Inflammation and disease were
more severe in viral/bacterial infection [3–6]. In nonimmunocompromised
adults the demonstrated viral etiology is less common. Roux et al. [7] iden-
tified a pathogen only in 38% of the cases with a proportion of viral infec-
tions of approximately 20% (single viral infection 9%) with influenzavirus
Viral pathogens and epidemiology, detection, therapy and resistance 29
Table 1. Major respiratory viruses
Virus family / Genus Virus type

Subfamily
Adenoviridae Mastadenovirus Human adenovirus (1 – 7, 14 and 21)
Coronaviridae Human coronavirus (HCoV-OC43, -229E,
-NH, -NL63, -HKU1)
SARS-coronavirus
Orthomyxoviridae Influenzavirus A, B, C
Avian influenzavirus A (e.g. H5N1)
Paramyxoviridae
Paramyxovirinae Respirovirus Parainfluenzavirus 1, 3
Rubulavirus Parainfluenzavirus 2, 4a/b
Pneumovirinae Pneumovirus Respiratory syncytial virus (A, B)
Metapneumovirus Human metapneumovirus (A, B)
Picornaviridae Rhinovirus Human rhinovirus (HRV 1-102)
Enterovirus Human enterovirus (Cox A10, 16, 21, Cox B1-
6, ECHO 4, 9, 11, 25, 32, HEV 68, 69, HRV 87)
Table 2. Other respiratory viruses
Virus family / Genus Virus type

Subfamily
Bunyaviridae Hantavirus e.g. Sin-Nombre virus, Andes virus, Choclo
virus
Paramyxoviridae Morbillivirus Measles virus
Herpesviridae
Alphaherpesvirinae Herpes simplex virus (HSV) (rare)
Varicella-zoster virus (VZV)
Betaherpesvirinae Cytomegalovirus (CMV)
Gammaherpesvirinae Human herpes virus 6 (HHV6)
Epstein-Barr virus (EBV) (very rare)
as the most frequently identified viral agent. In this study chronic heart
failure (CHF) was a risk factor for virus infections.
Viruses causing CAP
Adenovirus (Adv)
The virus is composed of nonenveloped, icosahedral particles containing a

double-stranded linear DNA genome. They are 70–90 nm in diameter and
consist of 252 capsomers (hexons and pentons) with filamentous glycopro-
teins (fibers) on the penton bases, which display characteristic different
lengths. Human adenoviruses have been classified into six species (subgen-
era) A-F on the basis of antigenicity and other biological properties (hem-
agglutination, tumorogenicity in animals). This is in quite good agreement
with classification based on genomic differences, like DNA homology of
less than 20% between viruses of the different subgenera. Serotype-spe-
cific epitopes are predominantly found on the hexon capsomere and the
terminal part of the fiber, and are defined by the quantitative neutraliza-
tion test. At present, 51 serotypes have been identified by neutralizing
antibodies and nine of these are documented as respiratory pathogens.
Adenoviruses are very resistant, also against proteolytic enzymes in the
intestinal tract.
The species C adenoviruses (1, 2, 5 and 6) are endemic and are respon-
sible for approximately 60% of all human adenovirus infections (Adv 6 only
for 4%) and for more than 80% of the adenovirus infections (most com-
monly Adv 1 and 2) early in life, whereas they cause 15% of symptomatic
lower respiratory tract infections. After primary infection, C viruses may be
shed in feces for months or even years. Following the initial infection the
C viruses establish a lifelong, asymptomatic, persistent infection, with cur-
rently unknown state of viral persistence. Probable places of persistence are
tonsils and adenoids. New data suggest that human mucosal T-lymphocytes
may harbor C adenoviruses in a latent form [8].
Premature infants are at high risk to develop disseminated neonatal
adenovirus infection with pneumonia and high lethality [9]. In infants and
children adenovirus infections primarily occur between 6 month and 5
years and are responsible for 4–10% of childhood pneumonias. Outbreaks
in predominantly healthy children are most frequently associated with type
7, followed by types 3 and 21 [10–12]. For Adv 7-infected children at high
risk the mortality rate is up to 40% and 12% in healthy children [13]. Adv
differ in their ability to induce inflammatory response in lung tissue, but
particularly Adv 7 is involved in severe lung inflammation and neutrophil
infiltration [14].
The incidence of Adv infection may vary and depends on the detection
assays and the study population. In general it is higher in children (21%)
than in adults (9%) [15, 16]. In 50% of the described cases a viral coinfec-
tion was recognized [8, 17]. It is likely that Adv infections were caused also
by reactivation of endogenous virus. Further, these infections are not linked
to seasonal pattern.
Adenovirus epidemics do occur in human populations living in crowded
conditions with poor hygiene. A large outbreak, predominantly caused by
types 4 and 7, was observed between 1950 and 1960 in 10% of the recruits
in the US military. Ninety percent of the Adv-infected persons developed
pneumonia. Vaccination against adv 4 and 7 since 1971 has effectively
reduced illness from these serotypes [18]. Recent epidemiological studies
have shown that latent subgroup C viruses are involved in some chronic dis-
eases in immmunocompetent patients which are at high risk for pneumonia
induced by other viral pathogens [19, 20].
Table 3. Examples of epidemiological data of adenovirus pneumonia
Patients Age / Risk factor Viral Pneumonia Adv sero- Study

no manifestation infections % % type
7588 < 5 years / ARD mostly none ~ 40 0,4 ? [17]
1640 < 5 years / LRTI mostly yes 43–50 4–10 7, 3, 2 [13]

immuno-
deficient
75 5–14 years / None 65 12 ? [21]
CAP
Therapy and resistance
Disseminated adenovirus infections are difficult to treat and the agents

used, ribavirin and cidofovir, yielded variable results. The benefit of intrave-
nous ribavirin (a synthetic guanosine analog) treatment in life-threatening
disease especially in immunocompromised patients, seemed to be better if
antiviral therapy was started early. Other reports could not show a clear
beneficial effect. In vivo as well as in vitro data suggest that susceptibility
to ribavirin is highly dependent on the virus species [22, 23]. Viruses of sub-
group C were shown to be sensitive to ribavirin, whereas serotypes of the
subgroups A, B, D, E and F were resistant.
Despite a high level and long treatment in some patients no changes in
sensitivity of the virus isolates against ribavirin to date have been report-
ed.
Cidofovir is a nucleotide analogue of cytosine with an effective in vitro
activity against different DNA viruses, adenoviruses included. Treatment is
indicated for severe, disseminated Adv infections, but results are varying
and treatment is limited by severe nephrotoxicity. Emergence of resistant
Adv has been observed only in experimental systems.
Human coronavirus (HCoV)
Coronaviruses are spherical, pleomorphic enveloped viruses with a diam-

eter of 80–200 nm. They possess the largest genomes of all RNA viruses.
The single-stranded positive RNA is associated with the nucleoprotein N
forming the helical nucleoprotein complex. It is surrounded by the enve-
lope, which contains three characteristic surface structures, the S protein,
the membrane protein M and the envelope protein E. Oligomeres of the
S protein are formed to spikes on the virion surface and resemble a solar
corona. The S protein determines cell tropism, is responsible for pathoge-
nicity and is the strongest inducer of neutralizing antibodies. Some coro-
Table 4. Examples of epidemiologial data of coronavirus pneumonia
Patients Age / Risk factor Viral Pneumonia HCoV Study

no manifestation infections % %
501 < 2 to > 65 years mostly none 37 0,4 -OC43 [25]
ARTI
316 < 65 years CHF, COPD ~ 30 0,6 -OC43, [24]
ARTI -229E
418 > 65 years cardio- ? 2,4 -HKU1 [29]
CAP pulmonary
naviruses contain a fourth envelope protein with a hemagglutinating and

esterase activity. Four serogroups have been distinguished containing three
human pathogens: group 1 with the prototype HCoV-229E, group 2 with
the known HCoV-OC43. In 2003 the severe acute respiratory syndrome
(SARS) led to the detection of a novel coronavirus. The consecutive exten-
sive research in this field led to the discovery of further coronaviruses (see
the following).
After rhinoviruses, coronaviruses are most frequently associated with
the “common cold” (15–30%) in young adults, and they seem not to pose a
risk for healthy elderly. Non SARS coronaviruses rarely cause pneumonia.
However, they may cause diseases of the lower respiratory tract (LRT) in
infants, immunocompromised patients, patients with underlying diseases
and in frail older adults. During endemic outbreaks of HCoV-OC43 and
-229E only elderly patients at risk rarely develop pneumonia [24, 25].
HCoV-NL63 (first described in 2004 in the Netherlands), but although
widespread within the human population it is seldom responsible for pneu-
monia in children [26–28]. Recently, a novel coronavirus HCoV-HKU1
from an 71-year-old patient with COPD and pneumonia was described in
Hong Kong [29]. HKU1-associated pneumonias so far described, occur from
winter to spring, predominantly in the elderly (80% were > 65 years) with
comorbidity [30].
In November 2002, a new emerging disease, SARS was described in
China as a contagious, potentially lethal atypical pneumonia. The SARS-
CoV originated from animal viruses, which could be the result of recom-
bination between mammalian and avian coronaviruses. By June 2003,
worldwide 8447 cases of this illness from > 30 countries were registered with
more than 800 deaths (lethality 9.5%). SARS-CoV seems to have a distinct
cell entry pathway. The initial infection is possible with an extreme low
infectious dose resulting in the generation of proteases in the lung, which
are responsible for a 100- to 1000-fold more efficient rate of infection [31].
In younger children SARS often induces a relatively mild and nonspecific
respiratory illness [32, 33]. In a Chinese study the overall mortality rate was
19.7%, but increased to 78.6% in the patient group with serious underlying
diseases [34].
There is no information about antiviral treatment of HcoV infections and

the rare cases of HCoV associated pneumonia. At present also no standard
antiviral therapy can be recommended for SARS. Ribavirin and cortico-
steroids used in severely ill patients seemed to be effective and a better
outcome was reported after combination therapy with lopinavir/ritonavir,
ribavirin plus steroid [35]. Nothing is known about emergence of resistant
virus variants.
Human influenzavirus / avian influenzavirus
Human and avian influenzaviruses are described in separate chapters within

this book. Only the epidemiological association with CAP and the virologi-
cal diagnosis are discussed in this chapter.
Children (1 to 5 years) and adults with comorbidity, predominantly
chronic heart disease and broncho-pulmonary dysplasia, pregnant women
in the second or third trimester and persons > 65 years are at high risk
for influenza virus-associated pneumonia. Using sensitive methods it has
been shown that the prevalence of influenza infections in older children
with CAP may be higher, and mixed infections, mostly viral/bacterial infec-
tions were documented in up to 35% [3, 21]. Influenza A virus-associated
pneumonia in pregnancy is accompanied by higher morbidity and lethality
and infants may be born preterm with low weight. Although pneumonia in
elderly patients may present only with few respiratory signs, recovery is pro-
longed, especially in the frail elderly, where the incidence of pneumonia is
highest [7, 36]. Mixed infections are more common in the older age groups,
but single virus infections are seen increasingly in the patients over 65 years
[37]. Influenza B virus-associated pneumonia is rare and has been reported
in single cases in children [38, 39].
Table 5. Examples of epidemiologial data of influenzavirus pneumonia
Patients no Age / Risk factor Viral Pneumonia Study

manifestation infections % %
514 CAP 10% premature [40]
23% comorbidity
126 0–1 year 42 0,8
241 1–< 5 years 21 5,8
147 5–16 years 6,8 1,4
75 5–14 years none 65 6,7 [21]
CAP
338 > 65 years CHF 18 11,5 [7]
CAP
Parainfluenza virus (HPIV)
HPIV are pleomorphic enveloped viruses of between 150 and 300 nm.
They contain a helical nucleocapsid with single-stranded negative RNA.
It encodes at least six structural proteins and two nonstructural proteins.
Important are the two envelope glycoproteins HN (hemagglutinin-neur-
aminidase) and F (fusion), responsible for neutralizing antibodies. Within
the family of the paramyxoviruses only HPIV express neuraminidase activ-
ity. Four major HPIV serotypes exist which are divided into subtypes and
genotypes with steadily occurring antigenic variations. Types 1, 2 and 3 are
distributed worldwide, while type 4 is predominantly spread in America.
The four serotypes are distinct in their epidemiological and clinical
behavior. HPIV 3 is endemic throughout the year, but there are yearly epi-
demic outbreaks from winter to spring. HPIV 1 and 2 are associated with a
biennial epidemic pattern with the peak from fall to winter. Parainfluenza-
viruses 1 to 3, particularly type 3, seem to have the highest virulence and the
capacity to persist.
The majority of type 1 infections occur in children between the second
and third year of life, whereas 60% of type 2 are in children younger than
5 years with a peak incidence between the first and second year. They are
most commonly associated with croup or laryngitis, but may also cause
pneumonia. Type 1 can be found in hospitalized, previously healthy adults
and may be involved in bacterial pneumonias.
HPIV-3 infections occur in 40% of young infants in the first year of
life and in most children in the first two years. Pneumonia with type 3 is
seen primarily in the first 6 months of life, similar to RSV, but with lower
frequency [41]. Like RSV HPIV can reinfect both children and adults with
predominantly mild respiratory tract symptoms accompanied by low and
short virus shedding [42, 43].
In childhood CAP parainfluenzavirus 1–3 infections beside RSV and
influenzaviruses are the most common viral pathogens, which are involved
in up to 10% of the diseases [3, 4, 40, 44]. Parainfluenza virus infections
have been reported in the elderly with pneumonia in up to 12% of cases
[43]. Viral CAP in adults with comorbidity like COPD or CHF is character-
ized by a more severe clinical outcome [7, 45]. Viral/bacterial coinfections
(approximately 20%) are observed frequently.
Ribavirin has in vitro activity against HPIV and both aerosolized and intra-
venous ribavirin have been used. There are anecdotal reports about reduc-
tion of clinical signs and viral load in immunocompromised patients, when
treatment was started early after onset symptoms. Ribavirin resistant virus
variants have not been described.
Respiratory syncytial virus (RSV)
RSV is a negative-strand RNA virus. It is pleomorphic and has a size of

150–300 nm in diameter. The genome encodes eight structural and two
nonstructural proteins. The helical capsid is surrounded by an envelope with
three surface glycoproteins, the fusion (F) protein which mediates mem-
brane fusion with the host cell resulting in viral penetration, the G protein
which is responsible for attachment to the host cell, and an SH protein with
unknown function. In contrast to other paramyxoviruses a hemagglutinin
and neuraminidase function do not exist. The G protein has the highest
degree of antigenic diversity in RSV. It accounts for the strain-specific
epitopes and allows the classification into two antigenic groups RSV-A
and RSV-B with multiple genotypes [46]. Immunologically important are
the F and the G glycoproteins inducing protective neutralizing antibodies.
However, immune-protection is not complete and early reinfections occur.
The G glycoprotein is produced as a membrane bound and a secretioned
form. The secretioned form is able to modulate the innate immune response
to RSV and priming with this protein increases the severity of illness after
RSV reinfection.
In children with severe disease caused by RSV the type 2 Th-cell
response seems to be dominant. The G protein induces an unbalanced Th-
cell response, which in the lung could result in airway hyperresponsiveness,
mucus hypersecretionion, and inflammation, and may contribute to periph-
eral blood and pulmonary eosinophilia [47].
RSV infections occur worldwide peaking in the winter months in tem-
perate climates and in the rainy season in tropical climates. A and B viruses
and their multiple variants generally circulate simultaneously within epi-
demical outbreaks. Yearly outbreaks are possible, because the pattern of
the circulating RSV strains changes, depending on the local strain-specific
immunity in the human population [48].
RSV is the most important cause of acute respiratory tract viral infec-
tion in infants. Primary infections are symptomatic with a spectrum of
clinical manifestations from mild upper tract illness to life-threatening
pneumonia. RSV accounts for 50% of all cases of pneumonia during the
first 2 years of life. The peak incidence of RSV lower respiratory tract
infection (LRTI) is between 1 and 6 months of age. Maternal RSV-spe-
cific antibodies rapidly decrease after birth to approximately 6% at 3
months. This may be the reason for the high frequency of RSV infections
before 3 months of age [49]. Premature infants (28 to 32 weeks) are at
risk for 12 to 6 months after birth. All children get infected once until the
age of 2 years, but 50% of them already had experienced re-infections [4,
21, 40, 50–52].
There are numerous independent risk factors for severe RSV infection
including genetic factors that are under discussion. In immunocompromised
children, RSV-related mortality (with very high RSV load) was 15%, and
for children with primary immunodeficiencies 40% [53].
In immunocompetent adults younger than 60 years RSV reinfections are
generally mild and may contribute to 2–4% of the lower respiratory tract
infections. In elderly people reinfections can induce life-threatening pneu-
monitis. Similar to children there may exist specific risk factors for severe
RSV disease. In different studies in adult and elderly patients 3–10% devel-
oped RSV diseases depending on risk factors. The infections accounted for
11% of hospitalizations for pneumonia [54-56].
The development of a prophylactic vaccine against RSV was pursued
with a high priority. The formalin-inactivated RSV vaccine developed in the
1960s unfortunately led to a more severe lung disease in vaccinated children
after a subsequent RSV challenge [57].
Ribavirin is licensed to treat severe RSV-associated diseases in children.

Immune globulin for intravenous administration and a humanized mono-
clonal antibody preparation (palivizumab (Synagis)) are designed to pre-
vent or reduce the severity of RSV infection. It has been shown that in
RSV-infected infants with lower respiratory tract disease ribavirin aerosol
therapy improves clinical outcome, but may be effective in adults as well.
Despite high dose and prolonged treatment in some patients no ribavirin-
resistant RSV variants have been isolated [58].
Human metapneumovirus (HMPV)
This recently identified paramyxovirus is most closely related to the pneumo-

virus RSV, but HMPV differs from RSV in two aspects: it is lacking the non-
structural proteins (NS1 and NS2) and it has a different gene constellation.
HPMV is classified into two main lineages, A and B, based on sequence analy-
ses of the F gen. Further sequence analysis of different HMPV genes includ-
ing the G gene are required for refined characterization of the virus isolates.
The two major groups with numerous genotypes cocirculate throughout the
year. Sometimes even genetically distinct strains of HMPV are circulating
during the same year. Most infections have been detected during late winter
and early spring following the peak activity of both RSV and influenzavirus.
The virus was first detected in young children in 2001 in the Netherlands
[59], but serological studies showed that the virus has been circulating in
Table 6. Epidemiologial data of metapneumovirus pneumonia

90 0–1 years none 47 10 [61]
ARD
208 < 3 years 10% 78 1 [62]
ARD
145 adults COPD / CHF / 19 2,8 [64]
CAP+EA asthma
humans for at least 50 years. HMPV has a worldwide distribution and asso-
ciation with respiratory illness in all age groups. It causes upper respiratory
tract infections, but is also associated with lower respiratory tract infections.
It has been suggested that most severe HMPV infections occur in children
< 2 years of age and seem to peak in the third and fifth months of life, some-
what later than RSV. It is found less frequently in hospitalized children
than RSV and the clinical course may be milder. Based on the presence of
HMPV antibodies approximately 55% of children at the age of two and
100% at the age of 5–10 years had a HMPV history. HMPV like RSV may
cause clinical important reinfections in late childhood and adult life, but the
highest infection rate was found in young adults.
HMPV also can be responsible for pneumonia in premature born babies
[60] and other persons at risk. Although in the study of Maggi et al. [61]
the number of infants with age less than 2 years is small, the majority of
the HMPV-infected children developed pneumonia. The incidence in this
study was higher (33%) than in other reports, but the difference is related
to the difference in the population of children studied [62, 63]. The rate of
bronchopneumonias was higher in children with isolated HMPV infection
than in children with mixed infections. Surprising in this study [61] was the
detection of HMPV RNA in plasma of 41% of HMPV-infected children,
like was shown also for RSV infections.
HMPV pneumonia clinically cannot be distinguished from RSV and
influenza, but the disease seems to be somewhat less severe and there is a
greater percentage of cases with underlying diseases (25%) compared to
the influenza- or RSV-associated pneumonia (< 10%). Small studies demon-
strated that HMPV is a relatively important viral pathogen, which can also
lead to pneumonia (14%), especially in elderly and adults with underlying
disease like COPD, CHF, or asthma [64].
Additional studies will be needed, especially year-long active surveil-
lance over consecutive years with analysis of more data from future respi-
ratory seasons to fully define the clinical and epidemic impact of HMPV
infections.
No antiviral agents or antibody preparations are currently available for the

treatment of HMPV infection. It was shown that ribavirin and a polyclonal
intravenous immunoglobulin had equivalent in vitro activity against both
HMPV and RSV. Treatment may be considered for severe HMPV infection
in immunocompromised patients [65]. No reports about resistant HMPV
exist.
Picornaviruses (human rhinovirus (HRV) / human enterovirus

(HEV))
Picornaviruses are nonenveloped particles and with a diameter of 30 nm

they are very small. They consist of a single positive-strand RNA genome
surrounded by an icosahedral capsid composed of 60 protomers. Each pro-
tomer consists of three nonglycosylated surface proteins (VP1 to VP3) and
internal proteins (VP4). In the center of each protomeric unit is a canyon
where antigenic sites and structures can be found binding to receptors of
the target cells. There are over 100 immunologically distinct rhinovirus
serotypes and two species A and B can be distinguished. Rhinoviruses are
acidlabile and differ in membrane receptor recognition. Further, more than
60 enterovirus serotypes exist.
Rhinoviruses are ubiquitous and infections by relatively low infectious
doses occur throughout the year. They are the most important pathogens of
the “common cold,”, in 80–90% of humans during the peak season in late
autumn. Rhinoviruses are able to infect the lower respiratory tract. It has
been shown in experimental infections that a lower respiratory infection
with rhinoviruses (detected in bronchial biopsy samples) during “common
cold” is not unusual.
Picornaviruses are the most frequently detected virus in respiratory tract
infections in the first year of life. In hospitalized infants with acute expira-
tory wheezing illness respiratory picornaviruses (rhino- and enteroviruses)
are found in 42% of the cases. In older children picornaviruses are predomi-
nant with 65% at the age of 1–2 years and with 82% in children older than
3 years [66, 67].
Earlier studies have not included rhinoviruses, but current data sug-
gest that they are rarely involved in pneumonia in infants, young children
and in older adults. Rhinoviruses can cause pneumonia in children of the
age group 0–6 months, but the highest isolation rate was found in children
between 6 and 12 months with the same frequency as RSV [4, 24, 68, 69].
Coinfections of rhinoviruses and bacteria were found in pediatric patients
with CAP in about 10%. The inflammatory response to rhinovirus infec-
tion is strong and several cytokines are related to pneumonia like IL-6,
which may be an important factor in rhinovirus pathogenesis [68, 70]. In
Table 7. Examples of epidemiologial data of picornavirus pneumonia

502 56% 43 3 [69]
ARTI passive smoking
178 0–6 months 46 2,8
184 6–12 months 40 4.9
96 > 12 months 38 1
254 0,1–17 years ? 62 23 [4]
CAP
316 > 65 years CHF, COPD 30 0,5 [24]
ARTI
a recent review about enterovirus-associated respiratory tract diseases

Rotbart et al. [71] reported, that 13% of infected patients presented with
pneumonia. In contrast, Kellner et al. [69] in a prospective study recovered
enteroviruses only sporadically from children with upper respiratory tract
disease. However, case reports exist about fatal pneumonias in congenital
and neonatal echovirus-infected infants. In these rare and sporadic cases
a maternal disease has been reported in 59 to 68% [71–75]. Epidemic out-
breaks of hand, foot, and mouth disease with enterovirus 70/71 in children
have been observed, where after CNS involvement pulmonary edema
appeared [76].
Using modern diagnostic methods it is increasingly recognized, that
rhinovirus and enterovirus infections are the most common reasons for
unnecessary antibiotic therapy.
The most promising antiviral of the so called WIN compounds is pleconaril

with a broad potent anti-EV and anti-RV activity. It binds to hydrophobic
sites in the base of the capsid canyons and inhibits uncoating of the capsid in
all enteroviruses. Rhinoviruses of the species B, have a significant reduced
susceptibility to pleconaril. For therapeutic application it will be important
to differentiate between natural occurring resistance to pleconaril in B rhi-
noviruses and the emergence of RV resistance under pleconaril treatment
[77].
Pleconaril has been shown to be effective in experimental studies and
to significantly reduce clinical symptoms in treated adult volunteers. It is
available for life threatening enterovirus infections in immunocompromised
patients. Ten percent of the enterovirus isolates have been shown to be
resistant to pleconaril and some patients did not respond (resistant virus
has been identified). Ruprintrivir is an inhibitor of the virally encoded 3C
protease, which is still under investigation for treatment of rhinovirus infec-

tion in immunocompetent patients [78].
Hantavirus (HV)
Hantaviruses are enveloped, predominantly negative-strand RNA viruses

(ambisense). The genome of these viruses consists of three different single-
stranded RNA segments. The segments L, M and S encode the viral poly-
merase, a glycoprotein processed into G1 and G2 glycoproteins located in the
envelope, and a nucleocapsidprotein. Virus particles are of spherical shape
with a diameter of 80–120 nm, but also elongated forms are seen (170 nm).
Hantaviruses are important zoonotic pathogens, primarily of rodents.
The infections are per-sistent and most of them seem to be asymptomatic
in the natural rodent hosts. Antibodies against hantaviruses are also present
in nonnatural hosts, other wild and domestic animals. Beside the transmis-
sion to humans through human/rodent contacts, infections are acquired by
inhalation of virus-contaminated aerosols of rodent excreta (saliva, urine or
feces) [79]. An occasional transmission may occur from person to person as
was documented for Andes virus in Argentina.
Some hantaviruses belong to the so called emerging pathogens. They
cause an influenza-like acute pulmonary disease in North, Middle and
South America, the hantavirus pulmonary syndrome (HPS). The first HPS
was recognized in 1993 in the USA and was caused by a number of hanta-
virus variants, e.g. Sin Nombre virus (SNV). Symptoms of HPS may vary,
depending on the virus genotype. In 1995, cases of HPS were reported in
South America and a new strain – the Andes hantavirus – was identified. In
late 1999 and early 2000 first outbreaks through an again novel hantavirus,
the choclo virus, were documented in Central America. The overall mortal-
ity was about 44%, but is declining as a consequence of better recogniz-
ing less severe forms of the infections and a better medical management.
Confirmed cases of HPS so far include children, but in the majority of cases
adults (19 to 58 years). SNV accounts for a small number of pediatric cases
in the US, whereas Andes virus was found in pediatric patients (Chile, South
Argentina) in a higher proportion (16%) [80–82].
The incubation period for Andes virus in Chile was 5 to 25 days. Febrile
prodromi last for approximately 4 days followed by a rapid progression to
moderate to severe respiratory distress.
For hantavirus diseases no established specific therapy is currently available.

Ribavirin shows activity against HV, but this has not been well documented
in studies. Emergence of resistant HV is unknown.
Nonconventional respiratory viruses
Measles virus
In developing countries the frequent complications of measles virus infec-

tion are responsible for the mortality rate. Measles virus infection causes
a transient and strong immunosuppression, which is the reason for the
increased susceptibility to other viral or bacterial infections. In 3-4% of the
infected patients measles virus can cause pneumonia, which can be pres-
ent either as primary measles virus pneumonia or as atypical measles virus
pneumonia, but most patients develop secondary bacterial pneumonia.
Measles pneumonia rarely occurs in young adults as shown in 1976 to 1979,
when a measles outbreak in US Air Force recruits was responsible for 106
cases with pneumonia (3.3%), of which two-thirds had a secondary bacterial
infection [83]. During pregnancy measles virus infections induce a higher
number of pneumonias in the mother [84, 85]. There are a few reports about
preterm and newborn infants in which measles pneumonia was observed
[86].
Herpes viruses
After primary infection herpes viruses are able to persist lifelong in their
human host, typically as latent infection, but they also reactivate under
immunosuppression and then some of them, like CMV, HSV, VZV, EBV
and HHV6, may induce pneumonia.
HSV-1 pneumonia may occur occasionally in high-risk persons. Rarely
disseminated HSV infections during pregnancy have been reported, as in
the case of a previously healthy womn with a fatal progressive HSV-2 pneu-
monia in the third trimester of pregnancy [87]. Ramsey et al. reported 20
patients with pneumonia, in which they could differentiate between cases
with focal HSV pneumonia as a result of HSV spreading to the lung paren-
chyma and cases with interstitial pneumonia as a result of a hematogenous
dissemination of HSV [88].
In immunocompetent adults primary VZV infection is uncommon, but
the incidence is increasing (5-10%) and VZV pneumonia is the main com-
plication (incidence 5.5%–16.5%) with high mortality. Some patients may
develop secondary bacterial pneumonia. Most patients (76.7%) do have
at least one known risk factor like pregnancy, smoking or chronic obstruc-
tive pulmonary disease [84, 89–91]. VZV beside influenzavirus is the most
common pathogen, causing pneumonia during pregnancy, predominantly in
the second and third trimester. The risk of primary VZV infection for VZV
pneumonia during pregnancy (0.1–18.3%) is higher if patients are smokers
or manifest multiple (> 100) skin lesions. The mortality rate before a possi-
ble antiviral intervention was significantly higher in pregnancy (41% versus
1.5–12.1%) [89, 92, 93]. VZV pneumonias as consequence of a primary VZV

infection in childhood are rare [94].
CMV-infected preterm infants can develop a chronic lung disease, which
may be associated with CMV pneumonia with high mortality. Ganciclovir
treatment has been shown to rapidly improve symptoms, a fact that sup-
ports CMV causality of pneumonia [95–97]. Extremely rare are reports
about CMV pneumonia in previously healthy adults [98]. In patients with
lymphoma CMV pneumonia is less common and the incidence after chemo-
therapy and corticosteroid application is approximately 1% with a mortality
rate of 30% [99].
Two genetically distinct variants HHV-6A and -6B do exist, but in
primary infection the variant -6B is dominant. During primary infection
a severe respiratory disease is an extremely rare complication. In a case
report from Knox et al. [100] a fatal pneumonitis due to HHV-6 infection
is documented in an infant with severe T lymphocytopenia. Whereas for
immunocompromised patients (BMT) HHV-6 pneumonia is documented,
in immunocompetent patients an etiologic role of the reactivated HHV-6
infection in pneumonia is not clearly defined [101, 102]. Another report of
an extremely rare case with HHV-6-associated pneumonia is documented
by Merk et al. [103], where an apparently immunocompetent young women
developed a fatal pneumonia.
A mild asymptomatic pneumonitis is described in 5–10% of the cases
with infectious mononucleosis, but a severe pneumonitis as result of pri-
mary EBV infection is rare in immunocompetent patients [84, 104, 105].
In patients with lymphocytic interstitial pneumonia EBV DNA has been
found in lung tissues of infants, children and of adults as well [106–108].
In autopsy cases with diffuse interstitial pneumonia EBV DNA could be
detected in leukocytes and pneumocytes and frequently in airway epithe-
lial cells [109]. The EBV-associated lymphocytic interstitial pneumonia
without HIV infection has been reported predominantly in adults. This
is also true for a rare chronic interstitial lung disease due to EBV, mainly
seen in adults, but also shown in two infants in early months of life [106,
110].
Diagnosis of viral pulmonary infection
Especially in adult patients respiratory virus infections are extremely under-

diagnosed since adequate and possible virological diagnosis is not routinely
performed.
For diagnosis of acute respiratory infections the detection of infec-
tious viruses or viral components (proteins, nucleic acid) is the method
of choice, whereas antibody detection is not relevant and should only be
used in selected patients at later timepoints after infection using paired
sera.
Specimen
Nasopharyngeal aspirate (NPA) is the gold standard for the detection of all
major respiratory viruses, predominantly adopted in infants and children.
NPA is taken by suction of cell-containing mucosal secretion from the naso-
pharyngeal area. Approximately 0.5 ml fluid should be collected into 2 ml
of viral transport medium.
Nasal lavage can be obtained with less discomfort for the patient by gently
instilling 2 ml PBS at room temperature into each nostril and by simultane-
ously suctioning into a sterile trap; the sensitivity for virus detection is compa-
rable to that of NPA, but is controversially reported for RSV [111, 112].
Nasopharyngeal swab, taken usually from adults, is obtained by deep
bilateral nasal and posterior pharynx swabs with sterile cotton swabs, which
should then be placed in 3 ml viral transport medium (VTM) [113]. Calcium
alginate swabs or swabs with wooden sticks may contain substances which
inactivate some viruses and inhibit PCR testing and should not be used!
Tracheal aspirate can be collected from intubated patients after instil-
lation of 4–10 ml sterile normal saline into the endotracheal tube and by
suctioning into a sterile trap; this is the most easily obtained specimen from
lower respiratory tract in these patients [114].
Induced sputum should be collected in the morning after rinsing mouth
and throat with sterile hypertonic saline. Thereafter sputum has to be col-
lected in a sterile container. Induced sputum represents a more complete
sampling of the respiratory tract and is comparable with nasopharyngeal
aspirate specimens. While induced sputum might be used for virus detection
from the lower respiratory tract, the obligate admixture of saliva with the
accompanied flora is an obvious disadvantage. Nevertheless, with minimal
saliva contamination it is a valuable alternative material to BAL [115, 116].
Bronchioalveolare lavage (BAL) has a high diagnostic yield for infec-
tious pathogens predominantly in immunocompromised patients with lung
infiltrates.
Transbronchial biopsy (TBB) / lung biopsy. This invasive technique can
in addition to BAL significantly improve the diagnostic yield [117].
EDTA blood in immunosuppressed patients may facilitate diagnosis of
some viruses (e.g. adenovirus, CMV) and disseminated virus infections and
determination of the virus load may be predictive for disease and outcome;
furthermore, virus load is important for monitoring of therapy.
Methods for detection of viruses
Direct virus detection (rapid antigen detection / nucleic acid assay)
Enzyme/immunoassays (EIA) for rapid viral antigen detection in airway

secretions exist for almost all major respiratory viruses, but have been often
evaluated only for children and may vary in sensitivity and specificity (range
between 60 and 95%). Results from EIAs for detection of single respira-
tory viruses are usually available between 10 min to 3 h. Alternatively a
screening for respiratory viruses in an airway secretion can be achieved by
indirect immunofluorescent antigen assay (IFA) (sensitivity 85–95%; speci-
ficity 95–99%) using a pool of monoclonal antibodies, whose specificity is
directed against RSV, influenzaviruses A, B, parainfluenzaviruses 1, 2, 3 and
adenovirus. The examination of several cell spots of a cytospin preparation
on slides with pooled and single antisera allows viral antigen detection and
typing within 2 h. The results are strongly dependent on the quality of the
clinical material. For elderly hospitalized and immunocompromised patients
with virus associated LRTI the rapid virusantigen detection is insensitive.
When rapid conventional methods are not available, like for coronavi-
ruses, metapneumoviruses, rhinoviruses or enteroviruses, the PCR has to be
established.
PCR
The PCR may be used alternatively to the mentioned antigen detection

assays. It offers a great sensitivity and can be used for a wider range of viral
pathogens. In principle PCR can detect and differentiate several viruses
simultaneously in a single reaction mixture as multiple RT-PCR (multiplex
PCR), but it provides a lower sensitivity than PCR for single pathogens and
is more difficult to establish. For early detection and monitoring of viral
infections the quantitative PCR represents a sensitive technique that deliv-
ers results within 3 h.
For diagnosis of disseminated virus infections with pulmonary manifes-
tations frequently caused by adenoviruses, RSV in infants or nonconven-
tional respiratory viruses, like CMV additional analysis of plasma or EDTA
blood is helpful.
Detection of infectivity (short term culture / isolation)
Clinical specimen namely secretions of the respiratory tract can be used for
an infectivity assay in centrifuge enhanced shell vial culture (SVC). After
inoculation of airway specimens on different cell systems, which are sus-
ceptible for many of the respiratory viruses, cell cultures are evaluated for
early virus infection after 24–72 h with a pool of virus-specific monoclonal
antibodies and afterwards for typing with the respective individual mono-
clonal antibodies by IFA.
Once isolated, the virus must be typed again preferentially with IFA
using virus-specific monoclonal antibodies or otherwise increasingly by
PCR.
Conventional cell culture with virus isolation has no impact on clinical

decision and management of patients during hospitalization. Virus isola-
tion is useful for a characterization of virus strains involved in epidemic
outbreaks.
Antibody detection
Serology is not useful during the acute phase of infection. Due to the
delayed onset of the antibody response, approximately at the end of the
first week of disease serological methods can detect antibodies. To use
only serological methods for diagnosis of viral infections in CAP is not at
all sufficient. Paired serum samples may be used to detect seroconversion
or a fourfold increase in antibody titer comparing the first and the second
serum.
Serology in addition is of limited value in newborns with maternal anti-
bodies, in immunocompromised patients, in the elderly and patients receiv-
ing blood products. Antibody detection can be done with the complement
fixation test (CFT, increasingly obsolete), ELISA-IgG, -IgA, -IgM, immuno-
fluorescent assays, immunoblot and neutralization test.
Diagnostic comments on specific viruses
Adenovirus
Viruses can be detected in 25 to 72% of patients with disseminated infection

in peripheral blood for more than 3 weeks. Detection of virus from multiple
sites correlates with more severe disease and higher mortality. It is essential
to monitor viral load during antiviral therapy. Phenotyping of isolates can
be done by neutralization test, by hemagglutination test or by serotype-spe-
cific monoclonal antibodies and finally by PCR.
Group-specific antigens are used in CFT for antibody detection in the
second week of disease. CFT is insufficient to detect antibodies in infants.
IgG antibodies are detectable by ELISA at the end of the second week,
whereas IgM antibodies are not detectable in all cases with primary infec-
tion [10].
Coronavirus
HCoVs antigen can be detected by IFT using rabbit antisera (no monoclo-
nals available). Coronaviruses RNA can be detected with RT-PCR also in
stool. SARS-CoV can be detected in plasma, but in urine and stool longer
than 4 weeks. Virus load is unusually low in the early phase of SARS. A
chip-based test detecting 10 respiratory viral pathogens including coronavi-

ruses has been developed. Non-SARS human coronaviruses are difficult to
isolate. With SARS-HCoV no IgG and IgM antibodies can be found within
the first 7 days of disease, seroconversion may be delayed up to 8 weeks and
IgG does not persist [34, 118, 119].
Influenzavirus
Rapid antigen detection tests are available (15 min) which differentiate
between A and B viruses and some now include avian influenza virus H5N1.
Sensitivity and specificity range from 60–95% and 52–99% respectively. RT-
PCR is much more sensitive and allows virus typing. Virus isolation is still
important for characterization of circulating influenza strains.
Antibody detection with ELISA-IgG, -IgA and IFA-IgG, -IgA is pos-
sible, but shows low levels of both IgG and IgA, a delayed peak antibody
titer and shorter persistence of antibodies in elderly patients.
Parainfluenzavirus and respiratory syncytial virus
In adults and elderly people virus shedding is lower and shorter. RT-PCR
is more sensitive in patients with parainfluenzavirus and RSV infections,
particularly reinfections, and it might be useful especially for rapid diagnosis
in elderly.
For rapid RSV antigen detection in pediatric patients different antigen
detection assays (20 min) with sensitivities between 61 to 92% and specifici-
ties between 93 to 98% are available [120]. Alternatively the rapid immuno-
fluorescent antigen assay can be used.
Since RSV is thermolabile, it is important to use a qualified transport
medium, and to have a cooled and short transport until processing (30
min). The shell vial culture assay shows the highest sensitivity (94.3%) and
specificity (96.9%) for detecting RSV from NPA in children younger than
1 year.
Only 41% of RSV-infected children could be identified by serology and
antibodies titers that develop are usually low, although both serum and
secretory antibodies are produced [121].
Human metapneumovirus
Commercial monoclonal antibodies are available for IFA. The sensitivity

and specificity of this test is 73.3 and 97% respectively and results agree
with RT-PCR in 89.6% [122].
Isolation of HMPV is difficult and regardless of CPE the use of RT-PCR

to enhance the HMPV identification in cell culture is indicated.
No commercial tests are available for antibody detection.
Picornaviruses (rhinovirus / enterovirus)
Due to more than 100 distinct serotypes a rapid detection of rhinovirus

capsid antigen cannot be developed. The application of molecular assays has
markedly increased the detection rate of picornaviruses in acute respira-
tory infections. Rhinovirus culture is the “gold standard,” but it takes 3 to
7 days. The sensitivity of PCR is superior to the infectivity detection in the
cell culture.
No reliable typespecific serological test for rhinovirus infections exists.
Entervoirus serology by neutralization tests is possible but difficult and not
completely reliable.
Hantavirus
Currently no antigen detection assay is available. RT-PCR for hantavirus

RNA detection is useful to detect infection and to identify the viral geno-
type. RT-PCR for viral RNA detection can be done from whole blood or
serum during the acute phase in the first 10 days of illness.
Virus isolation is inefficient and there is no routine assay.
Serum specimens were tested for IgM and IgG antibodies by ELISA
using Sin Nombre virus antigen, according to the guidelines of the US
Centers for Disease Control (CDC). Positive results indicate infections with
new world hantaviruses. IgM antibodies can be detected in all acute cases,
maximal IgG level occurs during the first week of illness and are detectable
after disease for a relatively long period.
HSV, VZV and CMV
Since HSV, VZV and CMV pneumonia can be effectively treated it is

important to rapidly investigate specimens from the lower respiratory
tract, because pneumonia can only be established on the basis of BAL or
lung tissue examined by PCR (HSV, VZV) and/or shell vial culture (HSV).
Serologic tests are not relevant in case of HSV reactivation.
Primary VZV infection can be confirmed by IgM detection and by IgG
seroconversion.
In premature infants with risk for CMV pneumonia CMV DNA can be
found directly by PCR in urine and in the pharynx. Virus detection in air-
way specimens (virus load in BAL) and lung tissue will be positive before
seronconversion.
Summary
In various studies and case reports it has been shown that respiratory viral
pathogens are frequently involved in community-acquired pneumonia. Age
groups at risk are the very young and elderly, as well as persons with co-
morbidity, where immune response is restricted.
The frequency of detection of respiratory viruses varies in different stud-
ies due to methodological problems, patient selection and the fact that only
detection of specific viruses was performed. With modern diagnostic tools
it has been increasingly realized that rhinovirus and enterovirus infections
are the most common reasons for unnecessary antibiotic therapy, often also
adenovirus infections. Future studies have to consider the whole range of
viral agents that can be involved in community-aquired pneumonia.
In recent decades several new respiratory viruses have been described
and it is reasonable to assume that new viruses or virus types from differ-
ent virus families will emerge in the future, often after having crossed a
host species barrier with high pathogenic potential for severe pulmonary
diseases.
Most of the relevant infections can be diagnosed by virus detection.
Several studies in children have documented a significant proportion
(25 to 30%) of viral/bacterial infections beside isolated virus infections.
Children with isolated virus infection tended to be younger than coinfected
children. The implications of coinfecting agents in epidemiology, pathoge-
nicity and clinical outcome have to be elucidated.
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Resistance in Streptococcus pneumoniae
Mathias W. R. Pletz1, Lesley McGee2 and Tobias Welte1

1Department of Respiratory Medicine, Hannover Medical School, Carl- Neuberg-Str.1,
Hannover, 30625, Germany; 2Hubert Department of Global Health, Emory University, 1518
Clifton Road, Atlanta, GA, 30322, USA
Abstract
Streptococcus pneumoniae is a leading cause of community-acquired lower respiratory
tract infections, sinusitis, meningitis, and bloodstream infections. Pneumococci are Gram
positive, encapsulated bacteria and exhibit more than 90 different capsular serotypes.
Resistance to penicillin in clinical isolates was reported anecdotally as early as 1965,
but was not considered a major concern until the mid-1990s. In the 1990s, there was a
tremendous global increase in resistance to penicillins and this led to the increased use
of macrolides and tetracyclines to treat infections. After several years, the resistance rates
to these antibiotics began to increase as well. Currently, fluoroquinolones are used most
frequently to treat community-acquired respiratory infections in adults and resistance
rates globally are still low.
Pneumococci are naturally competent bacteria and frequently acquire resistance by
intraspecies or interspecies gene transfer. Resistance to `-lactams is due to the acquisi-
tion of different mutations within the pencillin-binding proteins that have been demon-
strated to originate from the less pathogenic viridans streptococci. Other mechanisms of
antibiotic resistance include enzymes and efflux pumps on mobile genetic elements (e.g.
erm and mef), or resistance arising through spontaneous mutations. Clinical studies show
that resistance, particularly to penicillins, is not always related to clinical failure.
The global increase in resistance rates in pneumococci is in part due to the spread of
a limited number of highly sucessful multiresistant pneumococcal clones. Isolates belong-
ing to a specific clone, defined by sequence types according to multilocus sequencing,
often exhibit the same serotype. However, capsular switching due to genetic rearrange-
ments within the same clone has been observed. The recently introduced seven-valent
conjugated pneumococcal vaccine has been shown to decrease disease and carrier rates
of the included serotypes. Since some of the multiresistant clones exhibit vaccine sero-
types, resistance rates to penicillin, macrolides and fluoroquinolones have been decreas-
ing since the introduction of this vaccine.
INTRODUCTION
S. pneumoniae is the most frequent pathogen causing community-acquired

pneumonia and for many years penicillin or ampicillin were the drugs of
choice for all pneumococcal infections.
58 Mathias W. R. Pletz et al.
In contrast to other bacterial species, such as staphylococci or Gram-

negative bacteria, pneumococci had remained susceptible to almost all
antibiotics for decades. Penicillin-resistant strains were selected in the labo-
ratory already in the 1940s, but it took more than 30 years before the first
clinical isolates with penicillin-resistance were detected. The subsequent
global emergence of penicillin-resistance in the 1980s and 1990s led to the
increased use of macrolides, other non-beta lactam antibiotics and the fluo-
roquinolones. Due to these new selective pressures, resistance to macrolides
and tetracyclines emerged rapidly. For both classes it could be demonstrated
that the increase in resistance rates paralleled the use of these individual
classes of antibiotics [1]. It had also been shown that this trend could be
reversed when the use of the concerned antibiotic was restricted [2]. This
data explains, in part, the considerable differences in rates of antibiotic
resistance published between different countries and reflects the antibiotic
prescription behavior. In general, antibiotic resistance in countries where
antibiotics can be purchased without a prescription (over the counter) as in
Spain and Mexico, are higher than in countries where antibiotics can only
be prescribed by physicians. Recently, resistance to fluoroquinolones has
emerged, but there are global differences reported with individual cases
described in Austria and rates as high as 13.3% in Hong Kong [3, 4]. Due to
the tremendous cost for the development of antibiotics and the challenges
associated with the safety of newly developed drugs, many pharmaceutical
companies have discontinued their development of new antibiotics. During
the last few years only a limited number of new antibiotics were licensed,
such as telithromycin, linezolid and quinupristin-dalfopristin.
Clinical pneumococcal isolates with resistance to quinupristin-dalfopris-
tin, telithromycin and linezolid have already been reported [5–7]. In this
context it is also of concern that pneumococci with tolerance to vancomycin,
an antibiotic used as a reserve against multi-resistant Gram-positive bacte-
ria, have already been documented [8].
Resistance rates parallel antibiotic usage and exhibit therefore tre-
mendous regional differences and trends [3, 9]. Besides numerous local
and regional surveillance studies, several large longitudinal surveillance
projects monitor globally the trends of antibiotic resistance in pneumococci
(i.e. the Alexander project, Prospective Resistant Organism Tracking and
Epidemiology for the Ketolide Telithromycin – PROTEKT). Table 1 shows
resistance rates in eight European countries according to results of the
PneumoWorld study from 2001–2003 [3].
Genetic background
Bacterial resistance may be “intrinsic” or an inherent – naturally occurring

property of an organism, i.e., these organisms may lack the appropriate
drug-susceptible target or possess natural barriers that prevent the agent
Resistance in Streptococcus pneumoniae 59
Table 1. MIC90 and antibiotic resistance of 2,279 isolates of S. pneumoniae in eight European
countries according to Reinert et al. [(3] (Abbreviations: I, intermediate; R, resistant)
Country (no. of strains) Antibiotic MIC90 % Resistance (I+R)

Austria (n = 160) Penicillin G 0.03 4.4
Cefuroxime 0.06 0.6
Clarithromycin 0.25 10.0
Levofloxacin 1 0
Tetracycline 4 10.6
Belgium (n = 148) Penicillin G 0.125 11.5
Cefuroxime 0.25 8.8
Clarithromycin 32 23.7
Levofloxacin 1 0.7
Tetracycline 32 23.7
France (n = 443) Penicillin G 2 47.6
Cefuroxime 8 39.1
Levofloxacin 1 0.9
Germany (n = 530) Penicillin G 0.03 6.0
Cefuroxime 0.06 1.5
Levofloxacin 1 0.4
Tetracycline 4 11.3
Italy (n = 462) Penicillin 0.25 13.0
Cefuroxime 0.5 7.4
Levofloxacin 1 1.3
Portugal (n = 174) Penicillin G 2 19.0
Cefuroxime 8 14.4
Levofloxacin 1 1.2
Tetracycline 8 12.1
Spain (n = 310) Penicillin G 2 61.9
Cefuroxime 8 43.9
Levofloxacin 1 1
Switzerland (n = 52) Penicillin G 0.5 17.3
Cefuroxime 2 11.5
Clarithromycin 0.25 17.3
Levofloxacin 1 0
Tetracycline 4 13.5
from reaching its target. Acquired resistance results either from spontane-
ous mutations in existing DNA or the acquisition of foreign genetic material
by genetic elements such as plasmids and bacteriophages (horizontal gene
transfer).
To date, no plasmids have been detected in any resistant S. pneumoniae
strain and genetic changes conferring resistance are therefore all chromo-
somal. Pneumococci are one of the few bacterial species that are naturally
competent and are able to acquire resistance genes through horizontal gene
transfer, usually via the process of transformation.
Pneumococci are able to bind, internalize and integrate free DNA by
recombination without antecedent stimulation as it is, for example, neces-
sary for E. coli. The origin of the genetic material for these transformations
may be from the same (intraspecies recombination) or a different – but
closely related – species (interspecies recombination). The results of inter-
species recombination result in mosaic genes, which are genes consisting
of both pneumococcal sequences and sequences from other species. The
higher the degree of homology between the donor and recipient DNA the
higher is the likelihood of successful homologous recombination. Therefore
the amino acid sequence of the donor and recipient DNA is often identical.
In contrast, due to the degeneration of the genetic code the 3rd nucleotide
of a codon coding for the same amino acid can be different. The resulting
“codon usage bias” is species specific. A characteristic feature of integrated
DNA derived from another species is a repeated mismatch of the 3rd
nucleotide leading to a decreased similarity of the recombined sequence
when compared to a reference sequence (Fig. 1). Viridans streptococci have
been frequently observed as a donor species for the transfer of genetic
resistance determinants to S. pneumoniae. Viridans streptococci belong to
the commensal flora and are exposed to the antibiotics administered to the
host and resistant strains should therefore be easily selected. Since viridans
streptococci are to a high degree homologous with S. pneumoniae, recom-
bination between the two species exhibits a high success rate. In addition,
both species colonize the human nasopharynx and so contact between the
two species is guaranteed, which is necessary for the exchange of genetic
material. Since commensal bacteria rarely cause disease, they are not rou-
tinely tested for resistance and surveillance studies addressing resistance in
commensal bacteria are rare. In pneumococci acquisition of foreign genetic
material and the consequent spread of resistant strains seems to be the pri-
mary cause for the increase in resistance to different antibiotics.
Spread of resistance in pneumococci – the clone concept
The global spread of penicillin-resistant pneumococci has been documented

by numerous surveillance studies. Genetic fingerprinting using techniques
like pulsed-field gel electrophoresis of pneumococcal DNA digested by
Figure 1. Alignment of PBP 2b of a penicillin-resistant S. pneumoniae strain (#402) with the sus-
ceptible pneumococcal laboratory reference strain R6. The red part of the sequence is derived
from S. oralis. (Courtesy of Dr B. Beall, CDC)
the restriction enzyme SmaI, has revealed that the majority of the resistant
clinical isolates belong to a small number of highly successful clones, some
of which have spread globally [10]. More recently, another tool to investi-
gate the genetic relatedness of resistant isolates became available, namely
multi-locus sequence typing (MLST) [11]. MLST is based on the sequencing
of seven highly conserved “housekeeping genes.” The sequences at each of
the seven loci are compared with all of the known alleles at that locus which
is available at the pneumococcal MLST web site (http: //spneumoniae.mlst.
net/). The alleles at each of the seven loci define the allelic profile of each
isolate and their sequence type (ST). If isolates have identical sequence
types, or differ at less than three loci, they are considered to be related.
Many studies have used PFGE for the rapid clustering of genetically
related isolates and MLST to assign nonsubjective and electronically por-
table clone identifiers to clusters of related isolates.
In order to create a nomenclature to monitor the international spread of
resistant pneumococci, in 1997 the Pneumococcal Molecular Epidemiology
Network (PMEN) was established under the auspices of the International
Union of Microbiological Societies with the aim of characterizing, stan-
dardizing, naming, and classifying antibiotic-resistant pneumococcal clones
(http: //www.sph.emory.edu/PMEN/) [12]. Currently, the PMEN describes 26
international clones, some of which have a global distribution and have been
reported in many countries with varying antibiograms. The global spread of
the PMEN-clone 1, Spain 23F-1, is illustrated in Figure 2. Pneumococci have
the ability to switch their capsular serotypes which can be due to mutations
or the exchange of capsular genes. For surveillance it is therefore not appro-
priate to equal serotype and sequence type (clone) since capsular variants
of clones exist, e.g., 19F capsular variants of PMEN-clone 1 is designated
Spain 23F-1-19F.
Figure 2. Global distribution of the PMEN strain Spain23F-1.
Serotype switching serves also as a mechanism to avoid the selective

pressure due to vaccination with vaccines containing only a limited number
of serotypes.
PMEN clones are spreading more successfully than other pneumo-
cocci. The reason for that fitness advantage is not yet clear, but it has been
observed that resistance to some classes of antibiotics has emerged rapidly
once it has been introduced into these clones [13].
Beta-lactams
Beta-lactams bind and inactivate the so-called penicillin-binding proteins

(PBPs). These proteins are enzymes that catalyze the terminal stages of
murein synthesis which is the main component of the bacterial cell wall [14].
The action of `-lactams leads to cell death, i.e. the action is bactericidal. Most
of the bacterial species exhibiting `-lactam resistance secrete or contain `-
lactamase enzymes that cleave the `-lactams. In contrast to other bacterial
species, `-lactam resistant pneumococci do not express any `-lactamases.
The pneumococcal mechanism of resistance to `-lactams consists of mul-
tiple mutations within several penicillin-binding proteins (PBPs)[15]. These
mutations mediate changes in the structure of the penicillin-binding domain
of the PBP leading to a decreased affinity to `-lactams. Six types of PBPs
are found in pneumococci: PBP 1a (92–100 kDa), PBP 1b (89–95 kDa), PBP
2x (85 kDa), PBP2a (80–81 kDa), PBP 2b (77–78 kDa), and PBP 3 (43–52
kDa) (K 64, 117). High-level resistance to third-generation cephalosporins
has occurred primarily by the development of altered forms of PBP1a and
2x, whereas high-level penicillin resistance additionally requires alterations
of PBP2b [16]. It has been found that the genes of low-affinity forms of
PBPs 1a, 2b and 2x from clinical isolates contain sequence blocks with up
to 20% divergence in their nucleotide sequence. Such sequence blocks have
not evolved by spontaneous mutations and the majority of these altera-
tions do not confer amino acid substitutions (silent mutation). The mosaic
blocks are shared between resistant S. pneumoniae and the closely related
viridans streptococci (S. oralis, S. mitis). Based on these observations, it has
been assumed that `-lactam resistance developed originally in viridans
streptococci and was transferred to pneumococci by interspecies recom-
bination [17]. This hypothesis is supported by the fact that genes adjacent
to the pbps (e.g., ddl) are frequently part of the recombination and appear
in resistant strains. This was recently observed in very high level penicillin
resistant (MIC * 8 mg/l), invasive pneumococcal isolates from the CDC’s
ABC Surveillance study. The ddl locus in two of these strains was from S.
oralis, suggesting that the origin of very-high-level resistance in some strains
may result from the transformation and incorporation of resistance deter-
minants from viridans group streptococci [18].
There is evidence that altered PBPs from resistant pneumococci have a
preference for branched peptides in regard to the cell wall synthesis [19].
This is in contrast to PBPs from susceptible strains which use primarily
linear stem peptides. A newly identified protein MurM which, together
with MurN, is involved in the synthesis of short peptide branches in the
pneumococcal cell wall. Cells in which MurM was inactivated produced cell
walls without branches and also completely lost penicillin resistance [20].
Currently, the role of MurM in `-lactam resistant strains is the subject of
further studies.
Macrolides
Macrolides consist of saccharides that are attached to 14-membered (eryth-

romycin, clarithromycin), 15-membered (azithromycin) or 16-membered
rings. The use of the latter is usually restricted to veterinary purposes.
Macrolides act bacteriostatically by binding to the 23S ribosomal RNA
of the 50S subunit of the bacterial ribosome and consequently blocking
the elongation step of protein synthesis. There are two main mechanisms
of macrolide resistance: active efflux and target site modification. Active
efflux is performed by an energy dependent cell membrane transporter
protein that is encoded by the mef gene, which is located on a conjugative
transposon. The mef gene provides lower levels of resistance and confers
the so-called M-phenotype, i.e. resistance primarily to macrolides. Two vari-
ants of the mef gene, mef(A) (primarily found in S. pyogenes) and mef(E)
have been identified in pneumococci. However, due to confusion in the
nomenclature many authors do not differentiate between the two and call
it mef(A) regardless of the variant. Target site modification is performed by
a methylase enzyme which adds a CH3-group to an adenine residue on the
23S rRNA. The methylation blocks the binding site of not only macrolides,
but also lincosamides and streptogramins and the resulting phenotype is
called MLSB. The methylase enzyme is encoded by the erm gene which
is also located on a transposon. Among bacterial species there exists sev-
eral variants of the erm gene, in pneumococci primarily the erm(B) gene is
found. Some macrolide resistant pneumococci have been documented to
exhibit both the erm and mef genes. These strains appear to be highly clonal
and most belong to the PMEN clone Taiwan19F-14 [21].
A third, currently emerging mechanism of resistance to macrolides
consists of mutations conferring amino acid substitutions in the ribosomal
proteins and nucleotide mutations in the 23S rRNA itself. Frequently, the
ribosomal proteins L4 and L22 are involved and mutations in the rRNA and
ribosomal proteins can confer new resistance phenotypes with combined
resistance to macrolides and lincosamides (ML) or macrolides, ketolides
and streptogramins (MKSB). Usually, the decrease in susceptibility to
ketolides (for example telithromycin) is less than the decrease in suscepti-
bility for other MLKS(B) agents [22].
Tetracyclines
Tetracyclines are antibiotics derived from Streptomyces species and inhibit

bacterial protein synthesis by blocking attachment of the aminoacyl-tRNA
to the ribosome. Resistance to tetracyclines in pneumococci is conferred
by ribosomal protection proteins Tet(M) and Tet(O). These proteins are
homologous to the elongation factors EF-Tu and EF-G. It is assumed that
Tet(M) and Tet(O) induce the detachment of tetracyclines from the bacte-
rial ribosome, the detailed mechanism of action is not known. Tet(M) and
Tet(O) are encoded by the tet(M) and tet(O) genes that are located on a
transposon. In species other than pneumococci, resistance to tetracyclines is
frequently mediated by plasmids.
Fluoroquinolones
Resistance to fluoroquinolones in pneumococci is caused by efflux and/or

by mutations in the quinolone resistance-determining regions (QRDR)
of the genes coding for type II topoisomerase enzymes: DNA gyrase and
topoisomerase IV.
Mutations conferring resistance occur in a stepwise fashion, with
mutations observed in either parC or gyrA (depending on the selecting
fluoroquinolone) or both, leading to decreased fluoroquinolone suscepti-
bility [23]. Strains usually become fully fluoroquinolone resistant with the
addition of a mutation in the other target gene (either gyrA or parC) and
mutations in parE and gyrB may contribute to resistance in some isolates.
QRDR mutations can arise spontaneously or be transferred by the inte-

gration of foreign genetic material. In comparison to `-lactam resistance,
horizontal gene transfer seems to play a minor role in fluoroquinolone
resistance. Studies addressing that question found evidence for horizontal
gene transfer in 0–11% of fluoroquinolone resistant isolates and interest-
ingly, this ratio seems to be higher in respiratory isolates than in invasive
isolates [24–26]. This discrepancy might be explained by the hypothesis
that interspecies recombination takes place in the nasopharynx, which is
colonized by both pneumococci and viridans streptococci. One could spec-
ulate that the antecedent nasopharyngeal colonization period for invasive
strains is shorter than that of respiratory isolates. Therefore, there may be
less time for interspecies recombination in invasive isolates.
The impact of efflux on fluoroquinolone resistance seems to be more
limited and selective. To date, no highly resistant isolate has been found
with efflux as the only mechanism of resistance. Fluoroquinolones with
a small molecule size, e.g. ciprofloxacin, seem to be affected to a higher
extent than larger molecules such as moxifloxacin. A putative efflux pump,
PmrA was described by Gill et al., that exhibits homology to the multidrug
efflux pumps NorA and Bmr [27]. PmrA consists of 12 transmembrane
segments as efflux proteins of the proton-dependent pumps. In addition,
a recent article describes the presence of a non-PmrA pump in S. pneu-
moniae.
Many studies have addressed the epidemiology of PmrA by comparing
ciprofloxacin MICs in the absence and the presence of the pump inhibitor
reserpine. In contrast to Mef(A), PmrA seems not to be encoded by a resis-
tance gene but rather over expression. However, little is know about the
mechanism of the expression regulation of PmrA.
Resistance to new antibiotics
Since 1962, only a few classes of novel antibiotics have been introduced,
and all since 1999, including the streptogramins (quinupristin/dalfopristin),
the oxazolidinones (linezolid), and the lipopeptides (daptomycin). All other
recently introduced drugs, such as tigecycline and ertapenem are derived
from antibiotic classes that are already in use. The ketolides (e.g., telithro-
mycin) were developed from the macrolides to overcome antibiotic resis-
tance in pneumococci and are characterized by the lack of the L-cladinose
sugar at position 3 of the erythronolide A moiety, which is replaced by a
keto group.
Farrell and Felmingham recently reported only 10 isolates to be telithro-
mycin resistant among a worldwide collection of 13,874 S. pneumoniae iso-
lates isolated between 1999 and 2003. The strains isolated in France, Italy,
Spain, Hungary, and Japan had telithromycin MICs of 4 to 8 +g/ml and
showed an erm(B) genotype [28]. Reinert et al. recently described a telithro-
mycin resistant isolate in Germany (MIC 8 +g/ml) that exhibited in addition

to the erm(B) genotype, mutations in the L4 (S20N) protein [6]. The S20N
L4 alteration has been shown to contribute to the increase in macrolide
MICs in pneumococci. It may be assumed that a combination of erm(B) and
L4 mutations confer ketolide resistance. Also a single base deletion within
the 23S-rRNA has been detected to confer resistance to macrolides and
telithromycin [29]. Another clinical isolate resistant to telithromycin and
fluoroquinolones with a still unidentified mechanism was recently found in
Argentina [30].
Streptogramin (quinupristin-dalfopristin, Q/D) resistance among Gram-
positive cocci has been very uncommon [31]. Two clinical isolates among
8,837 (0.02%) S. pneumoniae isolates were discovered in 2001 to 2002
with Q/D MICs of 4 +g/ml. Each had a 5-amino-acid tandem duplication
(RTAHI) in the L22 ribosomal protein gene (rplV) preventing synergistic
ribosomal binding of the streptogramin combination [7].
Recently, two clinical S. pneumoniae isolates, identified as nonsuscep-
tible to linezolid and as resistant to macrolides and chloramphenicol, were
found to contain 6-bp deletions in the gene encoding riboprotein L4 [5].
Clinical relevance of resistance
MIC based breakpoints have been suggested by different organizations

to distinguish between susceptible, intermediate and resistant strains. The
breakpoints published by the CLSI (formerly the NCCLS) are widely used
in routine clinical laboratories and in surveillance studies. However, in some
cases these breakpoints are unable to predict clinical failure in patients
who were discordantly treated. Pharmacodynamic parameters are superior
to MIC in predicting bacteriological eradication that is related to clinical
outcome. To calculate these parameters, ideally antimicrobial susceptibil-
ity (measured by MIC) is compared to the achievable unbound fraction
of antibiotic at the site of infection. Since the latter is difficult to measure,
usually serum/plasma concentrations obtained from clinical studies with
healthy volunteers are used instead. This may lead to some limitations since
recent pharmacokinetic studies have clearly demonstrated the there can
be tremendous differences in the concentration of an antibiotic between
healthy subjects and severely ill patients, in regard to the patient’s age and
the body site.
Clinical relevance of `-lactam resistance
In pneumococcal pneumonia caused by penicillin-resistant strains, no

association between treatment failure and discordant therapy could be
observed in several studies, as long as the administration was intravenous
and drugs were penicillin, ampicillin, amoxicillin, cefotaxime or ceftri-

axone. The only studies that have found such an association were not
stratified for the severity of disease or did not document the susceptibility
of the pneumococci to the drug that failed. The basis of that seeming con-
tradiction is high serum concentrations of these `-lactams and relatively
low breakpoints defining resistance [32]. The main PK/PD parameter
of `-lactams which are time-dependent killing antibiotics is the propor-
tion of time of the dose interval during which the plasma concentration
exceeds the MIC (T > MIC) [33]. Pharmacodynamic calculations predict
that high doses of intravenous penicillin remain useful for the treatment
of pneumococcal pneumonia up to the MICs of 4 mg/l (non-susceptibil-
ity according to CLSI is defined by MIC, > 0.12 mg/l). Pharmacodynamic
also predicts that `-lactams with less anti-pneumococcal activity may lead
to failure. Examples of such failures have been documented for cefazolin,
cefuroxime and ticarcillin.
Only a few data are available on the clinical failure of oral `-lactam
therapy. However, due to the decreased bioavailability of ampicillin and
amoxicillin such failure could be expected and the failure of low-dose amox-
icillin prophylaxis to prevent penicillin-resistant pneumonia in patients
with sickle cell disease has been documented. In a recent cohort study
presented at the 45th Interscience Conference on Antimicrobial Agents
and Chemotherapy (45th, ICAAC, Washington, D.C., 2005) Iannini et al.
investigated treatment failure in outpatients with pneumococcal pneumonia
(abstract # 896). In patients treated orally with cephalosporines they found
cephalosporine resistance associated treatment failure, however, this was
not found in outpatients treated orally with penicillins. This might be also
caused by the unfavourable bioavailability of cephalosporins. In another
abstract presented at the same conference File et al. described the eradiac-
tion of penicillin resistant pneumococci (amoxicillin MIC, ) 4mg/l) in 43
out of 44 patients with pneumonie by amoxicillin/clavulanic acid (abstract
# 707). Since pneumococci do not express `-lactamases this effect has to be
contributed primarily to amoxicillin.
Macrolides
The clinical relevance of macrolide resistance has been a matter of debate.

After the first break through bacteraemias during macrolide treatment,
it was thought that only resistant strains harboring the erm(B) mecha-
nism could cause treatment failure [34]. It has since become clear that
also strains with the efflux mechanism can exhibit high MIC’s resulting
in treatment failure. Of particular concern is the recent observation that
mutations in the ribosomal proteins or the 23S rRNA itself can develop
during macrolide treatment and confer MIC’s high enough to cause treat-
ment failure [35].
Clinical relevance of fluoroquinolone-resistance and first step mutants
Whereas most studies have shown no increase in mortality in patients with

invasive disease infected with penicillin-resistant pneumococci, FQ resis-
tance has been associated with clinical failure [36, 37].
In 2002, Davidson et al. published a paper on a topic that was receiv-
ing broad attention [38]. They described four patients treated with levo-
floxacin with pneumococcal pneumonia in whom treatment failure due to
fluoroquinolone resistance was observed. Two of the patients died. Prior
to this publication several reports had documented treatment failure due
to fluoroquinolones resistance, however, most treatment failures occurred
with ciprofloxacin, a fluoroquinolone with weak antipneumococcal activity.
Meanwhile, several other reports of fluoroquinolone resistance associated
treatment failure have been published and were reviewed in a recent paper
by Fuller and Low [39].
During the stepwise acquisition of mutations leading to full resistance,
so called “first-step mutants” are evolving. These first-step mutants are
frequently phenotypically susceptible and therefore cannot be detected
by routine resistance testing [40]. Their detection therefore requires the
sequencing of the topoisomerase genes, a method outside the scope of
most routine susceptibility testing in clinical laboratories, but an important
method for surveillance laboratories. Once a mutation in one of the target
enzymes is present there is a significant increased likelihood for the acquisi-
tion of mutations in the second target enzyme leading to complete resistance
[41]. Croisier et al. exposed susceptible first-step mutants with mutations
within parC to levofloxacin and moxifloxacin in a rabbit model, matching
human pharmacokinetics and found that double mutants (i.e. containing
mutations in parC and gyrA) resistant to these agents were selected under
both treatment regimens [42]. This is particularly likely to occur when large
pneumococcal populations, as observed in COPD for example, are exposed
to fluoroquinolones [42].Therefore, FQ treatment of infections caused by
first-step mutants can lead to the selection of resistant isolates resulting in
treatment failure and a general increase in FQ resistance. Some authors
consider first-step mutants to be a key parameter for the spread of FQ resis-
tance and estimate that their prevalence may be several times higher than
the prevalence of phenotypically resistant pneumococci [40, 43].
Limiting the spread of resistance – the impact of vaccines
The main mechanism driving the increase of antibiotic resistance in pneu-

mococci is clonal spread. Even if “clone” does not equal “serotype”, the
most frequent isolates of the same clone exhibit also the same serotype.
The majority of the serotypes exhibited by the main antibiotic resistant
pneumococcal clones are covered by the 23-valent non-conjugated pneu-
mococcal polysaccharide vaccine. The vaccine was demonstrated to reduce

the severity of pneumococcal vaccine types but, because the vaccine does
only induce a B-cell response, it cannot prevent the colonization of the
nasopharynx by strains exhibiting the vaccine serotypes. In contrast, the
seven-valent conjugated vaccine does induce also a T-cell response. Studies
have shown that the overall prevalence of strains of vaccine serotypes
(both in the vaccine target group and all age groups) has been reduced
since the introduction of the conjugate vaccine in 2000 in the United States
[44]. Obviously the immunological response introduced by the vaccine
enables carrier to clear strains of vaccine serotypes. Since most of the vac-
cine serotypes are exhibited by antibiotic resistant clones, antibiotic resis-
tance should decrease. This has already been demonstrated for penicillin
and macrolide resistance [44–46]. Surveillance data indicate that the rise
of fluoroquinolone resistance seems also to plateau in the United States
after the introduction of the vaccine [47]. However, little is known about
the long-term impact of the vaccine, and the extent to which vaccine sero-
types will be replaced by nonvaccine types is still not clear. Pneumococci
are naturally transformable, and capsular switching is a well-documented
mechanism for the potential evasion of the host immune response. Reports
of the emergence of penicillin-nonsusceptible clones of NVT sharing
genetic relatedness with internationally established clones targeted by the
vaccine [48, 49] have indicated the need to track phenotypic and genotypic
changes within invasive pneumococci.
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Influenza
Hans-Dieter Klenk
Institut für Virologie, Philipps-Universität Marburg, Hans-Meerwein-Str. 3, 35043 Marburg,

Germany
Abstract
Influenza-A-viruses have a wide host range and occur with a wide spectrum of variants
defined by 16 HA and 9 NA subtypes. All of these subtypes occur in birds, whereas only
some of them have so far been observed in man, pig, horse, and a number of other mam-
mals. In contrast, influenza-B and C-viruses occur only in man, and there are no subtypes
of these viruses. Influenza-A-viruses occasionally can be transmitted from aquatic birds,
their natural reservoir, to terrestrial birds and mammals. On rare occasions, they adapt
to the new species and establish thus new virus lineages. Adaptation requires multiple
mutations and it may involve gene reassortment after co-infection with another virus.
By these mechanisms, viruses with new surface glycoproteins and therefore a distinct
change in antigenicity are generated. If a new virus with such an antigenic shift occurs in
man, it causes a pandemic. Antigenic drift, unlike antigenic shift, is characterized by slight
changes in antigenicity resulting from successive mutations in HA and NA. Antigenic drift
is responsible for the annual human epidemics. It occurs not only with influenza-A-viruses,
but also with influenza-B viruses.
Influenza is a highly contagious disease that is transmitted by aerosols. Virus replica-
tion occurs in airway epithelia and reaches its peak 2–3 days after infection. Symptoms
typically include high fever, chills, headache, sore throat, dry cough, myalgias, anorexia,
and malaise. Complications include primary viral pneumonia, secondary bacterial pneu-
monia, or combined bacterial and viral pneumonia. Serious complications of influenza
most often occur in people 65 years of age and older, in the very young, and in those of
any age with underlying chronic cardiac, pulmonary, or metabolic disease. Vaccination is
the most potent instrument for influenza control. Prime candidates for vaccination are
persons at risk for complications and individuals who might transmit influenza to such
persons. Inactivated vaccines obtained from infected chicken embryos are most commonly
used. Neuraminidase inhibitors are the influenza antivirals of choice. Application is lim-
ited to a relatively small time window shortly before or after infection.
Introduction
Influenza viruses are segmented negative stranded RNA viruses. They

have a high genetic variability and occur with a large number of different
74 Hans-Dieter Klenk
variants in man and animals. Human influenza viruses are unique in their
ability to cause recurrent seasonal epidemics of different severity as well
as global pandemics during which acute febrile respiratory disease occurs
explosively in large parts of the population. Influenza outbreaks can be
traced back to the Middle Ages and antiquity. Particularly disastrous was
the Spanish influenza which cost more than 40 million lives in 1918 and
1919. In the 1930s, influenza viruses have been identified in man and pigs.
Twenty years later it was discovered that influenza viruses occur also in
birds. It was then possible to systematically investigate these pathogens.
Despite detailed knowledge on structure and replication of influenza
viruses, many aspects of epidemiology and pathogenesis are still poorly
understood.
Classification and structure of influenza viruses
Influenzaviruses belong to the family orthomyxoviridae. There are three

genera or types: influenza-A-viruses, influenza-B-viruses, and influenza-C-
viruses. Influenza-C-viruses do not play a major role as human pathogens.
The genome of influenza-A and B-viruses consists of eight single-stranded
RNA molecules of negative polarity each of which encodes one or two
viral proteins. The entire genome of influenza-A and B-viruses has a length
of 13,600 and 14,600 nucleotides, respectively. The individual RNA seg-
ments have a circular structure resulting from base pairing at the 3’ and
5’ ends. The segmentation of the genome is of particular biological sig-
nificance, since it is a precondition for the high genetic variability of these
viruses. The RNA genome forms together with the polymerase proteins
PB1, PB2, and PA the helical nucleocapsid core of virus particles. It is sur-
rounded by the lipid-containing viral envelope that is lined on its inner side
by the matrix protein M1 and has glycoprotein spikes on its outer surface.
Influenza-A and B-viruses have hemagglutinin (HA) spikes with receptor
binding and fusion functions and neuraminidase (NA) spikes with recep-
tor destroying activity. The fusion activity of HA depends on proteolytic
cleavage by cellular proteases and a pH dependent conformational change.
This activation process results in exposure of the fusion domain of HA and
its penetration into the target membrane and thus in membrane fusion.
There are a number of other proteins. With influenza-A-viruses, these are
the M2 protein that forms an ion channel in the viral envelope, the NS2
protein present in small amounts in virions, and the non-structural protein
NS1 (Fig. 1).
The surface glycoproteins of influenza viruses are the subtype and
strain specific antigenic determinants. They are the major viral components
responsible for eliciting protective immunity. Matrix and nucleocapsid pro-
teins show type-specific antigenicity and allow the serological differentia-
tion of types A, B, and C [1].
Influenza 75
Figure 1. Structure of influenza-virus. Upper panel: Electronmicrograph of a virus particle

(diameter 100 nm). The spikes at the surface (length 10 nm) can be seen. Lower panel: Virus
model. The envelope contains hemagglutinin (HA) and neuraminidase (NA) spikes as well
the M2 protein. The interior of the particle is formed by ribonucleoproteins, consisting of the
genomic RNA-segments, the nucleocapsid protein NP, and the polymerase proteins PB1, PB2,
and PA. The inner side of the envelope is lined by the matrix protein M1. Virions contain also
small amounts of the NS2 protein.
Replication cycle
HA initiates infection by mediating binding to neuraminic acid-containing

receptors and membrane fusion following endocytosis. The M2 ion chan-
nel plays an important role in uncoating by lowering the pH within the
virus particle and, thus, allowing dissociation of the internal components.
The nucleocapsid complexes are then transported into the nucleus, where
transcription and replication take place. The genomic RNA (vRNA) serves
as template for two different RNA species: complementary RNA (cRNA)
which is a complete copy of vRNA, and mRNA with a cap structure at the
5’ end and with the 3’ terminal nucleotides of cRNA being replaced by a
poly-A-tail. Non-coding sequences at the 3’ end of vRNA serve as primers
for mRNA synthesis. The cRNA is the template for new vRNA molecules.
The cap structures of the viral mRNAs are derived from cellular mRNA
molecules. Viral mRNA utilizes the cellular translation machinery for the
synthesis of viral proteins. The virus enhances the coding capacity of its
genome by several mechanisms. These include splicing, expression of two
cistrons in tandem position, and start of translation at two different ini-
tiation codons. Translation of nucleocapsid protein, polymerase proteins,
matrix protein, and two non-structural proteins NS1 and NS2 occurs on
free polysomes. Ribonucleoproteins are assembled in the nucleus and sub-
sequently exported into the cytoplasm. The envelope proteins are translated
at the rough endoplasmic reticulum and then transported by the exocytotic
apparatus to the plasma membrane where virus particles are assembled in
a budding process. The neuraminidase mediates virus release by removing
receptors from the infected cell [2].
Epidemiology
The epidemiology of influenza is largely determined by its high genetic

variability. This is particularly obvious with influenza-A-viruses. They have
a wide host range and occur with a wide spectrum of variants defined by 16
HA and 9 NA subtypes. All of these subtypes occur in birds, whereas only
some of them have so far been observed in man, pig, horse, and a number of
other mammals. In fact, aquatic birds appear to be the natural reservoir of
influenza-A-viruses. In contrast, influenza-B and C-viruses occur only with
man, and there are no subtypes of these viruses.
The host barrier is not an insurmountable obstacle for influenza-A
viruses. Thus, they occasionally can be transmitted from aquatic birds, their
natural reservoir, to terrestrial birds and mammals with transient outbreaks
of disease. On rare occasions, they adapt to the new species and thus estab-
lish new virus lineages. Adaptation requires multiple mutations and it may
involve gene reassortment after co-infection with another virus. By these
mechanisms, viruses with new surface glycoproteins and therefore a distinct
change in antigenicity are generated. If a new virus with such an antigenic
shift occurs in man, it causes a pandemic. There were three pandemics in
the last century. After the Spanish influenza (H1N1) in 1918, an antigenic
shift in HA and NA gave rise to the Asian influenza (H2N2) in 1957, and
another shift in HA to the Hong Kong influenza (H3N2) in 1968. In 1977
the H1N1 subtype re-emerged and co-circulates now in man with H3N2
and influenza-B virus. More recently avian viruses of subtypes H5, H7, and
H9 have been transmitted, but not yet adapted to man. This includes H5N1
viruses that emerged in 1997 in South-East Asia and caused large outbreaks
in domestic and wild birds, and are now spreading into many other parts of
the world (Fig. 2).
Antigenic drift, unlike antigenic shift, is characterized by slight changes
in antigenicity resulting from successive mutations in HA and NA. Antigenic
drift is responsible for the annual human epidemics. It occurs not only with
influenza-A-viruses, but also with influenza-B viruses [1].
Influenza 77
Figure 2. Influenza-A periods in man. Circulation periods of H1N1, H2N2, and H3N2 viruses
are shown. In recent years, transmission of avian strains of subtypes H5, H7, and H9 to man
have also been observed. These viruses have not adapted yet to man. Of particular concern,
however, are the H5N1 viruses (“bird flu viruses”) that are now endemic in birds in Asia from
where they rapidly spread to other parts of the world.
Pathogenesis
Influenza viruses exhibit large differences in organ tropism and serverity

of disease. In man and other mammals, influenza usually causes respiratory
disease. In contrast, most avian viruses cause asymptomatic enteric infec-
tion, whereas a few H5 and H7 viruses lead to fowl plague or bird flu, a
highly lethal systemic disease.
Experimental studies at the molecular level have shown that many bio-
logical properties of influenza viruses contribute to their pathogenicity, such
as replication efficiency, organ tropism, spread of infection, and sensitivity
to host defense mechanisms. Thus, the immune status of the human popula-
tion and the extent of the antigenic differences between viruses are largely
the reason that new pandemic viruses induce more severe disease than
interpandemic ones. In each phase of the viral life cycle, there are specific
interactions between viral proteins and host factors, such as cell receptors,
nuclear proteins, and proteases. The role of these interactions in pathogen-
esis is particularly evident with proteolytic activation of HA. The highly
pathogenic avian viruses are activated by the protease furin that is ubiq-
Figure 3. Cells infected with influenza virus in human airway epithelium. Ciliae are stained
black. Cells infected with influenza virus A/Memphis/14/96 (H1N1) are stained brown. Virus
infection targets specifically non-ciliated cells [5].
uitous and therefore promotes rapid virus spread within the organism. In
contrast, the viruses causing local infection including the human influenza
viruses are activated by proteases confined to specific tissues. Staphylococcus
aureus, Streptococcus pneumoniae, and Haemophilus influenzae as well as
several other bacteria secrete also HA activating proteases. After co-infec-
tion with these microorganisms, influenzavirus infections show therefore a
particularly severe course of disease [3]. Other mechanisms that contribute
to pathogenicity are the interferon antagonism of the NS1 protein and an
activity optimum of the viral polymerase probably also depending on host
factors.
Examination of tissues obtained from patients revealed that viral repli-
cation can occur throughout the respiratory tract. These studies also indi-
cated that ciliated epithelial cells are a primary site of infection, whereas
recent studies carried out on cultured airway epithelia revealed that human
influenza viruses target specifically non-ciliated cells (Fig. 3). Inflammation
of the larynx, trachea, and bronchi, and desquamation of ciliated columnar
epithelium into the lumen of the bronchus was observed in individuals with
uncomplicated acute influenza infections.
Influenza 79
Regeneration of the respiratory epithelial cells takes ca. 3–4 weeks,

during which time pulmonary function abnormalities may persist. In these
typical cases of influenza in which infection is confined to the respiratory
tract, prostration, fever, and myalgia often seem to be disproportionate to
objective clinical signs or observed pathological changes.
Lungs from fatal cases of primary viral pneumonia most notably show
hyaline membrane coverage of alveolar walls together with extensive
intra-alveolar edema and hemorrhage. Tracheitis and bronchitis are also
observed. Patients with secondary bacterial pneumonia have changes char-
acteristic of bacterial pneumonia in addition to the tracheobronchial find-
ings of influenza.
Immune response
Innate immunity is an important defense mechanism early in infection.

There are profound changes in the expression of a large number of cel-
lular genes involved in interferon production and signaling, apoptosis,
and oxidative stress. Influenza-infected patients trigger the activation
of antiviral genes and production of cytokines, such as interleukin (IL)-
6, IFN-alpha, and IL-8. IL-8 is correlated with lower respiratory tract
involvement. Influenza viruses have evolved mechanisms to counter the
innate antiviral responses of the host. The viral NS1 protein antagonizes
interferon production and the activation of PKR in influenza-infected
cells [1].
The adaptive immune response to infection with influenza viruses leads
to induction of both virus-specific B- and T-cell immune responses that
clear infection and generate long-lasting specific immunologic memory.
Antibodies are made against the viral external glycoproteins HA and NA
as well as the internal type-specific proteins NP and M1. Neutralizing anti-
body directed against the HA is the primary immune mediator of protec-
tion from infection and clinical illness due to influenza viruses. Antibodies
and T-cells play complementary roles in clearing the infection and promot-
ing recovery. CD4 + (Th1 and Th2) and CD8 + T-cell responses to influenza
are type-specific and are largely cross-reactive among influenza A viruses
of different subtypes. In naturally infected humans the CD4 + cell response
recognizes epitopes on the internal proteins NP and M1 as well as the sur-
face proteins NA and HA. The CD8 + cytotoxic T lymphocyte response is
directed to multiple surface and internal influenza proteins, in which many
epitopes are potentially recognized, as determined by their binding to class
I MHC molecules. Recent animal model studies have shown that both
CD4 + and CD8 + T-cells can contribute to immunity to influenza viruses.
The increased severity of influenza infections in geriatric patients has been
correlated with a combination of functional impairments in their T-cell
responses to influenza [4].
Clinical manifestations
Influenza is a highly contagious disease that is transmitted by aerosols.

Virus replication occurs in airway epithelia and reaches its peak 2–3 days
after infection. Patients shed virus for about 7 days, with primary infections
for up to 2 weeks. Early symptoms in adults typically include fever, chills,
headache, sore throat, dry cough, myalgias, anorexia, and malaise. A fever
of 38–40°C that peaks within 24 h of onset is common, but peaks as high as
41°C can also occur. Pyrexia typically lasts 3 days, but may last from 1 to 5
days or longer. Other symptoms that occur less frequently include subster-
nal soreness, photophobia and other ocular symptoms, nausea, abdominal
pain, and diarrhea. Although most symptoms typically resolve within a
week, cough and malaise may persist for 1 or more weeks after fever has
subsided. In children, symptoms are similar to those in adults, but gastroin-
testinal symptoms such as vomiting, abdominal pain, and diarrhea are seen
more frequently. Maximum temperatures also tend to be higher in children
than in adults, and febrile convulsions can occur. In addition, myositis, croup,
and otitis media occur more frequently in children. Influenza infection of
neonates can be life-threatening and may be manifest only as an unex-
plained febrile illness.
Complications of the upper respiratory tract after influenza infection
include bacterial sinusitis and otitis media. Lower respiratory tract compli-
cations include exacerbation of chronic obstructive pulmonary disease and
chronic congestive heart failure, croup, bronchitis, bronchiolitis, wheezing
attacks in asthmatics, and pneumonia (primary viral pneumonia, secondary
bacterial pneumonia, or combined bacterial and viral pneumonia). Primary
influenza pneumonia develops abruptly and progresses rapidly. It has been a
frequent cause of death in the 1918 pandemic and is also responsible for the
high case fatality rate of the human infections with the ongoing H5N1 out-
break. However, with ordinary influenza epidemics, this type of pneumonia
is uncommon and occurs mainly among those at increased risk for complica-
tions of influenza. Rapid respiration rate, tachycardia, cyanosis, high fever,
and hypotension are frequent symptoms. Diffuse pulmonary infiltrates and
acute respiratory failure with a high mortality rate are also features of this
disease. Combined viral and bacterial pneumonia is more common than
primary viral pneumonia and may be clinically indistinguishable from it.
Secondary bacterial infections typically occur 5–10 days after initial onset
of influenza symptoms and are responsible for most pneumonias during
influenza epidemics. Productive cough, pleuritic chest pain, and chills are
common symptoms of this type of pneumonia. Streptococcus pneumoniae,
Staphylococcus aureus, and Haemophilus influenzae are the organisms most
commonly involved. These illnesses respond to appropriate antimicrobial
agents and have a lower case fatality rate than primary viral pneumonia.
Other reported but less frequent complications of influenza include
myositis, myocarditis and pericarditis, acute renal failure, encephalopathy,
Influenza 81
encephalitis, transverse myelitis, Guillain-Barré syndrome and a range of

other neurological complications, Reye’s syndrome, and toxic shock syn-
drome. Higher rates of spontaneous abortion, stillbirths, and premature
births were reported among pregnant women during the major pandemics
of 1918/19 and 1957/58.
Serious complications of influenza most often occur in people 65 years
of age and older, in the very young, and in those of any age with underlying
chronic cardiac, pulmonary, or metabolic disease. Complications in elderly
people, particularly among those with pulmonary, cardiovascular, or other
chronic diseases, account for most of the mortality in influenza epidemics
[4].
Laboratory diagnosis
Virus is isolated from nose or throat secretions by inoculation of cell cul-

tures (MDCK cells) or embryonated eggs. Isolation is usually only success-
ful within the first days of infection. Rapid diagnosis is possible by antigen
detection in cells obtained from throat swabs. Viral RNA can be detected by
PCR in clinical specimens. This approach is now replacing virus culture as
“gold standard” for virus detection, because it is more sensitive, more rapid,
and allows more precise characterization.
Serological diagnostics involves complement fixation, hemagglutina-
tion inhibition, neutralization, and enzyme immunoassays. Antibody titres
increase rapidly in the course of infection and decrease within a couple of
weeks to low levels. It is often impossible to detect a fourfold increase in
titre in blood samples taken with an interval of 2 weeks, since high antibod-
ies reach high titres already in the acute disease phase. Therefore a single
high antibody titre matched by the typical clinical symptoms is usually con-
sidered to be sufficient for the diagnosis of the infection.
Prophylaxis and therapy
Vaccination is the most potent instrument for influenza control. Prime can-
didates for vaccination are persons at risk for complications and individuals
who might transmit influenza to such persons. Inactivated vaccines obtained
from infected chicken embryos are most commonly used. They contain HA
and NA antigens as protective components and are each year formulated
according to the circulating influenza-A and B strains. Because of antigenic
drift, vaccination protects only against known viruses. Vaccinations should
therefore be done every year. Current vaccines protect 70–90 percent of
healthy adults. With older people, i.e. the most important target group, effec-
tiveness is lower. Live vaccines are only available in some countries, such as
the USA. Development of pandemic vaccines is a particular challenge, since
they will have to be manufactured within a very short time period in very
large quantities.
Antiviral compounds were previously available only in the form of
amantadine and rimantadine. Both compounds block the M2 ion channel
of influenza-A-viruses and are therefore not effective against influenza-B-
virus infections. Prophylactic application provides 70–90 percent protec-
tion. Concerns about side effects involving the central nervous system have
limited the use of amantadine. Development of amantadine resistant virus
strains, which have emerged recently with increasing frequency, is another
problem.
The neuraminidase inhibitors zanamivir and oseltamivir that interfere
with virus spread compare favorably with amantadine by having less side
effects and a wider therapeutic spectrum that includes also influenza-B
viruses. The neuraminidase inhibitors are therefore now the influenza anti-
virals of choice. However, as is the case with amantadine, application is lim-
ited to a relatively small time window shortly before or after infection, and
there is increasing evidence for the development of virus resistance to these
drugs, too. Nevertheless, neuraminidase inhibitors are expected to play an
important role in the control of a future pandemic.
References
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enza. In: BWJ Mahy, V ter Meulen (eds): Topley and Wilson’s microbiology and
microbial infections, Virology, Vol. 1. Hodder Arnold, London, 634–698
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ity. Trends Microbiol 2: 39–43
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Hay (eds): Textbook of influenza. Blackwell Science, Oxford, 219–264
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Pathogenesis of Chlamydophila pneumoniae infections –

epidemiology, immunity, cell biology, virulence factors
Matthias Krüll and Norbert Suttorp
Dept. Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité

Universitätsmedizin Berlin, Augustenburgerplatz 1, 13353 Berlin
Abstract
Chlamydophila (Chlamydia) pneumoniae, a Gram-negative obligate intracellular bacte-
rium, is a widespread respiratory pathogen causing sinusitis, pharyngitis, bronchitis and
pneumonia. Repetitive or chronic persistent infections have been associated with an
increased risk for asthma, chronic obstructive pulmonary disease (COPD) or vascular
lesions. Although the genome of C. pneumoniae has been sequenced completely this
information has not led yet to an understanding of the mechanisms of infection and target
cell activation nor to the identification of potential chlamydial virulence factors. In this
review we will give an overview on the pathogenesis of C. pneumoniae-induced acute and
chronic infections.
Epidemiology, diagnosis and clinical manifestation
Chlamydophila pneumoniae, a Gram-negative obligate intracellular bac-

terium, is a widespread respiratory pathogen causing sinusitis, pharyngitis,
bronchitis and pneumonia [1–3]. The majority of C. pneumoniae infections
are subclinical, but severe pulmonary infection and profound lymphocytic
alveolitis are observed [4]. In addition, chronic-persistent or recurrent infec-
tions may be an important risk factor for adult-onset asthma [5], chronic
obstructive lung disease (COLD, [6]) and development of vascular lesions
[7–10]. Regarding vascular diseases, the field, however, is troubled by the
“hen vs. egg” problem and causative proof is difficult because besides dif-
ferent anti-chlamydial isotypes of antibodies there are no good markers to
differentiate among new vs. old (IgM vs. IgG) as well as acute vs. chronic
persistent (IgM vs. IgA) C. pneumoniae infection.
Epidemiology
C. pneumoniae was first isolated 1965 from the conjunctiva of a Taiwanese

child (strain TW183, [11]). The first respiratory strain (AR 39) could be
84 Matthias Krüll and Norbert Suttorp
isolated 1983. Both strains had a DNA-homology of > 99.5% and were
established in 1986 as a new (third) human-pathogenic chlamydia species
“Chlamydia pneumoniae,” synonym TWAR [1]. Until now about 50 strains
have been isolated worldwide, DNA-homology between these strains is 94-
100%.
Chlamydiae have been placed in their own order, Chlamydiales, with one
family, Chlamydiaceae. Molecular evaluation of rRNA sequences confirms
that chlamydiae are bacteria, but with only very distant relationship to other
bacterial divisions [12, 13]. Although it has been proposed that Chlamydia
trachomatis and Chlamydia pneumoniae represent different genera [14],
their gene content and genome organization are extremely similar [15] as
are their structure and biology [16]. The newly proposed nomenclature – as
used throughout this chapter – “Chlamydophila pneumoniae”, however, has
not been generally accepted [17].
Chlamydophila pneumoniae is found worldwide. Seroepidemiologic
surveys have demonstrated that more than 70% of all adults have been
exposed to this organism during their lifetimes [18]. Age-specific prevalence
rates start to rise early in childhood, although there is only little disease
associated with these early timepoints of infection. The most rapid rise (in
the Western world) in age-specific prevalence occurs during the ages of 5- to
20-years.
In spite of a high percentage of C. pneumoniae-seropositive adults
reinfections are common. According to these seroepidemiologic studies,
C. pneumoniae infection seems to be both endemic and epidemic with fre-
quent reinfection during a lifetime. Currently available data suggest that
C. pneumoniae is primarily transmitted from human to human without any
animal reservoir. Transmission seems to be inefficient, although household
outbreaks with high transmission rates are reported [19].
Diagnosis
For differential diagnosis of an acute, repetitive or chronic/persistent C.

pneumoniae infection the kinetics of the antibody expression has to be
taken into consideration. In patients with primary infection IgM antibod-
ies appear about 2–3 weeks post infection and remain elevated/detectable
up to 2–6 months. IgG antibodies may not reach high titers until 6-8 weeks
after onset of clinical symptoms. C. pneumoniae does not induce a persistent
protective immunity and reinfections are common. In case of reinfection,
the level of IgG antibodies increase rapidly within 1–2 weeks while IgM
antibodies may not appear again [19]. Until now there is no reference test
for validating a chronic/persistent infection by means of serological testing.
Persistently elevated IgG or the presence of IgA antibodies has been sug-
gested as potential serological marker. Several studies have proposed that
high IgA titers might be a better marker for a chronic infection than IgG
Pathogenesis of Chlamydophila pneumoniae infections… 85
titers because serum IgA has a half- life of about 5–7 days, whereas IgG
has a half-life of weeks to months [9, 20]. Further evaluation, however, and
confirmation by other tests have to be awaited.
A lack of validated and standardized diagnostic techniques in diagnosis
of acute, recurrent or chronic persistent infection with C. pneumoniae has
made interpretation of published data difficult. Recommendations for stan-
dardized approaches were recently published by the Centers for Disease
Control and Prevention (CDC, Atlanta) and the Laboratory Centre for
Disease Control (Ottawa, Canada) in cooperation with members of the C.
pneumoniae-study group [21]. Diagnostic approaches include serological
testing, isolation/culture, nucleic acid-based amplification techniques such
as PCR, and tissue diagnostics like immunofluorescence, immunohisto-
chemistry (IHC) or in situ hybridization.
Serological testing
The microimmunofluorescence (MIF) test, developed by Wang et al. in the

1970s [22] is currently the serological testing method of choice for diagnosis
of acute C. pneumoniae infection. This test allows the quantitative deter-
mination of antibody reactivity to formalinized elementary bodies from C.
pneumoniae, C. trachomatis and C. psittaci fixed as dots on a single glass
slide. Dilution of sera are placed over the antigen dots and incubated. Use
of the MIF test allows definition of criteria for serologic evidence of acute
infection (defined by a four-fold rise in IgG between acute and convalescent
samples or an acute IgM titer * 1: 16), or past exposure (indicated by an
IgG titer * 1: 16). Kits based on the MIF format are commercially available,
however, it should be noted that the quality of commercially available MIF
kits varies and interpretation of the results is subjective [23]. Due to the
lack of species specificity or sensitivity, other serological tests like comple-
ment fixation (CF), whole fluorescence, ELISA and EIA cannot currently
be endorsed [24, 25].
Culture
Chlamydophila pneumoniae is an obligate intracellular pathogen and

must be cultivated on specific eukaryotic host cells. All currently estab-
lished culture techniques involve inoculation of specimens onto a human
cell line via centrifugation, incubation for up to 72 h and subsequent
staining with fluorescent-labeled species-specific antibodies to visualize
intracellular inclusion bodies [8, 26, 27] (see Fig. 1). Sensitivity is low due
to the complexicity of the assay. Specificity is dependent on the ability
of the lab staff to distinguish between true chlamydia inclusions and
artifacts.
Figure 1. Chlamydia pneumoniae strain TW183 inclusion bodies in freshly isolated human
airway epithelial cells. Inclusion bodies were visualized, using a fluorescein isothiocy-
anate (FITC)–conjugated, genus-specific monoclonal antibody (DakoCytomation, Hamburg,
Germany).
PCR
Although many in-house PCR techniques for detection of C. pneumoniae

have been published [28-31], sensitivity and specificity remain almost
unknown. Due to a broad variety of methods for specimen-collection and
specimen-processing, primer design, nucleic acid extraction, amplification
product detection and identification of false-positive and false-negative
results, no commercially standardized tests have been approved by the
FDA.
Tissue diagnostics/immunohistochemistry (IHC)
Tissue diagnostic methods offers the advantage of preserving tissue mor-

phology and permitting localization of the infectious agent to specific areas
and cells. IHC has been the most frequently used in published studies [32,
33]. Detection rates are higher than those of PCR. This is attributed to a
faster degradation of DNA, difficulties of extracting DNA from tissues and
the presence of PCR inhibitors. The most widely used IHC-technique is
the avidin-biotinylated immune-complex method. However, interpretation
of IHC-results is a critical challenge due to difficulties in distinguishing

between true- and false-positive results of the staining.
Specific recommendations for standardized culture/isolation or detec-
tion tests (e.g., specimen-collection, -transport and -processing conditions)
have been suggested elsewhere (s.a., [21]).
Taken together, diagnosis of C. pneumoniae infection is difficult because
cell culture techniques are not available for routine clinical use and non-
culture techniques using antigen detection methods (IHC) or DNA probes
have not been developed for commercial use. The MIF test is currently the
serological testing method of choice for diagnosis of acute C. pneumoniae
infection (“gold standard”), although the test is technically complex, requir-
ing experience in fluorescence microscopy, interpretation is therefore sub-
jective.
Clinical manifestation
Chlamydophila pneumoniae causes acute respiratory diseases and is

responsible for approximately 5% of bronchitis and sinusitis cases and 5-
10% of community acquired pneumonia (CAP) cases in adults worldwide
[34–37] (see Fig. 2). However, a broad geographic diversity and periodicity
with higher incidence rates of C. pneumoniae-mediated CAP-cases for 2
or 3 years followed by 4 or 5 years with low incidence has been suggested
[18]. A recently submitted study from the German “community acquired
pneumonia-network, CAPNETZ”-study group, including more than 4000
CAP-patients in Germany demonstrated, that between 2001 and 2004 using
recommended standardized diagnosis protocols (MIF test, PCR from BAL-
fluid) C. pneumoniae could be identified as causative agent in < 1 % of all
CAP-cases (personal communication N. Suttorp, speaker of CAPNETZ).
C. pneumoniae infection has also been implicated in the pathogenesis of
asthma in both adults and children. This hypothesis is based on clinical stud-
ies and on the evidence of specific IgE production, direct epithelial dam-
age, induction of T-cell immunopathologic diseases, and vascular smooth
cell infection. In addition, asthma patients, especially those with moderate
asthma, had significantly higher serum IgA antibody levels to chlamydial
heat shock protein 60 (cHsp60) than healthy controls [5, 38–41]. Moreover,
recurrent or chronic persistent C. pneumoniae infection seems to be com-
mon in patients with chronic bronchitis whether exacerbated or not, and is
characterized by a strong humoral immune response to this intracellular
pathogen, which is present in the majority of patients with severe chronic
bronchitis. Increased antichlamydial IgG and IgA indicated acute exacerba-
tions with C. pneumoniae in COPD patients [5, 42-44]. Some authors suggest
a possible role of this organism in the etiology of lung cancer; future studies,
using measures of chronic C. pneumoniae status, however are necessary, to
substantiate these data [45, 46].
Figure 2. Chest x-ray of a 28-year-old man reveals basal infiltration in both lungs as well as a
generally pronounced interstitial pattern. Chlamydophila pneumonia infection could be con-
firmed by MIF test and PCR from BAL-fluid.
Chronic-persistent or recurrent Chlamydophila pneumoniae infections

may be a trigger and promoter of inflammation which may cause vascular
lesions and atherosclerosis [8, 9, 31, 47, 48]. The theory is supported by a
serological association between C. pneumoniae infection and coronary heart
disease as well as other vascular diseases (arterial occlusive disease, carotid
artery stenosis and stroke [9, 20, 49, 50]), the demonstration of C. pneumoni-
ae in atherosclerotic plaques by electronmicroscopy, immunocytochemistry,
PCR, and isolation of viable chlamydia (indicating a productive chlamydial
infection [8, 10, 29, 31, 51, 52]), and different animal models, demonstrating
that intranasal infection of mice and rabbit with C. pneumoniae leads to
pneumonia, perimyocarditis, septic circulatory dysregulation and – more
delayed – systemic spread of chlamydia into spleen, lymphnodes, peritone-
um and atherosclerotic plaques of arterial blood vessels [7, 53–56]. Current
antibiotic treatment options for acute chlamydial infection, however, were
proven to be ineffective with respect to clinical outcome in different groups

of atherosclerotic patients (WIZARD, AZACS, ACES or PROVE-IT study
[55, 57–62]). Interpretation of these clinical trials is challenging as definite
markers of chronic vascular C. pneumoniae infection are still missing and
serologic testing seems to be inaccurate in patient populations with high
chlamydial IgG seroprevalence. Moreover and probably more important, C.
pneumoniae in the persistent state, as observed in blood monocytes, shows
high resistance against macrolides, tetracyclines and rifampicin [63–65]. This
observation may explain the ineffectiveness of all interventional studies in
atherosclerotic patients.
Several recent studies have suggested a chronic/persistent C. pneumoni-
ae infection as a possible risk factor for central nervous system diseases like
Alzheimer’s disease or multiple sclerosis, data, however, are poor and clear
evidence is still missing [66, 67].
Innate and adaptive immunity
Little is known about a humoral or cell-mediated immunity (“CMI”)

induced during acute or chronic/persistent infection with Chlamydophila
pneumoniae. Most of the data have been elaborated using other Chlamydiae
species (C. trachomatis, C. psittaci). As intracellular bacteria, Chlamydiae
pose an extra challenge for the defense mechanisms of the host. While
importance of anti-chlamydial antibodies still is controversially discussed
[68], cell-mediated immune responses are decisive, at least in mice. CMI
against C. pneumoniae, however, is weak since recurrent infections as well
as persistency of viable pathogens in different target cells are common
phenomena.
Innate immunity
C. pneumoniae is able to infect and to replicate in a multitude of target cells

[69–71]. Subsequently, different intracellular signal transduction pathways
are activated to induce a proinflammatory phenotype (for review see [72]).
However, there is still limited knowledge of the mechanisms of Chlamydiae
entry into host cells. Wuppermann et al. showed that heparan sulfate-like
glycosaminoglycans (GAG) might act as possible chlamydial receptors on
the surface of epithelial cells (HEp-2 cells), the importance of the estrogen
receptor complex is controversially discussed [73, 74]. In a recent paper,
Puolakkainen et al. demonstrated, that C. pneumoniae uses the mannose 6-
phosphate/insulin-like growth factor 2 receptor for infection of endothelial
cells, additional studies, however are necessary to confirm these results [75].
Several recent studies demonstrated the involvement of extracellular Toll-
like receptor-2 (TLR2) and -4 (TLR4) in initiation of innate immune cell
activation by C. pneumoniae or chlamydial components [76–80]. Moreover,

we were able to demonstrate that the recently identified nucleotide-binding
oligomerization domain (Nod) proteins might act as intracellular receptors
during C. pneumoniae-infection of target cells [81].
C. pneumoniae is internalized by macrophages as well as by nonprofes-
sional phagocytes (innate cells), where it survives and replicates [64]. In these
cells IFN-a synergizes with bacterial products to activate various bactericidal
or bacteriostatic mechanisms [82]. IFN-a is a strong activator of indoleamine
2,3-dioxygenase (IDO), limiting the availability of L-tryptophan to intra-
cellular microorganisms [83]. Induction of IDO has been demonstrated to
inhibit chlamydial growth in vitro [84, 85]. IFN-a can also activate inducible
nitric oxide synthase (iNOS) which catalyzes production of NO from L-argi-
nine. Inhibition of chlamydial growth through induction of iNOS has also
been reported [86]. Moreover, stimulation of neutrophils or monocytes with
IFN-a induced transcription of the gp91 component of NADPH oxidase (ox)
mRNA with subsequent enhanced respiratory burst of phagocytic cells [82].
In addition, Rottenberg et al. could demonstrate that IFN-a-receptor-double
knock-out mice (IFN-a-R–/–) showed a dramatically increased susceptibility
to C. pneumoniae, mediated via a reduced iNOS-mRNA accumulation, inde-
pendent of diminished levels of specific antibodies. An increased susceptibil-
ity of iNOS–/– mice substantiated the protective role of this enzyme-activity
during infection with C. pneumoniae [87]. These data suggested a relevant
protective role of IFN-a-dependent innate mechanisms of protection. For
a more extended view of the signal transduction pathways involved in
Chlamydia-mediated innate immune cell activation see Figure 3, in addition
we would like to refer to our recent review [72].
Adaptive immunity
In C. trachomatis infection models, both CD4+ and CD8+ cells have been
shown to confer protection, although the former are considered of major
importance [88, 89]. In C. psittaci infection, CD8+ rather than CD4+ cells
have been reported to confer protection in mice [90].
Since the overall DNA homology between C. pneumoniae and C. tracho-
matis or C. psittaci is less than 5 or 10%, respectively [15] the parameters of
infection identified with the later two cannot be directly extrapolated to C.
pneumoniae.
Most data have been acquired using mouse models for C. pneumoniae-
mediated pneumonia [91, 92]. The situation, however, is complicated due to
mouse strain-specific differences studying mechanisms of adaptive immu-
nity. In addition, importance for a protective role of T-cell subspecies during
primary infection and reinfection still is controversially discussed.
Using C57BL/6J mice genomically lacking T-cell coreceptors or cytokine
receptors, Rottenberg et al. demonstrated that CD4 + T-cells played a dual
Figure 3. Scheme of the supposed signaling cascades induced by C. pneumoniae infection in

human cells (TyrKi, tyrosin kinase; ERK, extracellular regulated kinase; PKC, protein kinase C;
reproduction from [75] courtesy of the authors and of Schattauer Verlag, Germany).
role, one deleterious, promoting bacterial growth and disease early after
infection, but also participating in the control of bacterial growth at later
timepoints as well as in protection against reinfection. The early damag-
ing effect of CD4 + cells in the absence of CD8 + cells was associated with
enhanced IL-4 and interleukin10 (IL-10) mRNA levels and delayed IFN-a
mRNA accumulation in the lungs of mice [87]. CD8 + T cells inhibited this
CD4 + activity. The CD8 + T-cell mediated protective immunity during early
stages of primary infection was perforin independent and associated with
an altered cytokine balance as indicated by increased IL-4 and IL-10 and
delayed accumulation of IFN-a mRNA in CD8–/– mice. The early higher
susceptibility of CD8-deficient mice correlated with an immune deviation
from a normal Th1 response to a Th2 cytokine pattern [87]. These results
were confirmed by Wizel et al. demonstrating the importance of peptide-
specific CD8 + T-cell lines in local and systemic compartments after primary
(intranasal) infection with C. pneumoniae. These CTL lines suppressed
chlamydial growth in vitro by direct lysis of infected target cells and by
secretion of IFN-a. In addition, they were able to identify 18 H-2(b) binding
peptides representing sequences from 12 C. pneumoniae antigens [93]. The
importance of CD8 + CTL during reinfection could be confirmed by Penttilä
et al. They demonstrated in a BALB/c nude mice model with absence of
all T-cells, that the overall clearance kinetic after primary C. pneumoniae
infection was not dependent on either CD4 + or CD8 + cells alone [94], but,
after reinfection, acquired immunity was strongly CD8 dependent with an
enhanced IFN-a-production and an increased local lymphoid reaction in the
lungs [94, 95].
Analyzing lymphocytes from male patients with respiratory tract infec-
tion Halme et al. demonstrated a C. pneumoniae-specific CMI response
during acute, primary infection early after onset of disease symptoms
and simultaneously with a humoral response (increase of C. pneumoniae-
specific IgM-antibodies). C. pneumoniae-induced lymphocyte activation
involved CD8 + T-cells in the early phase of infection and CD4 + cells in the
later stage [96].
The mechanism underlying the protection mediated by the CD8 + cells
in C. pneumoniae infection is unclear. CTL specific for C. trachomatis have
been demonstrated in C. trachomatis-infected mice [97, 98]. CD8 + cells may
function by secreting cytokines such as IFN-a. Rottenberg et al. showed
that C57BL/6 mice produce IFN-a in response to C. pneumoniae primary
infection and suggested a IFN-a-mediated protection mechanisms [82].
These results were supported by Vuola et al. demonstrating an altered bac-
terial load in anti-IFN-a-treated C57BL/6 mice with markedly exacerbated
signs of pulmonary inflammation [99]. Results are different in BALB/c
mice. Pentillä et al. and Vuola et al. demonstrated an IFN-a-independent
cellular response in BALB/c mice [95, 99]. Interestingly although BALB/c
mice appear not to be dependent on IFN-a during primary infection, they
do not develop a typical Th2 type response either [99]. During reinfection,
neutralization of IFN-a exacerbated the infection in both strains [82, 95].
CD8 + cells may therefore at least partially function through IFN-a produc-
tion, however actively IFN-a-producing cells have also been demonstrated
in CD8-depleted mice, in which the acquired immunity seen during reinfec-
tion is abolished [94]. Thus, IFN-a is an important, but not the only effector
mechanism in acquired immunity.
Overall, a different mouse model of C. pneumoniae-infection demon-
strated that immunity is critically dependent on CD8 + CTL. In a recent
study the group of Wizel et al. was able for the first time to successfully
immunize C57BL/6 mice with a CD8 + T-cell heptaepitope based DNA
vaccine to induce a protective immunity against C. pneumoniae [100].
These results were confirmed by Pentillä et al., demonstrating that DNA
immunization is a promising possibility for developing much wanted vac-
cines against important chlamydial pathogens [101]. Further studies are
now required to elaborate the optimal design of (multicomponent) anti-C.
pneumoniae vaccines for humans.
Infection with C. pneumoniae induces a strong serum response. Little
is known about the immunogenic antigens of C. pneumoniae. The 40 kDa
major outer membrane protein (MOMP) is the most important immuno-
dominant structure during C. trachomatis-infection; during infection with C.
pneumoniae it is a relatively immunorecessive antigen [102, 103]. This lack

of antigenicity has not been resolved yet. Among the different antigens of
C. pneumoniae the 60 kDa heat-shock protein 60 (cHsp 60, GroEL-1) has
been suggested to be a key player during C. pneumoniae-induced humoral
immunity, further studies, however, are required to identify the importance
of this antigen during acute and chronic infection (see also the chapter on
“virulence factors”) [102, 104]. Antibodies to different structures on the C.
pneumoniae elementary body can neutralize the organism in cell culture,
however, the epitope specificity of these neutralizing monoclonal antibodies
to specific C. pneumoniae proteins remains undefined at a molecular level
[105]. Moreover, in vivo, protective effects of antibodies seem to be weak
since reinfections or chronic chlamydial infections are common despite high
antibody titers [18, 19].
Under some circumstances, antibody response during C. pneumoniae-
infection might be immunopathological; for details please see chapter
“virulence factors”.
Cell biology
Monocytes, macrophages, smooth muscle cells, endothelial cells, human air-

way epithelial cells (HAEC), BEAS-2B (bronchoepithelial cell line), HEp-2
and Hela-229 cells have all been shown to be susceptible for C. pneumoniae
infection [69-71]. Following inhalation, bronchial epithelial cells, however,
are the first line of defense in getting in contact with C. pneumoniae and
respiratory epithelium has been identified as the primary target of infec-
tion [70, 71]. Little is known about Chlamydiae-induced epithelial cell
alterations and C. pneumoniae-mediated interactions among all cell types
involved in orchestrating airway inflammation (e.g. lymphocytes, macro-
phages, granulocytes).
Chlamydiae have a unique development cycle with two functionally
and morphologically distinct forms, the condensed, “spore-like” infectious
but metabolically inactive elementary body (EB, 0.3 +m) and the labile,
noninfectious metabolically active reticulate body (RB, 0.9 +m, see Fig. 4)
Infection or invasion is an active process requiring the existence of viable
chlamydia, heat- or UV-inactivated bacteria are not able to invade tar-
get cells. However, there is still limited knowledge of the mechanisms of
Chlamydiae entry into host cells. The chlamydial growth cycle is initiated
when an infectious elementary body (EB) attaches to a susceptible target
cell, promoting entry into a host cell-derived phagocytic vesicle. EB are
internalized, dissociated from the endocytotic pathway by actively modify-
ing the vacuole to become fusogenic with exocytic vesicles. Interaction with
this secretory pathway appears to provide a pathogenic mechanism that
allows chlamydia to establish themselves in a site that is not destined to
fuse with lysosomes [63, 106]. Coombes and Mahony suggested a receptor-
Figure 4. Transmission electron micrographs of Chlamydophila pneumoniae-infected HL cells

72 h postinfection. Large inclusions filled with C. pneumoniae particles are found in the HL
cells. Mature EBs are small with an electrodense core (thick arrow), while RBs are large with
a coarse inner structure (thin arrow, reproduction from [157] courtesy of the authors and the
American Society for Microbiology).
mediated induction of specific cell signaling by Chlamydiae as an essential

step in C. pneumoniae invasion of epithelial cells [107]. After internaliza-
tion EB then develop into reticular bodies (RB), a process which could
be detected metabolically within 15 min and microscopically 12-15 h after
addition of Chlamydiae to HEp-2 and Hela-229 cells [70, 71]. RBs are first
observed dividing by binary fission after about 12 h. As the RBs multiply,
the inclusion membrane expands to accommodate the increasing numbers
of bacteria. After about 18-24 hpostinfection, the first RBs begin differen-
tiating back into EB which accumulate in the lumen of the inclusion as the
remainder of the RBs continue to multiply. Forty-eight to 72 h after com-
pleting the development cycle, depending primarily on the infecting species,
infectious EB are released either by lysis of the host cell or by fusion of the
inclusion membrane with the plasma membrane to release the content of
the inclusion into the environment (for review see [108, 109]).
Under some conditions, however, such as in the presence of IFN-a, or
penicillin or depletion of essential nutrients (iron, tryptophan) Chlamydiae
can achieve a state of intracellular chronic, persistent infection in which they

remain viable, but metabolically quiescent and do not replicate [63, 110, 111].
Because of the reduced or negative ribosomal cell activities, these bacteria
have no adequate targets for the known chlamydia-targeting antibiotics [64,
112]. Persistent Chlamydiae fail to complete development from RBs into
EBs are enlarged and morphologically aberrant, and form small inclusions.
Moreover, they exhibit characteristic gene and protein expression profiles
showing reduced levels of outer membrane proteins like the major outer
membrane protein OmpA or OmcB and substantially increased levels of
Heat shock protein 60 (Hsp 60) [113]. Chlamydiae may be reactivated from
persistence by removal of the inducing stimulus.
A second strategy of Chlamydiae to prolong intracellular survival is
to inhibit pro-apoptotic pathways of the infected host cells. It is thought
that C. pneumoniae can protect infected cells by inhibiting the release of
cytochrome c from mitochondria and upregulate the expression of the anti-
apoptotic mediators IAP and MCL-1. Thus, protection against apoptosis
may be another strategy that the Chlamydiae use to maintain a persistent,
chronic infection. For a more extensive view of the mechanisms involved in
Chlamydia interference with apoptosis signaling in host cells please refer to
the review of Byrne et al. [114].
Virulence factors
Although the genome of C. pneumoniae was sequenced completely in 1999

(1.23 × 106 nt encoding for approx. 1052 proteins, Gen-Bank No.: AE001363,
[15]), until now little is known about structures on the chlamydial surface
(proteins, glycolipids) initiating and mediating bacterial contact to target
cells and inducing subsequent target cell activation. A multitude of the
identified sequences from the chlamydial genome encode for proteins of
bacterial metabolism. Other sequences demonstrated homologies to known
proteins and virulence factors of other bacterial pathogens. Most of these
factors of C. pneumoniae and C. trachomatis have now been identified by
proteom-analysis of both species [115, 116]. Proteins of the “outer mem-
brane complex” (OmpA/B, Omp3, OmcB, POMP), chlamydial lipopolysac-
charide (cLPS), chlamydial heat-shock-proteins (e.g. chsp60/GroEL-1), a
type III secretion apparatus (TTS), the “chlamydial protease- or protea-
some-like activity factor” (CPAF) or peptidoglycans and peptidoglycan-like
structures are likely candidates as possible virulence factors.
Chlamydial envelope/outer membrane complex
The chlamydial outer membrane complex is composed primarily of three

proteins specifically the outer membrane protein A, OmpA, formerly
referred to as major outer membrane protein (MOMP) and two cysteine-

rich proteins, the outer membrane complex B protein (OmcB) and the outer
membrane complex A protein (OmcA) [16]. The gene that encodes MOMP,
ompA [117] exhibits extensive DNA sequence variations that is confined
mainly to four variable segments or domains (VS or VD 1 to VD 4) that
contain subspecies- and serovar-specific antigenic determinants [118]. Until
recently, OmpA was the only protein unequivocally shown to be expressed
on the surface of all chlamydia. The protein is the most important immu-
nodominant structure in all chlamydial strains except C. pneumoniae [102,
103]. Until now, this lack of antigenicity has not been resolved. It might be
possible that either the exposed variable regions of C. pneumoniae OmpA
are not recognized by human or animal immune systems, or that surface
exposure of the OmpA is somehow masked, perhaps by one of the poly-
morphic outer membrane proteins (POMP) as suggested by Christiansen et
al. [103]. OmpA of the chlamydial elementary body is the main component
for chlamydial protection against the environment outside the host, defense
against the host immune response, and attachment to host cells [108].
OmcB, encoded by omcB, does not appear to be surface exposed but is
thought to form a supramolecular lattice in the periplasm. Another impor-
tant difference is that OmcB is extremely highly conserved (for review see
[119]). Stephens et al. could demonstrate that the 60 kDa cystein-rich outer
chlamydial membrane complex protein OmcB is also able to bind heparin
[120]. This protein therefore might be the link mediating chlamydial attach-
ment and initial steps of invasion into target cells.
Although recent studies indicated a functional peptidoglycan (PG)
pathway in chlamydia [121, 122], a clear cut biochemical evidence for the
synthesis of peptidoglycans in chlamydia is missing [123, 124]. Chlamydia,
however, are sensitive to antibiotics that inhibit peptidoglycan synthesis
[125]. This phenomenon has been referred to the “chlamydial anomaly,”
Recent studies, using genomics or proteomics suggested the expression of
peptidoglycan-like structures, not on the surface of elementary bodies, but
– after invasion of the target cells – during cell division on the subsequently
developed reticular bodies [121, 126]. Elucidating the existence of PG in
Chlamydia is of significance for the development of novel antibiotics target-
ing the chlamydial cell wall.
Chlamydial heat shock protein 60 (chsp, GroEL-1)
Heat shock proteins (Hsps) belong to a family of evolutionarily highly con-

served proteins, which are produced by eucaryotic and procaryotic cells dur-
ing a variety of conditions such as heat shock, nutrient deprivation, infec-
tions, and inflammatory reactions, functioning to stabilize cellular proteins.
Several studies using neutralizing monoclonal antibodies or puri-
fied recombinant chlamydial heat-shock protein 60 (cHsp60, GroEL-1),
however, suggested that this protein can act as an extracellular agonist

and might be a key player in activation of different intracellular signal
transduction pathways with a subsequent expression of a profound and
prolonged proinflammatory phenotype in treated cells [76, 79, 127].
Moreover, it has recently been demonstrated, that GroEL-1 is highly
expressed in IFN-a-induced persistent infections of tissue culture cells [63,
128]. Sasu et al. demonstrated that GroEL-1 is a potent inducer of human
vascular smooth muscle cell proliferation and that this effect is mediated
by rapid TLR4-mediated activation of ERK1/2 [80]. We were able to show
that purified recombinant GroEL-1 induced a rapid phosphorylation of
ERK1/2 and p38-MAPK with subsequent enhanced release of IL-8 from
human umbilical vein endothelial cells (HUVEC) [72]. Moreover, GroEL-
1 protein has been shown to stimulate a hyperinflammatory response in
animal models [129–131]. The responses were mediated via a TLR2- and
TLR4-dependent fashion similar to the whole microorganism and differed
markedly from responses induced by endotoxin or CpG oligonucleotides
[77, 78].
Several groups have demonstrated that elevated IgA antibody-titers
of chlamydial heat shock proteins are predictors of chronic chlamydial
infections like bronchial asthma, COPD, arteriosclerosis or pelvic inflam-
matory disease, PID [41, 104, 127, 131]. The molecular mechanisms, how-
ever, by which C. pneumoniae might contribute to development of chronic
diseases remain unclear. Due to the close structural homology between
human and bacterial Hsps and their highly immunogenic nature, Hsps
– especially Hsp60 – have been proposed as key antigens in the devel-
opment of autoimmune diseases [132]. Bachmaier et al. showed that a
peptide from the murine heart muscle-specific _-myosin heavy chain that
has sequence homology to the Hsp60 of C. pneumoniae, C. psittaci, and C.
trachomatis, was shown to induce autoimmune inflammatory heart dis-
ease in mice, suggesting that Chlamydia-mediated heart disease is induced
by antigenic mimicry of a heart muscle-specific protein [133]. In addition,
Kol et al. demonstrated that cHsp60, produced in large amounts during
chronic chlamydial infections, colocalizes within plaque macrophages
with human Hsp60 [127]. Human Hsp60, when expressed by heat-shocked
endothelial cells, can provoke an autoimmune reaction mediating endo-
thelial cytotoxicity [134]. Chlamydial Hsp60 might therefore augment ath-
erosclerosis and/or stimulate humoral and cellular immunity in atheroma
[127, 134].
Chlamydial LPS
Although chlamydia are Gram-negative bacteria, the common LPS group

antigen of all chlamydial species (cLPS) differs significantly from LPS of
other Gram-negative pathogens. In 1998, a first monoclonal antibody was
isolated which recognizes LPS and neutralizes the infectivity of C. pneu-

moniae strain TW183 [135]. This antibody, however, does not neutralize
other strains of C. pneumoniae suggesting the presence of more than a
genus-specific epitope on cLPS. Until now, at least two important differenc-
es between cLPS and LPS from of , for example, enteric bacteria have been
demonstrated: 1) the core trisaccharide 3-deoxy-D-manno-octulosonic acid
(KDO) structure of chlamydial LPS contains a 1-8 linkage, a genus specific
epitope as well as a 1–4 linkage similar to that of other bacteria, encoded
by a single multifunctional KDO transferase [136–138], and 2) the chla-
mydial LPS has low endotoxic activity, although inducing some cytokines
[139, 140]. Immunogold studies suggested that surface exposure of LPS
is greater on RBs than on EBs [141, 142]. Moreover, using different anti-
bodies which recognized either RB or EB Birkelund et al. suggested that
epitope exposure, or the chemical structure of LPS might differ during the
development cycle [143]. cLPS can be released from intracellular, intrain-
clusion Chlamydiae to the inclusion membrane, to the host cell cytoplasm
and surface, and to surrounding infected cells [144–146]. Although this
release might have an impact on the pathogenesis of chlamydial infections
and the host’s immune disposition of infected cells, several studies have
demonstrated that cLPS plays only a minor role for target cell activation
[79, 138].
Chlamydial protease- or proteasome-like activity factor (CPAF)
In 2001, Zhong et al. demonstrated for the first time that a chlamydial
species, C. pneumoniae, secreted a protease into the cytoplasm of infected
target cells. This protease, “chlamydial protease- or proteasome-like
activity factor” (CPAF) split host cell transcription factors necessary for
MHC-I (RFX5) and -II (“upstream stimulatory factor 1”, USF-1) antigen
presentation. Shaw et al. and Dong et al. showed that CPAF is secreted by
different chlamydial species [147, 148]. Heuer et al. suggested that CPAF
could be located in the inclusion lumen or associated with bacteria during
the first 48 h of an acute infection. Seventy-two hours and later, CPAF was
present predominantly in the cytoplasm of the infected cells. Translocation
of CPAF into cytoplasm correlated in time with degradation of the tran-
scription factor RFX5 [149]. CPAF does not preexist in chlamydial organ-
isms and synthesis requires organism replication in cells. Moreover, mice
inoculated with viable chlamydial organisms produced a strong antibody
response to CPAF. In addition, sera from women diagnosed with C. tra-
chomatis cervicitis displayed higher levels of antibodies to CPAF than to
either chlamydial major outer membrane protein or heat shock protein 60.
This sera neutralized the proteolytic activity of CPAF in vitro, suggesting
that CPAF is both produced and immunogenic during human chlamydial
infection [150, 151].
Type III secretion system (TTS)
The first genetic evidence that Chlamydia might have a type III secretion
(TTS) system was presented by Hsia et al. in 1997, when they described
four genes homologous to structural and regulatory components of a con-
tact-dependent or TTS apparatus share high homology with TTS systems
of other bacterial pathogens [152]. Subsequently, these results could be
confirmed by genome and proteom analysis as well as by microscopic obser-
vations [15, 115, 153, 154]. Since TTS systems have been shown to play a
major role in the pathogen-host interaction in several other pathogens like,
for example, Shigella or Salmonella, the TTS may also act as a key virulence
mechanism of Chlamydia [155].
One can speculate about a role for TTS, both in the initial stages of infec-
tion where Chlamydia first comes into contact with the host cell as well as
in the intracellular phase of chlamydial development using the structure of
the TTS system to translocate different effectors into the host cell, depend-
ing on the phase of the developmental cycle. TTS genes expressed in the
mid- to late stage of the developmental cycle appear to be down-regulated
by IFN-a treatment [156]. This suppression may play a role in maintaining
C. pneumoniae in a persistent or altered state within the host cell. It will
therefore be important to determine what structures are present in C. pneu-
moniae and what role each of them plays in the development and possible
persistence of Chlamydia.
Future research directions
Overall, the data presented suggest that Chlamydophila pneumoniae are able
to infect a multitude of target cells and subsequently to activate and trig-
ger a cascade of early and prolonged signal transduction events. Additional
studies are required to determine the relationship between distinct steps
of initial attachment, the chlamydial development cycle, importance of
different chlamydial virulence factors and initiation of host cell signaling
pathways that could lead to target cell damage and inflammation which in
turn may result in acute diseases like bronchitis or (community-acquired)
pneumonia or may promote chronic diseases like COPD, bronchial asthma
or atherosclerosis. New techniques of biochemical and genetical analysis
(genomics, proteomics) are now available to improve our understanding
about pathomechanisms of infection and inflammation and offer unprec-
edented opportunities to address many fundamental questions regarding
chlamydial interactions with the host cells. These results subsequently will
direct new research avenues in terms of diagnostics, therapeutics, as well as
vaccination strategies. Especially chronic persistent infections with meta-
bolically aberrant chlamydiae that are refractory to current antimicrobial
treatment schemes are a challenge for the development of new therapeutic
strategies. In addition, vaccination against Chlamydophila pneumoniae (e.g.,

via DNA immunization as recently demonstrated [101]) could be a benefi-
cial approach for either preventing or controlling infection by this human
respiratory pathogen.
Acknowledgements
The authors apologize for not citing more original manuscripts due to
space limitations and hope that the cited reviews will provide more detail.
This work was in part supported by the Deutsche Forschungsgemeinschaft
to M.K. and N.S. (Kr 2197/1-2), as well as by the Bundesministerium für
Bildung und Forschung (BMBF) to N.S. (CAPNETZ).
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Legionnaires’ disease and its agent

Legionella pneumophila
Dina M. Bitar1, Marina Santic2, Yousef Abu Kwaik3 and Maëlle Molmeret3
1Department of Microbiology and Department of Medical Microbiology and Immunology,
Faculty of Medicine, Al-Quds University, Jerusalem, 19356, Israel; 2Department of Microbiology
and Parasitology, Medical Faculty, University of Rijeka, Croatia; 3Department of Microbiology
and Immunology, Room MS-410, University of Louisville, Louisville, KY 40209, USA
Abstract
Legionella pneumophila, the agent responsible for Legionnaire’s disease, is a facultative
intracellular pathogen that can replicate within protozoa and macrophages. Protozoa are
considered to play a central role in the pathogenesis and ecology of L. pneumophila. In
humans, L. pneumophila reaches the lungs, where it is ingested by alveolar macrophages.
Unlike phagosomes containing inert particles or avirulent bacteria, the L. pneumophila-
containing vacuoles avoid fusion with lysosomes, recruiting rough endoplasmic reticulum
and mitochondria. The formation of this specialized vacuole is directed by the type IV
secretion system encoded by the dot/icm genes in mammalian and protozoan cells. Killing
of mammalian cells by L. pneumophila has been proposed to occur through induction
of apoptosis during the early stages of the infection. A rapid induction of necrosis by L.
pneumophila also occurs upon entry into the post-exponential phase of growth within
both macrophages and protozoa, when the bacteria become cytotoxic. Before the lysis of
the mammalian or protozoan plasma membrane, the bacteria egress into the cytoplasm.
In vivo, clearance of Legionella from the lungs depends on the host production of IFN-a
in A/J mice, while in BALB/c mice IFN-a is not produced. Intracellular replication of L.
pneumophila is inhibited in IFN-a-activated mouse and human primary macrophages.
Both antigen-specific humoral and cell-mediated immune responses are induced dur-
ing Legionella infection. Although Legionella-specific antibodies are produced during
human or murine infection, acquired cell-mediated immune response is believed to play
a stronger role in Legionella clearance. Both macrophages and DCs are able to pres-
ent microbial antigens on major histocompatibility class I and class II molecules, which
stimulate antigen-specific T-cell response. Identification of antigens and determination of
vesicular trafficking mechanisms involved in processing and presentation remain to be
understood in greater detail.
Introduction
The first recognized outbreak of pneumonia due to Legionella pneumophila

occurred in Philadelphia, during the summer of 1976 among 180 persons
attending the 56th annual American Legion Convention. Twenty-nine
patients died and the disease became known as Legionnaires’ disease [1].
112 Dina M. Bitar et al.
Guinea pigs were infected with postmortem lung tissue from the patients
with fatal Legionnaires’ disease, and embryonated yolk sacs were inoculated
with spleen homogenates from the infected guinea pigs. In January of 1977,
a Gram-negative bacterium was isolated and designated L. pneumophila [2].
The source of the infection during the Legionnaires’ convention was later
found to be the air conditioning system in the hotel.
It has been documented that the hallmark of Legionnaires’ disease is
the intracellular replication of L. pneumophila in the alveolar spaces. At
least 48 species of legionellae have been identified, some of which are asso-
ciated with disease while others are environmental isolates and whether
they can cause disease is not known [3]. L. pneumophila is responsible
for more than 80% of cases of Legionnaires’ disease, and among the 13
serogroups of L. pneumophila, serogroup 1 is responsible for more than
95% of Legionnaires’ disease cases. It is estimated that L. pneumophila is
responsible for at least 25,000 cases of pneumonia/year in the US, which is
most probably an underestimate due to the difficulty in bacterial isolation
from clinical samples. Since L. pneumophila is the most frequent cause of
Legionnaires’ disease, most pathogenic and environmental studies have
focused on L. pneumophila.
Free-living amoebae are important predators controlling microbial com-
munities. They are ubiquitous and have been isolated from various natural
sources such as soil, freshwater, salt water, dust, and air. Although their
presence in soil is limited, they have been implicated in the stimulation
of phosphorus and nitrogen turnover and thus play an important role in
soil ecosystems [4]. Free-living amoebae are also frequently isolated from
anthropogenic ecosystems, such as tap water, air conditioning units and cool-
ing towers, feeding on the microbial biofilms present in those systems [5, 6].
However, several bacteria have developed mechanisms to survive phagocy-
tosis by free-living amoebae and are able to exploit them as hosts [7, 8]; see
review in [9]. Transient association with amoebae have been reported for a
number of different bacteria including Legionella pneumophila, mycobacte-
rium sp., Francisella tularensis, or Escherichia coli O157, among others [8, 10–
12]. As most of these bacteria are human pathogens, amoebae have been sug-
gested to represent their environmental reservoirs, acting as “Trojan horses”
of the microbial world [9, 13]. To date only the interaction of L. pneumophila,
a facultative intracellular pathogen of humans causing Legionnaire’s disease,
with free-living amoebae has been studied in greater detail.
L. pneumophila has a very similar intracellular fate within both mam-
malian and protozoan cells. Intracellular multiplication of Legionella with
protozoa such as Acanthamoeba polyphaga and macrophages requires the
Dot/Icm secretion system for biogenesis of the phagosome and intracellular
replication [14, 15]. The dot/icm genes are located in two different regions.
The region I includes seven genes (dotA–D, icmV,W,X) and the larger
region II contains the remaining 17 members (icmT,S,R,Q,P,O,N,M,L,K,E,
G,C,D,J,B,F) [16–19]. The dot/icm genes of the Type IV secretion system are
Legionnaires’ disease and its agent Legionella pneumophila 113
thought to encode proteins involved in the translocation of effector mol-

ecules into the host cell that will prevent the bacteria from being killed [16,
20–23]. The dot/icm loci are highly similar to the transfer region of plasmid
R64 and other IncI1 plasmids, which suggests that dot/icm virulence genes
share a common ancestor with plasmid conjugation system [15, 24–28]. It is
not known whether the dot/icm genes derive from a single plasmid, which
has been separated into the two gene clusters, or there were the result of
multiple gene transfer events. However, only Coxiella burnettii has homo-
logs of the full icm/dot genes, which are contained in a single locus [29-33].
Besides the dot/icm Type IVB secretion system, L. pneumophila possess a
second Type IV secretion system, the lvh/lvr genes that surprisingly are not
involved in its virulence [26]. The Type IV secretion system has been shown
to be the main virulence system of L. pneumophila.
Legionellae facultative intracellular pathogen of free-living amoebae
In 1980, Rowbotham described the ability of L. pneumophila to multiply

intracellularly within protozoa [7, 8]. Since then, L. pneumophila has been
described to multiply in many species of protozoa, and this host-parasite
interaction is central to the pathogenesis and ecology of L. pneumophila.
At least 14 species of amoebae and 2 species of ciliated protozoa have
been shown to support intracellular replication of L. pneumophila [34, 35].
Among the most predominant amoebae in water sources are Hartmannellae
and Acanthamoebae, which have also been isolated from water sources
associated with Legionnaires’ disease outbreaks [34]. Interaction between
L. pneumophila and protozoa is considered to be central to the pathogen-
esis and ecology of L. pneumophila [8, 9, 36]. In humans, L. pneumophila
reaches the lungs after inhalation of contaminated aerosol droplets [34, 37].
The main sources of contaminated water droplets are hot water and air con-
ditioning systems, but the bacteria have been isolated from fountains, spas,
pools, dental and hospital units and other man-made water systems [37]. No
person to person transmission has ever been described. Once in the lungs,
L. pneumophila are ingested by alveolar macrophages, which are thought to
be the major site of bacterial replication. This results in an acute and severe
pneumonia. Approximatively one-half of the 48 species of Legionella have
been associated with human disease. L. pneumophila is responsible for 95 %
of cases of Legionnaires’ disease. However, all the Legionella species under
appropriate conditions may be capable of intracellular growth and infliction
of human disease. Infections due to less common species of legionellae are
not frequently diagnosed and reported, and are less studied than L. pneu-
mophila (review in [38, 39]).
In addition to recognized Legionella species, a number of Legionella-
related bacteria designated Legionella-like amoebal pathogens (LLAPs) [8,
40] have been described [41]. Interestingly, many LLAPs have been asso-
ciated with Legionnaires’ disease [42, 43]. In contrast to other Legionella

species, however, most of LLAPs cannot be cultured in vitro on artificial
media, but are isolated by co-culture with protozoa [34]. Considering that
approximately 50% of the 0.5 million annual cases of pneumonia in the US
are of unknown etiology, the LLAPs may be responsible for at least some
of these cases. The recent developments in using the polymerase chain reac-
tion for bacterial identification in environmental samples will facilitate bet-
ter identification of legionellae and LLAPs. Further cellular and molecular
biology studies are needed to better understand the intracellular life of
these endosymbionts.
The role of amoebae in persistence of Legionella in the environment
It is most likely that the association of legionellae with protozoa is a major

factor in continuous presence of the bacteria in the environment. Many
strategies have been used to eradicate legionellae from sources of infec-
tion in water and plumbing systems that have been associated with disease
outbreaks. These strategies include chemical biocides such as chlorine,
overheating of the water, and UV irradiation [44–46]. Such interventions
have been successful for short periods of time after which the bacteria reap-
pear in these sources [46, 47]. Thus, eradication of L. pneumophila from the
environmental sources of infection requires continuous treatment of the
water with agents such as monochloramine or copper-silver ions in addi-
tion to maintenance of the water temperature above ~55 °C [45, 48–50].
Compared to in vitro-grown L. pneumophila, amoebae-grown bacteria have
been shown to be highly resistant to chemical disinfectants and to treat-
ment with biocides [51]. Amoebae-grown L. pneumophila have been shown
to manifest a dramatic increase in their resistance to harsh environmental
conditions such as fluctuation in temperature, osmolarity, pH, and exposure
to oxidizing agents [52]. Protozoa have been shown to release vesicles con-
taining L. pneumophila that are highly resistant to biocides [53]. The ability
of L. pneumophila to survive within amoebic cysts, further contributes to
resistance of L. pneumophila to physical and biochemical agents used in
bacterial eradication [51, 54]. It is likely that eradication of the bacteria from
the environment should start by preventing protozoan infection, an integral
part of the infectious cycle of L. pneumophila. Extracellular L. pneumophila
is more susceptible to environmental conditions and is not protected from
biocides and disinfectants.
The role of amoebae in pathogenesis of Legionella
There are many lines of evidence to suggest that protozoa play major roles
in transmission of L. pneumophila as infectious particles for Legionnaires’
disease ([7]; review in [9]). First, many protozoan hosts have been identi-
fied that allow intracellular bacterial replication, the only documented
means of bacterial amplification in the environment [34, 36, 55]. Second, in
outbreaks of Legionnaires’ disease, amoebae and bacteria have been iso-
lated from the same source of infection and the isolated amoebae support
intracellular replication of the bacteria [56]. Third, following intracellular
replication within protozoa, L. pneumophila exhibit a dramatic increase
in resistance to harsh conditions including high temperature, acidity, and
high osmolarity, which may facilitate bacterial survival in the environment
[57-59]. Fourth, intracellular L. pneumophila within protozoa are more
resistant to chemical disinfection and biocides compared to in vitro-grown
bacteria [51, 54, 60]. Fifth, protozoa have been shown to release vesicles
of respirable size that contain numerous L. pneumophila. The vesicles are
resistant to freeze-thawing and sonication, and the bacteria within the
vesicles are highly resistant to biocides [53]. Sixth, following their release
from the protozoan host, the bacteria exhibit a dramatic increase in their
infectivity for mammalian cells in vitro [61]. In addition, it has been dem-
onstrated that intracellular bacteria within H. vermiformis are dramatically
more infectious and are highly lethal in mice [62]. Seventh, the number of
bacteria isolated from the source of infection of Legionnaires’ disease is
usually very low or undetectable, and thus, enhanced infectivity of intracel-
lular bacteria within protozoa may compensate for the low infectious dose
[63]. Eight, viable but non-culturable L. pneumophila can be resuscitated
by co-culture with protozoa [64]. This observation may suggest that fail-
ure to isolate the bacteria from environmental sources of infection may
be due to this “dormant” phase of the bacteria that cannot be recovered
on artificial media. Ninth, there has been no documented case of bacte-
rial transmission between individuals. The only source of transmission is
environmental droplets generated from man-made devices such as shower
heads, water fountains, whirlpools, and cooling towers of air conditioning
systems [34]. These findings indicate a rather sophisticated host-parasite
interaction and a tremendous adaptation of legionellae to parasitize pro-
tozoa. This host-parasite interaction is central to the pathogenesis and
ecology of these bacteria.
Intracellular infection of L. pneumophila
Entry of L. pneumophila into protozoa
The attachment and entry mechanisms of L. pneumophila into its proto-

zoan hosts and variations in their mechanisms have been reported for both
amoeba Hartmanella vermiformis and Acanthamoeba spp. Attachment of
L. pneumophila to H. vermiformis is mediated by adherence to a protozoan
receptor characterized as a putative galactose/N-acetyl-galactosamine (Gal/
GalNAc) lectin [65–67]. Host protein synthesis by A. polyphaga is not

required for invasion by L. pneumophila whereas it is required for inva-
sion of H. vermiformis [66]. Integrins are heterodimeric protein tyrosine
kinase receptors that undergo tyrosine phosphorylation upon engagement
to ligands, which subsequently results in recruitment and rearrangements
of the cytoskeleton. Interestingly, attachment of L. pneumophila to the
Gal/GalNAc of H. vermiformis triggers signal transduction events in H.
vermiformis that are manifested in dramatic tyrosine dephosphorylation
of the lectin receptor and other proteins [65, 68]. Similar observations have
been obtained upon infection of H. vermiformis by another species. of
legionellae, L. micdadei [69]. Among the L. pneumophila-induced tyrosine
dephosphorylated proteins in H. vermiformis are the cytoskeletal proteins
paxillin, vinculin, and focal adhesion kinase [65, 68]. Tyrosine phospha-
tases have been shown to disrupt the cytoskeleton in mammalian cells.
Thus, the induced tyrosine phosphatase activity in H. vermiformis is prob-
ably manifested in disruption of the protozoan cytoskeleton to facilitate
entry through a cytoskeleton-independent receptor-mediated endocytosis.
Interestingly, in addition to these manipulations of the signal transduction
of H. vermiformis by L. pneumophila, bacterial invasion is also associ-
ated with specific induction of gene expression in protozoa, and inhibi-
tion of this gene expression blocks entry of the bacteria [70]. Following
this initial host-parasite interaction, uptake of L. pneumophila by A.
castellanii occurs by coiling phagocytosis [11, 12]. However, the uptake
of L. pneumophila by H. vermiformis occurs mainly through cup-shaped
invaginations (or zipper phagocytosis) on the surface of the amoeba, in
addition to occasional coiling phagocytosis [11, 12]. Human macrophages
are able to phagocytose heat- or formalin-killed L. pneumophila by coiling
phagocytosis [71], which indicates that coiling phagocytosis does not play
a role in the intracellular fate of L. pneumophila. However, the infectiv-
ity of A. castellanii and macrophages by L. pneumophila has been shown
to be similar [72]. In addition, the adherence receptors of macrophages
used by L. pneumophila does not seem to affect its intracellular survival
profoundly as the bacterium multiplies within phagocytes after entry
under different opsonizing or non-opsonizing conditions [73–76]. Uptake
of L. pneumophila by another protozoan host, A. polyphaga, is not com-
pletely blocked by Gal or GalNAc and is associated with partial tyrosine
dephosphorylation of a 170 kDa protein, which may be related to the Gal/
GalNAc lectin of H. vermiformis [66]. Thus, entry of the bacteria into A.
polyphaga is partially mediated by the Gal/GalNAc lectin and additional
receptors may be involved for bacterial attachment and entry. The hetero-
geneity in the uptake mechanisms of L. pneumophila into H. vermiformis
and A. polyphaga has been confirmed using invasion defective mutants of
L. pneumophila. Several mutants that were severely defective in attach-
ment to A. polyphaga exhibited minor reductions in attachment to H.
vermiformis [66].
The mode of entry into mammalian and protozoan host cells have been
shown to occur by both dot/icm dependent and independent mechanisms
[72, 77, 78]. Studies have shown that phagocytosis of wild-type L. pneu-
mophila is more efficient than uptake of dot/icm mutants within macro-
phages and A. castallanii, indicating that this mechanism is independent of
adherence receptors [72, 77]. However, when the hosts are infected with sta-
tionary-phase cultures that have been incubated overnight in pH 6.4 buffer,
a treatment which enhances the resistance to acid, hydrogen peroxise and
antibiotics stress, entry into A. castellanii and macrophages do not require
functional dot/icm genes [78]. In addition, a “repeats in structural toxin”
(RTX) gene, rtxA has also been shown to play a role in adherence and entry
and replication within human macrophages, A. castellanii and in vivo [79,
80]. These data indicate the remarkable adaptation of L. pneumophila for
attachment and invasion into different host cells.
Bacterial invasion of macrophages cells and genetic susceptibility
Invasion and intracellular replication of L. pneumophila within pulmo-

nary cells in the alveoli is the hallmark of Legionnaires’ disease [81].
Uptake of L. pneumophila by monocytes and macrophages has been
shown to occur through conventional and coiling phagocytosis [71, 82-85].
It has been recently shown that the enhanced phagocytosis of L. pneu-
mophila by mammalian cells is dot/icm-dependent [72]. Interestingly, the
dot/icm genes delay uptake and induce macropinocytosis in A/J mice mac-
rophages [77]. With the exception of A/J mice, most of the inbred mouse
strains are not permissive to L. pneumophila infection, neither are macro-
phages isolated from these mice [86, 87]. Macropinosomes containing L.
pneumophila in A/J mice macrophages are induced transiently and shrink
rapidly (5-15 min) [77], and this mode of uptake is linked to the lgn1 locus
on chromosome 13 of mice [77, 88, 89]. In macrophages of non permissive
strains of mice, the macropinocytic uptake of L. pneumophila is reduced
[77]. The lgn1 allele causes the bacteria to behave as if they are lacking
the dot/icm system [77]. Thus, the lgn1 allele is required for dot/icm-
dependent macropinocytosis and delayed uptake by mice macrophages
[77]. Whether this mode of uptake plays a role in subsequent trafficking
of L. pneumophila is not known. The intracellular growth of others spe-
cies than pneumophila are not under lgn1’s control [90]. The mouse lgn1
region includes six copies of the neuronal apoptosis inhibitory protein
(naip) gene. These Naip proteins have been shown to be direct inhibitors
of caspase 3 and 7 [91]. Recently, naip5, also known as birc1e (baculoviral
inhibitory apoptosis protein repeat-containing 1), has been shown to be
the allele within lgn1 responsible for susceptibility to Legionella [92, 93].
The mechanism by which naip5 regulates susceptibility to L. pneumophila
is not yet known.
Manipulation of vesicle traffic by the Dot/Icm system and biogenesis

of a replicative ER-derived vacuole
L. pneumophila utilize the Dot/Icm secretion system to transfer macromol-

ecules into the host cell to evade endocytic fusion [25, 27]. The dot/icm loci
may be involved in the insertion of a pore in the host cellular membrane
through which the effector proteins are exported into the host cell [94, 95].
The effector molecules involved in intracellular trafficking and evasion of
the lysosomal fusion within mammalian cells are cis-acting on the phago-
some, but do not alter endocytic fusion in the rest of the cell [96]. With few
exceptions, the function of individual Dot/Icm proteins is unknown.
Several studies demonstrated that upon internalization of L. pneumophila
by the host cell, the Legionella-containing vacuole recruits organelles such as
vesicles, mitochondria and ER [11, 12, 97, 98] (Fig. 1). Within 5 min following
entry of the bacteria into host cells, the L. pneumophila phagosome is sur-
rounded by host cell vesicles, and RER [98-100]. It has been shown that the
Legionella-containing phagosome within mammalian macrophages and pro-
tozoa does not fuse to lysosomes [12, 71, 101, 102]. Interestingly, examination
of the intracellular infection of macrophages, alveolar epithelial cells, and
protozoa by another legionellae spp., L. micdadei, showed that within all of
these host cells, the bacteria were localized to RER-free phagosomes [103].
Whether other legionellae species replicate within RER-free phagosomes is
still to be determined. Recent studies suggest that fusion [104], or exchange
of lipid bilayer with ER vesicles on the L. pneumophila-containing phago-
some [99] allows the phagosomal membrane to become as thin as the ER
membrane with similar characteristics [99, 104]. Within 5 min of uptake, host
vesicles come into contact with wild-type Legionella-containing phagosomes
and flatten along the surface of the phagosome, and this process is completed
within 15 min [99], and is dot/icm-dependent [99]. This is consistent with
earlier studies that have shown that after 4 h of infection, there are only few
vesicles associated with the phagosomal membrane, but there are ribosomes
studding the phagosomal membrane [98]. It is likely that the recruitment of
the ER may be involved in the biogenesis of the phagosome that is depen-
dent on the type IV secretion system, since the dot/icm mutants are unable to
recruit RER and their phagosomes fuse to the lysosomes [105]. In addition,
it has been shown that the L. pneumophila-containing phagosome is a transi-
tional ER (tER)-derived organelle [106]. Its biogenesis involves intercepting
early secretory vesicles exiting from tER [106].
Similar to its intracellular fate within macrophages, L. pneumophila is
enclosed, after entry into amoebae, in a phagosome surrounded by host
cell organelles such as mitochondria, vesicles, and a multilayer membrane
derived from the rough endoplasmic reticulum (RER) of amoeba [11,
98–100]. A few hours after internalization and formation of the ER-derived
replicative organelle, bacterial replication is initiated. Both, evasion of
endocytic fusion and recruitment of early secretory vesicles as they exit
Figure 1. L. pneumophila are contained within intact phagosome at 8 h post-infection.

Representative electron micrographs of L. pneumophila-infected macrophages 8 h post-infec-
tion are shown in (a) and (b). The membrane of the LCP is intact as indicated by the thin
arrows. Lpn for L. pneumophila, N for nucleus. Adapted from [140].
the ER is controlled by the Dot/Icm type IV secretion system [11, 98, 99,
106–108]. Most or all of the Dot/Icm structural proteins have been shown
to be essential for biogenesis of the LCP and for intracellular multiplication
within both protozoa and mammalian cells [25, 27]. The role of the RER
in the intracellular infection is unknown, but the RER is not required as a
source of proteins for the bacteria [109].
Recently, the soil amoeba, Dictyostelium discoideum has been studied as
a new host model for Legionella, in particular to understand how Legionella
establish its replicative niche within phagocytic host cells. The advantages of
using this model for the infection of Legionella is that D. discoideum can be
genetically manipulated, that cellular markers are commercially available,
and more importantly, the growth of L. pneumophila is similar to that in
macrophages and fresh-water amoebae including the requirement for a func-
tional dot/icm Type IV secretion system [110, 111]. D. discoideum is found in
soil as a unicellular free-living amoeba that feeds on bacteria. Under starva-
tion conditions, the organism undergoes a complex developmental cycle dur-
ing which it aggregates to form a multicellular motile phototactic slug. This
slug can develop into a fruiting body forming viable spores supported by a
column of stalk. In a rtoA mutant of D. discoideum where vesicle trafficking
event is lowered, the intracellular growth of L. pneumophila is depressed
[112]. In addition, Cytoskeleton-associated proteins and calcium-binding
proteins of the ER, calnexin and calreticulin, specifically influence the

uptake and the intracellular growth of L. pneumophila within D. discoideum
[113]. Therefore, ER recruitment plays an important role in the intracellular
multiplication of L. pneumophila within this host. One of the hypotheses
regarding the role of the recruitment of ER to the LCP is that autophagy
mechanism involved in cellular homeostasis was highjacked by Legionella in
order to establish its replicative niche. As macroautophagy genes have been
identified in D. discoideum, they have been used to show that macroautoph-
agy is dispensable for the intracellular multiplication of L. pneumophila in
D. discoideum [114]. Therefore, it is rather clear that ER-derived vesicles and
proteins are part of a system that leads to the establishment of the replicative
vacuole of L. pneumophila but autophagy does not appear play a role in this
mechanism within Legionella protozoan hosts.
Role of the dot/icm genes in evasion of the endocytic pathway
The main virulence system of L. pneumophila is the dot/icm Type IV secre-

tion system. Because the Dot/Icm secretion system is ancestrally related to
Type IV secretion systems that mediate conjugal DNA transfer between
bacteria [16], L. pneumophila may utilize this transporter to transfer macro-
molecules into the host cell to evade endocytic fusion [104]. The Dot/Icm-
mediated transfer is thought to occur through the insertion of a pore in the
host cellular membrane through which the effector proteins are transported
[94, 95]. With few exceptions, the function of individual Dot/Icm proteins is
unknown. However, the dot/icm genes are present in all tested Legionella
species [115].
Most of the dot/icm genes required for intracellular growth within
human cells, are also required for intracellular growth in the protozoan host
Acanthamoeba castellanii [15]. Although some loci have been shown to be
only essential for the intracellular growth of L. pneumophila in macrophages
[116], numerous loci have been identified as essential for survival and intra-
cellular replication of L. pneumophila in A. polyphaga or H. vermiformis
and in macrophages [14, 117, 118]. D. discoideum has been shown to support
intracellular multiplication of L. pneumophila [110, 111, 119]. As stated ear-
lier, the intracellular fate of L. pneumophila is very similar in infected D. dis-
coideum to that in macrophages, including the recruitment of RER, evasion
of lysosomal fusion [119], and dependence of intracellular growth on dot/icm
gene functions [119]. The similarity between the infection by L. pneumophila
of different protozoa, supports the idea that the ability of L. pneumophila to
parasitize macrophages and hence to cause human disease is a consequence
of its prior adaptation to intracellular growth within protozoa.
The Type IV secretion system of L. pneumophila is though to be an effec-
tive apparatus to translocate effector molecules into the host cell and modu-
late the host cell physiology in order for the bacteria to establish a replicative
niche by recruiting ER-derived vesicles and evading the endocytic pathway.

Genetic screens that enable the identification of the set of 26 dot/icm genes
have failed to identify the genes encoding the Type IV secretion system
substrates secreted into host cells [17, 19, 27]. Different strategies have been
used for this purpose. First, strategies, which are not based on intracellular
growth defect of the bacteria, but based on homology to eukaryotic protein
have allowed the identification of Type IV secretion system effectors such as
RalF, LepA and LepB [20, 21]. Most of these substrates do not express an
intracellular replication deficient phenotype (or a minor one) within both
mammalian and protozoan host cells, which explain why they had not been
isolated previously in genetic screens for intracellular growth mutants. RalF
protein, containing a Sec7-homology domain is produced by L. pneumophila
and is injected into the host cells by the Dot/Icm transporter, functioning as
an exchange factor that activates members of the ARF protein family [21,
120]. LepA and LepB, which have a weak homology to SNAREs have been
shown to be delivered to host cells by a Type IV secretion system-depen-
dent mechanism. The double mutant of the paralogues lepA lepB exhibited
a defect in release of L. pneumophila-containing “fecal” or “respirable”
vesicles from A. castellanii and D. discoideum [20]. Second, a strategy based
on that fact that some translocated proteins also function to maintain the
integrity of the Dot/Icm translocator and mutations that destroy this func-
tion are predicted to result in a Dot/Icm complex that poisons the bacterium,
resulting in reduced viability, has allowed identifying an effector called LidA.
This substrate LidA, identified by a complex genetic screen has been shown
to be associated with the cytoplasmic face of the L. pneumophila-containing
phagosome [23]. Third, an interbacterial protein transfer assay is another
strategy used to identify the substrate SidC (substrate of icm/dot complex)
identified among other Sid substrates also secreted into macrophages [22].
Finally, some substrates have been identified using chaperone proteins
belonging to the dot/icm genes secretion system, as bait. IcmS has been
hypothesized to serve as a chaperone for secreted substrates since it is pre-
dicted to be located in the bacterial cytoplasm, has no homology to bacterial
conjugal transfer proteins and has similar biochemical properties of secre-
tion chaperones from the type II secretion system [121, 122]. IcmS has been
used as a bait to identify potential secreted substrates such as SidE family
proteins (SdeA, SdeB, SdeC, SidE) [123] family proteins have been shown to
be secreted by L. pneumophila at early stages of infection of macrophages
across the phagosomal membrane and have been shown to be required for
full virulence in Acanthamoeba castellanii although individual deletions of a
number of substrates had a modest or no effect in intracellular replication
[22] probably resulting from the export of functionally redundant proteins by
the Type IV secretion system. In addition, as the complex IcmS-IcmW form a
stable protein complex and play an important role in subtrate translocation,
IcmW has also been used as a bait in a yeast two-hybrid system to identify
substrate proteins translocated into host cells by the type IV secretion sys-
tem [124]. IcmW-interacting proteins (Wips) have been recovered including

SidG and SidH identified previously with the interbacterial protein transfer
screen [22] as well as WipA, which is translocated into mammalian host cells.
A paralogue called wipB, which is also translocated into macrophages by the
Type IV secretion system, was found in the Legionella genome data base and
the double mutant wipA wipB had no growth defect in A. castallanii [124].
The functions of the identified substrates are under investigation. There are
probably hundred of effectors that need to be identified in order to under-
stand how Legionella establish a successful replicative niche within its host
cells. Some of these substrates may also be host-dependent and be functional
only in one of the various host cells of Legionella.
Growth phase-regulated virulence
L. pneumophila obtained from post-exponential cultures, expresses traits

that are correlated with virulence, in contrast to exponentially-growing
bacteria, [7, 8, 57, 58]. During the replication phase, L. pneumophila are
sodium resistant and aflagellated [7, 8, 57, 58]. When L. pneumophila egress
from host cells, the bacteria are flagellated and sodium-sensitive [7, 8, 57,
58]. It has been hypothesized that amino acid limitation in vitro induces the
virulent phenotype [57, 58]. When L. pneumophila enters into post expo-
nential growth phase or is subjected to amino acid limitation, the bacteria
accumulate the stringent response signal, guanosine 3’,5’-bispyrophosphate
(ppGpp) through the ppGpp synthetase, RelA [125]. The accumulation of
ppGpp increases the amount of the stationary-phase sigma factor RpoS,
which triggers the expression of the stationary-phase genes [125]. A rpoS
mutant of L. pneumophila replicates within HL60 and THP-1 monocytic
cell lines, but is attenuated in A. castellanii [126]. In bone marrow-derived
macrophages from A/J mice, L. pneumophila rpoS mutants replicate poorly
because they traffic rapidly to a late endosome-like compartment [125].
These phenotypic differences in different host cells may be due to differ-
ent parental strains used to construct the rpoS mutants. It has been shown
that some genotypic and phenotypic differences exist between the AA100,
JR32 and LP01 stains, which are the most commonly used in virulence
studies [127] and AA100 is clearly the most virulent. Therefore, the differ-
ences between L. pneumophila strains and/or between the host cells may
explain the different intracellular growth observed for the rpoS mutants.
Sodium sensitivity and maximal expression of flagellin also requires RpoS
[125]. Therefore, some RpoS-regulated traits could be critical for efficient
transmission or infection [126]. L. pneumophila in post-exponential phase
becomes cytotoxic by an RpoS-independent pathway [125]. It is proposed
that when nutrient levels and other conditions are favorable, L. pneumoph-
ila replicates within host cells, and when amino acids become rare, intracel-
lular bacteria express several traits that permit escape from the host cell,
survival in the environment and the transmission to a new host. However,

the intracellular signals that trigger phenotypic transition at the post-expo-
nential phase are still to be determined.
Although a previous study has shown that RpoS, which accumulates
when RelA is activated, is required for intracellular growth in A. castellanii
[126], Zusman et al. have shown that relA gene product is dispensable for
intracellular growth in HL-60-derived human macrophages and in A. cas-
tellanii [128]. Moreover, it has also been shown that RelA and RpoS have
minor effects on expression of some of the dot/icm genes [128]. Thus, the
role of RpoS in the intracellular infection seems to be host cell-specific, but
that may be due to differences between the parental strain used to construct
the rpoS mutants.
Interestingly, it has been shown that the conserved RNA binding protein
CsrA is a global repressor of phenotypic transition of L. pneumophila at the
post-exponential phase [129]. Overexpression of CsrA in L. pneumophila
blocks many phenotypic traits that are expressed at the post-exponential
phase such as flagellation and reduction of cell size [129]. This is also asso-
ciated with reduction in transcription of fliA and flaA [129]. In addition,
the global response two-component regulator of L. pneumophila, LetA/S,
is involved in regulation of phenotypic transition, since letA mutants of L.
pneumophila exhibit reduced infectivity and are more resistant to oxidative
and acid stress in addition to a severe defect in intracellular replication in
Acanthamoeba [130, 131]. However, the letA mutant is not defective for
intracellular replication in human-derived macrophages, similar to the rpoS
mutant and mutants in the type II secretion system [131]. Thus, RpoS and
LetA/S regulate phenotypic transition of L. pneumophila at the post-expo-
nential phase and both are essential for expression of L. pneumophila genes
required for replication in the protozoan host but not in human-derived
macrophages. Since both regulatory systems are triggered by ppGpp syn-
thesized by RelA, and RelA is not required for the intracellular infection
of macrophages or amoeba, it is not clear how other regulatory pathways
contribute to the regulation of phenotypic transition.
Cytolysis of the host and bacterial egress
A fundamental step in the pathogenic life cycle of intracellular bacteria is

the ability to lyse the host cell and to egress. Apoptosis and necrosis are
the two commonly observed types of cell death. Necrosis is characterized
by physical damage causing cell death. Apoptosis is a regulated suicide
program of the cell manifesting morphological and biochemical features
distinct from those of necrosis [132]. Killing of mammalian cells by L.
pneumophila has been proposed to occur in two phases [133, 134]. In the
first phase, L. pneumophila induces apoptosis in macrophages, monocytes
and alveolar epithelial cells during the early stages of the infection [133,
Figure 2. L. pneumophila is cytoplasmic at 24 h post-infection. Representative electron micro-

graphs of L. pneumophila-infected macrophages at 24 h post-infection are shown in (c) and (d).
L. pneumophila (Lpn) are cytoplasmic where bacteria are surrounded by numerous vesicles
(V), lysosomal contents (white arrows), mitochondria (M), and amorphous elements (A). No
distinguishable phagosomal membrane surrounds the bacteria. N for nucleus. Adapted from
[107].
135–137], which is mediated through the activation of caspase-3 [134].

Induction of apoptosis is largely independent of the bacterial growth phase
[134]. The second phase is mediated through rapid induction of necrosis by
L. pneumophila upon entry into the post-exponential phase of growth when
the bacteria become cytotoxic [138, 139]. Our working model of bacterial
egress can be presented in three steps. First, upon exiting the exponential
phase of intracellular growth, an „egress pore“ is inserted into the phago-
somal membrane leading to its disruption. Second, the bacteria egress into
cytoplasm. Third, disruption of organelles and the plasma membrane occurs,
culminating in lysis of the host cells and bacterial egress.
A detailed ultrastructural analysis of late stages of intracellular replica-
tion has been performed to examine egress of L. pneumophila from both
macrophages and amoebae by electron microscopy [140]. The membrane
of the L. pneumophila-containing phagosome (LCP) within both macro-
phages and Acanthamoeba polyphaga is intact up to 8 h postinfection [140].
However, at 12 h, the majority of the LCPs are disrupted within both hosts,
while the plasma membrane remains intact [140]. At 18 and 24 h postinfec-
tion, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and
amorphous material are dispersed among the bacteria and these bacteria
are considered cytoplasmic [140] (Fig. 2). Thus, by 18 h–24 h postinfection,
the majority of the remaining host cells harbor cytoplasmic bacteria and
this transient cytoplasmic presence of L. pneumophila precedes lysis of the
plasma membrane. Interestingly, within both macrophages and amoebae,
bacterial replication proceeds in the cytoplasm.
Therefore, the phagosomal membrane is disrupted first, rather than by

simultaneous lysis of both the phagosomal and the plasma membranes.
These disruptions of LCP may be the result of multifactorial events linked
to apoptosis, the pore forming activity (PFA) of the Type IV secretion system
and mechanical pressure due to the increase of the phagosomal size [95].
Immunity to intracellular L. pneumophila
The Toll-like receptors
As the interaction between host phagocytes and Legionella results in the

induction of the host cell response that is thought to activate the innate
immune system through the stimulation of TLRs on the surface. It leads
to the production of pro-inflammatory cytokines that recruit lymphocytes
to the infection sites and activate macrophages. Macrophages express both
TLR2 and 4, which can recognize different bacterial products (peptido-
glycan, LPS, etc.). However, surprisingly TLR2 plays a major role in the
response to Legionella, whereas TLR4, which recognizes predominantly
LPS, is less involved in the process [141, 142]. In addition, TLR5 may also be
important in immunity to Legionella as TLR5 recognizes a conserved region
in most bacterial flagellin including Legionella flagellin.
The cytokine production
Neutrophils, macrophages and dendritic cells have been shown to produce

cytokines after Legionella infection, which is comparable to the cytokine
production involved in the Th1 response [143, 144]. IL-12 is produced by
many cells, including macrophages, DC and neutrophils after exposure
to bacteria and has been shown to have an important role in eliminating
Legionella in vivo [145]. IL-18 is also crucial in the induction of IFN-a
production from T-cells, B cells and NK cells in vitro and during in vivo
infection with Legionella [146]. When they are depleted independently or
together, the levels of IFN-a drop significantly, which lead to a decreased
ability of clearing Legionella from the lungs of the mice [146]. Intracellular
replication of L. pneumophila is inhibited in gamma interferon (IFN-a)-
activated bone marrow-derived mouse macrophages and IFN-a-activated
human monocyte-derived macrophages in a dose-dependent manner. This
inhibition of intracellular replication is associated with the maturation of
the LCP into a phagolysosome [147]. Together with IFN-a, TNF-_ plays
also a role in the clearance of Legionella from the lungs via activation of
macrophages. Added independently or together, both IFN-a and TNF-_
can also significantly restrict the intracellular growth of Legionella [148,
149].
The clearance of Legionella from the lungs depends on the host produc-
tion of IFN-a in A/J mice [150]. Additionally, Legionella infection cannot be
cleared efficiently in BALB/c mice, which do not produce IFN-a compared
to infection in mice producing IFN-a [151]. Similarly, when human mono-
cytes and alveolar macrophages are treated with IFN-a, it results in dose-
dependent restricted growth of Legionella [152]. Restriction of Legionella
growth is, in part, due to low availability of intracellular iron, a process
mediated by the transferring receptor which is downregulated in IFN-a
activated monocytes [153]. A recent study also showed that activated mac-
rophages infected by L. pneumophila can downregulate T-cell responses via
production of prostaglandins, which may play a role in limiting unnecessary
immune-mediated damage of host tissues [154].
Activated macrophages can also produce nitric oxide after bacterial
infection, which has a direct lethal effect on many pathogens. In the case of
Legionella infection, the role of NO may not be direct. Inhibitors of NO syn-
thesis had an effect intracellular replication of L. pneumophila in BALB/c
alveolar macrophages, but not in A/J mice macrophages. It had, however, a
effect on the Legionella infection of A/J mice model [149, 151, 155-157].
Acquired immune response
Both antigen-specific humoral and cell-mediated immune responses are

induced during Legionella infection. Although Legionella specific antibod-
ies are produced during human infection, or in the guinea pigs model, their
role in controlling Legionella infection has not been clearly demonstrated
[158]. Antibody opsonization does not inhibit intracellular multiplication of
L. pneumophila [159].
However, when CD4 and CD8 T-cells are depleted in mice, susceptibility
to Legionella infection is enhanced, suggesting that acquired cell-mediated
immune response play a role in Legionella clearance [160]. Both macro-
phages and DCs are able to present microbial antigens on major histo-
compatibility (MHC) class I and MHC class II molecules, which stimulate
antigen specific T-cell response. DCs appear to play an important role in
producing antigen-specific immune responses and in priming T-cells. DCs
are able to restrict the intracellular growth of Legionella without prevent-
ing traffic of Legionella-containing vacuole or bacterial proteins synthesis.
Therefore, resctriction of growth is possible without the Legionella vacuole
fusing to lysosomes, suggesting that the range of proteins presented by DCs
to naïve T-cells is very similar to those presented by macrophages [161, 162].
Taken together, these data showed that Legionella antigens synthesized
in a non-degradative ER-derived vacuole are most likely processed and
loaded onto MHC class II molecules for presentation to CD4 + T-cells [161].
In addition, it has recently been shown that in mice primary macrophages,
trafficking of Legionella-containing phagosome to lysosomes is required for
optimal antigen processing and MHC-II presentation to CD4 T-cells [163].

Identification of antigens and determination of vesicular trafficking mecha-
nisms involved in processing and presentation remain to be understood in
greater detail.
Conclusions
L. pneumophila has become a paradigm of an intracellular pathogen that

manipulates several processes involved in endocytosis and vesicle traffic
between host cell organelles, and many of these manipulations are con-
trolled by the Dot/Icm secretion system and the effectors exported through
this system. This organism avoids phagosomes-lysosomes fusion within the
first few minutes of the intracellular infection and the phagosomes are con-
verted into endoplasmic reticulum-derived organelles that support intra-
cellular replication [99]. This process involves intercepting early secretory
vesicles exiting from the tER [106]. After 4 h, L. pneumophila starts to repli-
cate within this replicative organelle. Between 8 h and 18 h, the phagosomal
membrane is gradually disrupted and the bacteria become cytoplasmic and
are dispersed among cytoplasmic organelles such as mitochondria and lyso-
somes prior to lysis of the host cell [140]. Legionella infection triggers both
antigen-specific humoral and cell-mediated immune responses, but the cell-
mediated responses appear to play a greater role in Legionella clearance
from the lungs. Understanding the unique intracellular fate at the molecular
and the cellular level will unravel fascinating aspects of the intricate balance
in the evolution of this host-parasite interaction and the ability to control
Legionella propagation in human and in environment.
Acknowledgements
Our work is supported by Public Health Service grant RO1AI43965 award-

ed to Y.A.K.
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Pathogenesis of Streptococcus pneumoniae infections:

adaptive immunity, innate immunity, cell biology,
virulence factors
Sven Hammerschmidt1, Gavin K. Paterson2, Simone Bergmann1 and

Timothy J. Mitchell2
1Research Center for Infectious Diseases, University of Würzburg, Röntgenring 11, 97070
Würzburg, Germany; 2Division of Infection and Immunity, Institute of Biomedical and Life
Science, Joseph Black Building, University of Glasgow G12–8QQ, UK
Abstract
During the past two decades the intense study of the infection process of Streptococcus
pneumoniae has elucidated multifaceted interactions of the human pathogenic bacterium
with the host. A broad spectrum of pneumococcal virulence factors, which are adapted
successfully to different host niches, is involved either predominantly in nasopharyngeal
colonization or subsequently in dissemination and transmigration of host tissue barriers.
The severe course of infections becomes manifest in invasive diseases like pneumonia,
meningitis and septicaemia.
To escape the risk of increasing antibiotic resistance and to combat the threat of
pneumococcal infections pneumococcal vaccines have been developed. The carrier pro-
tein of the current available heptavalent vaccine is not derived from pneumococci there-
fore it is thought to substitute this carrier by a highly conserved and immunogenic pneu-
mococcal-specific protein. S. pneumoniae is a versatile microorganism and has evolved
numerous successful strategies to colonize its host and to evade host defence mechanisms.
In this report we discuss the bacterial repertoire of virulence factors and provide insights
into the surface protein variability. In addition, we show the impact of these virulence
factors on interactions with host components, including cellular receptors and how the
function of these proteins contributes to colonization and virulence of S. pneumoniae.
The non-invasive and invasive infections are accompanied by immune responses of both
the innate and adaptive immune system. These two systems operate in concert to combat
infections, but pneumococci have developed highly sophisticated mechanisms to subvert
the host immune system. We introduce pattern recognition receptors that recognize spe-
cific structures of pneumococci and stimulate thereby host defence mechanisms.
Introduction
Streptococcus pneumoniae (the pneumococcus) is a serious human patho-

gen causing local infections including otitis media and sinusitis and life-
threatening invasive diseases, such as lobar pneumonia, sepsis and men-
140 Sven Hammerschmidt et al.
ingitis [1]. The burden of disease is highest in the youngest and elderly
population and in patients with immunodeficiencies. The pneumococcus is
the prime cause of community-acquired pneumonia in adults and accounts
for 50–75% cases. The incidence of pneumococcal pneumonia remains high,
between 68–260 cases per 100,000 and year [2]. Despite the use of antibiot-
ics and availability of vaccines the mortality rate remains high. Each year,
1 million children younger than 5 years die from pneumonia and invasive
diseases [3]. Even more, community-acquired pneumococcal meningitis has
a very high case-fatality rate. The survivors often develop long-term clinical
sequelae including hearing loss, neurological deficits, and neurophysiologi-
cal impairment [4]. The infections caused by pneumococci are preceded by
an asymptomatic carrier status and accompanied by the transmigration of
tissue barriers by the pathogen. The clinical outcome of disease has been
shown to be dependent on both the pathogen and host susceptibility for
the individual pathogen. Pneumococci are endowed with a multitude of
factors that contribute to the pathogenic potential of this versatile patho-
gen. Biological activities that have been attributed to these virulence fac-
tors include the subversion of host immunity and adoption of host-protein
functions to facilitate adherence and invasion. Pneumococci disseminate
and gain access to the ear, lungs, blood or meninges. The adaptation of
pneumococci to different host milieus including the nasopharynx has been
correlated with a differential expression of several pneumococcal factors
[5–9]. A comprehensive understanding of critical steps during pneumococ-
cal pathogenesis including colonization, progression to pneumonia, dis-
semination in the bloodstream, and transition of the blood-brain-barrier is
crucial to combat the threat of pneumococcal infections and hence, reduce
the mortality exacted by this pathogen. This review will evaluate our current
understanding of the mechanisms employed by pneumococci to encounter
the human host.
Nasopharyngeal colonization
S. pneumoniae is a complex microorganism divided into over 90 serotypes

depending on the antigenic structures of their capsular polysaccharides [10].
Pneumococci of different serotypes are able to simultaneously colonize the
nasopharyngeal cavity of healthy individuals [11]. The rates of carriage and
acquisition depend on age, genetic background, socioeconomic conditions
and geographical area [12]. Europe and the United States show similar
serotype distributions with minor differences in several serotypes. Disease is
most commonly due to strains representing 20 of the > 90 different pneumo-
coccal serotypes and these 20 serotypes have been covered by the 23-valent
polysaccharide (PS) vaccine [13, 14]. By contrast, the serotype distribution
in Asia is slightly different, which had resulted in only < 70% effectiveness
of the 23-valent conjugate PS vaccine [15]. The current vaccine, a seven-
Pathogenesis of Streptococcus pneumoniae infections… 141
Figure 1. Adherence of Streptococcus pneumoniae to human epithelial cells after 4 h infec-

tion as seen by high-resolution field emission scanning electron microscopy. (Image courtesy
Manfred Rohde, German Research Centre for Biotechnology, Braunschweig, Germany.)
valent conjugate vaccine (Prevnar, Wyeth, USA), covers the most prevalent
serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. It is noteworthy that invasive
disease originates from nasopharyngeal colonization with the homologous
serotype [16]. Interestingly, certain serotypes including 14 and 18C clones
have a high potential to cause invasive disease, whereas the most commonly
carried serotypes 6B, 19F, and 23F are least invasive. By contrast, infrequent
colonizers and non-vaccine serotypes including serotypes 8, 38, 33F appear
to be more invasive [12, 17].
Transition from colonization to pneumonia and invasive infections
The mechanisms of how the pneumococcus makes the transition from a

commensal with asymptomatic carriage to a virulent pathogen causing dis-
ease have not yet been fully explained. It is, however, known that the local
immune response has an important regulatory role during colonization and
subsequent infections [18]. The successful conversion of the commensal to
an invasive microorganism is accompanied by the transmigration of tissue
barriers and the subsequent adaptation of the pathogen to different host
niches. A prerequisite for transmigration is the ability of the pneumococ-
cus to adhere to mucosal cells of the respiratory tract. It has been shown
that pneumcoccal colonization of the nasopharynx correlates with observed
differences in colony phenotypes. S. pneumoniae undergoes spontaneous,
reversible opacity phase variation with a frequency of 10–3 to 10–6 resulting
in opaque and transparent colonies [19]. Differences in colonial opacity
have previously been shown to correlate with different levels of capsular
polysaccharide (CPS) [19]. The transparent phenotype produces lower
amounts of CPS and has an enhanced ability to reside on mucosal surfaces
when tested in several animal models [20, 21]. By contrast, the opaque
variant is more virulent in systemic infections [22]. The higher amounts of
CPS produced by these variants make them more resistant to complement-

mediated opsonophagocytotic killing [22]. Although higher amounts of CPS
have been demonstrated to block pneumococcal adherence, a basal level
of encapsulation is essential for colonization [23, 24]. Strikingly, electron
micrographs illustrated that the intimate contact of pneumococci with host
cells is associated with a reduction of encapsulation [25].
Pneumococci spread presumably to the lungs by aspiration and attach-
ment of pneumococci has been indicated to bronchial epithelial cells [26]
or components of the basement membrane including laminin, collagen and
fibronectin [27, 28]. Binding is promoted by an impaired ciliary beating
frequency due to the damage caused by smoking or the release of pneumo-
lysin, a cytolysin of pneumococci. Pneumolysin inhibits the normal beating
of cilia on epithelial and endothelial cells thereby facilitating penetration
of pneumococci into the bloodstream [29, 30]. The effect of the hydrogen
peroxide released by pneumococci, which is as toxic as pneumolysin to
ependymal ciliary cells, is masked in the presence of pneumolysin [31].
Within the alveoli pneumococci adhere to type II pneumocytes and can
spread rapidly into the blood by crossing the vascular endothelium [32].
Three stages of lesions were distinguished during pneumococcal pneu-
monia: (1) engorgement, (2) red hepatization, and (3) gray hepatization.
Engorgment is associated with the accumulation of a serous exudate in
the alveoli and is followed by a leakage of erythrocytes into the alveoli.
At the stage of gray hepatization bacterial multiplication peaks, fibrin is
formed through the procoagulant activity induced by pneumococci and
polymorphonuclear leukocytes (PMNs) recruited to the infection site start
to control pneumococcal multiplication [32]. Clearance of pneumococci
by opsonophagocytosis is further dependent on complement and facili-
tated by anti-capsular antibodies [15]. One of the remarkable features of
pneumococcal pneumonia is that the lungs of patients who withstand the
inflammatory response of the host and survive almost invariably return to
normal, irrespective of the severity of the systemic or pulmonary condition
when the disease was at its peak [33].
Cell wall structures and virulence factors of S. pneumoniae
Pneumococci are encased by a capsular polysaccharide that has been recog-

nized as a sine qua non of virulence. Survival in the bloodstream depends on
the expression of capsule. The degree of activation of the classical or alter-
native pathways of complement, the deposition and degradation of comple-
ment components, and the degree of protection against complement-medi-
ated opsonophagocytosis is determined by the biochemical structure of the
CPS rather than by the thickness of the CPS [15].
The layer underneath the capsule, the pneumococcal outer cell wall, is
comprised of peptidoglycan, teichoic (TA) and lipoteichoic acids (LTA),
Figure 2. Infections caused by pneumococci. Depicted are encapsulated pneumococci (arrow-

heads) in the cerebrospinal fluid (top left panel) and the histopathology of purulent meningitis
caused by pneumococci (top right panel). The photomicrograph of a section of the subarach-
noid space, in hematoxylin-eosin stain, shows the infiltration of leukocytes (arrowheads) and
purulent meningitis (arrows). Image courtesy Roland Nau, University of Göttingen, Germany.
Chest X-ray of a lobar pneumonia (bottom, left panel) and section of lung representing a stage
of hepatization.
and phosphorylcholine (PCho). The PCho is covalently linked to the TA

and LTA, which differ only in their attachment to the pneumococcal cell
wall. Although PCho is part of the cell wall of other respiratory pathogens
including Neisseria spp., Haemophilus influenzae, and Pseudomonas, only
pneumococcal PCho anchors a special class of proteins, the choline-bind-
ing proteins (CBP) non-covalently on the surface. CBPs have a modular
organization and consist in general of a leader peptide, a biologically active
N-terminal domain and the conserved choline-binding domain (CBD) that
targets the aminoalcohol PCho. The CBD, generally located at the carboxy
terminus and preceded by a proline-rich sequence, consists of highly homol-
ogous 20-amino acid long repetitive sequences.
Pneumococci can produce 13 to 16 different CBPs and the number of
produced CBPs depends on the pneumococcal strain. To date, the extensive-
ly characterized CBPs include the pneumococcal surface protein A (PspA),
the pneumococcal surface protein C (also referred to as CbpA or SpsA),
and four cell wall hydrolases. In general, the bacterial cell wall hydrolases
(CWHs) are endogenous enzymes that specifically cleave covalent bonds of
the cell wall. The pneumococcal CWHs are: the major autolysin LytA (N-
acetyl-muramoyl-L-alanine amidase), a `-N-acetylglucosamidase (LytB), a
`-N-acetylmuramidase (LytC; lysozyme), and a phosphorylcholine esterase
(Pce or CbpE). LytA has been characterized in detail and recently, the struc-
tural analysis of the CBD of LytA demonstrated that this module adopts a
peculiar solenoid structure [34]. LytB is highly expressed in the early expo-
nential growth phase and has been shown to be important for cell separa-
tion of pneumococci. In contrast to other CBPs, LytB and LytC possess the
CBD as an N-terminal domain [34]. Apart from these, CbpD, CbpG, CbpJ,
CbpI and, PcpA represent further members of this family [35, 36].
Three clusters of surface proteins can be distinguished by genome analy-
sis: the lipoproteins including peptide permeases and ATP binding cassette
(ABC) transporter (42 in R6 and 47 in TIGR4), the above mentioned family
of CBP (10 in R6 and 15 in TIGR4) and proteins with an LPxTG motif (13
in R6 and 19 in TIGR4). The latter represent typical Gram-positive surface
proteins that are covalently anchored in the cell wall after cleavage of the
LPxTG sequence by a transpeptidase, designated sortase. Strikingly, many
of these LPxTG proteins contain enzymatic activity including neuramini-
dases, hyaluronidase, IgA1-protease and zinc metalloproteases (ZmpB,
ZmpC, and ZmpD). In addition to these predicted surface proteins, non-
classical surface proteins lacking a classical leader peptide and membrane
anchoring motifs, have been identified on the pneumococcal surface. These
proteins have received considerable attention for their contribution to viru-
lence of pneumococci and other pathogenic bacteria. To date, the mecha-
nism of secretion and anchoring of these proteins that include the enolase,
the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the PavA
(pneumococcal adherence and virulence factor A) remain unknown for
pneumococci and many other Gram-positive pathogens.
Another important molecule that is associated with pneumococcal

virulence, but not surface-exposed due to the lack of secretion signal,
is pneumolysin. This hemolysin is intracellularly produced by pneumo-
cocci and released by the action of cell-bound autolysin. Pneumolysin is a
sulfhydryl(thiol)-activated pore-forming cytolysin that binds to cholesterol
in the plasma membrane of host cells [37]. Soluble pneumolysin monomers
form ring-shaped oligomeric pores [38, 39]. The observation of the pore-
forming process by cryo-electron microscopy has indicated that the mem-
brane bound form with an intact bilayer is the prepore and that the confor-
mational transition from the prepore to the pore form is accompanied with
a separation of monomers and a substantial refolding of protein domains
[40]. The multiple biological activities of pneumolysin have been shown to
interfere with eukaryotic host-cell function and the immune system.
Cellular biology of pneumococcal infections
Bacteria attach to eukaryotic host cells via surface-exposed adhesins which

specifically interact with cellular host receptors or adhesive glycoproteins
of the extracellular matrix (ECM) which connect the microorganism with
cellular receptors [41]. It has been shown that pneumococci translocate the
respiratory barrier and gain access to the blood circulation through the
intracellular route [42]. However, the strategies used by pneumococci for
translocation are not yet fully elucidated and a paracellular route of entry
cannot be excluded as an alternative route.
Adherence to host cells
In the early stages of the infectious process pulmonary epithelial cells and
vascular endothelial cells are targeted by pneumococci. Attachment to rest-
ing lung cells and vascular endothelial cells occurs via the recognition of two
classes of glycoconjugates. The disaccharides N-acetyl-D-galactosamin `1-
3/4 galactose (GalNAc (`1-3/4) Gal) and sialylated N-acetyl-D-glucosamine
`1-3 galactose (GlcNAc (`1-3) Gal) are recognized by pneumococcal viru-
lence determinants that have not been identified so far [43, 44]. Binding of
pneumococci to resting cells was completely abolished by a combination of
these two carbohydrates as represented by asialo-GM and globoside [45].
It is has been suggested that the multiple neuraminidases (NanA, NanB
and probably NanC) of pneumococci cleave terminal sialic acid (N-acetyl-
neuraminic acid) from glycolipids, glycoproteins, and oligosaccharides on
host-cell surfaces and body fluids thereby enhancing intimate adherence. In
fact, NanA has been shown to cleave the terminal sialic acids of lipooligo-
saccharides from Haemophilus influenzae and Neisseria meningitidis [46].
Because terminal sialic acids protect these respiratory pathogens against
complement mediated phagocytosis, desialylation of competitors may pro-

vide an advantage during colonization of host niches. In addition, NanA has
been shown to be implicated in desialylation of human proteins exhibiting
sialic acid including the secretory component, lactoferrin and IgA2 [47].
These human proteins are pneumococcal host targets and the removal of
sialic acid may facilitate bacterial persistence in the respiratory tract. NanA
sequence diversity is restricted to regions that are not required for enzy-
matic activity and has been suggested to provide an important advantage in
evading the adaptive immune response [48]. In a chinchilla infection model,
loss of NanA has been shown to impair pneumococcal persistence in the
nasopharynx and middle ear [49]. In contrast, the nanA knockout was not
attenuated in an intraperitoneal infection model [50]. A similar role is sug-
gested for the hyaluronidase (Hyl), which hydrolases primarily hyaluronan.
Although hyl knockout strains are attenuated in an intraperitoneal mouse
infection model [51], the precise function of the Hyl during pathogenesis
has yet to be clarified.
S. pneumoniae produce up to four zinc metallo proteases including IgA1-
protease, ZmpB, ZmpC, and ZmpD, which are anchored to the cell wall by
an N-terminal LPxTG motif. The IgA1-protease is produced virtually by all
pneumococci and Weiser and co-workers [52] demonstrated that cleavage
of surface-bound serotype-specific IgA1 by the IgA1-protease markedly
enhanced adherence of pneumococci to host cells. It has been assumed that
bound Fab fragments neutralize the negatively charged capsule and negate
the anti-adhesive effects of the capsule.
A role in colonization has also been suggested for the lipoprotein SlrA,
and the choline-binding proteins CbpD and CbpG, which is thought to be a
serine protease [35]. Recent reports indicated that CbpD is a competence-
stimulating-peptide-inducible protein and a function as a murein hydro-
lase has been proposed. CbpD has been demonstrated to assist LytA in
competence-induced cell lysis [53]. A further study provided experimental
evidence that the biological activity of CbpD is involved in the ability of
competent bacteria to trigger release of virulence factors from non-com-
petent S. pneumoniae [54]. The surface-exposed lipoprotein SlrA is a func-
tional, cyclophilin-type peptidyl-prolyl isomerase. The deficiency in SlrA
caused a less efficient nasopharyngeal colonization of mice, and this has
been attributed to the decreased ability of the knockout mutants to adhere
to non-professional cells [55].
The pneumococcal surface adhesin A (PsaA), which is the substrate-
binding lipoprotein of an ATP binding cassette (ABC)-type manganese
transport system [56] has also been demonstrated to affect adherence
of pneumococci. Mutations in psaA caused pleiotropic effects including
reduced adherence of pneumococci to host cells, attenuation in an intra-
nasal and intraperitoneal mouse infection model and increased sensitivity
to oxidative stress [9, 57] . After detecting the pleiotropic effects it was
assumed that PsaA does not function itself as an adhesin, however, a recent
report has demonstrated that antibodies against PsaA reduce the adherence
of pneumococci to nasopharyngeal epithelial cells [58]. This is consistent
with the finding that mucosal immunization of mice with PsaA is highly
protective against pneumococcal carriage [59] and that psaA is upregulated
during atttachment of pneumococci to epithelial cells [7, 9].
The major pneumococcal adhesin
The pneumococcal surface protein C (PspC; also known as SpsA or CbpA)

is a multifunctional CBP of pneumococci; PspC promotes uptake of pneu-
mococci into nasopharyngeal epithelial cells and interferes with components
of the innate immune system. PspC promotes pneumococcal adherence via
a human specific interaction with the secretory component (SC), which is
found on the polymeric immunoglobulin receptor and secretory forms of
IgA and IgM on mucosal surfaces [60–62]. The SC consists of five Ig-like
ectodomains (D1 to D5) and independent reports have indicated that the
human specificity of the PspC-SC interaction is determined by amino acid
differences in the ectodomains D3 and D4 of the SC [63, 64]. Despite such
human specificity, loss of function in PspC has been shown to reduce colo-
nization of infant rats [65] and pIgR knockout mice [62]. The underlying
mechanisms of these in vivo effects are poorly defined. However, PspC has
also been shown to bind to immobilized sialic acid and lacto-N-neotetraose
[65] and to interact with complement components including C3 and factor
H [66, 67].
The amino-terminal part of most of the PspC molecules contains repeat-
ed domains, designated R1 and R2. Structural analysis of R1 (aa175 to 285)
and R2 (aa327 to 442) of PspC derived from TIGR4 (PspC allele PspC3.4
[68]) demonstrated that the R domains adopt an unusual and simple struc-
ture comprised of three _-helices. These R domains of PspC contain the
minimal and conserved SC-binding site Y/RRNYPT [61]. The structure
indicated that bundling of the helices through _-helix/_-helix interactions
results in a flat, raft-like structure in which the critical residues YPT of the
minimal SC-binding motif are located in a loop between helix 1 and helix 2
and form a “tyrosine fork” structure [69].
Interaction with ECM components and recruitment of proteolytic

activity
There is experimental evidence that binding of microorganisms to ECM

components may be of importance for pathogenesis. Pneumococci have
been shown to bind to various ECM components, however, the impact of
these interactions on colonization are not yet clarified. The PavA protein
(pneumococcal adherence and virulence factor A) has been identified as a
pneumococcal adhesin for fibronectin and, probably more important, as a

crucial virulence determinant in pneumococcal infections. In a systemic and
experimental mouse meningitis model of infection the pneumococcal pavA
knockout of strain D39 was substantially attenuated [70, 71]. In addition,
PavA is most likely involved indirectly and in a fibronectin-independent
manner in pneumococcal adherence to host cells. However, the precise
function of PavA has yet to be clarified, because deficiency in PavA does
not affect expression and function of known virulence factors [71].
S. pneumoniae acquires host proteolytic activity by binding plasmin(ogen),
and interestingly, the glycolytic enzymes enolase and GAPDH were identi-
fied as plasmin(ogen)-binding proteins displayed on the cell wall [72, 73].
The enolase has been shown to potentiate degradation of extracellular
matrix (ECM), dissolution of fibrin and pneumococcal transmigration [74].
The key pneumococcal binding site in enolase responsible for plasmin-
mediated ECM degradation has been attributed to the nonameric peptide
“FYDKERKVYD” [75]. Crystal structure analysis indicated an octameric
composition of the pneumococcal enolase and depicted the nonameric plas-
minogen-binding site on the surface of the protein [76]. The importance of
enolase in virulence has been demonstrated in intranasal mice infections of
mice. Enolase mutants with functionally inactive plasminogen-binding sites
were significantly attenuated compared to the isogenic D39 parental strain
[75].
Invasion of host cells by pneumococci
The proportion of internalized pneumococci in resting pulmonary cells was

only 0.1 % of the adherent bacteria [77]. In contrast, activation of vascular
endothelial cells with thrombin or tumor necrosis factor-_ (TNF-_) caused
a substantial increase in pneumococcal uptake. The cell activation is associ-
ated with an increase in expression of novel cell adhesion molecules such as
receptor for the platelet-activating factor (PAF). The PAF receptor is rap-
idly internalized after interaction with its ligand PAF and pneumococci have
been shown to engage the upregulated PAF receptor for internalization
[77]. It has been shown that the cell wall structural component phosphor-
ylcholine (PCho), which is also present in PAF, function as an adhesin for
the PAF receptor. Binding and uptake of pneumococci to activated endo-
thelial cells was inhibited by PAF antagonists, purified pneumococcal cell
wall components, or anti-PCho antibodies. The absence of PCho in pneu-
mococci reduced adherence to levels indicated for resting cells, indicating
that PCho directly interacts as an adhesive molecule with the PAF receptor
[77]. Binding of pneumococci via PCho to activated cells was inhibited
by glycoconjugates N-acetyl-glucosmamine or lacto-N-neotetraose. These
sugars showed no effect on adherence of pneumococci to resting cells. This
implicates that pneumococci most likely bind to the PAF receptor at two
Figure 3. Surface proteins of pneumococci contributing to colonization and invasion of host

cells.
A. The extracellular matrix (ECM) of the mucosal cavity represents the first mechanical bar-
rier for pneumococcal colonization. Colonization of pneumcocci is facilitated by cleavage of
bound IgA1 by the IgA1-protase and by removing of terminal sialic acids by NanA. The glyco-
lytic enzymes enolase and GAPDH bind human plasminogen (PLG), which is converted to the
protease plasmin (PA) and promotes degradation of various ECM compounds. ZmpC activates
the matrix metallo protease 9 (MMP-9) which has collagenase activity and degrades the ECM.
The non-classical surface associated protein PavA has been shown to bind to immobilized
fibronectin (Fn) and to modulate pneumococcal adherence.
B. The intimate contact of pneumococci with cellular receptors is mediated by adhesins. The
major adhesin of pneumococci identified is PspC, which interacts with the ectodomain of the
human polymeric immunoglobulin receptor (pIgR). PCho mediates pneumococcal adher-
ence to stimulated cells via an interaction with upregulated platelet activating factor receptor
(PAFr). It is thought that the lipoprotein PsaA may also function as an adhesin, but the cellular
receptor is not defined yet. Reprinted from Microbiology SGM [209] with permission of the
publisher.
sites: one shared with the natural ligand PAF and the other at a site of gly-
cosylation of the receptor. Alternatively, the glycosyl determinant can be
located on a putative co-receptor that interacts with the PAF receptor. The
PCho-PAF receptor interaction represents further a specific mechanism for
pneumococcal targeting of the blood-brain barrier (BBB) and transmigra-
tion of pneumococci across the BBB [24].
The efficiency of colonization and invasion of brain microvascular

endothelial cells correlates with the higher amounts of PCho on transpar-
ent pneumococci. In addition, the amount of PCho is modulated by the
phosphorylcholine esterase activity of Pce (also referred to as CbpE) which
removes PCho from the cell wall and causes changes in colony phenotype
[78, 79]. The crystal structure reveals that Pce belongs to the metallo-`-
lactamase family and that only PCho residues that are located at the end
of the teichoic acid chains are accessible to the catalytic center of Pce [80,
81]. It is thought that Pce may have a dual function and favor both colo-
nization and invasive infection by modulating the amount of PCho on the
pneumococcal cell wall. The physiological significance of the PCho-PAF
receptor interaction was demonstrated in a rabbit model of pneumonia.
The PAF-receptor antagonist blocked activation-dependent adherence and
transition of pneumococci from the alveolus into the blood. This resulted in
reduced nasal colonisation and attenuated development of pneumococcal
bacteremia [77].
Cell signaling induced by S. pneumoniae
The PAF receptor is a G-protein coupled receptor and binding of PAF acti-
vates phopholipase C [82]. In contrast, internalization of pneumococci via
the PAF receptor was independent of the G-protein pathway and failed to
induce signal transduction [77]. The endocytosis of pneumococci requires
both the PAF receptor as a portal of entry and `-arrestin. It has been dem-
onstrated that pneumococci induced the translocation of `-arrestin at the
plasma membrane, where it colocalized with the PAF receptor. This event
caused a G-protein independent activation of the MAP kinase ERK-1/
ERK-2 and pneumococi moved into clathrin-coated vesicles. At least half
of the pneumococci proceeded through Rab5 to Rab7 marked endosomes
toward lysosomes. Other vacuoles acquire Rab11, which is consistent with
the known recycling of the bacteria to the apical surface [83].
In pneumococcal pneumonia massive leukocyte recruitment to the lung
is observed [84]. Invasion of leukocytes is accompanied by secretion of pro-
inflammatory and chemotactic cytokines by lung epithelium and cells of the
innate immune response including alveolar macrophages. It has been shown
that the pneumococcal cell wall mediated signaling induces the expression
of transcription factor NF-kB and induces the production of TNF-_, IL-8,
IL-6 and IL-8 [85-87]. Schmeck and co-workers demonstrated that in pneu-
mococcal pneumonia the induction of NF-gB and p38 MAPK signaling
pathways contribute to the secretion of IL-8 and GM-CSF. Activation of
NF-gB was IgB-kinase dependent, but activation was independent of p38
MAPK. It has been demonstrated that p38 MAPK did not affect inducible
nuclear translocation of NF-gB/RelA to the IL-8 promotor but regulates
IL-8 transcription on the level of phosphorylated RelA at the promoter.
Both, p38 MAPK and NF-gB have been shown to be required to modulate
the host response at the transcriptional level in response to pneumococci
infection [88].
Pulmonary virulence determinants
Large-scale in vivo screens including signature-tagged mutagenesis (STM),

differential fluorescence induction (DIF) and whole-genome microarray
analysis have provided an extended list of putative pulmonary virulence
factors [5–9]. These approaches confirmed already known virulence factors
including CbpD, ComD, Hyl, IgA1-protease, LytA, NanA, Pneumolysin,
PcpA, PsaA, PspA, PspC, sortase, and ZmpB. In addition, a role in patho-
genesis has been suggested for ABC transporters such as the glutamine
transporters, proteases such as HtrA and PrtA, proteins involved in meta-
bolic pathways and regulatory elements such as the transcription factor
RlrA. The importance of individual virulence determinants for pneumococ-
cal pneumonia has further been assessed in in vivo studies.
Pneumococci produce several proteases that are surface-exposed and
implicated in pneumococcal virulence. Intranasal infection experiments
confirmed the significant contribution of IgA1-protease and ZmpB to pneu-
mococcal virulence [89]. ZmpC was characterized in TIGR4 as a bacterial
zinc metallo protease cleaving human matrix metalloproteinase 9 (MMP-9)
and inactivation of the zmpC gene in serotype 19F impaired virulence in
a pneumonia mouse model [90]. The HtrA (high-temperature require-
ment A) functions in a temperature-dependent manner as a molecular
chaperone or heat shock-induced serine protease, and is regulated by the
CiaRH two-component system. HtrA has been shown to be implicated in
resistance against oxidative stress, colonizing the nasopharynx of rats and in
pneumococcal pneumonia. Moreover, htrA knockouts induced lower levels
of inflammatory cytokines IL-6 and TNF-_ in the lungs during pneumonia
compared to the isogenic wild type D39 [91–94]. PrtA is a further surface-
exposed serine protease and prtA knockouts have been shown to be attenu-
ated in an intraperitoneal mouse infection model [95].
The highly variable pneumococcal surface protein A (PspA) is expressed
virtually by all important clinical serotypes and has significant immune pro-
tective potential. Loss of function has been shown to attenuate virulence
and increase complement receptor-mediated clearance of pneumococci
[96]. PspA is a lactoferrin-binding protein [97] and Shaper et al. [98] dem-
onstrated that expression of PspA protects against the bactericidal effect of
apolactoferrin. In the presence of antibodies recognizing PspA this effect
is abrogated, suggesting that binding of apolactoferrin to PspA blocks the
bactericidal activity of apolactoferrin. Regarding the cell wall hydrolases of
S. pneumoniae, loss-of-function of LytB or LytC significantly reduced naso-
pharyngeal colonisation of rats [35].
Table 1. Pulmonary virulence factors
Biological activities Refs.

Toxins Pneumolysin inhibition of ciliar mobility, cytotoxic [29, 30, 122]
and cytolytic activities
enzymes/ HtrA resistance against oxidative stress, [91–93]
proteases induction of inflammatory cytokines
Hyaluronidase hydrolysis of hyaluronan [51]
IgA1-protease enhancement of bacterial adherence to [52]
host cells
LytA, LytB, LytC cell wall hydrolases [34, 35]
Neuraminidase cleavage of terminal sialic acids of [46–49]
(NanA) human IgA secretory component, lacto-
ferrin and IgA2
PrtA serine protease [95]
ZmpB impact on pneumococal virulence [89]
ZmpC cleavage of metalloproteinase 9 [90]
(MMP-9)
choline-binding CbpD competence-induced cell lysis by [53, 54]
proteins murein hydrolytic activity, impact in
allolysis
Pce phosphorylcholine esterase [77, 78]
PcpA unknown [36]
PspA binds lactoferrin and protects against [97, 98, 127,
bactericidal effects of apolactoferrin, 128]
inhibition of complement-induced
clearance
PspC pneumococcal adhesin for pIgR [60]
regulatory com- ComD transformation competence factor [5, 211]
ponents RlrA transcriptional regulator [5]
lipoproteins PsaA Mn-transporter and putative adhesin [56]
Regulation of gene expression and virulence
The pneumococcal virulence determinants can be regulated by one-compo-

nent regulatory systems including luxS, rlrA, regM/R, and mgrA, or by one
of the 14 two-component regulatory systems (TCS) that have been identi-
fied in S. pneumoniae [99, 100]. TCS respond to environmental changes and
mediate, therefore, the adaptation of pneumococci to their different micro-
environments. Throup et al. [101] and others have demonstrated the impact
of TCS on pneumococcal virulence in a mouse infection model. The CiaRH
was the first TCS identified (and is required for efficient nasopharyngeal
colonization and important for protecting cells from stress of competence
development [102, 103]). As mentioned above, CiaRH regulates HtrA,
which is thought to be the key component in the contribution of CiaRH

to virulence [93]. The major adhesin PspC is upregulated during coloni-
zation [7] and pspC expression has been shown to be influenced by the
TCS RR06/HK06 [104]. However, in vitro and in vivo infections suggested
that this TCS is also important for the regulation of other yet unknown
virulence factors interfering with colonisation and invasion. Recently it
has been shown that the phosphorylated VicR (YycF) response regulator
of the TCS VicRK (YycF) binds to a region upstream of pspA and regu-
lates expression of pspA [105]. As opacity variation accounts for changes
of a number of cell surface-exposed components including capsule, PspC,
PspA and PCho it is implicated in pneumococcal colonization and invasive
diseases. Capsule regulation has been indicated under in vivo and in vitro
conditions. Recombinant exchanges and spontaneous sequence duplications
within type 3- and 8 specific capsule genes cause high-frequency serotype
and phase variations, respectively [106-108]. Proteins such as CpsB and
CpsD which influence production of capsular polysaccacharide regulate
the amount of capsule at the post-translational level. Both have been dem-
onstrateed to modulate the phosphorylation of CpsD or other substrate
molecules [109–112].
The pneumococcus and the innate immunity of the host
Innate immunity covers a diverse array of host defenses including muco-

cilliary clearance, complement, neutrophils and macrophages. It acts as a
non-specific defense able to recognize and respond rapidly against a broad
range of microbes. Unlike adaptive immunity, which involves the clonal
expansion of T- and B-cells specific to the pathogen, innate recognition is
achieved through a limited set of germline encoded receptors and does not
possess immunological memory. The two systems do not however operate
in isolation as the innate immune system plays crucial roles in the initiation,
development and effector stages of adaptive immunity.
Complement and the pneumococcus
The complement system comprises over 30 serum and membrane pro-

teins which, when activated, form a cascade of reactions contributing to
the elimination of invading microorganisms. The binding and activation
of complement components to the surface of a microbe leads to opso-
nophagocytosis and the induction of inflammation. For some organisms,
but not the pneumococcus, complement can destroy the microbe directly
through lysis by the membrane attack complex. Complement contributes
to and links both innate and adaptive immunity. Three pathways of activa-
tion exist (reviewed in [113], in brief these are: 1) The classical pathway
activated by antibody: antigen complexes for example, antibody binding to

the microbial surface. Non-antibody-dependent activation can also occur,
such as the binding of acute phase proteins to the microorganism, 2) the
lectin pathway, which is triggered by mannose-binding-lectin recognition
of carbohydrate on the microbial surface, and 3) the alternative pathway
is continuously activated at low levels, but only amplifies on foreign sur-
faces.
The crucial contribution of complement to innate and adaptive
responses to pneumococcal infection has been long established both in
animal models and humans with genetic complement deficiencies (for
recent overview see [114]). However, only recently, through the use of
a panel of gene knock-out mice lacking various complement compo-
nents, has the relative importance of individual activation pathways been
assessed in innate immunity to pneumococcal infection [115]. The classical
pathway of complement activation was found to be the dominant comple-
ment pathway for innate immunity to the pneumococcus in mice [115]; the
specific loss of which resulted in significantly increased disease severity.
Natural IgM antibodies, possibly to C polysaccharide (teichoic acid), con-
tributed to this activation of the classical pathway as shown with the use of
+s–/– knock-out mice which lack such antibodies [115]. However, the activa-
tion of the classical pathway during this innate immune response was only
partially dependent on natural antibodies and other activation pathways
also contributed. These were proposed to include acute phase proteins
such as C-reactive protein or direct binding of complement component
C1q to the pneumococcal surface [115]. The alternative pathway also con-
tributed to protective innate responses, but to a lesser degree than seen
for the classical pathway, while the role for the lectin pathway appeared
negligible. In line with this latter finding, genetic mannose binding lec-
tin deficiency is associated with only a small (but significant) increased
susceptibility to pneumococcal disease in humans [116]. Regardless of
the activation pathway, the deposition and activation of complement
component C3 on the bacterial surface is a key step in the complement
cascade leading to elimination of the microbe. In accordance with this
crucial role of complement in innate immunity, the pneumococcus has
evolved several mechanisms to resist its affects. The capsule is a key factor
in this resistance, acting not only to limit access to cell bound comple-
ment, but also reducing the amount of complement deposited [117]. The
pneumococcal surface protein, PhpA (also called PhtB and BVH-11 [118],
has been found to possess C3 degrading activity [119, 120] and so PhpA
may contribute to preventing complement-mediated clearance. In addi-
tion to its cytolytic activity the toxin pneumolysin, a major pneumococcal
virulence factor, has multiple other biological activities (for review see
[121]). Recently pneumolysin has been shown to confer protection from
complement mediated clearance [122]. Deletion of pneumolysin caused
an increase in C3 deposition on pneumococcal cells in vitro. Showing that
this activity of pneumolysin contributed to virulence, absence of comple-

ment in gene knock-out mice reduced the importance of pneumolysin to
pneumococcal virulence. Furthermore, this effect of pneumolysin in vitro
and in vivo was specific to the classical and not the alternative activation
pathway [122]. The mechanisms by which pneumolysin achieves this affect
are as yet unconfirmed. However, this specific evasion of the classical
pathway is in agreement with the ability of pneumolysin to activate this
pathway in the complement; evasion in this instance may ironically be the
result of complement activation rather than inhibition. Released pneumo-
lysin may result in complement activation away from the bacterial cell thus
protecting it and also consuming the available complement components.
In addition, increased complement activation may contribute to host tis-
sue damage thereby promoting bacterial pathogenesis. Furthermore, the
surface proteins, PspA and PspC also contribute to complement resistance.
The PspC molecules are divided into different groups. Interestingly, both
the classical PspC proteins containing a CBD and the PspC-like Hic (PspC
11.4) bind the complement factor H [123]. Hic is produced predominantly
by serotype 3 pneumococcal strains that are negative for SC binding. Hic
contains an LPxTG motif that anchors the protein in a sortase-dependent
manner to the peptidoglycan of the cell wall. Factor H is a fluid phase
regulator of the alternative complement pathway and consists of 20 short
consensus repeats (SCRs). Hic interacts with the SCRs 8-11 and 12-14 of
factor H [124], whereas a role for the SCRs 6 to 10 and 13-15 of factor
H was suggested for the interaction with PspC [67, 125]. Recruitment of
factor H by Hic has been shown to efficiently prevent activation of C3b
and complement mediated opsonophagocytosis of pneumococci [124]. The
improved survival of pneumococci expressing PspC or Hic in a systemic
mouse infection model provides further evidence for the versatility and
importance of PspC in different host niches [126]. PspA contributes to
inhibition of complement receptor-mediated clearance of pneumococci.
Tu et al. [127] have demonstrated a delay of wild type pneumococci clear-
ance compared to the isogenic pspA mutant. It is thought that PspA prob-
ably inhibits the factor B-mediated complement activation and functions
as an inhibitor of C3b deposition [127]. The inhibitory effect by PspA has
been shown for PspA from family 1 and family 2 [128]. In addition, PspA
expression decreases C3 binding on pneumcococi, indicating that PspA
may also inhibit complement deposition via the classical pathway [96].
Pattern recognition receptors
Key components of the innate immune system are so-called pathogen

recognition receptors (PRRs). These can be located on the hosT-cell sur-
face, intracellularly, or be secreted and act to initiate the recognition and
response to microbes and in some cases host products [129]. The microbial
components recognized by PRRs are referred to as pathogen-associated

molecular patterns (PAMPs) so-called because they are typically invariant
structures found among many microbes, but absent in eukaryotes. Examples
include lipopolysacchride (LPS) and peptidoglycan from bacteria, double-
stranded RNA from certain viruses and mannan from fungi. C-reactive
protein (CRP) is a well-known example of a PRR involved in the response
to this pneumococcus. This soluble protein binds PCho in the pneumococ-
cal cell wall, inducing complement activation leading to bacterial clearance
[130, 131]. Additional PRRs, important in pneumococcal infection have
recently been described including members of the toll-like receptor (TLR)
family, LBP, and the cytosolic PRR Nod proteins.
Consequences of pneumococcal recognition by Toll-like receptors
The TLR family of PRRs has received much attention due to their impor-
tance in the response to a wide range of microbes (for review see [132,
133]). Their major function is as PRRs to recognize microbes and initiate
an inflammatory response leading to eradication of infection. At least ten
TLRs have been described in humans and mice and several have been
implicated in pneumococcal infection in animal models. Furthermore,
descriptions of genetic defects in TLR signaling associated with increased
susceptibility to pneumococcal disease show these receptors have relevance
to human infection [134, 135].
TLR2 recognizes both pneumococcal lipoteichoic acid (LTA) and cell
wall peptidoglycan [136-139]. Interestingly, despite numerous studies sup-
porting the recognition of bacterial peptidoglycan via TLR2, this interac-
tion has recently been challenged [140]. Knock-out TLR2–/– mice display
increased disease severity and decreased survival times compared with wild
type mice in a pneumococcal meningitis model [141]. This greater suscepti-
bility correlated with heightened bacterial levels in the brain, but appeared
independent of systemic disease as both strains showed similar bacterial
levels in the blood. In agreement with these data, Koedel et al. [142] also
found TLR2–/– mice had enhanced disease and increased bacterial levels in
the brain in their meningitis model, which in turn have contributed to an
enhanced inflammation in the brain later in infection.
The role of TLR2 has also been investigated in experimental pneumo-
coccal pneumonia [143]. Comparison between wild type and TLR2–/– mice
following intranasal infection revealed only a modest contribution for this
receptor in the host response and no changes in bacterial clearance and
morbidity with compared with their wild type counterparts. On the basis
of these data, TLR2 does not appear to play a key role in host resistance
to pneumococcal pneumonia. Interestingly, the stimulation of isolated
alveolar macrophages in vitro to produce TNF-_ in response to heat-killed
pneumococci was entirely dependent on TLR2. However, immunohisto-
chemical staining of infected lungs from TLR2–/– mice showed these cells
were producing TNF-_ comparable with wild type mice. Presumably in the
setting of the intact animal other host innate immune factors such as other
PRRs and complement mask the loss of TLR2, rendering its influence
minimal in pneumococcal pneumonia. Following intraperitoneal infection
TLR2 knock-out mice have slightly reduced survival times compared with
wild type [144]. Thus, TLR2 has a protective role in this model of systemic
infection, although, as with pneumonia, the defect was arguably minor.
Furthermore, in a model of nasopharyngeal colonization TLR2 knock-out
mice had impaired clearance of pneumococci [145] showing that TLR2 is of
relevance not only to disease states, but also carriage.
The recognition of pneumococcal peptidoglycan probably involves
interaction between TLR2 and 6 as shown for Staphylococcus aureus pepti-
doglycan [146]. Indeed, confirming a role for this receptor in pneumococcal
recognition, expression of a double negative TLR6 mutant inhibited TNF-_
production in response to stimulation by the pneumococcus in a macrophage
cell line [146]. A role exists for TLR1 in the recognition of pneumococcal
LTA, whereby monoclonal antibodies against this receptor inhibited LTA
induced TNF-_ production from human peripheral blood mononuclear
cells [136]. The importance of these interactions between the pneumococcus
and TLR1 and 6 has not yet been assessed in an infection model.
Recognition of pneumolysin by TLR4
Through the recognition of LPS, TLR4 is a key component of the innate

response to Gram-negative infections. A role for this receptor has also been
extended to the pneumococcus with the finding that the in vitro pro-inflam-
matory effect of pneumolysin on macrophages was TLR4-dependent [147].
Subsequently, pneumolysin has been shown to directly interact with TLR4
[148]. This inflammatory activity was not dependent on the pore-forming or
complement-activating activities of pneumolysin because the PdT pneumo-
lysin mutant, that lacks these properties, was also active in these studies. The
significance of this interaction during colonization was studied by compar-
ing wild type and TLR4 deficient mice in a nasopharyngeal carriage model
[149]. In the absence of functional TLR4, mice were more heavily colonized
and much more likely to develop invasive disease. Thus, through its recogni-
tion of pneumolysin, TLR4 acts in the nasopharynx to limit pneumococcal
proliferation. While the inflammatory response to pneumolysin may con-
tribute to this protection, it has also been shown that pneumolysin-TLR4
signaling can induce hosT-cell apoptosis in vitro and in vivo [148]. This
also appears to be a protective host response as the inhibition of apoptosis
rendered mice more susceptible to death following pneumococcal infection
[148]. Interestingly, a similar model of colonization using the same mouse
strains found no difference in the clearance of pneumococci between wild
Fig. 4: Key pathogen recognition receptors involved in the recognition and initiation of the
immune response towards the pneumococcus. Abbreviations: LBP, LPS-binding protein; LTA,
lipoteichoic acid; TLR, toll-like receptor. Reproduced with permission from reference [210].
type and TLR4 deficient mice [145]. The use of different bacterial strains
in these studies may explain this apparent conflict, but this remains to be
tested. In pneumococcal pneumonia TLR4 also plays a protective role [150].
In this experimental model the absence of functional TLR4 rendered mice
more susceptible to morbidity with increased bacterial counts in the lungs.
The effects however, were modest with the effect on death rate only appar-
ent at low doses and with no significant impact on pulmonary inflammation.
Furthermore, the significance of TLR4 in pneumococcal infections appears
restricted to the airway surfaces as earlier work found the absence of TLR4
made no difference to survival rates and blood bacterial counts after intra-
venous infection of mice [151].
TLR signaling
Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the

signaling cascade activated by engagement of TLRs or IL-1 family recep-
tors [152]. In agreement with a role for TLRs in innate protection against
pneumococcal infection, MyD88–/– mice show enhanced susceptibility to
S. pneumoniae in different infection models [144, 153, 154]. Providing rel-
evance to human infection, deficiency in IL-1 receptor-associated kinase
4 (IRAK4), also a mediator in the TLR/IL-1 receptor signaling pathway,
results in increased susceptibility to pneumococcal disease [135]. As does a
distinct but as yet undefined mutation in this signaling pathway [134].
Pneumococcal cell wall recognition by LBP
In addition to TLR2, the pneumococcal cell wall (PCW) is recognized by

the soluble acute phase protein, LBP (LPS binding protein) [138]. This pro-
tein has previously been found to bind LPS and enhance its inflammatory
activity. It has now also been found to bind purified PCW and whole pneu-
mococci and the addition of LBP accentuated the inflammatory activity of
PCW in vitro [138]. In a mouse meningitis model LBP gene-knock out mice
showed significantly reduced meningeal inflammation following challenge
with purified cell wall or live pneumococci. Recognition of PCW by LBP
appears independent of cell wall phosphorylcholine and teichoic acid, with
the glycan backbone seemingly a crucial structure for this interaction [138].
Significance to human infections was shown by enhanced LBP levels in the
CSF of pneumococcal patients compared with controls. Furthermore, PCW
coprecipated with LBP in the CSF from a patient showing this interac-
tion occurs in human infection [138]. LBP also contributes to the response
to pneumococcal LTA [137]. However, LBP gene knock-out mice do not
have altered susceptibility or responses to pneumococcal pneumonia [155].
Given that LBP acts to facilitate recognition of TLR2 ligands, this pheno-
type is compatible with the modest effect seen with TLR2 knock-out mice
in similar infections [143]. Interestingly, LBP levels in lavage fluid increased
seven-fold during experimental pneumococcal infection [155]. This was a
much greater increase than seen with parallel Klebsiella pneumoniae pneu-
monia [155]. However, unlike pneumococcal pneumonia, endogenous LBP
contributed significantly to protection against K. pneumoniae pneumonia
[155]. Clearly, this highlights that functional importance cannot easily be
predicted merely from expression levels alone.
CD14
Membrane bound and soluble CD14 act as a co-receptor to enhance the

response to LPS [117, 156, 157]. CD14 also contributes to the recognition
of the pneumococcus in vitro [136, 139, 158]. A role in infection has also
been confirmed by the recent finding that CD14 gene knock-out mice show
exacerbated pneumococcal meningitis [159].
Nod proteins and the pneumococcus
The cytosolic proteins, Nod1 and Nod2 are additional PRRs acting within
host cells to recognize and respond to microbial products [160]. In addition
to toll-like receptors, a role for Nod proteins in the recognition and response
to pneumococci has also been shown [161]. Transfection with Nod2, but not
Nod1, conferred responsiveness to cells following pneumococcal exposure
as judged by NF-gB activation [161]. In line with the role of Nod proteins in
the response to intracellular material, the recognition of pneumococcus by
Nod2 was dependent on internalization of the bacteria. Nod2 has previously
been shown to be responsive to a muramyldipeptide conserved in multiple
peptidoglycans and this was the suggested mechanism for its recognition
of the pneumococcus [161]. The availability of Nod1 and 2 knock-out mice
will allow a fuller appreciation of these genes in the host response to the
pneumococcus [162–164].
Recognition of capsule
Recognition of the pneumococcal polysaccharide capsule by PRRs has

received much attention of late. Kang et al. [165] showed that the C-type
lectin SIGN-R1 expressed by macrophages, particularly in the marginal
zone of the mouse spleen, bound capsular polysaccharide from several dif-
ferent serotypes as well as whole pneumococcal cells. Lanoue et al. [166]
demonstrated a functional significance of this interaction with the genera-
tion of SIGN-R1 gene knock-out mice. Following intraperitoneal infection
with either serotype 2 or serotype 14 pneumococci, these mice displayed
increased susceptibility to infection compared with their wild type coun-
terparts. Absence of SIGN-R1 caused a decrease in survival rate, shorter
time to death, increased sickness scores and increased bacterial levels in the
blood. A defect in the ability of macrophages in the peritoneum and spleen
to bind and phagocytose the pneumococcus was likely to be a major con-
tributor to the increased susceptibility of the knock-out mice [166].
The role of SIGN-R1 in pneumococcal pneumonia has also been
investigated with the use of SIGN-R1 deficient mice [167]. When infected
intranasally with a serotype 3 strain, the SIGN-R1 knock-out mice showed
increased bacterial levels in the lungs compared with wild type. This was
accompanied by a higher incidence of bacteremia and increased bacterial
counts in the blood and spleen. Interestingly, alveolar macrophages do not
express SIGN-R1 and expression was not induced following pneumococcal
infection [167]. This suggests the protective role of SIGN-R1 in pneumonia
does not occur within the lungs themselves. One potential mechanism for
the increased bacterial growth in the absence of SIGN-R1 was found to be
reduced levels of anti-phosphorylcholine IgM. In addition, systemic disease
in this model was probably exacerbated by defective phagocytosis by mac-
rophages in the peritoneum and spleen as described by [166]. Therefore, in

both pulmonary and systemic infections, SIGN-R1 is instrumental in host
resistance to pneumococcus.
Another macrophage receptor, MARCO has also recently been iden-
tified as important in pneumococcal infection [168]. Genetic deletion of
this scavenger receptor rendered mice more susceptible to pneumococcal
pneumonia with impaired bacterial clearance from the lungs and increased
morbidity [168]. Isolated alveolar macrophages from the knock-out mice
were impaired in their ability to bind and phagocyctose the pneumococcus
in vitro and this was likely a key factor in the increased susceptibility to
infection. Interestingly, reduced phagocytosis was not due solely to reduced
bacterial binding and so a role for MARCO appears to exist not only in
binding, but in subsequent bacterial uptake. The pneumococcal ligands rec-
ognized by MARCO have not yet been identified. Previously pneumococcal
capsular polysaccharide has been shown to activate macrophages, [169] an
activity partially dependent on CD14, whether or not it also involves SIGN-
R1 and MARCO remains to be determined.
Surfactant proteins
Pulmonary surfactant is a mixture of lipids and proteins that act to prevent

alveoli from collapsing during expiration. In addition, the surfactant proteins
(SP) SP-A and SP-D play a role in innate immunity against a variety of
pathogens acting by binding microbes and promoting their phagocyctosis or
by modulating immune cell function [170, 171]. In the case of pneumococcal
infection, SP-D knock-out mice show enhanced susceptibility to intranasal
infection [172]. While SP-A has recently been shown to promote phagocycto-
sis of S. pneumoniae by rat and mouse alveolar macrophages in vitro [173].
Innate immunity and interaction between the pneumococcus and

other microbes
For ease of study, most work on the interaction of the pneumococcus with
the innate immune system has employed pure cultures. However, the muco-
sa of the upper respiratory tract is colonized by a diverse array of microbial
species. Indeed, analysis of DNA from human airway surface fluid suggested
the presence of > 500 bacterial species [174]. Concurrent stimulation of the
innate immune system by multiple species appears to have distinct effects
from single species interactions [175, 176] and this has recently been shown
to have relevance to the pneumococcus [177]. Co-stimulation of human
respiratory epithelia cells in vitro by S. pneumoniae and Haemophilus influ-
enzae, also an inhabitant of the upper respiratory tract, resulted in synergis-
tic production of IL-8 [177]. This extended to a mouse colonization model
with a synergistic effect on MIP-2 production and inflammatory influx into

the upper airways. This synergy was independent of TLR2 and TLR4 but
involved NF-gB translocation to the nucleus and phosphorylation of p38
MAPK. With regards to the microbial products involved, pneumolysin
could substitute for the pneumococcus, but the PdB pneumolysin toxoid,
lacking cytolytic activity, was inactive. It was therefore speculated that the
pore forming activity of pneumolysin lead to enhanced delivery of microbial
products such as the soluble inflammatory protein, SCF from H. influenzae,
into the host cell where recognition by Nod1 and 2 would result in increased
stimulation [177]. This pro-inflammatory activity of pneumolysin is there-
fore distinct from its effects on macrophages that were mediated through
TLR4 and were independent of pore-forming activity.
The significance of pneumococcal H. influenzae interactions have
recently be examined in a co-colonization mouse model [178]. In contrast
to what might be expected based on in vitro studies with these bacteria [46,
179], co-coloniation in vivo resulted in rapid clearance of the pneumococ-
cus. This effect was dependent on the innate immune system in the form of
neutrophils and complement, with the depletion of either abolishing the
competitive effect. Activation of peritoneal neutrophils with heat-killed H.
influenzae caused an increase in their ability to kill the pneumococcus, but
had no effect on their ability to kill H. influenzae [178]. The basis of this
activity is not yet clear. Thus, interactions with the innate immune system
can have a significant effect during competition between the pneumococcus
and other microbes in the nasopharynx.
Another important microbial interaction is that of the pneumococcus
and influenza A. Subsequent to influenza A outbreaks secondary pneu-
mococcal infection is an important cause of morbidity and mortality. This
heightened susceptibility to pneumococcal disease can be reproduced in
animal models, allowing investigation of the mechanisms involved. While
viral neuraminidase contributes to this phenomenon by exposing pneumo-
coccal receptors [20, 180], alterations in the immune response also seem
to contribute. Prior influenza A infection in mice primes for an exagger-
ated inflammatory response to subsequent pneumococcal infection [181].
Increased levels of IL-10 in this response likely contribute to increased
susceptibility as neutralization of this cytokine improved disease outcome
[181]. In vitro exposure to both influenza A and pneumococcus results in a
synergistic inflammatory response from human middle ear epithelial cells
[176]. Microarray gene expression analysis of these cells following influenza
A infection provides insight into the possible mechanisms behind this syn-
ergy [182]. For example, it was found that tlr2 expression was up-regulated
by influenza A infection. This may make the cell more responsive to stimu-
lation by pneumococcal peptidoglycan and LTA [182].
It is therefore clear that the interaction of the pneumococcus with the
innate immune system is greatly influenced by the presence of other organ-
isms such as H. influenzae and influenza A.
CD4 +cells in immunity to the pneumococcus
The function of CD4 + T-cells in adaptive immunity is well established.

Interestingly, they appear also to contribute to early resistance to pneu-
mococcal infection independently of their role in adaptive antigen-specific
responses [183]. Intranasal infection of MHCII knock-out mice, which dis-
play a significant decrease in CD4 + T-cell levels, revealed a key role for
these cells in the early response to pneumococcal pneumonia. These mice
displayed increased susceptibility to infection as evidenced by increased
bacterial counts in the lung and blood compared with their wild type coun-
terparts. Indeed, the increased susceptibility was so great that it resulted in
100% mortality in the knock-out mice by three days post-infection, whereas
all wild type mice survived the challenge. In accordance with previous data
showing T-cell migration to infected lung tissue in pneumococcal pneumonia
[184, 185], purified CD4 + T-cells migrated to the pneumococcus in vitro. This
migration was associated with T-cell activation and interestingly occurred
only in response to in vivo and not to in vitro grown bacteria. Pneumolysin
plays a significant part in this migration as pneumolysin-deficient pneumo-
cocci stimulated significantly less cell migration. How pneumolysin stimu-
lates these T-cells is unclear but the recent description of TLR4 expression
by T-cells may be of relevance [186]. Recently Malley et al. [147] have dem-
onstrated a crucial role for CD4 + T-cells in antibody independent acquired
immunity to pneumococcal colonisation. How CD4 + T-cell migration and
activation in response to the pneumococcus as described in pneumonia
relates to this acquired immunity to colonisation is as yet unclear.
The pneumococcus and the adaptive immunity of the host
During carriage and invasive disease the pneumococcus encounters the pro-
tective effect of host immunoglobulins. The pneumococcal surface is covered
by a capsular polysaccharide which is involved in pneumococcal coloniza-
tion and, more importantly, is a key virulence factor in invasive diseases [23,
187]. A correlation between decrease in pneumococcal carriage with rising
levels of both mucosal and serum antibodies to pneumococcal surface poly-
saccharides has been described [145]. However, capsular polysaccharides
do not yield an anamnestic response, due to the inability of polysaccharides
to recruit cognate CD4 + T-cell help through T-cell receptor recognition of
peptide-major histocompatibility class II complexes (MHCII) on the sur-
face of antigen-presenting cells [188]. McLay and coworkers demonstrated
in mouse infection studies that the lack of memory response by capsular
polysaccharides can be overcome by the use of conjugated vaccines that
elicit a different IgG subclass response to polysaccharides. Co-expression of
surface polysaccharides with proteins has been assumed to mediate cognate
CD4 + T-cell help for polysaccharide-specific B-cells [188]. The development
of a seven-valent conjugate vaccine has resulted in high immunogenicity,

but immune protection was restricted only to the seven serotypes, which
were represented by the polysaccharides [189].
Efforts have been made to overcome the problem of variable polysac-
charide determinants and to develop potent protein vaccines that have
the capability to cover most pneumococcal serotypes. A number of pneu-
mococcal cell-surface or secreted components have been shown to induce
opsonophagocytic antibodies or to offer at least partial protection in murine
infection models. The protein antigens currently under investigation include
the phosphorylcholine epitope found on lipoteichoic acid (LTA), PspC,
pneumolysin, PpmA, PsaA, PspA and some surface proteins identified by
whole-genome-approaches [97, 123, 190-198].
Some of the identified protein vaccine candidates have been investigated
in an experimental model of human carriage within the nasopharynx to cir-
cumvent the limitations given by murine models of pneumococcal infections
[196]. In contrast to the low level of antibody response to pneumococcal
polysaccharides, serum IgG and secretory IgA-response was detected to an
N-terminal region of PspA and also to PspC of the inoculum strain during
experimental carriage of type 23F and 6B pneumococci in adults. The spe-
cific antibody titers even exceeded basal levels of humans with pre-existing
antibody response [196, 199]. Former studies described serum IgG titers to
PsaA and pneumolysin emerging with age and exposure to pneumococci. In
contrast, no antibody response to PsaA and pneumolysin was detected in
the carriage studies with adults [196, 200, 201].
A stated problem of the human carriage model concerns the high vari-
ability of pneumococcal surface proteins including PspC that has been
described as strain-to-strain diversity and may account for the low IgG titers
against different serotypes. The functional organization of PspC proteins
among different strains is similar, but the molecular weight of PspC has
been shown to vary between 59 kDa to 105 kDa. PspC is divided into 11
groups due to differences in the N-terminal domain [68, 202]. Further stud-
ies are required to evaluate the impact of the described interaction of PspC
with the secretory IgA and with free secretory component for the immune
status of the host. A further example of the high strain-depending variability
in protein structure and in immunogenicity is PspA, which is divided into
two different protein families and subdivided into six clades [203].
To further explore the mechanism contributing to natural carriage and
clearance of carriage, a mouse model of pneumococcal colonization was
developed. In this model pneumococcal carriage induced a mucosal and
serum antibody response to pneumococci and to PspA. But no correlation
was detected between the density of colonization and amounts of detected
mucosal or serum antibodies [145].
The relationship between systemic and local antibody production and
carriage in children was studied in in vitro studies after antigen stimulation
by Zhang and co-workers [204]. Serum, saliva and cell culture superna-
tants of adenoidal mononuclear cells of children undergoing adenectomy

were assayed for antibodies to the pneumococcal proteins: PspC, pneu-
molysin, PsaA, PspA. In this study carriage rates fell with age and serum
levels of anti-PspC, Ply and PspA were rising. The results revealed that
antibody production to PspC and pneumolysin have the potential to pro-
tect children aged 2 years and older against pneumococcal colonization.
Other highly conserved vaccine candidates like LTA, PsaA, and PpmA
were tested, but no antibody response was detected [196]. Further studies
are required to correlate the described data of immunogenicity of vac-
cine candidates during human carriage with protective potential against
colonization.
The development of antibodies against pneumococcal surface associated
proteins including enolase, IgA1.protease, SlrA and PpmA has recently
been investigated in relation to pneumococcal carriage and otitis media in
children [205]. These studies have indicated that enolase, IgA1-protease,
SlrA and PpmA are immunogenic proteins, but no significant correlation
between antibody titers and pneumococcal carriage or infection has been
found. In contrast to the high structural variability of PspC and PspA, eno-
lase and IgA1- are highly conserved proteins that share strong homology
to other bacterial species colonizing the same nasopharyngeal niche like
N. meningitidis and H. influenzae. The presence of cross-reactive epitopes
enhances the basal titer of antibodies and might explain the little impact of
current pneumococcal colonization on serum concentrations of anti-IgA1-
protease and anti-enolase antibodies [205].
Future challenges of vaccine development might be the identification
of a surface exposed pneumococcal protein with serotype-unspecific con-
served immunogenic domains that show no cross reaction with other bacte-
rial or eukaryotic components.
Global analysis of host responses
The advent of microarray technology allows greater insight into the host-
cell response to the pneumococcus. The response of the human monocytic
cell line THP-1 has been assessed by microarray analysis following expo-
sure to S. pneumoniae and an isogenic mutant lacking Pneumolysin [206].
After 3-hour exposure to the pneumococcus, expression differences were
revealed in 182 host genes from the 4133 examined, illustrating the potential
for large-scale expression changes induced by the pneumococcus [206]. Of
these 182 genes, 142 were responsive to pneumolysin showing the dominant
nature of this virulence factor in host responses. While this study will not
be comprehensive in fully documenting the host response it illustrates the
complexity of the interaction between host-cells and the pneumococcus. An
important future challenge will be to understand the significance of these
expression changes in the disease process.
Host gene expression changes in an infection model has been investi-

gated in a rat model of otitis media [207]. Twelve hours following pneumo-
coccal challenge, 280 genes in the middle ear (effusion and mucosa) showed
a greater than two-fold change in expression compared with mock infected
controls. This represented ~24% of the genes examined, again showing the
ability of the pneumococcus to induce large-scale changes in host gene
expression. Such data allows the pneumococcal response pathways to be
mapped and for the identification of previously unrecognised responses.
For example, it was found that the transcription factor fra-1, implicated in
bone proliferation was upregulated during experimental otitis media. This
provides a candidate mechanism to explain clinical features of otitis media
involving the bone seen in both human patients and animals [207]. The glob-
al response to nasopharyngeal colonization has also been investigated in a
mouse model [208]. Up-regulation of siderocalin, an iron sequestering host
defense protein was noted in the nasal mucosa. How the pneumococcus
causes this up-regulation of siderocalin is unclear with the effect still seen
in mice deficient for either TLR2 or 4. Interestingly, this response could not
be replicated in vitro, again showing the complexity of the immune response
and the value of whole animal systems [208]. S. pneumoniae is resistant to
siderocalin and its upregulation may be advantageous to the pneumococcus
by inhibiting potential competitors in the nasopharynx [208].
Conclusion
Pneumococcus is a versatile microorganism causing local infections and

severe invasive diseases as well. The dramatic clinical outcome of pneu-
mococcal pneumonia, bacteriaemia and meningitis, respectively, is the
result of massive inflammatory host responses. The infections caused by
this pathogen can be controlled by the innate and antibody-mediated host
defense mechanisms. However, the pneumococcus has developed several
sophisticated mechanisms to overcome the defense mechanism and is able
to subvert host protein functions for its survival and dissemination. Another
threat is the increasing rate of antibiotic resistant isolates that requires the
development and use of cost effective pneumococcal vaccines or new thera-
peutics. A further understanding of the pneumococci-host interaction may
aid to develop a better vaccine against pneumococcal infections.
Acknowledgements
The work in the group of Sven Hammerschmidt is supported by grants of

the German Research Foundation (DFG-SFB 479 to S.H.) and Federal
Ministry of Education and Research (grant 01KI0430 to S.H., Competence
Network CAPNETZ). The work in the group of Tim J. Mitchell is supported
by the Wellcome Trust, MRC, BBRSC, the Egyptian government and the
European Union. Our apologies to authors of primary articles we have
failed to discuss in detail or to cite due to limitations on space. The authors
are grateful to Roland Nau (University of Göttingen, Germany) for provid-
ing histopathological micrographs and Manfred Rohde (German Research
Centre for Biotechnology, Braunschweig, Germany) for providing electron
micrographs.
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Pathogenesis of Mycoplasma pneumoniae infections:

adaptive immunity, innate immunity, cell biology, and
virulence factors
Ken B. Waites1, Jerry W. Simecka2, Deborah F. Talkington3

and T. Prescott Atkinson4
1Department of Pathology, University of Alabama at Birmingham, Department of Pathology,
WP 230, 619 19th St. South, Birmingham, AL 35249, USA; 2Department of Molecular Biology
and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107,
USA; 3Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention,
Atlanta, GA 30333, USA; 4Department of Pediatrics, University of Alabama at Birmingham,
Birmingham, AL 35249, USA
Abstract
Mycoplasmas represent the smallest self-replicating organisms. They are unique among
bacteria in that they lack a cell wall and require sterols for growth. The limited metabolic
and biosynthetic activities of mycoplasmas have complicated development of accurate
means for laboratory detection and hampered understanding of their roles as human
pathogens. Mycoplasma pneumoniae was first identified and characterized in the 1960s
and shown to be a common cause of upper and lower respiratory disease in children and
adults. Serious infections requiring hospitalization, while rare, occur in persons of all age
groups, and may affect multiple organ systems. Severity of disease appears to be related
to the degree to which the host immune response reacts to the infection. Extrapulmonary
complications involving all of the major organ systems can occur in association with M.
pneumoniae infection as a result of direct invasion and/or autoimmune response. Evidence
is accumulating for this organism’s contributory role in chronic lung conditions such as
asthma. Serology has been the most common means for laboratory detection of M. pneu-
moniae infection due to the slow growth that makes culture impractical. Newer diagnostic
methods utilizing nucleic acid amplification offer the advantages for rapid detection and
are likely to become increasingly important in the future, but these techniques have not
achieved widespread utilization thus far due to the lack of commercially sold products
and non-standardized methodology. Management of M. pneumoniae infections can usu-
ally be achieved with macrolides, ketolides, tetracyclines, or fluoroquinolones. As more is
learned about pathogenesis and immune response elicited by M. pneumoniae, improved
methods for diagnosis and prevention of disease due to this organism are anticipated.
184 Ken B. Waites et al.
Cell biology of Mycoplasma pneumoniae
Mycoplasmas represent the smallest self-replicating organisms capable of

cell-free existence, both in cellular dimensions and genome size. Individual
spindle-shaped cells of M. pneumoniae are 1–2 +m long and 0.1–0.2 +m
wide. Accordingly, the M. pneumoniae cell volume is less than 5% of that of
a typical bacillus. Typical colonies of M. pneumoniae rarely exceed 100 +m
in size and require examination under a stereomicroscope to visualize their
morphologic features. The M. pneumoniae genome was sequenced in 1996
and shown to consist of 816,394 basepairs with 687 genes [1], about one-
sixth the size of Escherichia coli.
The small genome of M. pneumoniae and its limited biosynthetic
capabilities are responsible for many of the biological characteristics and
requirements for complex medium supplementation in order for the organ-
ism to be cultivated in vitro. Mycoplasmas cannot synthesize peptidoglycan
cell walls. Lack of a rigid cell wall makes them pleomorphic and unable to
be classified in the manner of conventional eubacteria. Mycoplasmas are
not found freely living in nature since they depend on a host cell to supply
the necessary nutrients. Another characteristic of the genus Mycoplasma
is the requirement for sterols in artificial growth media, supplied by the
addition of serum. Sterols are necessary components of the triple-layered
mycoplasmal cell membrane providing structural support to the osmotically
fragile organisms.
Although mycoplasmas can flourish within an osmotically stable envi-
ronment in their eukaryotic host, they are extremely susceptible to desicca-
tion. This explains the need for close contact for transmission of infection
from person to person by airborne droplets. Another structural component
that is important for extracellular survival is a protein network that provides
a cytoskeleton to support the cell membrane. M. pneumoniae also produces
capsular material that may have a role in cytadherence.
M. pneumoniae possesses very limited metabolic and biosynthetic activi-
ties for proteins, carbohydrates, and lipids. It scavenges for nucleic acid pre-
cursors and apparently does not synthesize purines or pyrimidines de novo.
Fermentation of glucose to lactic acid by means of substrate phosphoryla-
tion mediated by phosphoglyceric acid kinase and pyruvate kinase activities
are means of ATP generation. M. pneumoniae possesses all reactions of
glycolysis, but the tricarboxylic acid cycle and a complete electron transport
chain containing cytochromes are absent. M. pneumoniae reduces tetrazo-
lium and this property has been used historically to distinguish it from com-
mensal oropharyngeal mycoplasmas. Reproduction occurs by binary fission,
during which the attachment organelle migrates to the opposite pole of the
cell during replication and before nucleoid separation.
Neither genomic analysis nor electron microscopy has demonstrated the
presence of structures such as flagella or pili, suggesting that gliding motility
occurs by an unknown mechanism involving the attachment organelle [2].
Pathogenesis of Mycoplasma pneumoniae infections… 185
In addition to the crossing of the mucus barrier that protects the respiratory
epithelium, gliding motility may contribute to the ability of M. pneumoniae
to travel down respiratory cilia to attach to respiratory epithelial cells.
Epidemiology and transmission of infection
M. pneumoniae infections can involve both the upper and lower respiratory
tract and occur both endemically and epidemically worldwide in persons of
all ages. Climate, seasonality, and geography are not thought to be of major
significance, although most outbreaks in the USA tend to occur in the late
summer and early fall. Foy [3] reported that M. pneumoniae was responsible
for 15-20% of all cases of community-acquired pneumonia (CAP) between
1962 and 1975 in Seattle, Washington, USA. Additional retrospective sero-
logical studies performed in Denmark showed a pattern of M. pneumoniae
infections over a 50-year period from 1946 through 1995 with endemic
disease transmission punctuated with cyclic epidemics every 3 to 5 years
[4]. The long incubation period and relatively low transmission rate have
been implicated in the prolonged duration of epidemics of M. pneumoniae
infections.
A study performed in the USA during the 1990s detected M. pneu-
moniae in 23% of CAP in children 3-4 years of age [5]. Another study
from France [6] documented its occurrence in children less than 4 years
of age without significant differences in infection rates for other children
or adults. These findings may reflect the greater number of young children
who attend day care centers on a regular basis than in previous years, and
the ease with which young children share respiratory secretions with older
household members or contacts. Marston [7] reported that M. pneumoniae
was definitely responsible for 5.4% and possibly responsible for 32.5% of
2,776 cases of CAP in hospitalized adults in Ohio, USA. Extrapolation of
data nationally provides an estimated 18,700 to 108,000 cases of CAP in
hospitalized adults due to M. pneumoniae annually. Since the majority of
CAPs are treated as outpatients, the total number of pneumonias due to M.
pneumoniae is almost certainly many times greater. An additional striking
finding was their observation that mycoplasmal pneumonia in hospitalized
adults increased with age and it was second only to Streptococcus pneu-
moniae in elderly persons.
The P1 adhesin is a 170 kD transmembrane protein that is concentrated
on the adhesive tip of M. pneumoniae and serves an essential function in
cytadherence. Different P1 subtypes may operate in cycling times of M.
pneumoniae epidemics. Gene divergences within the P1 adhesin and devel-
opment of subtype-specific antibodies following initial infection might also
contribute to the frequency of reinfections due to another subtype. Studies
using a variety of genotypic methods to characterize over 200 M. pneu-
moniae isolates collected over several years from multiple countries showed
that most of the isolates could be classified into two subtypes based on the
sequences of the P1 adhesin gene, the ORF6 gene, the P65 gene, and by a
typical DNA restriction fragment pattern [8, 9]. One or the other of the two
subgroups tended to predominate in specific regions.
M. pneumoniae is not considered part of the normal flora and its
detection by culture can usually be considered abnormal and of etiologic
significance if detected in a person with a clinical condition known to be
caused by the organism. However, it can persist for variable periods in the
respiratory tract following infections that resolved clinically with appropri-
ate antimicrobial therapy, providing a reservoir for spread of the organism
to others.
Cytadherence and other virulence factors
The initial step in the pathogenesis of mycoplasmal respiratory disease

involves M. pneumoniae adherence to ciliated respiratory epithelium.
This intimate interaction damages respiratory epithelial cells through the
production of toxic substances, such as hydrogen peroxide and superoxide
radicals, leading to oxidative stress. Subsequent development of the host
inflammatory response may actually have the greatest impact on disease. In
fact, the host’s bronchoepithelial cells may contribute to the development
of the inflammatory lesions through the release of cytokines in response to
infection or other stimuli. Release of interleukin (IL)-8, from human bron-
choepithelial cells may occur in response to stimulation with mycoplasma
membranes [10]. Similarly, human lung alveolar type II pneumocytes (A549
cells) infected with M. pneumoniae show an increase IL-8, tumor necrosis
factor-alpha (TNF-_), and IL-1` mRNA [11], supporting the idea that the
adherence to human airway epithelial cells leads to production of cytokines
and recruitment of lymphocytes and other inflammatory cells, and that
these cytokines subsequently modulate the activity of the inflammatory
infiltrates.
The P1 protein adhesin is also immunogenic, and it is the target for
antibodies that develop in the course of natural infection. The host-cell
ligand for mycoplasmal adhesins has not been characterized conclusively,
though sialoglycoconjugates and sulfated glycolipids have been implicated
[12]. At least six other proteins (HMW1, HMW2, HMW3, P90, P40 and
P30) are known to participate in adhesion. All are localized to the terminal
tip of the organism, and most are likely involved in the architecture of the
attachment organelle and localization of P1. P30 appears to be involved
with gliding motility. HMW1, HMW2, and HMW3 are critical in the forma-
tion and stabilization of the attachment organelle, including localization
of other adhesin-related proteins. Once this polar structure is established,
an independently assembled complex of proteins B, C and P1 is drawn to
the structure to complete formation of the functional terminal attachment
Figure 1. Transmission electron micrographs of Mycoplasma pneumoniae grown in culture

demonstrating flask-shaped morphology and the prominent adhesin tip (Courtesy Kristin
Hoek and Leigh Milligan, UAB High Resolution Imaging Facility).
organelle shown as an electron dense region in the flask-shaped organism

by electron micrography (Fig, 1). Two proteins, elongation factor TU and
pyruvate dehydrogenase E1` are involved in binding M. pneumoniae to
fibronectin [13].
Mammalian cells parasitized by M. pneumoniae can exhibit a number
of cytopathic effects as a result of the local damage following cytadher-
ence. Cells may lose their cilia entirely, appear vacuolated, show a reduc-
tion in oxygen consumption, glucose utilization, amino acid uptake, and
macromolecular synthesis, ultimately resulting in exfoliation of all or parts
of the infected cells. These subcellular events result in some of the clinical
manifestations of respiratory tract infection such as the persistent, hacking
cough.
Dallo [14] recently described the ability of M. pneumoniae to survive,
synthesize DNA, and undergo cell replication in artificial cell culture sys-
tems over a 6-month period. Intracellular sequestration could facilitate the
establishment latent or chronic states, circumvent mycoplasmacidal immune
mechanisms, facilitate the ability to cross mucosal barriers and gain access
to internal tissues, and impair efficacy of some drug therapies, accounting
for difficulty in eradicating the mycoplasmas in clinical conditions. However,
the extent to which M. pneumoniae invades and replicates intracellularly in
vivo is not known. High-frequency phase and antigenic variation of surface
adhesin proteins made possible by DNA rearrangements in truncated and
sequence-related copies of the P1 adhesin genes that are dispersed through-
out the genome may also be a means for M. pneumoniae to evade the host
immune response [15].
Innate immunity to M. pneumoniae disease
Elements of the host innate immune system are activated by M. pneu-

moniae and the attendant inflammation accounts for many of the initial
signs and symptoms accompanying the early stages of the evolving infec-
tion. Once M. pneumoniae reaches the lower respiratory tract, the organ-
ism may be opsonized by complement or antibodies. Macrophages become
activated, begin phagocytosis, and undergo chemotactic migration to the
site of infection. There is in vitro evidence that the organism is susceptible
to complement-mediated cytolysis, probably through both the alternative
and classical pathways [16, 17]. Therefore, it is possible that complement
plays a significant role in inhibiting growth of the organism, particularly
after inflammation induces an exudate. While there are reports of invasive
infections with M. pneumoniae in hypogammaglobulinemic individuals,
there are none relating similar occurrences in patients with complement
deficiencies alone. A recent study identified a highly significant association
between mannose binding lectin deficiency and invasive infections with M.
pneumoniae in patients with concomitant antibody deficiency [18]. A series
of surfactant proteins (SP-A–SP-D) are also capable of binding and regulat-
ing the growth of microorganisms through lectin-like activity. SP-A exhibits
calcium-dependent binding and growth inhibition of M. pneumoniae [19].
Interaction of M. pneumoniae with mast cells via a sialic acid-dependent
binding mechanism was reported to induce cytokine production, especially
IL-4, IL-5, and TNF-_ [20, 21], and is dependent upon the presence of the P1
adhesin. M. pneumoniae lipoproteins may interact with toll-like receptors 2
(TLR2) and/or 6 (TLR6) of respiratory epithelial or other cells, as found
with other mycoplasmas [22, 23]. As a result of TLR interaction, cells are
stimulated to produce cytokines or begin apoptosis, thus contributing to the
pathogenesis of disease.
M. pneumoniae may induce cellular activation during co-culture with
a variety of other immunologic cells, including lymphocytes, macrophages,
and respiratory epithelial cells. The nonspecific nature of the cellular targets
of activation by M. pneumoniae could explain the broad range of inflamma-
tory and autoimmune phenomena following acute infection.
Adaptive immunity and interaction of M. pneumoniae with host

immune cells
Adaptive immunity, characterized by both B and T lymphocyte responses,

has a major impact on the progression of M. pneumoniae respiratory dis-
ease. While data are limited on the role of lymphoid responses in human M.
pneumoniae infections, studies of other mycoplasmal respiratory diseases in
animals indicate that some immune responses will be beneficial in control-
ling or preventing infection, while others contribute to disease severity.
Local (mucosal) immune responses may be most effective in protect-

ing the host from mycoplasma infection since resistance to M. pneumoniae
seems to be most closely associated with IgA responses, but immunity is
often short-lived, as humans are susceptible to repeated infections, despite
the development of complement fixing and growth inhibiting antibody
responses [15]. Immune responses that develop after infection often fail to
eliminate the mycoplasma, indicating that adaptive immunity apparently
has a limited effect on clearance of an established infection, thus leading to
asymptomatic carriage for variable periods of time. M. pneumoniae infec-
tions in adults may be asymptomatic, perhaps reflecting some degree of
protective immunity against reinfections over time.
Persons with impaired ability to produce antibody, such as in congenital
hypogammaglobulinemia, can suffer from chronic respiratory disease due
to M. pneumoniae, suggesting antibody and other immune responses have
a limited, but significant, role in recovery from disease [24]. In addition,
hypogammaglobulinemic persons are more susceptible to extrapulmonary
complications [25]. These observations suggest that antibody responses are
important in controlling infection and preventing dissemination of myco-
plasmas from the respiratory tract.
Cytokine production and lymphocyte activation may either minimize
disease through the enhancement of host defense mechanisms, or exac-
erbate disease through the development of immunologic hypersensitivity,
worsening damage to the respiratory epithelium. The more vigorous the
cell-mediated immune response and cytokine stimulation, the more severe
the clinical illness and pulmonary injury. Lymphoid infiltration characteristic
of M. pneumoniae disease suggests that lymphocyte activation contributes
to the development of inflammatory responses. M. pneumoniae infection of
laboratory rodents reveal CD4 + T helper (Th) cells within the inflamma-
tory infiltrates in lung [26], and mycoplasma-specific Th cell responses are
found in peripheral blood from humans [27]. CD8 + T cells also increase in
lungs of mice after intranasal infection with M. pneumoniae [28] and T-cell-
depleted hamsters develop less severe M. pneumoniae respiratory disease
[29]. Mycoplasma pulmonis infection of severe combined immunodefi-
ciency (SCID) mice or T-cell-deficient mice results in milder lung lesions
[30], and both T-cell populations modulate disease severity without directly
affecting the number of mycoplasmas in the lungs. CD4 + Th-cells promote
the inflammatory responses in rodent lungs, whereas CD8 + T-cells dampen
these responses [31]. Most likely, a similar dichotomy of T-cell activity
occurs in human M. pneumoniae disease. Depletion of CD4 + T-cells in mice
results in a decreased cytokine responses and lymphocyte accumulation in
the lungs after experimental M. pneumoniae infection [28]. Thus, popula-
tions of CD4 + T- and CD8 + T-cells most likely have contrasting roles in M.
pneumoniae pulmonary disease, with the actions of a population of proin-
flammatory CD4 + Th-cells being suppressed by mycoplasma-specific CD8 +
T-cells. Furthermore, CD4 + T-lymphocyte, B lymphocyte, and plasma cell
accumulation in the lung is responsible for the radiographic manifestation

of pulmonary infiltrates and is associated with lymphocyte proliferation,
production of immunoglobulins, and release of TNF-_, interferon gamma
(IFN-a), and various interleukins [15].
Host responses that develop after M. pneumoniae infection likely con-
tribute to extrapulmonary complications. Examples are the association
between M. pneumoniae infection and increased severity of asthma and
production of anti-erythrocyte autoantibodies “cold agglutinins,” once used
as a diagnostic indicator of M. pneumoniae infection.
Overall, adaptive immune responses generated after M. pneumoniae
infections have contrasting impacts on the pathogenesis of infection.
Beneficial effects of host resistance lead to containment of the infection and
ultimately recovery in many persons. Relative persistence of M. pneumoniae
in the host leads to the provocation of ineffective immune-mediated inflam-
matory responses, regulated by opposing T-cell activities. Complicating the
impact of these responses is their contribution to other adverse reactions,
such as asthma and autoimmune-like effects.
Clinical and laboratory aspects of M. pneumoniae infections
Following a 2–3-week incubation period, symptoms may develop over

1–2 days and consist of worsening nonexudative pharyngitis, nasal and
sinus congestion, low-grade fever, and cough. In 1–2 weeks, more seri-
ously affected individuals develop tracheobronchitis and primary atypical
pneumonia (PAP), a syndrome consisting of fever, cough, musical inspira-
tory rhonchi reflecting mucus secretion in large airways, and occasion-
ally bilateral expiratory wheezing. Acute lower respiratory symptoms may
progress to respiratory distress with hypoxemia and necessitate hospital
admission, but such severe cases represent only 5–10% of affected indi-
viduals. Patients with pre-existing pulmonary disease such as asthma or
chronic bronchitis may be more severely affected. Antibiotic therapy with
appropriate drugs results in clinical improvement, but the organism tends
to persist in the airways. The cough, often productive, typically continues
for several weeks and abnormalities in pulmonary hyperresponsiveness can
persist for months.
Diagnosis of PAP is often presumptive because of difficulties in micro-
biological confirmation. Chest radiographs typically reveal only perihilar
and bibasilar streaky infiltrates, findings less impressive than symptoms
would suggest. Small pleural effusions occur in 5-20% of patients. Clinical
presentation may be similar to what is also seen with other pathogens, par-
ticularly Chlamydophila pneumoniae, various respiratory viruses, and even
bacteria such as S. pneumoniae. M. pneumoniae may also be present in the
respiratory tract concomitantly with other pathogens. About one-third of
persons with mycoplasmal infections have leukocytosis and an elevated
erythrocyte sedimentation rate. Sputum gram stain may show mononuclear

cells or neutrophils and normal flora. There are no hepatic or renal abnor-
malities typical of M. pneumoniae infection, although the hemolytic anemia
that develops in some patients may be reflected in the hemogram.
Autoimmunity and extrapulmonary manifestations
Autoimmune reactions are believed to be responsible for many of the

extrapulmonary complications that are associated with as much as 25% of
M. pneumoniae infections. They may occur as a result of molecular mim-
icry in the ascending paralysis of Guillain-Barré Syndrome that results
from autoantibody-mediated destruction of peripheral nerve fibers. These
antibodies may also recognize glycolipids extracted from M. pneumoniae.
Another mechanism is illustrated in hemolytic anemia due to cross-react-
ing antibodies against erythrocyte antigens as seen in cold-agglutinin
disease. The PCR assay has greatly enhanced understanding of how M.
pneumoniae can disseminate throughout the body. The presence of M.
pneumoniae in blood, synovial fluid, cerebrospinal fluid, pericardial fluid,
and skin lesions has been documented by PCR and/or culture. Thus, direct
invasion must always be considered [15]. The frequency of direct invasion
of these sites is unknown because the organism is rarely sought and most
reported extrapulmonary syndromes have been attributed to M. pneu-
moniae infection based on serology. Extrapulmonary manifestations have
been reviewed in depth elsewhere with original citations [15] and are sum-
marized in Table 1.
Pathologic aspects of lung disease due to M. pneumoniae
Acute perivascular and peribronchial cell infiltration results in destruc-

tion of respiratory epithelium. There are also neutrophilic accumulations
within the airways early in disease. At later stages, massive mononuclear
cell infiltration, of which T-cells are a major component, occurs. Ulceration
with destruction of ciliated epithelium of bronchi and bronchioles, edema,
bronchiolar and alveolar infiltrates of macrophages, lymphocytes, and
neutrophils, increased numbers of plasma cells, and deposition of fibrin.
Type II pneumocyte hyperplasia with diffuse alveolar damage and bron-
chiolitis obliterans have been described [15]. Pleura may contain patches
of fibrinous exudates. Pleural effusions sometimes occur in association
with more severe cases complicated by long-term sequelae such as pleural
scarring, bronchiectasis, and pulmonary fibrosis. Lung abscesses may also
occur. Immunosuppressed persons may lack pulmonary infiltrates, further
attesting to the importance of the host immune response in lesion develop-
ment.
Table 1. Extrapulmonary manifestations of M. pneumoniae infection resulting from autoim-

mune reaction and/or direct invasion
Organ system Manifestations Comment

Neurologic Ascending paralysis, encepha- Nervous system complications are
litis, aseptic meningitis, trans- among the most common and most
verse myelitis, cerebellar syn- severe manifestations. They often
drome, polyradiculitis, cranial occur within 1–2 weeks of respiratory
nerve palsies, optic neuritis, infection and in some cases without
choreoathetosis, leukoen- any apparent preceding respiratory
cephalopathy, acute psychosis symptoms.
Musculoskeletal Myalgias, arthralgias, polyar- Invasive joint infections occur more
thropathies, septic arthritis, commonly in persons with antibody
reactive (inflammatory nonin- deficiency, but they have also been
fectious) arthritis, osteomyeli- described in immunocompetent per-
tis, rhabdomyolysis sons. Recent data suggest a possible
association with adult and juvenile
rheumatoid arthritis.
Skin Erythematous maculopapular Dermatological disorders are the
and vesicular rashes, Stevens- most common type of extrapulmonary
Johnson Syndrome, conjuncti- manifestations.
vitis, ulcerative stomatitis
Cardiovascular Pericarditis, myocarditis, Cardiac involvement occurs in up to
pericardial effusion, cardiac 8.5% of serologically diagnosed infec-
tamponade tions and is more common in adults
than children.
Hematologic Hemolytic anemia, cold agglu- Cross-reactive antibodies against
tinin disease, aplastic anemia, erythrocyte antigens or plasma von
thrombotic thrombocytopenic Willebrand factor-cleaving protease
purpura, disseminated intra- have been implicated as causative fac-
vascular coagulation tors.
Renal Acute glomerulonephritis, Antibody-mediated pathogenesis is
renal failure, tubulointerstitial believed to predominate, but myco-
nephritis, IgA nephropathy plasma antigen has been detected in
damaged kidney tissue by immunohis-
tochemistry.
Gastrointestinal Nausea, vomiting, diarrhea, These nonspecific complaints can be
and other cholestatic hepatitis, pancreati- associated with respiratory disease.
tis otitis media, myringitis Mechanisms have not been carefully
investigated.
Chronic lung disease associated with M. pneumoniae infection
M. pneumoniae has been isolated in increased frequency from stable asth-

matics. It has been linked with exacerbations of existing asthma, as well as
the subsequent development of asthma in previously healthy persons. Kraft
[32] detected M. pneumoniae by PCR in respiratory secretions of 42% sta-
ble adult asthmatics versus 9% of healthy controls. In another study, throat
cultures for M. pneumoniae were positive in 24.7% of children and adults
with asthma exacerbation, compared with 5.7% of healthy controls [33]. In

another study, 21% of adults having a flare of their asthma were found to
have high levels of M. pneumoniae-specific IgM, suggesting exacerbation
occurred in the context of acute infection [34].
Macrolide treatment of asthma patients in whom M. pneumoniae has
been detected resulted in improvement in pulmonary function tests in
comparison with asthma patients who did not have evidence of the organ-
ism in their airways [35], owing perhaps to both the antibacterial as well as
anti-inflammatory effects of these drugs. Mycoplasmas have been detected
by PCR in airways even when culture and serology are negative, suggest-
ing that low numbers of organisms may evade detection by the immune
system [35]. Lack of a measurable serologic response may also facilitate the
organism’s persistence in the lower respiratory tract.
M. pneumoniae is known to induce a number of the inflammatory
mediators implicated in the pathogenesis of asthma that may play a role in
exacerbations. IgE triggering of mast cell degranulation is a key event in
allergic asthma, and mycoplasma-specific IgE responses may elicit a similar
response [20]. Mycoplasma IL-4 stimulation may enhance the asthma-pro-
moting IgE responses against potential allergens. M. pneumoniae can be
associated with significantly greater numbers of mast cells in humans with
chronic asthma [36], and experimental evidence from a rodent mast cell line
suggests that the organism can induce activation of mast cells [20, 21]. Koh
[37] showed that levels of IL-4 and the ratio of IL-4/IFN-a were significantly
higher in children with M. pneumoniae than those with pneumococcal
pneumonia or uninfected controls, suggesting a TH2-like cytokine response
representing a favorable condition for IgE production.
Gump [38] reported that mycoplasmal infections could be associated
with some cases of chronic obstructive pulmonary disease (COPD) exac-
erbation. Subsequent studies, summarized in a recent review [15] have
reported serologic evidence of acute M. pneumoniae infection in 6-14% of
COPD exacerbations. Evidence of concomitant respiratory pathogens in
some cases complicates understanding of the significance of M. pneumoniae
in this context.
Bacterial infection is the main cause of progressive pulmonary failure
in patients with cystic fibrosis. Serological examinations have been the
sole means of assessing the presence of M. pneumoniae in persons with
cystic fibrosis and the methods employed present some difficulty in proper
interpretation to define a recent infection. Limited findings to date suggest
mycoplasmas may occur but are fairly uncommon causes of respiratory
complications in persons with cystic fibrosis [15]. More work must be done
to clarify the importance of M. pneumoniae in the epidemiology and patho-
genesis of exacerbations of chronic lung diseases using a more comprehen-
sive diagnostic strategy that would include direct tests for the presence of
the organisms by PCR, culture, and serology.
Measurement of the immune response as a means for diagnosis
Serology has been the most common laboratory means for diagnosis of M.
pneumoniae infections. Although culture and PCR are also used, persis-
tence of the organism for variable lengths of time following acute infection
makes it difficult to assess the significance of a positive culture or PCR
assay without additional confirmatory seroconversion. The length of time
necessary for culture (sometimes 6 weeks or more) makes it impractical
for patient management and it is not widely available except in specialized
reference laboratories. Detailed information on laboratory diagnosis of M.
pneumoniae infection is available elsewhere [15] and only pertinent sum-
mary information is discussed here.
M. pneumoniae has both lipid and protein antigens which elicit antibody
responses that can be detected after about 1 week of illness, peaking at 3-
6 weeks, followed by a gradual decline, allowing several different types of
serological assays, based on different antigens and technologies. Serology
is a useful epidemiologic tool in circumstances where the likelihood of
mycoplasmal disease is high, but it is less suited for assessment of individual
patients. Its main disadvantage is the need for both acute and convalescent
paired sera collected 2 to 3 weeks apart that are tested simultaneously for
IgM and IgG to confirm seroconversion. This is especially important in
adults over 40 years of age who may not mount an IgM response, presum-
ably because of reinfection. Moreover, IgM antibodies can sometimes per-
sist for several weeks to months, making it risky to base diagnosis of acute
infection on a single assay for IgM alone. Antibody production may also
be delayed in some infections, or even absent if the patient is immunosup-
pressed. False-negative tests for IgM can also occur if serum is collected too
soon after onset of illness. Since M. pneumoniae is a mucosal pathogen, IgA
is typically produced early in the course of infection. Measurement of serum
IgA may therefore be a better approach for diagnosis of acute infection
because of its rapid rise and decline, but very few commercial assays include
reagents for its detection.
Complement fixation (CF) was the first method developed for sero-
logical testing for M. pneumoniae. CF measures mainly the early IgM
response and does not differentiate among antibody classes, which is
desirable to differentiate acute from remote infection. CF suffers from
low sensitivity and specificity because the glycolipid antigen mixture used
may be found in other microorganisms, as well as human tissues, and even
plants. Cross-reactions with Mycoplasma genitalium are well recognized.
In most clinical laboratories CF has been replaced by alternative tech-
niques with greater sensitivity and specificity, many of which have been
developed and sold as commercial kits. Immunofluorescent antibody
(IFA) assays, direct and indirect hemagglutination using IgM capture, and
other particle agglutination antibody assays (PAs) have been developed
to detect antibody to M. pneumoniae. Enzyme immunoassays (EIAs)
have become the most widely used commercial methods for detection
of M. pneumoniae. EIAs are more sensitive for detecting acute infection
than culture, and can be comparable in sensitivity to PCR, providing a
sufficient time has elapsed since infection for antibody to develop and
the patient has a functional immune system. These assays may be qualita-
tive or quantitative, may or may not require specialized equipment for
performing the assay and reading the results, and can be performed with
very small volumes of serum. The need for acute and convalescent sera
has remained the obvious limitation for prompt point-of-care diagnosis.
However, qualitative rapid point-of care serologic assays that detect both
IgM and IgG or IgM alone in an easy-to-read format without the need
for any instrumentation have been developed and shown to compare
favorably with other commercial assays [15]. The variability of results
from comparative studies underscores the need for improved serologi-
cal reagents for detecting acute M. pneumoniae infection [39]. The best
commercial EIA for individual patient diagnosis depends on the age of
the patient, timing of serum collection, whether paired sera are avail-
able, equipment available, and experience of the laboratory personnel.
However, maintaining a large variety of different assays within one labo-
ratory is not practical or cost-effective.
Use of the PCR assay for detection of M. pneumoniae
Gene targets used in PCR assays for M. pneumoniae include 16S rRNA,
P1 adhesin, ATPase operon gene, and tuf gene. Real-time PCR assays
have also been described [15]. The sensitivity of PCR is very high, cor-
responding to a single organism when purified DNA is used. Other
advantages are the potential ability to complete the procedure in one day,
the requirement of only one specimen containing organisms that do not
have to be viable, as well as the ability to detect nucleic acid in preserved
tissues. Comparison of PCR with culture and/or serology has yielded
varied results that are not always in agreement. Positive PCR results in
culture-negative persons without evidence of respiratory disease suggest
inadequate assay specificity, persistence of the organism after infection,
or asymptomatic carriage, perhaps in an intracellular compartment that
does not yield culturable organisms. Quantitative studies may be useful
in drawing conclusions. Positive PCR results in serologically-negative
persons may be due to an inadequate immune response, early success-
ful antibiotic treatment, or to the collection of specimens before specific
antibody synthesis could occur. Negative PCR results in culture or sero-
logically proven infections raise the possibility of inhibitors or other tech-
nical problems with the assay. If antibiotics have been administered, PCR
results may be negative even though serology is positive. Ideally, positive
PCR assays should be confirmed by a second unrelated target gene. Thus
far, there are no commercial PCR kits for detection of M. pneumoniae,

but several are in development.
Treatment of M. pneumoniae infections
Administration of antimicrobials to patients with M. pneumoniae infections

will generally produce satisfactory results with a marked reduction in dura-
tion of respiratory symptoms. Management has been guided primarily by
well-known and consistent susceptibilities to a variety of drugs. Macrolides
are the treatments of choice, but tetracyclines and fluoroquinolones are also
effective, as is the new ketolide telithromycin. Most clinical trials evaluating
treatments for CAP identified small numbers of cases proven to be due to
M. pneumoniae by serologic diagnosis, though some recent studies incorpo-
rated culture and/or PCR. Use of serology alone precludes determination as
to whether a treatment regimen actually eradicates the organism, thus very
little data are available regarding microbiological efficacy of any regimen.
A summary of clinical trials evaluating various treatments for CAP in which
outcome data specific for M. pneumoniae were included can be found in a
recent review [15]. A recent study from Japan found that macrolide resis-
tance in M. pneumoniae due to transition mutations in domain V on the 23S
rRNA gene occurred in 6% of 195 clinical isolates from patients with acute
infections [40]. The clinical significance of this resistance is uncertain, but it
suggests the need to monitor clinical isolates for such resistance. High-dose
steroids have been reported to be useful in treatment of encephalitis in chil-
dren with complicated M. pneumoniae infection [41]. Plasmapheresis and
intravenous immunoglobulin therapy might be considered if steroid therapy
is ineffective in these settings.
Summary
M. pneumoniae is a common cause of CAP in both children and adults.

Serious infections requiring hospitalization, while rare, occur in persons of
all age groups, and may affect multiple organ systems. Severity of disease
appears to be related to the degree to which the host immune response
reacts to the infection. Extrapulmonary complications involving all of the
major organ systems can occur in association with M. pneumoniae infection
as a result of direct invasion and/or autoimmune response. Evidence is accu-
mulating for this organism’s contributory role in chronic lung conditions
such as asthma. Effective management of M. pneumoniae infections can
usually be achieved with macrolides, ketolides, tetracyclines, or fluoroquino-
lones. As more is learned about pathogenesis and immune response elicited
by M. pneumoniae, improvement in methods for diagnosis and prevention
of disease due to this organism are anticipated.
Acknowledgments
Portions of the work summarized in this chapter were supported by U.S.

Public Health Service grants HL73907-01A1 to T.P.A. and grants HL069431,
AI42075, and AI055907 to J.W.S.
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Community-acquired pneumonia: paving the way

towards new vaccination concepts
Pablo D. Becker and Carlos A. Guzmán
Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7,

38124 Braunschweig, Germany
Abstract
Despite the availability of antimicrobial agents and vaccines, community-acquired pneu-
monia remains a serious problem. Severe forms tend to occur in very young children
and among the elderly, since their immune competence is eroded by immaturity and
immune senescence, respectively. The main etiologic agents differ according to patient
age and geographic area. Streptococcus pneumoniae, Haemophilus influenzae, respira-
tory syncytial virus (RSV) and parainfluenza virus type 3 (PIV-3) are the most important
pathogens in children, whereas influenza viruses are the leading cause of fatal pneumonia
in the elderly. Effective vaccines are available against some of these organisms. However,
there are still many agents against which vaccines are not available or the existent ones
are suboptimal. To tackle this problem, empiric approaches are now being systematically
replaced by rational vaccine design. This is facilitated by the growing knowledge in the
fields of immunology, microbial pathogenesis and host response to infection, as well
as by the availability of sophisticated strategies for antigen selection, potent immune
modulators and efficient antigen delivery systems. Thus, a new generation of vaccines
with improved safety and efficacy profiles compared to old and new agents is emerging.
In this chapter, an overview is provided about currently available and new vaccination
concepts.
Introduction
The mucosa of the human respiratory tract represents a primary target for
a large number of microbial pathogens. Typically, colonization is an asymp-
tomatic process, resulting from the interplay between bacterial factors and
host clearance mechanisms. Clinical illness may result from either the local
release of bacterial toxins or the systemic dissemination of the pathogen
after breaching the mucosal barrier. In the course of respiratory infections
adaptive immune responses could be significantly impaired. This might
lead to more severe forms of disease or to super-infections, which in turn
complicate the clinical management of the patient. The most severe forms
of respiratory infection tend to occur in very young children and among the
202 Pablo D. Becker and Carlos A. Guzmán
elderly, in whom immune competence is eroded by immaturity or immune-

senescence, respectively. In addition, patients who are immunocompro-
mised, as a result of disease or therapeutic interventions, have the greatest
risk of developing a fatal infection.
Despite the availability of new antimicrobials and effective vaccines,
community-acquired pneumonia remains a common and serious illness. In
fact, it is a leading contributor to the nearly 4 million deaths occurring each
year due to respiratory infections, especially in children from developing
countries [1, 2]. The main causative agents of pneumonia differ according
to the patient age and the geographic area. In addition, there are relatively
few comprehensive studies on the specific aetiology of pneumonia [2] due
to (i) overlaps in the clinical manifestations of the different syndromes, (ii)
difficulties in establishing the precise aetiology, and (iii) frequent occur-
rence of co-infections. However, Streptococcus pneumoniae, Haemophilus
influenzae, respiratory syncytial virus (RSV), and parainfluenza virus type 3
(PIV-3) have been identified as the main agents responsible for acute respi-
ratory infections in children, whereas influenza virus related pneumonia is
the leading cause of disease-related deaths in the elderly. In addition, the
availability of new and more sensitive diagnostic tests have contributed to
the identification of hitherto unknown lower respiratory pathogens, such as
the human metapneumonovirus (hMPV) and novel coronaviruses causing
the severe acute respiratory syndrome (SARS).
Significant efforts have been invested in the last two decades to develop
new diagnostic tools, to elucidate the molecular mechanisms of microbial
pathogenesis and to understand host clearance mechanisms. This resulted in
an improved knowledge on host responses to infection and immuno-patho-
genesis, which in turn have facilitated the establishment of new prophylac-
tic and therapeutic interventions. However, despite our accomplishments
in vaccine development, there are many pathogens for which vaccines or
adequate therapies are not available or the existent ones are suboptimal.
The main approach applied for vaccine development has radically
changed in recent years. Whole cell vaccines are systematically being
replaced by subunit vaccines, in which purified antigens or their coding genes
are exploited in combination with new adjuvants and/or delivery systems. As
a result, many of the vaccines under development will exhibit consistently
improved stability, safety and efficacy profiles. They will also be amenable
for mucosal administration, thereby mimicking natural infections.
Currently available vaccines
Influenza vaccines
Influenza A viruses are the most commonly responsible for severe respira-
tory illness in humans, followed by influenza B. The population’s susceptibly
New vaccination concepts for CAP 203
to infection is renewed annually, because of the rapid antigenic variation of

this virus. The antigenic variation is due to the accumulation of point muta-
tions in the two major surface glycoproteins of the virus, haemagglutinin
(HA) and neuraminidase (NA). This can lead to an antigenic drift of the
virus, which often leaves current influenza vaccines outdated and ineffec-
tive. Antigenic shift can also occur due to the segmented nature of the viral
genome that favours the emergence of re-assorted strains, in which an entire
glycoprotein can be acquired from a different animal influenza virus. Both
types of variation represent a critical bottleneck for the establishment of
a robust vaccination strategy against influenza. In fact, when an influenza
virus with the capacity to spread from person-to-person and a complete new
glycoprotein subtype suddenly emerges, a worldwide pandemic outbreak
can result [3].
Inactivated vaccines against influenza
The earliest vaccines against influenza were whole cell vaccines obtained in
the 1940s by inactivating viruses grown in the allantoic cavity of embryonat-
ed chicken eggs with formalin. While contemporary inactivated influenza
vaccines are still produced in embryonated eggs, improvements in manu-
facturing have resulted in a highly purified and less-reactogenic detergent-
split product. Three viral strains are selected on the basis of the previous
year’s surveillance data on the most prevalent subtypes, therefore, vaccine
composition may vary from year-to-year. Vaccination has a high benefit:cost
ratio, since influenza-related illness (e.g., hospitalizations and deaths) are
effectively prevented [1].
The world’s total vaccine production is approximately 300 million
doses, with a maximum capacity of 900 million doses. However, the World
Health Organization (WHO) estimates that there are about 1.2 billion
people at high risk for severe influenza outcomes (e.g., elderly over 65
years of age, infants, health care workers, children and adults with under-
lying cardiopulmonary disease). Furthermore, the global infrastructure
would not be able to handle the timely manufacturing and distribution
of a vaccine for a pandemic outbreak [4]. One alternative would be to
lower the quantity of antigen per dose and add adjuvants to the vaccine
formulation, but this needs to be tested in clinical trials [5]. Another solu-
tion would be to improve current vaccine production technologies (i.e.,
egg-derived vaccines). However, there is the limited number of egg pro-
ducers and viral strains can emerge, which could not be easily adapted to
embryonated eggs. To overcome these problems, several pharmaceutical
companies have embarked themselves on projects for the development of
vaccines produced by growing the virus on cell lines. The influenza virus
can be adapted to grow on a variety of mammalian cell lines, including
Vero, PER.C-6, and Madin-Darby canine kidney (MDCK) cells [6–8].
This strategy would also improve the possibility of up-scaling vaccine pro-
duction in face of a pandemic spread. Alternatively, it would be possible
to develop a vaccine against any influenza virus, such as the avian H5N1
strain, by using reverse genetics techniques [9] (see below in advances in
vaccinology).
Live attenuated vaccines against influenza
Cold adaptation was found to be a reliable and efficient procedure for the
derivation of live attenuated viral vaccine strains for humans. Cold-adapted
(ca) virus strains can grow in primary chick kidney cells or embryonated
eggs at 25–33°C, however, they exhibit a reduced replication at 37°C. The
process of genetic re-assortment with the transfer of the six internal genes
from a stable attenuated ca master donor strain of influenza A or B to the
new prevailing wild-type epidemic strain has been used to generate attenu-
ated cold-reassorted vaccines with the proper level of attenuation, genetic
stability and immunogenicity, which show low or absent transmissibility
[10]. MedImmune and Wyeth have developed along these lines a trivalent
live ca vaccine (Flumist) for intranasal spray delivery, which was licensed
in 2003. In contrast, to parenterally-administered vaccines, this formulation
triggers immune responses resembling those observed after natural infec-
tions [11]. Despite the moderate hemagglutination-inhibiting antibody titres
observed in vacinees, Flumist showed 92% efficacy over a 2-year period in
children, including protection against antigenic variants that circulated dur-
ing the second year [12–14]. This ca vaccine also stimulated the production
of nasal IgA, as well as T-cell and interferon responses [15]. The cell-medi-
ated immunity against virus matrix and nucleoprotein antigens may favour
viral clearance and early recovery from illness [3]. The Advisory committee
on Immunization Practices has recommended its use only in persons from
5 to 49 years of age, since side-effects were observed in young children
(wheezing, nasal congestion) and there are no data available for elderly
[16, 17]. Despite its remarkable genetic stability, this vaccine has to be kept
at – 18°C. Thus, a new heat stable derivative has recently been developed,
which showed good efficacy in clinical trials [1]. A live vaccine based on
a master virus strain developed at the Institute of Applied Microbiology
(Austria) by growing wild influenza virus in Vero cells at 25°C was also
demonstrated to be safe, well-tolerated and immunogenic after intranasal
immunization in young adults [18].
Subunit and DNA vaccines against influenza
A number of subunit- or DNA-based vaccines are also in various stages

of development. An influenza vaccine formulated in virosomes has been
commercialized by Berna Biotech (Inflexal® V); it contains the surface

spikes of the three currently circulating influenza virus strains inserted
in vesicle membranes of the three corresponding virus types (for more
details see section “Pseudoviruses as antigen delivery systems”) [19].
This company has also developed a virosome-based nasal formulation.
However, it was withdrawn from the market due to the presence of side-
effects (i.e., Bell’s palsy), which was assumed to be linked to the presence
of the Escherichia coli heat labile toxin (LT) as adjuvant. Two companies,
Yeda and BionVax, are also developing a peptide-based influenza vaccine
for nasal administration, which showed protective efficacy in humanized
mice [1]. A subunit vaccine containing recombinant HA protein produced
using a baculovirus system was successfully tested in a phase II trial in
64 to 89-year-old volunteers. An epidermal DNA-based influenza vac-
cine, which contained the HA gene from A/Panama/2007/99 delivered
by particle-mediated epidermal delivery was also tested in humans by
PowderJect [20]. Serum haemagglutination-inhibition antibody responses
were observed in volunteers receiving a single dose of 1, 2 or 4 +g of
DNA, with the strongest and most consistent responses in subjects vac-
cinated with the highest dosage.
Some immunization approaches aim at the development of a univer-
sal vaccine with a broad spectrum of protective activity against different
influenza strains [21]. Among them, the use of the highly conserved trans-
membrane M2 protein of the virion can be mentioned. A recombinant par-
ticulate vaccine has been engineered by genetically fusing copies of the M2
to the hepatitis B core antigen (HBc). The M2-HBc fusion protein sponta-
neously assembled into virus-like particles (VLP), which provided complete
protection against a lethal challenge with influenza virus A in mice [22, 23].
Promising results were also obtained after vaccination with a M2 peptide
conjugated with a Neisseria meningitidis outer membrane protein complex
(OMPC) in monkeys [24].
Vaccines against the parainfluenza virus
The human PIV (hPIV) consist of four serotypes, with hPIV-3 being the
second leading cause of bronchitis and pneumonia in infants. No vac-
cine has been licensed to date against PIV, however, several approaches
are currently under investigation. The initial attempts to provide pro-
tection by using vaccines based on formalin-inactivated viruses failed.
Subsequent work demonstrated that the glycoproteins haemagglutinin-
neuraminidase (HN) and F, which are responsible for virus attachment
and fusion, are able to stimulate the elicitation of neutralizing antibodies
in animals. However, their poor immunogenicity in naïve subjects led to
the currently favoured approach, which is based on the use of live attenu-
ated PIV.
Live attenuated PIV vaccines have been developed from both human
and bovine strains, which are amenable for delivery by the intranasal
route. Candidate vaccines should be able to replicate and induce a protec-
tive immune response in young infants, even in the presence of maternally
acquired antibodies. Two main attenuated strains have been studied in
detail. One is the hPIV-3 strain cp45, which was selected after 45 pas-
sages of the virus in African green monkey cells at low temperature. The
other is a bovine PIV (bPIV)-3 strain, which is antigenically related to the
hPIV-3, and replicates poorly in humans. Both cp45 and bPIV-3 have been
evaluated in phase I/II trials in sero-positive and sero-negative children
and in young infants. They were found to be over-attenuated in sero-posi-
tive children, but immunogenic in sero-negative children and infants [25].
However, the magnitude of the anti-HN response was lower in children
who received the bPIV-3 vaccine [25]. This prompted the engineering of
chimeric bovine/human PIV-3 candidates (e.g., hPIV-Nb strain in which
the human nucleocapsid is replaced by the bovine counterpart, or a bPIV-
3 strain that expresses the F and HN proteins of hPIV-3). Attenuated,
chimeric viruses that contain PIV-3 cp45 internal genes with the F and HN
genes from either PIV-1 or PIV-2 have also been tested in hamsters [26].
Berna Biotech is also developing a virosomal formulation of the PIV-3
[1].
Vaccines against the respiratory syncytial virus
Using the successful approach of the influenza vaccine, a formalin-inacti-

vated candidate against the respiratory syncytial virus (RSV) was tested in
children in the 1960s. The consequence was the hospitalization of 80% of
vaccinees and two deaths [1]. Moreover, vaccinated children also suffered
more severe disease on subsequent exposure to the virus, as compared
to unvaccinated controls [27]. This demonstrated that the elicitation of
a strong immune response is not sufficient to confer protection against
disease, and can even lead to immuno-pathological reactions. Thus, it is
essential to stimulate the “right” type of immune response. In the particu-
lar case of RSV, host responses play an important role in the pathogenesis
of the disease, thereby making the development of a preventive vaccine
extremely difficult. In addition, naturally acquired immunity to RSV is
neither complete nor long-lasting, and recurrent infections often occur
[28]. However, older children and adults are usually protected, suggesting
that protection against severe disease develops after several consecutive
infections. Passive immunization with RSV-neutralizing immune globulins
was also shown to prevent RSV infection in newborns with underlying car-
diopulmonary disease [29]. This demonstrates that antibodies play a major
role in protection against this disease, whereas T-cell immunity targeted to
internal viral proteins appears to contribute to clearance.
Subunit vaccines against RSV
Although live attenuated vaccines seem to be preferable for immunization

of naïve infants, subunit vaccines may be of choice for elderly, high-risk chil-
dren and pregnant women. Candidate subunit vaccines based on purified F
proteins (PFP-1, -2 and -3) were demonstrated to be safe and immunogenic,
even during pregnancy [30]. Maternal immunization using a PFP-based
vaccine could be an interesting strategy to protect infants younger than 6
months of age [25]. However, no significant protection was reported in a
phase III trial performed on children 1–12 years of age with cystic fibrosis
after vaccination with a subunit vaccine based on PFP-3 [31]. A formulation
based on surface glycoproteins F and G together with the virion matrix
protein M from RSV-A was tested in healthy adult volunteers in the pres-
ence of either alum or polyphosphazene as an adjuvant. Short-live neutral-
izing antibody responses to RSV-A and RSV-B were detected in 76–93% of
the vaccinees, suggesting that annual boosting will be needed [32–34]. The
central domain of the G protein of RSV-A is relatively conserved among
viruses from the groups A and B. Thus, a recombinant vaccine candidate,
BBG2Na, was developed by fusing the G2Na domain to the albumin bind-
ing region of streptococcal protein G. This candidate was shown to be mod-
erate immunogenic in adult human volunteers, but its clinical development
was interrupted due to the appearance of purpura in vaccinees [1].
Live attenuated vaccines against RSV
The main two difficulties associated with the generation of live attenuated
vaccines against RSV are over- or under-attenuation of the virus and lim-
ited genetic stability. Temperature-sensitive (ts), ca and cold-passaged (cp)
mutant viral strains have been generated. Despite the attenuation shown in
adults and sero-positive children, cpts mutants still caused moderate conges-
tion in the upper respiratory tract of sero-negative infants (1–2 months old)
[35]. Recombinant RSV vaccines with deletions in non essential genes (e.g.,
SH, NS1 or NS2), which also carry cp and ts mutations in essential genes are
currently being evaluated [1]. Through recombinant DNA technology chi-
meric viruses were engineered, which contain the genes of hPIV-3 surface
glycoproteins F and NH together with those of RSV glycoproteins F and G
in a bPIV-3 genetic background. One of these candidates was found to be
attenuated and able to induce the elicitation of immune responses against
both hPIV-3 and RSV in rhesus monkeys [36]. Similarly, a bPIV-3 genome
was engineered to express hPIV-3 F and HN proteins and either native
or soluble RSV F protein [37]. The resulting strain, which induced RSV
neutralizing antibodies and protective immunity against RSV challenge in
African Green monkeys, needs to be tested for safety and efficacy against
RSV and PIV-3 in infants.
Vaccines against the severe acute respiratory syndrome (SARS)-

associated coronavirus
This emerging disease was originally described in the Guangdong prov-

ince of China in 2002. Even when the global outbreak of SARS was under
control in 2003, new infections were reported in persons who had contacts
with animals in 2003 and 2004 [38]. The typical SARS-CoV-like virus is not
transmitted from animals to humans. However, under certain conditions
the virus can evolve into the early human SARS-CoV, which has the abil-
ity to be transmitted from animals to humans or even humans to humans,
thereby leading to localized outbreaks of mild disease. The early human
SARS-CoV, under selective pressure in humans, may further evolve into
the late human SARS-CoV, which can cause local or global outbreaks of
typical SARS [39].
SARS can be easily grown in cell cultures [38]. Thus, there is an urgent
need for vaccines, not only to prevent naturally occurring epidemic out-
breaks, but also as a tool against the threat of biological weapons. Several
structural proteins are expressed by SARS-CoV, including nucleocapsid,
envelope and spike (S) proteins [38]. The latter is a type I trans-membrane
glycoprotein, which is responsible for virus binding, fusion and entry, and
being the major target of neutralizing antibodies [38, 40]. The extracelullar
domain of the S protein consists of two subunits, S1 and S2 [40]. The S1
subunit possess a receptor-binding domain (RBD), which is responsible for
viral binding to one of its receptors [41, 42]. Vector-based vaccines express-
ing the S protein, as well as DNA vaccines encoding full-length S protein
have been assessed in preclinical studies [43, 44]. When modified vaccinia
virus Ankara (MVA) coding for full-length S protein was administered
by either intranasal or intramuscular route, neutralizing antibodies were
elicited [45]. However, vaccination of ferrets resulted in liver damage after
challenge, raising some concerns about the safety of this approach [46].
Vaccines formulated using different synthetic peptides encompassing lin-
ear B cell epitopes from the S protein, which were identified using sera from
convalescent patients, stimulated high antibody titres. Nevertheless, none
of them triggered the elicitation of neutralizing activity. On the other hand,
some studies demonstrated that although antibodies against S protein of the
late SARS-CoV (Urbani strain) exhibit neutralizing activity, they can also
enhance infection by an early human SARS-CoV isolate (GD03T0013) and
the civet SARS-CoV-like viruses. A derivative of the S protein with a trun-
cation at amino acid (aa) 1153 fails to cause antibody dependent enhance-
ment of infection, but retains the ability to induce neutralizing antibodies.
These findings suggest that the elimination of the putative heptad repeated
2 (HR2, aa 1153-1194), which is implicated in viral fusion, might abrogate
the stimulation of virus infection-enhancing antibodies [47, 48]. The use of
the nucleoprotein of the coronavirus in a DNA vaccination protocol also
led to the stimulation of a protective response [49]. In contrast, protection
was not achieved when a recombinant PIV-3 expressing the nucleoprotein

alone or together with the matrix protein was used [50]. This demonstrates
that the selection of the delivery system and immunization strategy play a
critical role in vaccine efficacy.
Vaccines against adenovirus
The human adenoviruses are divided into six subgroups (A–F). The adeno-
virus can cause large-scale epidemics of acute respiratory disease, and
dissemination is especially favoured under conditions in which persons
are housed communally. The subgroup A viruses, such as Ad31, have been
associated with pneumonia in immunocompromised patients. Neutralizing
antibodies directed against the capsid (hexon and fiber proteins) seems
to be the main effector mechanism to prevent re-infections by adenovirus
[3]. Until 1998, military recruits in USA were administered enteric-coated
capsules containing live viruses from the serotypes 4 and 7. The virus, which
was not attenuated if delivered by respiratory route, was able to replicate
in the gastrointestinal tract without causing disease, thereby stimulating
protective responses in the respiratory tract [51]. When the vaccine went out
of production, outbreaks of respiratory diseases caused by adenovirus re-
emerged among the military recruits [3]. Since serotypes 1, 2, 3 and 5 cause
the 80% of adenovirus associated respiratory disease in young children, the
development of a tetravalent vaccine similar to the above mentioned might
solve the problem in children [52]. However, the implementation of a vac-
cine (live or attenuated) against adenovirus should be carefully evaluated,
since recombinant adenoviruses are proposed both as vaccine vectors and
as tools for the transfer of foreign genes in gene therapy protocols.
Vaccines against Streptococcus pneumoniae
Polysaccharide-based vaccines against S. pneumoniae
In 1945, MacLeod et al. [53] reported the protective efficacy of a capsular

polysaccharide (PS) vaccine in military personnel during an outbreak of
pneumococcal pneumonia. The immunization with purified PS showed a
drastically reduced reactogenicity, in comparison with the previously used
inactivated whole cell vaccines. This was a major breakthrough, not only in
terms of safety, but also because it demonstrated that a specific virulence
factor can be purified and effectively implemented for the prevention of an
infectious disease, thereby paving the road for modern non toxoid-based
subunit vaccines.
Although the serological correlates of immunity are poorly defined,
type-specific anti-capsular antibodies are responsible for protective immu-
nity. However, immunity is serotype specific, rendering extremely difficult

the development of a universal vaccine. This is in part due to the elevate
number of serotypes, the regional variations in dominant serotypes and the
lack of updated sero-prevalence data for certain regions. These problems
have been partially solved by the use of PS-based polyvalent vaccines. The
currently licensed formulations contain 23 serotypes of S. pneumoniae,
which cover approximately 90% of serious pneumococcal disease, but only
in Western industrialized countries. Relatively good antibody responses
(60–70%) are elicited in healthy adults 2–3 weeks after a single intramus-
cular or subcutaneous immunization [54]. Unfortunately, they are poorly
immunogenic in children aged less than 2 years, in immune compromised
individuals (e.g., AIDS patients) and in elderly people with concomitant
disease, and they do not induce good immunological memory. Randomized
controlled trials in healthy elderly and young men also failed to show a ben-
eficial effect against pneumonia [55]. However, vaccination is recommended
for healthy people over 65 years of age to confer protection against inva-
sive disease [54]. PS-based vaccines can be also used in pregnant women to
stimulate the production of antibodies, which are transferred to the foetus
via the placenta or to the newborns by breast-feeding. However, it is still
a matter of controversy whether maternal vaccination can indeed protect
newborns against pneumococcal infections [56].
Conjugate vaccines against S. pneumoniae
The second generation of PS-based conjugate vaccines stimulates stronger

antibody responses, even in infants, young children and immune deficient
individuals, as well as immunological memory. These vaccines also suppress
nasopharyngeal carriage of the pathogen and reduce bacterial transmis-
sion in the community leading to herd immunity, which adds considerable
value to their implementation. The introduction of these vaccines in USA
in 2000 resulted in a dramatic decline in the rates of invasive pneumococcal
disease [1, 57, 58]. A significant reduction in the incidence rates among non
vaccinated individuals was also observed as a result of herd immunity [59,
60]. However, the licensed seven-valent vaccine does not contain some of
serotypes that cause severe disease in developing countries (i.e., serotypes 1
and 5). New conjugate vaccines including more serotypes, such as the nine-
valent vaccine (Wyeth) and two 11-valent vaccines (GlaxoSmithKline and
Sanofi-Pasteur), should provide better serotype coverage.
Protein-based subunit vaccines against S. pneumoniae
New approaches to develop protein-based subunit vaccines against S. pneu-

moniae are currently being pursued by different research groups. This is
expected to enable the generation of a universal vaccine conferring protec-

tive immunity against a large number of serotypes, as well as to avoid the
complexity of manufacturing a conjugate vaccine [61]. There are different
pneumococcal candidate antigens, such as the pneumolysin, neuraminidase,
autolysin, pneumococcal surface protein A (PspA) and adhesin A (PsaA),
which are in an early phase of clinical development [1]. In addition, several
promising candidates have been identified, which are currently being tested
in pre-clinical experimental models [1]. Among them, the two iron uptake
ABC transporters of S. pneumoniae (PiaA and PiuA), which trigger protec-
tive immunity against invasive pneumococcal disease in mice. Through the
screening of S. pneumoniae genomic expression libraries with sera from
convalescent patients, bacterial surface proteins were identified (e.g., BVH-
3 and BVH-11) that promote the elicitation of protective anti-pneumococ-
cal antibodies in mice [1]. A recombinant hybrid protein, BVH3/11V, has
successfully been tested in toddlers and elderly volunteers. This candidate
vaccine should be able to trigger serotype-independent responses, since
the BVH3 and BVH11 antigens are common to all serotypes of S. pneu-
moniae.
Vaccines against typeable and non typeable Haemophilus influenzae
Conjugated Haemophilus influenzae type b vaccines
The major obstacle for developing an effective vaccine against H. influen-

zae capsular PS was related to the inherently poor immunogenicity of this
T-cell-independent antigen. Antibody responses against PS are age-related,
with extremely poor immunogenicity in infants during the first 18 months of
life. Unfortunately, this age group exhibits the highest risk for invasive infec-
tions caused by H. influenzae. A PS-based vaccine against the H. influenzae
type b (Hib) was licensed in the United States in 1985, for children more
than 18 months old [62, 63]. The protective efficacy after licensure studies
showed the inefficacy of this vaccine not only in infants, but also in older
children [64]. This problem was solved by the generation of a conjugate Hib
vaccine. To this end, the Hib PS (i.e., polyribosylribitol phosphate; PRP)
was covalently linked to an immunogenic carrier protein, thereby lead-
ing to T-cell-dependent responses against the PS. Different conjugate Hib
vaccines currently exist. These vaccines are HbOC, PRP-T and PRP-OMP,
which make use of the mutant diphtheria toxin CRM197, the tetanus toxoid
and the outer membrane protein from group B N. meningitidis as carriers,
respectively. All of them trigger similar immune responses at the recom-
mended doses. However, the dynamic of the elicited response may vary for
each of them [65, 66].
Efficacy studies of these vaccines showed that they confer protection
not only against meningitis, but also against pneumonia [67–69]. Although
Hib vaccines are highly effective, their cost is still prohibitive for the world’s
poorest nations. However, with the establishment of the Global Alliance for
Vaccines and Immunization (GAVI), we have moved consistently ahead in
making them also available for developing countries. GAVI has approved
the establishment of a Hib initiative to support countries wishing to sustain
Hib vaccination, as well as those exploring whether their introduction could
be considered a priority in the near future.
H. influenzae typeable and non typeable: vaccination perspectives
Although the introduction of conjugated PS vaccines has significantly

decreased the prevalence of invasive Hib disease, paediatric infections due
to non typeable H. influenzae (NTHi) are still highly prevalent. NTHi is
most often associated with otitis media, sinusitis and bronchitis. In addition,
NTHi is an important cause of lower respiratory infection in adults with
chronic obstructive pulmonary disease (COPD). Thus, the development of
a vaccine against NTHi is considered an important goal in public health. In
contrast to Hib, vaccines against the non-encapsulated NTHi strains must
be directed against alternative virulence factors. The lipoproteins D and
P6 are widely distributed and antigenically conserved among H. influenzae
strains, and also trigger the elicitation of protective immunity in animals
vaccinated by mucosal route [70–73]. Thus, their incorporation in vaccine
candidates might facilitate the generation of a universal vaccine against all
typeable and non typeable H. influenzae.
Vaccines against Bordetella pertussis
Even in the age of vaccine availability, B. pertussis continues to be a major

cause of childhood morbidity and mortality (i.e., approximately 50 million
cases and 300,000 deaths occur annually worldwide). Since the late 1940s,
the incidence of whooping cough has dramatically decreased in most devel-
oped countries, as a result of widespread immunization. The first vaccine
formulations, which are still in use, consist of preparations based on killed
B. pertussis. The frequent incidence of minor adverse effects (e.g., fever,
protracted crying and local erythematous reactions), as well as concerns
raised by reports of serious neurological side-effects, resulted in a decline
in vaccine acceptance and use [74]. This in turn led to a re-emergence of
whooping cough and its complications. This serious problem prompted the
development of a new generation of acellular vaccines.
In 1981 Japan was the first country to successfully introduce acellular
vaccines against whooping cough in its immunisation programme [75], lead-
ing to a consistent reduction in the reported side-effects. In the mid 1980‘s
a major phase III trial of acellular vaccines was undertaken in Sweden, at a
time when the banning of the whole cell vaccine had resulted in a pertussis
epidemic in that country [76]. The first vaccine trials contained chemically
detoxified pertussis toxin (PT) and filamentous haemagglutinin (FHA), or
detoxified PT alone. The results of these trials showed that whilst producing
good antibody responses, the vaccines failed to give an adequate level of
protection in infants. The mono-component vaccine conferred no protec-
tion against infection, whereas the use of the two component candidate only
gave incomplete protection against infection [77]. The results obtained in
Japan and Sweden stimulated vaccine companies in the USA and Europe
to establish vigorous research programmes aimed at the development of a
new generation of acellular vaccines with higher efficacy. Currently avail-
able vaccines have incorporated chemically or genetically inactivated PT
and additional virulence factors, such as FHA, the outer membrane protein
pertactin (PRN) and fimbrial proteins (FIMs).
The efficacy studies of this second generation of acellular vaccines have
demonstrated that they confer levels of protection equivalent to the whole
cell vaccines. The advent of improved techniques for antigenic characterisa-
tion and the introduction of acellular vaccines containing genetically defined
components also resulted in a reduction of lot-to-lot variation in compari-
son with conventional whole cell vaccines and the acellular formulation
originally introduced in Japan. However, despite the wide implementation
of vaccination campaigns in infants and children, the disease continues to
be endemic. In addition, in countries with high vaccine coverage we are now
observing a consistent increment in the cases of pertussis in adolescents
and adults [78–80]. These patients can then transmit the disease to infants,
thereby now representing a primary reservoir for bacterial transmission and
cycling in the community.
The above-mentioned observations can be explained by one or more of
the following factors: (i) improved detection techniques, (ii) major aware-
ness on the possibility that bacteria may affect these age groups, (iii) vaccine-
driven antigenic changes in circulating isolates, and (iv) reduction in vaccine
efficacy over time. In this context, concerns have been raised about genetic
variation between the strains used for vaccine preparation and circulating
isolates. This seems to be true, since the currently used whole cell and acellu-
lar vaccines are prepared with strains that were isolated before mass vaccine
introduction and show clear mismatches with respect to circulating strains.
There is a steady tendency to decrease diversity in recent isolates, together
with clonal expansion during epidemic outbreaks [81, 82]. Over time, at
least two surface proteins (PT and PRN) may have changed sufficiently to
allow for an increase in the incidence of disease. Unfortunately, our global
information on antigenic variation and disease in adults and adolescent is
extremely limited. Thus, despite widespread introduction of pertussis vac-
cines, it is essential to continue surveillance studies and collection of circu-
lating strains. The present view is that successful control of pertussis in the
community may require routine immunization of adolescents and adults
with the new acellular vaccines, perhaps in combination with the diphtheria
and tetanus toxoids (DTaP). This intervention might help in turn to reduce
the burden of disease and transmission to infants.
Vaccines against Chlamydia pneumoniae
Chlamydia pneumonia is an intracellular bacterium transmitted person-to-

person via respiratory droplets. This pathogen is a common cause of pneu-
monia, with infections usually being oligosymptomatic or asymptomatic in
young age groups. However, the rate of asymptomatic carriage in the nor-
mal population is unknown. There is also a tremendous gap in our under-
standing of host response to infections caused by C. pneumoniae. Most of
the studies have been focused on the development of efficient diagnostic
methods. However, less work has been done on vaccine development, and
there is a paucity of knowledge on the microbial components which may
serve as target antigens. In fact, at present there are no licensed vaccines
against C. pneumoniae. However, the potential of different antigens, such as
the major OMP2 [83] have been assessed in experimental animal models.
Nevertheless, mice vaccinated with OMP2 using a protocol based on prim-
ing with DNA and boosting with recombinant VLP showed only partial
protection [84]. Recent studies also suggested that CTL responses play a
role in protection and clearance [85]. Animals immunized with a mini-gene
encoding seven H-2(b)-restricted CTL epitopes fused to a endothelial retic-
ulum-translocation signal showed protection following intranasal challenge
with a virulent C. pneumoniae [85].
The current view is that multi-component vaccine will be required in
order to induce a protective response [86]. Using the promising approach
of reverse vaccinology combined with proteomics (see section “Reverse
vaccinology”), the whole-genome of C. pneumoniae was screened search-
ing for vaccine candidate antigens among exposed and immune accessible
surface proteins [87]. The selected candidates were then expressed in a
heterologous system and used in immunization studies. Approximately 53
proteins were able to trigger the elicitation of C. pneumoniae-binding anti-
bodies. When tested in secondary screenings, six of them were also able to
neutralize bacteria in vitro, and four inhibited systemic dissemination of C.
pneumoniae in a hamster model [86].
Vaccines against Moraxella catarrhalis
Moraxella catarrhalis is the third most common bacterial etiologic agent

of otitis media in children. Furthermore, M. catarrhalis is an important
cause of respiratory infections in patients with COPD. Thus, different stud-
ies have been carried out to characterize potential protective antigens. In
this context, two major OMP (CD and E) have been identified, which are
considered prime candidate antigens for vaccine development. These pro-
teins are expressed on the surface and show a high degree of conservation
among circulating strains. Both OMP triggered the elicitation of bactericidal
antibodies and protective immunity in preclinical models [88]. Additional
candidates are the UspA1 protein [89], which seems to be required for bac-
terial colonization of the human upper respiratory tract, the iron-induced
OMP B1 and LBP, and the iron-repressed OMP B2 [90]. A conjugate vac-
cine based on detoxified lipo-oligosaccharide was also tested in mice by
intranasal route with encouraging results [91, 92]. Some of these candidates
are planned to be tested in clinical studies soon [90].
Vaccines against Mycoplasma pneumoniae
Mycoplasmas are commensal microorganisms, as well as opportunistic

pathogens. Mycoplasma pneumoniae is one of the causative agents of acute
and chronic human respiratory diseases and the main responsible for prima-
ry atypical pneumonia, accounting for approximately 20–30% of all commu-
nity-acquired pneumonia [93]. There is a considerable underreporting for
M. pneumoniae-associated diseases. This is in part due to the wide diversity
of clinical manifestations, the difficulties associated with its cultivation from
clinical specimens and the lack of adequate diagnostic tools. No vaccines
are currently available against this pathogen. However, studies conducted
in human volunteers in the late 1960s demonstrated that a formalin-inacti-
vated whole cell vaccine and an acellular extract were able to confer mod-
erately protective immunity against M. pneumoniae [94]. Unfortunately,
immune pathological reactions were observed following challenge with live
organisms. Therefore, studies are still needed to understand the underlying
mechanisms to the observed autoimmune responses [95]. More specifi-
cally, we need to elucidate the specific role played by humoral and cellular
response in protection against M. pneumoniae. M. pneumoniae is one of the
smallest self-replicating prokaryotic pathogens (approximately 800 kb). The
complete genome sequence is now available. This is expected to expand our
knowledge on the physiological and virulence properties of this agent, as
well as new hints for vaccine development.
Vaccines against Legionella pneumophila
A previously unrecognized bacterium was isolated after the outbreak of

Legionnaires disease in 1976, which was designated Legionella pneumophila
[96, 97]. The spreading of L. pneumophila is increasing due to the use of
air-conditioners and humidifiers, since infections can occur by inhala-
tion of aerosolized contaminated water sources. Several approaches have
been developed in the fight against this facultative intracellular pathogen.

Infection and immunization induce a rapid increase of antibody titres.
However, antibodies do not seem to play a significant role in host resistance,
particularly after aerosol challenge [98–100]. Some authors also suggested
that these antibodies can promote bacterial phagocytosis, thereby favour-
ing invasion and subsequent intracellular replication [101]. In contrast,
cellular responses appear to be important for protection. Different vaccine
candidates were tested in the past. Heat-, acetone- and formalin-killed
L. pneumophila vaccines were not able to confer protective immunity in
guinea pigs, whereas animals immunized with L. pneumophila membranes
survive an aerosol challenge with virulent bacteria [98, 99]. Additional work
demonstrated that also purified antigens, such as the major secretory pro-
tein [98], the major cytoplasmatic membrane protein [102], the peptidogly-
can-associated lipoprotein [103], OmpS [104] and flagella [100] can confer
protection against challenge with virulent L. pneumophila. Finally, different
live attenuated mutants of L. pneumophila were used in animal infection
models with promising results [105].
Vaccines against Pseudomonas aeruginosa
Cystic fibrosis (CF) patients are particularly susceptible to severe bacte-

rial infections of the lung, being Pseudomonas aeruginosa one of the most
prominent etiologic agents. Thus, significant efforts have been invested to
develop a vaccine against this pathogen. Surface PS are among the anti-
gens that were most intensively assessed. Berna Biotech have developed
an octavalent vaccine against the eight most prevalent serotypes based on
O-PS conjugated with the exotoxin A [106–113]. A consistent reduction in
the number of CF patients with chronic P. aeruginosa lung infection was
observed in a cohort receiving the basic immunization protocol, followed by
yearly boosters over a period of 10 years [112, 113]. The conjugate vaccine
induced the production of specific IgG antibodies and increased the number
of IgG memory B cells. It is still unclear if cellular responses might contrib-
ute to the overall protection conferred by this vaccine. However, strong
proliferative responses of lymphocytes with a Th1 phenotype were observed
in vaccinated individuals in response to the carrier exotoxin A protein [113].
Alternative vaccination strategies are currently being tested in clinical tri-
als. Among them, formulations based on a fusion protein between the outer
membrane proteins F and I, which have been administered by parenteral
and mucosal routes [114, 115]. These formulations were demonstrated to
be safe in volunteers and conferred increased protection against P. aerugi-
nosa in CF patients. Cell-surface alginate, flagella, components of the type
III secretion system, inactivated toxins and proteases are other proposed
target antigens [116]. Some of them are already in clinical trials alone or in
combination [116].
New advances in vaccinology
When Pasteur returned from his summer holidays in 1881 to continue with
his studies on chicken cholera, he inoculated chickens with an old culture
of Pasteurella multocida, which was left during the whole summer on his
bench. The animals that received the preparation were protected against
a challenge performed with a fresh isolate. Thus, Pasteur developed the
hypothesis that pathogens could be attenuated by exposure to environmen-
tal insults (e.g., high temperature, oxygen and chemicals) [117]. The strategy
was then successfully extrapolated for developing anthrax vaccines in live-
stock in the 1880s, with significant economic benefits. This was followed by
the generation of attenuated vaccines against rabies and other important
pathogens towards the end of the nineteenth century. Pasteur’s approach
for “attenuating” or “inactivating” a pathogenic organisms still constitutes a
cornerstone in vaccine technology [117]. This exemplifies that until recently
the major achievements in vaccinology have been facilitated by technologi-
cal (e.g., adjuvants, delivery systems, reverse vaccinology, genetic engineer-
ing) rather than immunological advances [117–119]. However, it is expected
that the impressive knowledge accumulated in recent years in the fields of
immunology, immune pathology and microbial pathogenesis will pave the
road to a new golden era in vaccinology, in which knowledge and technol-
ogy will enable rational vaccine design.
New technologies and approaches for vaccine development
Reverse vaccinology
In the 20th century, pertussis vaccines progressed from crude bacterial

preparations to the highly purified antigens used for acellular vaccines. A
similar quantum jump in technology allowed the development of subunit
vaccines against influenza, Hib and S. pneumoniae, as well as the production
of antigens by recombinant DNA techniques (e.g., genetically inactivated
PT). Despite the fact that these techniques enable the production of almost
any foreseeable antigen, the identification of suitable targets still remained
as a main bottleneck for vaccine development [120].
The advent of genomics and its exploitation in the vaccinology field have
rendered possible the implementation of a systematic and holistic approach
for the screening, identification and prioritisation of candidate antigens.
This new approach, called “reverse vaccinology” [121], does not require
cultivation of the original pathogen, thereby being amenable for highly-
pathogenic or non culturable micro-organisms. It is possible to predict and
select the most promising candidates by the analysis of genomic sequences
in silico, which will then be cloned and expressed in heterologous systems.
The resulting proteins are then used to perform immunological and/or
functional studies to select the most promising candidates (e.g., able to

induce the production of microbicidal or neutralizing antibodies, capacity to
confer protective immunity). Flanking studies are usually carried out, such
as molecular epidemiological analysis to assess their degree of conserva-
tion among circulating strains, or transcriptional profiling to evaluate their
expression during natural infections [122].
The time-consuming process in which highly expressed components of
an in vitro cultivable organism are identified (one at a time) and separated
(different components between them) is one of the disadvantages that
reverse vaccinology has solved. The conventional method usually requires
15–20 years to arrive to a clinical trial, whereas reverse vaccinology reduces
the process to approximately 5 years. Reverse vaccinology also allows the
identification of hundreds of potential candidates in a few days, in compari-
son with the small number of antigens that conventional approaches have
provided after decades of research. Moreover, reverse vaccinology offers
the possibility to select potential candidates independent of their expression
levels or purification easiness.
The reverse vaccinology approach has proved its usefulness in the field
for both viral and bacterial pathogens (e.g. hepatitis C virus, Group B
meningococci, group B streptococci) [123, 124]. Reverse vaccinology has
also become an essential tool for several vaccine development projects
against agents causing community-acquired pneumonia (e.g., C. pneumoni-
ae, streptococci). The potential and speed of genomic-based approaches
was also shown when the nucleotide sequence of the coronavirus causing
SARS was made available in less than one month. In addition, the increas-
ing number of available genomes from bacteria and viruses would allow
comparative genomic studies, thereby providing hints on conserved protein
families and/or functional domains. This would facilitate the generation
of vaccines using immunogens covering multiple micro-organisms [125].
Despite the incredible potential of reverse vaccinology, this approach also
has some important limitations (Tab. 1). Among them is the fact that it is
not be possible to identify non-protein antigens (e.g., PS, glycolipids), which
are the cornerstone for many successful vaccines (e.g., pneumococcal and
Hib vaccines).
Reverse genetics
Currently available influenza vaccines (see above) are based on inacti-

vated viruses, and, more recently, attenuated ca viruses and virosomes. All
these vaccines exploit the same starting material (wild-type virus), which
is inactivated or attenuated. The last approach consists in the co-infection
of chicken eggs with the new isolate and a master attenuated strain, and
subsequent selection for re-assorted viruses with the desired genotype/phe-
notype. However, the virulence of certain virus strains, such as the H5N1,
Table 1. Classical vaccine development versus reverse vaccinology
Reverse vaccinology Classical approach

Time required to reach the clinical ~ 5 years ~ 20 years
phase
Type of organisms Culturable and non- Only culturable
culturable
Antigens Only proteins Proteins, lipoproteins,
polysaccharides and gly-
colipids
Genome Necessary Unnecessary
Target genes All Mainly in vitro expressed
Exclusion of known antigens Yes No
Need to handle microorganisms No Yes
(e.g., highly pathogenic)
Surface and structural antigens Yes (only proteins) Yes
Internal antigens Yes (only proteins) Rarely
Antigens with low expression Yes Rarely selected
levels
Number of candidate antigens Many (more than Few
hundred)
Selection of antigens Poorly or highly Mainly highly immunogenic
immunogenic
Antigens identification and Easy Could be difficult
separation
Need for genetic tools during the Not necessary Usually essential (e.g., to
initial discovery process create and complement
mutants)
Need for genetic tools for antigen Necessary for the initial Only in a late phase of
expression phase of development development
renders difficult the implementation of this traditional strategy. The use of

reverse genetics represents a valid alternative for the generation of vaccines
against RNA respiratory viruses, such as the influenza virus, PIV and RSV.
It consists in the production of the virus from cloned DNA [126], thereby
allowing the development of vaccines against any pandemic viral strain.
In some cases (e.g., avian H5N1) an additional mutagenesis step would be
required to attenuate its virulence [127]. Then, the new HA and NA seg-
ments would be transferred into an appropriate influenza A virus master
strain adapted to grow in a cell line. The final re-assorted virus will have the
antigenic specificity of the pandemic strain and the growth characteristics
of the master strain [128, 129].
This technology would also allow production of the influenza vaccine
in cells that are co-transfected with plasmids encoding for different frag-
ments of the virus [130]. Therefore, the complete genome is inside the cell
and virus can be produced and assembled. One of the main advantages is
that a plasmid encoding for HA and NA can be easily replaced. Therefore,
re-assortment and selection become unnecessary. This method would con-
siderably reduce the time for vaccine production, from many months to
only a few weeks. Another advantage would be the simple manipulation of
the genome (contained in plasmids), which would enable detoxification of
specific virulence factors. Similar approaches can be implemented for other
viruses, such as RSV, PIV and SARS-CoV. However, intellectual prop-
erty and liability issues are still obstacles for the industrial development
of reverse-genetics-based vaccines [131]. Furthermore, since the resulting
viruses are considered genetically modified organisms, additional problems
may arise from the regulatory stand point [131].
Mucosal delivery systems
Most of the infective agents are either limited to the mucosal membranes, or
need to transit across them in order to cause disease. Therefore, it is highly
desirable to elicit an efficient immune response at the local site in which the
first line of defence is laid. The stimulation of a pathogen-specific response
at the portal of entry is expected to impair infection (i.e. colonization),
thereby reducing the risk of transmission to susceptible hosts. Parenterally
administered vaccines mainly stimulate systemic responses, whereas vac-
cines given by the mucosal route mimic natural infections, thereby leading
to efficient mucosal and systemic responses. Thus, there is a considerable
interest in the development of mucosal vaccines. However, antigens admin-
istered by this route are usually poorly immunogenic. Different strategies
are being pursued to overcome this bottleneck, among them can be cited the
use of (i) advanced synthetic delivery systems, (ii) live attenuated bacterial
or viral vectors, (iii) bacterial ghosts, (iv) pseudoviruses and (v) mucosal
adjuvants [132–135].
Advanced synthetic mucosal delivery systems

Particulate antigens are more immunogenic than those in solution, due
to their vulnerability to degradation by enzymes and extreme pH. Thus,
it would be helpful to incorporate them into a protective vehicle. Often,
these vehicles do not serve only to protect them, but can also enhance
their uptake, promote targeting to antigen presenting cells and serve as
adjuvants [136]. The most commonly exploited delivery systems are: (i)
gelatine capsules, which are dissolved at alkaline pH in the intestine but
not in the stomach, (ii) muco-adhesive polymers that are highly viscous
inert PS, (iii) eldexomer and carboxymethyl cellulose, which have been
used for oral, nasal and vaginal delivery, (iv) lipid-based structures with
entrapped antigens, such as immune stimulating complexes (ISCOMs)

and liposomes, and (v) biodegradable micro/nano-spheres based on bio-
compatible materials such as starch, copolymers of lactic or glycolic acid
[137, 138]. Some of these approaches are currently being explored to
develop vaccines against agents causing community-acquired pneumonia.
Encouraging results have been obtained, among others, using surface
antigens from S. pneumoniae encapsulated in micro-spheres [139] and a
ISCOM-adjuvanted vaccine obtained by reverse genetics against the influ-
enza virus, in preclinical models [140].
Live attenuated bacterial or viral vectors

Attenuated viruses and bacteria can be used not only as vaccine candidates
per se, but also as delivery systems for heterologous antigens. Thus, many
attenuated microorganisms have been exploited as a scaffold for the devel-
opment of subunit vaccines against other agents, under the premise that the
expression of the recombinant antigen(s) does not increase their pathogenic
potential for humans or animals. The most frequently exploited bacterial
vectors are attenuated derivatives of Salmonella enterica and Shigella spp.,
and the Bacille Calmette-Guérin (BCG). For example, vaccination with an
attenuated Salmonella expressing the OprF-OprI was also shown to be able
to confer protection against P. aeruginosa in a murine experimental infection
model [141]. In addition, it was also demonstrated that a recombinant BCG-
based vaccine expressing the PspA confers protection against S. pneumoni-
ae in an infection animal model [142]. The use of commensals represents an
alternative to attenuated organisms (e.g., lactobacilli). In this context it was
demonstrated that oral administration of Lactobacillus expressing proteins
from coronavirus can protect against a gastric infection [143]. Thus, this
approach has been also proposed to combat SARS. Promising results were
also obtained using x Chlamydia psittaci [144]. On the other hand, different
attenuated viruses, such as MVA, bovine or attenuated hPIV-3 and adeno-
virus can be used as delivery systems for heterologous antigens [25, 145]. In
fact, MVA has recently been exploited for antigens of the SARS associated
coronavirus [146].
Bacterial ghosts
An alternative approach to the use of live attenuated carriers is given by the
use of bacterial ghosts. Ghosts are generated by the conditional expression
of the lethal lysis gene E from bacteriophage PhiX174 in Gram-negative
bacteria [147–151]. This leads to the formation of a trans-membrane tun-
nel through the bacterial cellular envelope [147]. Due to the high internal
osmotic pressure, the cytoplasm content is expelled through the tunnel,
thereby leading to an empty bacterial cell envelope [152]. The presence of
envelope components in the ghosts provides a strong danger signal through
the activation of pattern recognition receptors [153]. In addition, bacterial
ghosts are efficiently taken up by antigen-presenting cells, stimulating their

maturation and activation [154].
Bacterial ghosts retain all morphological, structural, and antigenic fea-
tures of the cell wall and can be used as vaccine candidates per se. Ghosts
can also be externally loaded with purified antigens. Alternatively, ghosts
can be generated from recombinant bacteria expressing heterologous anti-
gens, hence avoiding the difficulties associated with the purification steps.
This technology also offers the possibility to manipulate the topology of
the recombinant antigen (e.g., the antigen can be bound to the inner mem-
brane, secreted into the periplasmic space or associated to the surface).
Encouraging results has been obtained in preclinical models using ghosts
expressing chlamydial antigens [135, 155].
Pseudoviruses as antigen delivery systems

Promising results have been reported using different types of pseudoviruses,
such as virosomes and virus-like particles (VLP), which are non-replicating
viral-like structures. Virosomes are based on the principle of reconstitut-
ing empty viral envelopes through integration of viral envelope proteins in
liposomes. They offer the versatility of liposomes in terms of lipid composi-
tion, with the advantage of including viral membrane proteins. Virosomes
are produced by disassembling the viral membrane envelope with deter-
gents. Then, the viral nucleocapsid is removed by ultracentrifugation before
reconstitution (Fig. 1). In contrast, VLP exploit the capacity of recombinant
viral coat proteins to spontaneously self-assemble, thereby mimicking at
structural level the viral capsid. VLP can be isolated after protein expres-
sion in eukaryotic cells or by in vitro assemblage from subunits produced
in an heterologous system [156]. Their main advantages are the lack the
viral genetic material with an “intact” envelope, and the fact that they are
significantly more immunogenic than soluble proteins. They can be used
as vaccines per se, as well as a delivery system for protein- or nucleic acid
based vaccines, or as carriers for small molecules. Foreign antigens can
be expressed on their surface, or can be simply encapsulated. In addition,
amphiphilic adjuvants can be incorporated into their membranes, thereby
offering the advantage of combining an adjuvant and the antigen in one
entity without a covalent attachment.
Pseudoviruses are especially attractive for mucosal vaccination proto-
cols, since they offer the opportunity to use the natural route of transmis-
sion of the agents. Induction of serum antibodies, secretory IgA, T helper
and CTL responses, and protection against mucosal pathogen challenge
has been reported from studies in animals and humans [157–159]. The
virosomes generated using the influenza virus retain membrane fusion
properties very similar to the naïve virus. Therefore, they are able to deliver
material to the cytosol of target cells, offering the possibility to access the
MHC class I-restricted pathway of antigen presentation to prime CTL activ-
ity [160–162].
Figure 1. Virosomes are reconstituted viral envelopes, which incorporate the cell binding and
fusion proteins of native virus without its viral genetic material. (a) Virosomes are produced
by disassembling the viral membrane envelope with detergents. (b) The viral nucleocapsid is
then removed by ultracentrifugation, and (c) they are reconstituted by removing the detergent
with or without addition of lipids. (d) Electron-microscopy of an influenza virosome kindly
provided by Etna Biotech.
Mucosal adjuvants
Bacterial toxins and their derivatives are among the first molecules that
have been used as mucosal adjuvants. They are characterized by the pres-
ence of an A moiety with enzymatic activity, and a B moiety that medi-
ates toxin binding to the target cells. Cholera toxin and the closely related
Escherichia coli heat-labile toxin showed potent adjuvant activity when
co-administrated with different antigens by the mucosal route [163–165].
However, their use in humans is hampered by their intrinsic toxicity. Thus,
mutated derivatives were developed, in which the A subunit was modi-
fied to remove the ADP-ribosylating activity. The resulting polypeptides
retain their adjuvanticity, in the absence of detectable toxicity [166–168].
However, additional studies have demonstrated that even these deriva-
tives can lead to potential severe side-effects, such as retrograde homing
of adjuvant and antigen to neural tissues [169]. This might explain, at least
in part, the side-effects observed after intranasal vaccination against influ-
enza with a virosomes-based formulation containing heat-labile toxin (i.e.,
Bell’s palsy), which in turn led to its retraction from the market. However,
chimeric derivatives lacking the targeting moiety for neural tissues (i.e., B
subunit) are now available [170]. They might allow the exploitation of the
high potential of these molecules for the development of vaccines against
respiratory pathogens. In fact, preclinical studies provided the proof-of-con-
cept for the usefulness of derivatives of bacterial toxins in the generation
of acellular vaccines against microorganisms, such as S. pneumonia and H.
influenzae [171, 172].
Other bacterial components were also explored for their activity as adju-
vants. The monophosphoryl lipid A retains much of the immune stimulatory
properties of LPS, without the inherent toxicity [165]. On the other hand,
extracellular matrix binding proteins, such as the fibronectin binding protein
I of Streptococcus pyogenes, also exhibit adjuvant activity [173]. This offers
the possibility of using them as dual antigen/adjuvant moieties in the same
formulation. Recent reports also demonstrate that vaccine formulations
containing adamantylamide dipeptide, a non-toxic compound obtained by
linking the L-alanine-D-isoglutamine residue of the muramyl dipeptide to
the antiviral drug amantadine, confer protection against non typeable H.
influenzae in preclinical models [73].
The innate immune system plays a critical early role in host defence
against pathogenic microorganisms through the recognition of pathogen-
associated molecular patterns [174]. This is achieved through the stimula-
tion of pattern-recognition receptors (PRR) that sense a broad range of
exogenous and endogenous danger signals [153, 174]. Toll-like receptors
(TLR) represent the best-characterized family of PRR. Natural and syn-
thetic TLR agonists are being used as immune modulators to optimize
responses after vaccination. Since the identification of the TLR4, many
mammalian TLR homologues have been identified (i.e., 10 in humans
Table 2. Toll-like receptors (TLR) and their ligands
TLR Ligands
TLR1 (with TLR2) Mycobacterial lipoprotein (LP), triacylated lipopeptides, lipotei-
choic acid (LTA)
TLR2 LPS (P. gingivalis), fungal products (zymosan), peptidogy-
can (PGN), LP, GPI anchors (T. cruzi) , lipoarabinomannan,
muramyl dipeptide
TLR3 Viral dsRNA, synthetic Poly (I:C)
TLR4 Gram-negative bacterial products, LPS, respiratory syncytial
virus, synthetic lipid A, E5564, plant products, saturated and
unsaturated fatty acids, murine ß-defensin 2, BCG
TLR5 Flagellin
TLR6 (with TLR2) Mycoplasma LP, LTA, PGN, diacylated LP
TLR7 GU-rich ssRNA, resiquimod, imiquimod
TLR8 GU-rich ssRNA, resiquimod, imiquimod
TLR9 Bacterial and viral DNA, unmethylated CpG-ODN
TLR10 Unknown
TLR11 (in mice) Components from uropathogenic E. coli, and profiling-like from
Toxoplasma gondii
and 13 in mice) [175]. Each TLR member binds specifically to different

ligands (Tab. 2), alone or in combinations (e.g., heterodimers formed by
TLR2 with either TLR1 or TLR6). An example of TLR agonist is bacte-
rial DNA, but not vertebrate DNA, and synthetic oligodeoxynucleotides
containing unmethylated CpG motifs. They act on TLR9, thereby induc-
ing a strong Th1 responses by activation of dendritic cells [176, 177]. CpG
motifs have been successfully used as adjuvants in preclinical studies of
different candidate vaccines against agents causing community-acquired
pneumonia [178-180].
Another important adjuvant with TLR-binding capacities is the
Mycoplasma-derived macrophage-activating lipopeptide MALP-2, which
act a the level of the TLR heterodimer 2/6 [181, 182]. MALP-2 promotes a
global activation of cells from the innate and adaptive immune system [183,
184], such as macrophages, DC, T- and B-lymphocytes [183, 185]. When co-
administered with an antigen by either the parenteral or the mucosal route,
MALP-2 promotes the elicitation of humoral and cellular responses at
systemic and mucosal level [186]. Preclinical studies suggested that MALP-
2 could be exploited in vaccine formulations against the SARS-associated
coronavirus, M. catarrhalis and influenza virus, among others (unpublished
data).
DNA vaccines
DNA vaccination offers some advantage over the normal antigen vacci-
nation, such as the fact that it is not necessary to express any antigen. In
contrast, it is the biosynthetic machinery present in the cells of the vac-
cinees that takes care of this work. Furthermore, since eukaryotic cells are
in charge of protein synthesis, their glycosylation and folding are optimal.
However, the large-scale purification of DNA might be associated with high
costs. This can be solved by the use of attenuated or inactivated bacteria or
viruses as delivery systems [187]. This approach can also lead to an enhanced
induction of antibodies, which is otherwise poor using conventional naked
DNA vaccines. We have recently demonstrated that bacterial ghosts can be
also exploited as a delivery system for DNA vaccines for both in vivo and
ex vivo applications [188].
The potential of this approach is demonstrated by the fact that it is pos-
sible to optimize performance by a broad range of manipulations, such as (i)
choice of optimal promoters, (ii) use of codon optimized genes for expres-
sion in mammalian cells, (iii) addition of nuclear localization signals or ubiq-
uitination signals to improve expression and processing, and (iv) co-delivery
of DNA constructs coding for immune modulatory molecules [189]. In addi-
tion, by the presence of immune stimulatory CpG motifs, the DNA vaccine
constructs has built-in adjuvant properties. This vaccination approach is
particularly suited for the stimulation of cellular immune responses [190].
Interestingly, several reports suggest that DNA vaccines may represent a
valid alternative to prime the neonatal immune system, even in the pres-
ence of passive transferred maternal antibodies [191, 192]. In fact, promis-
ing results were also obtained in preclinical models of community-acquired
pneumonia, such as influenza [193] and S. pneumoniae [194]. Furthermore,
DNA coding for vaccine antigens appears to induce excellent immunologi-
cal memory, which can be reawakened by later immunization or exposure
to the pathogen.
New immunological concepts that need to be addressed to optimise

vaccine design
The knowledge generated in several basic disciplines, such as immunology

and microbial pathogenesis, has allowed the identification of critical bottle-
necks for establishing a successful vaccination strategy. It is expected that
in the coming years we will develop customized approaches to address each
of them, in order to stimulate efficient protection against infective agents
under specific clinical settings (i.e., newborns, aging individuals, immuno-
compromised patients).
The importance of immunological memory
B lymphocytes that have differentiated into plasma cells are the producers
of antigen-specific IgG antibodies. Bone-marrow (BM) plasma cells have a
short life, therefore, the BM reservoir needs to be replenished by the stimu-
lation of memory B cells [195, 196]. The maximal life span of BM plasma
cells is still debated. Only few factors have been identified that control the
differentiation of antigen-specific B cells toward short- or long-life plasma
cells or to memory B cells [119]. Beside the requirement of CD4 + T cells, the
nature of the antigen [197] and the dose are also important. Higher antigen
doses, as well as rapid vaccination schedules (closely spaced vaccine doses)
tend to favour the rapid induction of short-term effectors, whereas lower
doses of antigens preferentially support the induction of immune memory
[198-201].
It was demonstrated that neonatal vaccination (priming) and infant
boosting might be effective even when pathogen exposure occurs very early
in life. In children in whom vaccine-induced Hib antibody titres have fallen
to undetectable levels, memory is readily demonstrated [202]. However,
immune memory per se is not enough to protect against pathogens that
required high levels of neutralizing antibodies. The delay between memory
B-cell reactivation and differentiation may limit the ability to interrupt
pathogen invasion. Therefore, it is important to establish vaccination proto-
cols in which the population is boosted at different ages in order to maintain
the required levels of antibodies. This is particularly important in diseases in
which antibodies play a central role in microbial clearance or toxin neutral-
ization. In the particular case of community-acquired pneumonia, we should
consider that aging individuals are neglected in many vaccination programs.
However, the strategies proposed for elderly would be different from those
used for small children, since the main factors affecting vaccine efficacy are
immune senescence and immaturity, respectively. The attempts to give a
rational solution to this issue are discussed in the next sections.
The immune system in children and elderly
The immune system in children

Immune responses to bacterial and viral antigens usually increase with age
in a stepwise manner [203]. Prompt immunization after birth is required
to induce active immunity against diseases that may occur early in life.
Unfortunately, this strategy is limited by the relative immaturity of the
neonatal and infant immune system. Some factors implicated in this poor
response are the limited switch from IgM to IgG2 antibodies, impaired
complement-mediated reactions and deficient organization of the splenic
marginal zone. Vaccination studies performed in newborn mice suggested
that limited germinal centre reactions may results from the delayed devel-
opment of follicular DC and limit plasma cell differentiation [204]. It was

also showed that the neonatal BM has a limited capacity to support the
establishment of long-life antibody-secreting plasma cells [205].
Thus, the responses to glyco-conjugates and to most T-cell-dependent
antigens are usually affected [119]. Therefore, only few and highly immu-
nogenic vaccines show significant protective efficacy after a single dose in
infants. The limited IgG responses are extended all over the first year of
life. In addition, the immune responses, particularly antibodies, elicited in
the first year of life after vaccination rapidly decline [203]. However, the
problem observed in infants in terms of magnitude and duration of immune
response does not seem to affect efficient priming. In fact, the immune mem-
ory generated in neonates may be recalled later in life [119]. Nevertheless,
strategies to generate strong and long-lasting protective responses in infants
are still needed. This is in part due to the presence of maternal antibodies,
which inactivate and clear the vaccine antigens, thereby rendering difficult
the stimulation of an immature immune system [203]. In addition, the effects
of adjuvants reported in adults cannot be extrapolated to neonates [206]. A
potential strategy to overcome these problems would be to implement vac-
cination during pregnancy, to provide the required antibodies by placenta
and later by maternal feeding [30, 207–209]. This could be complemented
with an early priming of the “immature” immune system of the newborn
by DNA vaccination, followed by a boost during the second half of the first
year or later in life [203].
The immune system in the elderly

Poly-pathology and multiple organ failure is the rule rather than the excep-
tion in aging individuals. Thus, many systems are affected (e.g., endocrine,
cardiovascular), and the immune system is not an exception. The mecha-
nisms involved in the immune senescence process, which in turn may lead to
poor response to vaccination, are not fully understood. However, it is clear
that responses against certain vaccines are more affected by immune senes-
cence than others (e.g., PS-based vaccines against S. pneumoniae) [210]. In
contrast, the responses to a boost dose of the anti-tetanus vaccine are hardly
affected by age [211].
A rapid decline of antibody responses, together with a relative restriction
of the T-cell repertoire is characteristic of the immune senescence process.
This restriction and the reduction in the pool of naïve cells can explain the
poor CD4 + T cell responses against antigens that are cross-reacting with pro-
teins which were seen earlier in life. In contrast, T-cells responses of healthy
elderly individuals to new antigens are often unaffected. Nevertheless, the
overall response to vaccination in the elderly is less efficient than in young
adults, making more vigorous approaches necessary (Fig. 2).
In the case of influenza, the actual strategy is annual re-vaccination.
However, there are concerns regarding the capacity to increase antibod-
ies with proper specificity against re-assorted viruses in aging adults
Figure 2. Factors affecting the responses in young adults and aging individuals after vaccina-
tion. The process of immune senescence impairs host response to both infection and vac-
cination. This critical issue needs to be considered during vaccine design and will require the
development of special approaches.
who have been repeatedly infected or immunized. After exposure to a

new, but cross-reacting antigenic variant, such individuals may respond
by producing antibodies. However, these antibodies could be primarily
directed against influenza strains, which were encountered earlier in life.
For example, individuals previously exposed to the “old” H1N1 influenza

strain (i.e., 50 years ago), may respond differently from naïve adults who
are vaccinated with a “new” H1N1 strain which have accumulated differ-
ent mutations. The former might produce antibodies against the HA of the
“old” H1N1 strain rather than to the cross-reacting epitopes of the new
strain [212]. This is phenomenon is the so-called “original antigenic sin”
[119]. On the basis of this observations, it was proposed that variations
in vaccine efficacy might be due to differences in the antigenic distance
between the vaccine strains and the epidemic strains responsible for
influenza outbreaks [213]. However, this hypothesis was not confirmed by
epidemiologic studies [214]. Even more, individuals aged 65 years or older
who were annually vaccinated showed a significantly reduced mortality
risk. Therefore, until now, it seems that the antigenic sin does not represent
a major practical obstacle in influenza vaccination and additional strate-
gies may not be required.
Concluding remarks
Despite the broad availability of vaccines against agents causing com-

munity-acquired pneumonia, they still represent an important cause of
death, human suffering and economic losses. However, we have dra-
matically expanded our knowledge on the pathophysiology of diseases
caused by respiratory pathogens, their virulence factors and the effector
mechanisms responsible for their clearance. It is becoming clearer which
microbial components are attractive as vaccine targets, as well as the type
of immune response needed to confer protection against disease. Thus,
it is now possible to address vaccine development using rational rather
than empiric approaches. This is facilitated by powerful bioinformatics
tools for the accurate prediction of epitopes and proteasome trimming
[215–217], as well as by the availability of a broad palette of immune
modulators and delivery systems. Therefore, we can predict that new and
improved vaccines against the etiologic agents of community-acquired
pneumonia will considerably reduce the global impact of this disease in
the coming years.
Acknowledgments
This work was supported in part by grants from the DFG (GU482/2-3) and
the BMBF (“PathoGenoMik” – Competence Center for Genome Research
of Pathogenic Bacteria “Pathogenomik”, 031U213B) to CAG. We are par-
ticularly grateful to D. Felnerova from Etna Biotech, who provided us with
a micrograph from a transmission electron microscopy of a virosome, and
to M. Höfle for critical reading of the manuscript.
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Index
adaptive immunity 183, 188, 190 reinfection 84

adenovirus, vaccines 209 seroprevalence 84
alternative activation pathway 155 virulence factors 95
alveolar macrophage 150 vaccines 212
amantadine 82 Chlamydia, nomenclature 84
amantadine-resistant virus strain 82 Chlamydophila, nomenclature 84
antibiotics 7 ff choline-binding proteins (CBP) 144
penicillins 8 chronic obstructive pulmonary disease
macrolides 8 (COPD) 80, 193
fluorquinolones 9 classical pathway of complement
resistance 5 activation 154
antigen, lipid and protein 194 clathrin-coated vesicle 150
antigenic drift 76 clone, international (pneumococcal) 61
antigenic shift 76 clone, pneumococcal 68
antiviral compounds 82 co-infection with microorganisms
antiviral therapy 31, 33, 36, 45 (influenzavirus) 78
asthma, and M. pneumoniae 192 combined bacterial and viral pneumonia
autoimmune reaction 191 80
complement factor H 155
bacterial ghosts 221 complement fixation (CF) 194
biological characteristics 184 CRB-65 score 5
bird flu virus 77 C-reactive protein 154
blood cultures 6 cytokine production 189
blood-brain barrier (BBB) 149
Bordetella pertussis, vaccines 212 differential fluorescence induction
bronchiectasis 191 (DIF) 151
bronchitis 79, 80 DNA vaccines 226
Dot/Icm type IV secretion system 119
C. pneumoniae see Chlamydia pneumoniae
C3 degrading activity 154 efflux, PmrA 65
CAPNETZ 16 elderly, immune system 228
capsular polysaccharide 142 encephalitis 81
capsule regulation 153 encephalopathy 80
CD4+ 79 enolase 148
CD8+ T-cell responses to influenza 79 enzyme immunoassay (EIA) 194
cell wall hydrolase 144 extrapulmonary complication 190
cellular activation 188 extrapulmonary syndrome 191
chest x-ray 6
Chlamydia pneumoniae 15, 84, 86, 89–95 factor H (complement) 155
detection by PCR 86 failure, treatment 67
differential diagnosis 84 fluoroquinolone 196
immunity 89–93 fowl plague 77
248 Index
GAPDH 148 local (mucosal) immune response 189

gene target 195 LPxTG sequence 144
gene, mosaic 60 lung abscesses 191
ghosts (bacterial) 221 lymphocyte activation 189
G-protein pathway 150 lymphoid infiltration 189
Guillain-Barré syndrome 81, 191
macrolide 196
Haemophilus influenzae 3, 15, 78, 80 macrolide resistance 196
non typeable, vaccines 212 macrolide treatment 193
type b (Hib), conjugated vaccines 212 macrophage receptor, MARCO 161
vaccines 211 manipulation of vesicle traffic 118
healthcare associated pneumonia 2 mast cell degranulation 193
hemagglutinin (HA) 74–76 microimmuno-fluorescence (MIF) 22
hemolytic anemia 191 Moraxella catarrhalis, vaccines 212
hypogammaglobulinemia 189 mouse infection model 146
hypotension 80 mucosal adjuvants 224
mucosal delivery systems 220
IFN-alpha, in influenza 79 multi-locus sequence typing (MLST) 61
Immunoglobulin subclasses, and MurM protein 63
diagnosis 194 Mycoplasma pneumoniae 3, 15, 183, 184
immunologic hypersensitivity 189 adaptive immune response 190
immunological memory 227 adherence to epithelium 186
influenza infection of neonates 80 biological characteristics 184
influenza vaccines 202–204 cell biology 183
influenza virus 73 epidemiology 185
adaptive immune response to 79 genome 184
co-infection with microorganisms 78 pathogenesis 183
influenza, laboratory diagnosis 204 vaccines 215
innate immunity 79, 183, 188 myocarditis 80
interferon antagonism 78 myositis 80
interleukin (IL)-6 79
interleukin (IL)-8 79 naip5 117
natural reservoir of influenza-A-virus 76
ketolide, resistance 65 neuraminidase (NA) 74, 76
neuraminidase (NanA, NanB, NanC) 145
Legionella 15 neuraminidase inhibitors 82
dot/icm genes 112, 120 neuraminidase subtype 76
dot/icm mutants 117 neutralizing antibody directed against the
egress 123 HA 79
entry in host cells 117 nucleic acid amplification assay 15, 20, 195
growth phase 122
immunity 125 opsonophagocytosis 153
in free-living amoebae 113 orthomyxoviridae 74
Naip proteins 117 oseltamivir 82
persistence 114 otitis media 80
Toll-like receptors 125
transient association with amoebae 112 P1 adhesin 185
vesicles 118 pandemic (influenza) 76
Legionella pneumophila, vaccines 215 parainfluenza virus (PIV), vaccines 205
pathogen-associated molecular patterns
Legionnaires’ disease, outbreak 111 (PAMPs) 156
leukocyte recruitment 150 Pce 150
linezolid, resistance 66 PCR assay 15, 20, 195
lipoteichoic acid (LTA) 156 penicillin-binding protein (PBP) 62
Index 249
pericarditis 80 Reye’s syndrome 81

pharmacodynamics 66 ribosomal protein 64
phase variation 141 rimantadine 82
phospholipase C 150 risk stratification 5, 6
phosphorylcholine (PCho) 144
phosphorylcholine esterase 150 secondary bacterial pneumonia 79, 80
photophobia 80 secretory component (SC) 147
PK/PD 67 senescence, immune 229
plasmin(ogen) 148 seroconversion 194
platelet-activating factor (PAF) 148 severe acute respiratory syndrome (SARS),
pleural effusion 191 vaccines 208
pneumococcal clone 61, 68 signature-tagged mutagenesis (STM) 151
pneumococcal colonization 141 Spanish influenza 74, 76
pneumococcal adherence and virulence Staphylococcus aureus 78, 80
factor A (PavA protein) 147, 148 streptococci, viridans 60, 63
pneumococcal pneumonia 142 Streptococcus pneumoniae 3, 5, 15, 78, 80,
pneumococcal serotype 140 209, 210
pneumococcal surface adhesin A vaccines 140, 209, 210
(PsaA) 146 Streptococcus, erm gene 64
pneumococcal surface protein A Streptococcus, mef gene 63
(PspA) 151 streptogramin, resistance 66
pneumococcal surface protein C (PspC,
SpsA, CbpA) 147 telithromycin 196
pneumococcal molecular epidemiology telithromycin, resistance 65
network (PMEN) 61 terminal sialic acid 146
pneumolysin 142 tetracycline 196
polymeric immunoglobulin receptor 147 Toll-like receptor 156
primary atypical pneumonia (PAP), transmission 185
diagnosis 190 transverse myelitis 81
primary viral pneumonia 79, 80 treatment 7ff
progressive pulmonary failure 193 two-component regulatory systems
Pseudomonas aeruginosa 3 (TCS) 152
Pseudomonas aeruginosa, vaccines216
pseudoviruses 222 urinary antigen 6, 20
pulmonary fibrosis 191
pulmonary infiltrates 80 vaccine 140, 202–216
adenovirus 209
quinolone resistance-determining region B. pertussis 212
(QRDR) 64 C. pneumoniae 214
H. influenzae 211, 212
real-time PCR assay 195 influenza 202-204
receptor binding 74 L. pneumophila 215
recombination 60, 65 M. catarrhalis 214
requirement for sterols 184 M. pneumoniae 215
resistance, acquired 60 P. aeruginosa 216
resistance, fluoroquinolone 64 parainfluenza virus (PIV) 205
resistance, macrolides 63 respiratory syncytial virus (RSV) 206,
resistance, tetracycline 64 207
resistant virus variants 33, 35, 36, 39, 40 S. pneumoniae 209, 210
respiratory syncytial virus (RSV), severe acute respiratory syndrome
vaccines 206, 207 (SARS) 208
respiratory viruses 28, 29, 42–44, 48 vaccine design 226
reverse genetics 218 vaccine, pneumococcal 69
reverse vaccinology 217, 219 vectors, live attenuated bacterial 221
250 Index
vectors, live attenuated viral 221

vesicle traffic, manipulation 118
viral pneumonia 27, 32, 33, 37, 41, 42, 47
virosomes 222, 223
virosomes 223
virulence 183
virus detection 42-48
virus-like particles (VLP) 222
wheezing attacks in asthmatics 80
zanamivir 82
zinc metalloprotease 146
The BAID-Series
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Pathogenesis of Mycoplasma pneumoniae infections: Adaptive immunity,

Chapter · January 1970

Jerry William Simecka Norbert Suttorp

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Axel Schmidt Manfred H. Wolff

Stefan H.E. Kaufmann

Edited by N. Suttorp, T. Welte and R. Marre

Norbert Suttorp Reinhard Marre

Bibliographic information published by Die Deutsche Bibliothek

ISBN 3-7643-7562-0 Birkhäuser Verlag, Basel - Boston - Berlin

© 2007 Birkhäuser Verlag, P.O. Box 133, CH-4010 Basel, Switzerland

List of contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Walter Hampl and Thomas Mertens

Mathias W.R. Pletz, Lesley McGee and Tobias Welte

Matthias Krüll and Norbert Suttorp

Dina M. Bitar, Marina Santic, Yousef Abu Kwaik

Sven Hammerschmidt, Gavin K. Paterson, Simone Bergmann

Ken B. Waites, Jerry W. Simecka, Deborah F. Talkington

Pablo D. Becker and Carlos A. Guzmán

Yousef Abu Kwaik, Department of Microbiology and Immunology, Room

Timothy J. Mitchell, Division of Infection and Immunity, Institute of Bio-

Community-acquired pneumonia is a disease of high morbidity and mor-

Berlin/Hannover/Ulm, August 2006 Norbert Suttorp

Diagnosis and treatment of community acquired

Department of Pulmonary Medicine, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1,

Pneumonia is a worldwide, serious threat to health, and an enormous

The largest proportion of this amount is spent on elderly and hospitalized

Pneumonia is an infection of the alveolar space, with accumulation of

The spectrum of pathogens and resistances varies widely between conti-

Table 1. Clinical findings in patients with legionella infection

that vaccination of children reduced the incidence of severe pneumonia in

Problems with resistances of the most important pathogens, especially

insufficiency (PaO2/FiO2 < 250), multilobular infiltrates in the chest radio-

A chest radiograph (pa (posterior/anterior) and lateral view) is highly rec-

Serological testing (to screen for Mycoplasma, Chlamydia and viruses)

All guidelines distinguish patients with low risk of dying (CRB-65 ) 1,

– Previous antibiotic treatment increases the rate of resistant pathogens.

– Pulmonary concomitant diseases (structural defects in COPD stadium

Substance Dosage (per day) Duration of therapy

Depending on the number of risk factors, the probability of Pseudomonas

Substances Dosage Duration of therapy

# Can be applied as sequential treatment using the same drug

with Mycoplasma seem to be self-limiting in low-risk patients. In these sit-

Baseline (minor) criteria assessed at admission

Major criteria assessed at admission or during clinical course

Pa,O2, arterial oxygen tension; Fi,O2, inspiratory oxygen fraction;

Substances Dosage Duration of therapya

roquinolones). Gram-negative pathogens and Pseudomonas must be cov-

6 Arancibia F, Bauer TT, Ewig S, Mensa J, Gonzalez J, Niederman MS, Torres A

22 Goossens H, Ferech M, Vander Stichele R, Elseviers M (2005) Outpatient anti-

35 Janssens JP (2005) Pneumonia in the elderly (geriatric) population. Curr Opin

Detection of respiratory bacterial pathogens

University of Ulm, Institute of Medical Microbiology & Hygiene, Robert-Koch-Str. 8,

Community acquired pneumonia is caused by a wide variety of differ-

of a causative agent. In addition, clinical patterns often do not give a clear

Microscopy of respiratory secretions

Microscopy of respiratory sample may help in guiding initial therapy in

10x field, shaddowed columns: > 25 leukocytes per 10x field.

albicans, Escherichia coli and other enterobacteria) can be detected in high