SECTION 8 Clinical Microbiology: Bacteria
183 
Gram-Negative Coccobacilli
FIONA J. COOKE  |  MARY P.E. SLACK
KEY CONCEPTS
The pathogens covered in the chapter exemplify a
number of diverse aspects of the interaction between
man and microbes, as exemplified by:
• Zoonotic and imported infections, such as Brucella, Francisella,
Pasteurella and Yersinia.
• Mucosal infections, such as Moraxella and nontypeable Hae-
mophilus influenzae (NTHi), which emphasize the importance
of biofilms and microbial interactions in the pathogenesis of
mucosal disease.
• Environmental infections such as Legionella sources. Interna-
tional alerting systems are critical in many outbreak
investigations.
• Vaccine-preventable infections, notably pertussis and Hae-
mophilus influenzae type b (Hib).
• Commensal bacteria, which can cause serious disease, such as
the HACEK group causing endocarditis.
Introduction
The gram-negative coccobacilli (GNCB) that are important human
pathogens include Bordetella, Brucella, Francisella, Haemophilus, Legio-
nella, Pasteurella, Moraxella and Yersinia spp. Other GNCB including
Actinobacillus, Aggregatibacter, Cardiobacterium, Eikenella, and Kin-
gella spp. occasionally cause human disease. All of these genera except
Yersinia are fastidious, requiring special nutrients and growth factors
for isolation.
Other less common GNCB not discussed further in this chapter
include Oligella ureolytica, Psychrobacter phenylpyruvicus (formally
Moraxella phenylpyruvica) and Psychrobacter immobilis. Actinobacillus
ureae is an occasional opportunist human pathogen, associated with
pneumonia, lung abscess and invasive infections.
Bordetella spp.
Nature
Bordetella spp. are minute GNCB. All nine species in the genus are
strict aerobes, except for Bordetella petrii, and grow optimally at
35–37°C. Bordetella pertussis and B. parapertussis are nonmotile. B.
pertussis is the most fastidious and requires special media for isolation;
B. parapertussis is slightly less exacting, while B. bronchiseptica grows
on ordinary laboratory media. B. pertussis and B. parapertussis are
human pathogens of the respiratory tract and cause pertussis or
whooping cough. Bordetella holmesii causes both invasive infections
and pertussis-like respiratory symptoms.1 B. bronchiseptica, B. hinzii,
B. trematum, B. petrii, B. avium and B. ansorpii infrequently cause
infection in humans and tend to affect immunocompromised hosts.
B. bronchiseptica and B. avium are primary respiratory tract pathogens
of birds and mammals. Whole genome sequencing has confirmed the
close genetic relatedness of the three ‘classical’ Bordetella species (B.
pertussis, B. parapertussis and B. bronchiseptica) and genomic analysis
suggests that B. pertussis and B. parapertussis are probably human-
adapted subspecies of B. bronchiseptica.2
Epidemiology
Pertussis is the most prevalent vaccine-preventable disease in high-
income countries and an important cause of death in malnourished
children. In 2011 the World Health Organization (WHO) estimated
there were at least 16 million cases and 200 000 deaths annually, mostly
in low- and middle-income countries (LMIC). In most populations
the disease is endemic, with epidemics occurring every 4 years in
autumn and winter. There is no animal reservoir.
Pertussis is highly contagious, being transmitted via aerosolized
droplets of respiratory secretions. In the prevaccine era nearly all chil-
dren became infected between the ages of 1 and 5 years. Attack rates
range from 50% for school contacts to 80–90% for close family con-
tacts. Patients may disseminate organisms for weeks or months and
are highly infectious in the nonspecific catarrhal and early paroxysmal
stages of the infection. The infection can therefore be transmitted to
susceptible individuals before the possibility of whooping cough is
considered. There is little evidence of asymptomatic carriage.
Vaccination against pertussis has resulted in a decline in the inci-
dence of the disease in children aged 3 months to 9 years (Figure
183-1). However, infants less than 3 months old remain susceptible to
pertussis as they cannot be fully protected by immunization. Over the
last decade, pertussis has emerged in adolescents and adults in many
higher-income countries,3 probably due to waning immunity after
natural infection or vaccination. Other important factors include
improved molecular diagnostic testing, increased awareness of the
disease leading to increased reporting of cases, active surveillance,
changes in disease susceptibility and characteristics of the vaccine.3
Combination vaccines containing diphtheria toxoid, tetanus toxoid
and an acellular pertussis component (DTaP) are less potent than vac-
cines containing whole-cell pertussis (DTP)4 and vaccine-induced
immunity may wane more rapidly when DTaP is used.5 It has also been
postulated that genetic changes in circulating strains of B. pertussis may
be a factor in the recent resurgence4 though this is not proven. Older
patients who develop pertussis can act as a source of transmission to
very young children.
Vaccination schedules vary between countries, but administration
of boosters at different ages may reduce individual morbidity and
onward transmission, and increase herd protection. Rates of infection
in the 2011–2013 UK outbreak were greatest in infants less than three
months old, who are at most risk of complications and death (Figure
183-1). A temporary program to offer pertussis vaccination to preg-
nant women from 28–32 weeks of gestation was introduced in the UK
in 2013 and successfully reduced cases in very young infants.6 Due to
the success of the maternal immunization program in preventing per-
tussis in infants, the UK Joint Committee on Vaccines and Immunisa-
tion (JCVI) has decided to continue the temporary policy for at least
5 more years (https://www.gov.uk/government/publications/vaccine
-update-issue-217-july-to-august-2014).
The three serotypes of B. pertussis pathogenic for humans contain
agglutinins 1,2; 1,2,3; and 1,3. Strains may switch serotype both in vitro
and in vivo. Various genotypic methods, such as multiple locus vari-
able number tandem repeat analysis (MLVA), pulsed-field gel electro-
phoresis (PFGE), multiple antigen sequence typing (MAST) and more
recently whole genome sequencing have been applied to the epidemio-
logical typing and population analysis of B. pertussis.
1611
1612 SECTION 8  Clinical Microbiology: Bacteria

The epidemiology of pertussis in the UK

Notifications Coverage by 2nd birthday

Total notifications 200000 100 Coverage (%)
180000 90
Incidence per 20 160000 80 300 Incidence per
100,000 (all ages) 140000 70 100,000 (<6
18 120000 60 months of age)
100000 50 250
16
80000 40
14 60000 30
200
12 40000 20
20000 10
10 0
1940
0 150
1943
1946
1949
1952
1955
1958
1961
1964
1967
1970
1973
1976
1979
1982
1985
1988
1991
1994
1997
2000
2003
2006
2009
2012
8
100
6

4
50
2

0 0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

All ages <3 months 3–5 months

Figure 183-1  The epidemiology of pertussis in the UK. The main graph shows the incidence of confirmed pertussis in infants under 6 months old and at all ages,
England only, 1998–2013. The insert graph shows vaccine coverage data by second birthday for England between 1940 and 2012, and the incidence of confirmed pertus-
sis over this period. Data supplied by Public Health England.

Pathogenicity of pertussis occurred in the late 1970s (Figure 183-1). There is no

Bordetella pertussis, B. parapertussis and B. bronchiseptica all possess a
virulence control system regulated by BvgAS operon, which is a two-
component phospho-relay system that responds to environmental
conditions by switching between three phenotypic phases.7 The Bvg+
phase occurs with respiratory tract colonization and is associated with
the expression of a number of virulence factors. The Bvgi phase is
associated with respiratory transmission and Bvg− is an avirulent phase
that enables B. bronchiseptica to survive in the environment.7 Some
Bvg-regulated genes are expressed in all three subspecies. Others,
although they are present, are differentially expressed. Importantly the
ptx-ptl operon, which encodes pertussis toxin (PT), is present in B.
pertussis, B. parapertussis and B. bronchiseptica but only expressed in
the Bvg+ phase of B. pertussis.
Bordetella pertussis produces a number of adhesins and toxins,
which are important in pathogenesis (Table 183-1).8 Following inhala-
tion, B. pertussis adheres to ciliated epithelium in the trachea and
bronchi. Adhesion is mediated by filamentous hemagglutinin, pertac-
tin and, possibly, PT. The bacteria then begin to multiply and produce
various toxins: PT (disrupts cell function); tracheal cytotoxin (inhibits
ciliary motion); adenylate cyclase toxin (interferes with phagocytosis)
and dermonecrotic toxin (causes local necrosis). The organisms
remain localized on the respiratory epithelium, and do not invade.
Prevention
Prevention of pertussis depends on immunization. Whole-cell pertus-
sis vaccine, consisting of killed suspensions of whole bacterial cells
adsorbed with the adjuvant aluminum hydroxide, was introduced in
the UK during the 1950s and resulted in a steady decline in the size of
pertussis epidemics until the mid-1970s. Concerns about the safety of
whole-cell vaccines and fears of possible neurologic sequelae led to a
dramatic fall in vaccine uptake in the UK, and three large epidemics
strong evidence that whole-cell pertussis vaccine produces long-term
adverse effects but it may trigger the appearance of pre-existing neu-
rological problems.
A number of acellular pertussis vaccines, containing up to five B.
pertussis antigens, including PT, filamentous hemagglutinin, pertactin
and fimbrial antigens FIM2 and FIM3, have been developed. These
have replaced whole-cell pertussis vaccines in routine infant immuni-
zation programs in many high-income countries, including the UK.
They are generally less reactogenic than whole-cell preparations and
may be used for booster immunization in older children and adults.
There is no vaccine against B. parapertussis.
Continual review of vaccination strategies, together with the devel-
opment of new immunogenic and efficacious vaccines, is critical to
control the resurgence of this potentially fatal disease.
Diagnostic Microbiology
With careful sampling and culture techniques B. pertussis can be recov-
ered during the catarrhal phase and the first 2–3 weeks of the parox-
ysmal phase of the illness. Nasopharyngeal aspirates, pernasal swabs or
nasopharyngeal swabs are the specimens of choice, and are superior to
‘cough plates’. Material collected for bacterial culture should be plated
immediately onto appropriate agar, or placed in charcoal transport
medium. Charcoal blood agar, containing cephalexin to enhance selec-
tivity, has largely replaced the traditional Bordet–Gengou agar. Cul-
tures should be incubated in a moist aerobic atmosphere at 98.6°F
(37°C) for at least 7 days. After 3–5 days of incubation, typical ‘bisected
pearl’ colonies appear (Figure 183-2). Suspect colonies should be
Gram-stained and identified using slide agglutination with type-
specific antisera. B. pertussis, B. parapertussis and B. bronchiseptica can
be identified using a commercial test kit such as API 20NE. Matrix-
assisted laser desorption/ionization time-of-flight (MALDI-TOF)
mass spectrometry reliably identifies the classical Bordetella species.
 Chapter 183  Gram-Negative Coccobacilli 1613

TABLE
183-1  Virulence Factors of Bordetella pertussis*
Toxin Synonyms Composition Actions Comments

Filamentous hemagglutinin FHA Filamentous protein Adhesin, binds to ciliated respiratory Highly immunogenic component
tract epithelium, and macrophage of acellular pertussis vaccines
CR3 promoting phagocytosis

Pertactin PRN Outer membrane protein Adhesin, important in colonization of Enhances protective immunity
ciliated respiratory tract epithelium

Pertussis toxin PT A-protomer (S1 subunit) and
B-oligomer which consists
of four subunits (S2 to S5)
Attachment to ciliated respiratory Adjuvant component of acellular
epithelium pertussis vaccines
Activation of cAMP, HSF, IAP, LPF
Impairs leukocyte function
Hemolytic

Adenylate cyclase toxin ACT Protein Activation of cAMP Calmodulin-activated RTX toxin
Antiphagocytic
Anti-inflammatory
Hemolytic

Dermonecrotic toxin DNT Polypeptide, heat-labile Vascular smooth muscle contraction

Dermal necrosis
Activates host GTP binding protein Rho

Tracheal cytotoxin TCT Glycopeptide Ciliostasis

Inhibits DNA synthesis

Lipopolysaccharide LPS Lipopolysaccharide Endotoxin

Tracheal colonization factor TCF-A Proline-rich protein Cytotoxin

Adhesin predominantly in trachea
Contributes to destruction of respiratory
epithelium

Serum resistance factor BRK Protein Potential adhesin and serum resistance
factor
Complement resistance
Fimbriae FIM Filamentous proteins Facilitates persistent tracheal Component of some acellular
composed of subunits colonization pertussis vaccines

cAMP, cyclic adenosine monophosphate; HSF, histamine sensitizing factor; IAP, islet-activating protein; LPF, lymphocytosis promoting factor; RTX, repeats in toxin.
*With the exception of pertussis toxin, similar toxins are expressed in B. parapertussis and B. bronchiseptica.

in titer of IgG or IgA (twofold or greater) in acute and convalescent

phase samples taken at least 2 weeks apart is considered significant. In
adolescents and adults a single high titer of antipertussis toxin IgG has
a good predictive value of acute pertussis infection, with reported
sensitivity of 76% and specificity of 99%.9 Serology is unreliable in
young infants.

Figure 183-2  Colonies of Bordetella pertussis on charcoal blood agar.
Real-time polymerase chain reaction (PCR) is more sensitive than
culture, especially for specimens collected more than 3–4 weeks after
onset of the cough and following antibiotic treatment. A number of
different primers have been used, including IS481, which detects B.
pertussis and B. holmesii; IS1001, which detects B. parapertussis and
B. holmesii; and ptxA, which is specific for B. pertussis. Serotyping of
B. pertussis is based on the three major surface agglutinogens (1, 2 and
3). Type 1,3 is more common than serotypes 1,2 and 1,2,3, and
accounts for 90% of isolates. Typing by MLVA facilitates comparison
between laboratories.
Serological diagnosis of B. pertussis is based on enzyme-linked
immunosorbent assay (ELISA) of IgG and IgA antibodies to PT. A rise
Clinical Manifestations
Bordetella pertussis is the cause of pertussis (whooping cough). Typi-
cally, following an incubation period of 7–14 days, the patient develops
red eyes, a runny nose, mild cough and sneezing (‘catarrhal stage’).
Symptoms are clinically indistinguishable from many viral upper
respiratory tract infections. After approximately 1 week the cough
becomes more severe and the patient experiences paroxysmal bouts of
a severe, hacking cough (‘paroxysmal stage’). A paroxysm consists of
repeated coughing followed by an inspiratory gasp as the patient finally
inspires. This is the characteristic ‘whoop’. Paroxysms may be trig-
gered by a variety of stimuli, including cold air and loud noises. A child
may become cyanosed during a paroxysm and can suffer fatal hypoxia.
Often the child vomits at the end of a paroxysm and is left exhausted.
This paroxysmal stage may last for 1–4 weeks, and is followed by a
lengthy ‘convalescent stage’ as the paroxysms decline and the patient
slowly recovers.
Common complications include pneumonia (which may be
primary or secondary to a supervening infection with another patho-
gen) and otitis media. The child may suffer cerebral hypoxia during a
paroxysm, which can lead to encephalopathy or fitting. Other possible
complications include intraventricular hemorrhage, subconjunctival
hemorrhages, umbilical or inguinal herniae, fractured ribs, ruptured
diaphragm and rectal prolapse. Pertussis is most severe in very young
infants and in this age group the presentation may be atypical with no
1614 SECTION 8  Clinical Microbiology: Bacteria
characteristic paroxysmal whooping. Partially immunized children
and adults may have an atypical form of pertussis.10
Bordetella parapertussis is a cause of acute bronchitis or pertussis-
like infections in children. Bordetella bronchiseptica may rarely cause
pneumonia, meningitis or whooping cough in highly immunocom-
promised patients.10 Bordetella holmesii is associated with pertussis-
like symptoms, but may also cause invasive infections, including
meningitis, bacteremia, pneumonia, endocarditis and arthritis. While
respiratory infections with B. holmesii tend to occur in healthy adults
and adolescents, invasive infections generally affect patients with
underlying co-morbidities, including asplenia, HIV infection or
immunosuppression. Infection with B. holmesii may be misidentified
as being due to B. pertussis because routine diagnostic tests are not
species-specific.1
Management
Antimicrobial therapy for pertussis, when administered early in tissues of ruminants, which are rich in mesoerythritol. Erythritol stim-
the illness, can decrease transmission to susceptible contacts and ulates the multiplication of Brucella spp. Thus the main symptoms of
possibly ameliorate symptoms. Macrolides are the antibiotics of choice
and ap­­pear to reduce the severity and duration of the disease.
Co-trimoxazole or fluoroquinolones are alternatives. Corticosteroids
may be indicated in infants who have life-threatening disease. Second-
ary bacterial infections should be treated with appropriate antibiotics.
Antibiotic prophylaxis should be offered to all close contacts, regard-
less of their immunization status, where there is an unimmunized or
partially immunized vulnerable close contact.11 Immunization should
be considered for those offered antibiotic prophylaxis.11
Brucella spp.
Nature
Brucella spp. are small, nonmotile, nonsporing, noncapsulate
GNCB. They are aerobic, but some strains require 5–10% carbon
dioxide for primary isolation. Growth in vitro is slow and primary
isolation may require 4 weeks incubation. Growth may be improved
by addition of serum or blood. Brucella spp. are catalase-positive and
usually oxidase-positive.
Brucella is a monospecific genus (Brucella melitensis) with multiple
biovars. In practice it is more useful to refer to separate species, which
have different preferential host specificities. There are 10 ‘species’ of
Brucella: B. melitensis is the most virulent and is responsible for most
human infections (Table 183-2). B. melitensis, B. abortus and B. suis
cause considerable morbidity in countries where brucellosis persists in
domestic animals. Human infections with B. canis and the marine
species (B. ceti, B. pinnipedialis) are rare. Complete genome sequences
TABLE
183-2  Brucella species
Species (Number Of Biovars) Primary Host Humans As Secondary Host
B. abortus (6) Cattle, camels, yaks, buffalo
B. melitensis (3) Goats
B. suis (5) Pigs
B. canis Dogs
B. ceti Whales
B. pinnipediae Seals
B. neotomeae Desert rats
B. ovis Sheep
B. microti Voles
B. inopinata ? ?
of at least 25 Brucella strains are available, and analysis has confirmed
that members of the genus are genetically homogeneous. Investigating
Brucella diversity helps our understanding of evolution and taxonomy,
and also directs the development of new molecular typing tools.12
Epidemiology
Brucellosis is a true zoonosis, because humans acquire the infection
directly or indirectly from infected animals. It is the commonest zoo-
notic infection worldwide, with more than 500 000 cases of human
brucellosis reported annually, which is likely to be an underestimate.
Brucellosis is prevalent in Mediterranean countries, the Middle East,
the Indian subcontinent, Mongolia and Central and South America.13
Some countries have eradicated brucellosis, including the UK, much
of northern Europe, Australia, New Zealand and Canada.
In their natural hosts, Brucella spp. cause chronic infections which
are mild or asymptomatic. The bacteria localize in the reproductive
brucellosis in animals include sterility or abortion. Brucella organisms
are shed in large numbers in the products of conception, urine and
milk, and when infected animals are slaughtered. Humans are infected
either by direct contact with infected animals or animal products or
indirectly by ingesting infected milk or dairy produce.
In endemic areas, most cases occur in dairymen, herdsmen, abat-
toir workers, butchers and veterinary surgeons. Children may become
infected in rural areas of LMIC if they live in close proximity to domes-
tic animals. The general public is usually infected by ingesting unpas-
teurized milk and milk products such as fresh soft cheese. Case-to-case
transmission in humans is very rare. Self-inoculation with live Brucella
vaccine is a risk among veterinary surgeons, and laboratory workers
are at risk if they handle as-yet unidentified gram-negative coccobacilli
without adequate precautions. Brucellosis is considered a potential
biologic weapon via airborne transmission.
Pathogenesis
Brucella spp. are facultative intracellular parasites, their survival and
persistence within the body being dependent on their ability to survive
and multiply within cells of the reticuloendothelial system.14 The
organisms enter the body by inhalation, ingestion or after penetration
of intact skin, abrasions or the conjunctival mucosa. The lipopolysac-
charide (endotoxin) of smooth strains (S-LPS) is a major virulence
determinant. S-LPS is antiphagocytic, facilitates cell entry, inhibits the
fusion of Brucella-containing vacuoles (BCV) with lysosomes and
inhibits host cell apoptosis. A two-component regulatory system,
BvrR/BvrS is important for invasion and intracellular survival, and acts
by regulating the expression of outer membrane proteins, Omp3a
Notes Regarding Human Infections
++ Usually sporadic, in people who work with cattle
++++ Responsible for the majority of human infections. Primarily
food-borne
+ Usually sporadic, in people who work with pigs
+ Unusual
+ Very rare
+ Very rare
− −
− −
− −
1 case report of human infection in breast implant

Major events in the pathogenesis of brucellosis and host immune response
Macrophage Phagosome
Endoplastic
reticulum
Autophagosome
Antibody
production
T lymphocytes
B lymphocytes
Figure 183-3  Major events in the pathogenesis of brucellosis and the host immune response. (From Pappas G. et al. N Engl J Med 2005; 352:2325-2336.)
(Omp25) and Omp3b (Omp22). The altered expression of surface
proteins permits Brucella to bind to and penetrate host cells. The type
IV secretion system (VirB) of Brucella, encoded by the virB operon, is
also important for adherence, cell entry and bacterial survival and
replication within macrophages.
After ingestion by phagocytic cells, the organisms proliferate in
local lymph nodes. The infection spreads hematogenously to tissues
rich in elements of the reticuloendothelial system, including the liver,
bone marrow, lymph nodes and spleen. Organisms may also localize
in other tissues, including the central nervous system, joints, heart and
kidneys.
Endotoxin and hypersensitivity to Brucella antigens may explain
some of the symptoms of brucellosis, including fever and weight loss.
Antibodies against the Brucella LPS appear within a few days of onset
of the acute phase of the disease and are important in preventing
re-infection. However, cell-mediated immunity, particularly the pro-
duction of activated mononuclear phagocytes, is more important in
promoting recovery (see Figure 183-3).
Prevention
The ideal method of prevention of human brucellosis is elimination
of the disease from domestic livestock. There are effective vaccines for
cattle (S19) and goats (Rev-1). Where eradication has not been
achieved, individuals with at-risk occupations should wear protective
clothing, gloves, goggles and masks, especially when handling post-
partum animals.
Previously, live attenuated animal vaccines have been given to
workers at high risk of contracting brucellosis. However, these vaccines
may produce infection in humans and are far from ideal. Candidate
vaccines, including an LPS–protein conjugate vaccine and a subunit
vaccine against the outer membrane vesicle are under development.14
Boiling or pasteurizing milk eliminates the risk of transmission via
dairy products, as Brucella is killed by heating at 140°F (60°C) for 10
minutes. If laboratory workers are exposed to Brucella, postexposure
antibiotic prophylaxis, for example with doxycycline or co-trimoxazole,
should be considered.
Chapter 183  Gram-Negative Coccobacilli 1615
Brucellae
Inhibition
of TNF-
/ T lymphocytes
Interferon-
Natural killer cells production
Diagnostic Microbiology
Clinicians should inform laboratory staff if brucellosis is suspected (or
when investigating febrile patients who have visited or resided in
Brucella-endemic areas) to ensure samples are processed appropri-
ately. Definitive diagnosis depends on isolating Brucella spp. from
cultures of blood, bone marrow or tissue obtained early in the disease.
Bone marrow cultures reportedly give a slightly higher yield than blood
cultures. However, automated blood culture methods may produce
positive results in approximately 80% of cases, so bone marrow cul-
tures are rarely required. Cultures should be incubated at 98.6°F
(37°C) for at least 6 weeks, but commercial systems may show growth
in a few days. In LMIC where automated blood culture systems are
not available, blood clot culture is more sensitive than whole blood
culture and lysis centrifugation may increase yield.
Positive cultures should be subcultured onto serum dextrose agar
or similar agar. Selective media may be required for contaminated
material. After 48 hours of incubation at 98.6°F (37°C) in air plus
5–10% carbon dioxide, Brucella spp. produce small, smooth, translu-
cent colonies. Commercial galley-test identification systems (API) may
misidentify Brucella spp. as Moraxella phenylpyruvica. MALDI-TOF
MS is a rapid and reliable method to identify Brucella spp.
The biovar of the strain is usually determined by reference labora-
tories, based on production of hydrogen sulfide, urease activity, toler-
ance to bacteriostatic dyes and agglutination. Tbilisi (Tb) phage typing
may also be used. A number of PCR assays for diagnosing brucellosis
in clinical samples have been described and may rapidly diagnose acute
brucellosis and enable early detection of relapse.
Many serological tests have been described for brucellosis, includ-
ing rose bengal test, serum agglutination, Coombs, competitive ELISA
lateral flow immunochromatography for IgM and IgG detection and
immunoprecipitation with Brucella proteins. Interpretation of results
is often difficult since both IgG and IgM antibodies can persist for a
long time.
The serum agglutination test (SAT) using B. abortus strain 119 is
widely used, preferably on serum collected very early in the course of
the disease. False-negative results may occur if serum samples are not
1616 SECTION 8  Clinical Microbiology: Bacteria
diluted beyond 1:320, owing to a prozone phenomenon. False-positive
reactions may occur due to cross-reactivity with Escherichia coli, Vibrio
cholerae, Yersinia enterocolitica and Francisella tularensis. The SAT
measures both IgM and IgG. The best indicators of acute infection are
IgG and IgA antibodies. A rise in antibody titer is observed in more
than 97% of patients who have acute brucellosis and positive blood
cultures. An initial IgM antibody response is followed after 1–2 weeks
by a rise in IgG. During recovery the IgG antibody titers slowly decline
over a period of months but IgM antibodies may persist at a low level
in the serum for several years. Sustained IgG antibody titers or a
second rise in IgG antibody levels are seen in chronic infection or
relapse. The SAT test does not detect B. canis as it lacks surface LPS,
and specific serology for this organism should be requested.
Commercial ELISA tests based on antibodies to S-LPS are highly
sensitive but appear to be less specific than the SAT. Again, because B.
canis lacks surface LPS it provokes minimal anti-LPS-specific antibody
response.
Rapid point-of-care tests, including fluorescent polarization
immunoassay (FPA) and immunochromatographic Brucella IgM/IgG
lateral flow assay (LFA) are highly sensitive and specific and have been
used successfully to diagnose brucellosis in endemic areas. Ideally the
diagnosis of brucellosis should be based on clinical suspicion, isolation
of the organism and positive serology.
Clinical Manifestations (see also Chapter 129)
Brucellosis, also known as Malta fever or undulant fever, is a systemic
infection that can affect any tissue of the body, and tends to present
with nonspecific clinical symptoms.15 The possibility of brucellosis
should be considered in any fever of unknown origin, especially in
patients who have occupational exposure or relevant travel history. See
Chapter 68.
Management
Before the introduction of antibiotics, brucellosis was a chronic,
relapsing illness. In vitro antibiotic activity does not always correlate
with clinical efficacy, and agents that achieve adequate intracellular
concentrations should be used. A prolonged course of treatment is
recommended to reduce relapse. Most recommendations center on
dual or triple therapy, usually involving an aminoglycoside. Examples
include oral doxycycline plus rifampin (rifampicin) for at least 6
weeks, or oral doxycycline for 6 weeks supplemented by a parenteral
aminoglycoside for the first 2–3 weeks (see also Chapter 129). In and veterinary surgeons are at increased risk of infection. Laboratory
cases of accidental laboratory exposure, post-exposure prophylaxis
should be offered as soon as possible. Possible regimens include or cultures of the organism. Person-to-person transmission of Fran-
oral doxycycline or co-trimoxazole. In all such cases the patient should
TABLE
183-3  The Subspecies and Biovars of Francisella tularensis
Subspecies Geographical Distribution
F. tularensis (type A)
Clade AI (sub-groups AIa and AIb)* Central USA (east of Rocky Mountains)
Clade AII Western USA
F. holarctica (type B)
Biovar I North America,
Biovar II Europe, Siberia,
Biovar japonica Eurasia, Japan
F. mediasiatica Central Asia
F. novicida† North America, Australia, Thailand
*Human infections with F. tularensis subspecies tularensis AIb run a fulminant course with a high mortality rate compared with infections due to AIa, AII or Type B
strains.
†
Less virulent. Infection mainly occurs in immunocompromised hosts.
§
Infections associated with brackish water.
be monitored for 6 months for signs of infection, including the devel-
opment of fever, headache, malaise, myalgia, arthralgia, anorexia or
weight loss.
Francisella spp.
Nature
Francisella spp. are very small, faintly staining, pleomorphic, nonmo-
tile and nonspore-forming GNCB, surrounded by a thin lipid-rich
capsule. They are strictly aerobic, oxidase-negative and weakly catalase-
positive. They attack carbohydrates slowly, producing acid but no gas.
Francisella tularensis requires cysteine or cystine for growth and grows
slowly at 98.6°F (37°C) on suitably enriched media.
F. tularensis causes tularemia in animals and humans. Within the
species F. tularensis there are three subspecies, which differ in their
geographical distribution and virulence in man (Table 183-3). Com-
parative genomics suggest that these subspecies are genetically distinct
groups.16
Epidemiology
The majority of human infections with F. tularensis occur in the north-
ern hemisphere between latitudes 30° and 71°. It is well recognized in
North America, Scandinavia and Russia, and cases have been reported
from central and southern Europe, including Czech Republic, Kosovo,
Bulgaria, Germany, Spain and France. All cases in the UK are imported.
The organism is found in more than 250 host animal species including
wild and domestic animals, birds, fish, amphibians, protozoa and
blood-sucking arthropods. In the USA the most important reservoirs
of F. tularensis are cottontail rabbits, jackrabbits, hares and muskrats.
In Scandinavia and Russia, hares and rabbits are important reservoirs.
The modes of transmission to humans include tick or mosquito bites,
contact with infected animal tissues, inhalation of infectious aerosols
or ingestion of contaminated meat or water.
F. tularensis may survive in soil, water (possibly in association with
free-living amebae) and animal carcasses for several weeks. In endemic
areas, tularemia is seasonal with the majority of cases occurring during
the late spring, summer and autumn. There are approximately 200
cases each year reported in the USA, with over 60% occurring in the
southern and southern-central states of Missouri, Oklahoma, Arkan-
sas, Texas and Kansas.
People who handle infected animals, including hunters, farmers
workers are at high risk through handling infected laboratory animals
cisella tularensis has not been documented.
Virulence In Humans Main Animal Hosts
+++ Cottontail rabbits, jackrabbits, hares, ground
squirrels
++ Beavers, voles, muskrats
Hares
Voles
+ Rodents, rabbits
+ ?§

Pathogenicity
The infectious dose for humans depends on the subspecies and the
route of entry. Inhalation of fewer than 10 organisms of F. tularensis
subsp. tularensis can result in infection. The infecting dose rises to 108
when the organisms are ingested.
F. tularensis subsp. tularensis (type A) is more virulent to animals
and humans than the other subspecies. The low infecting dose, aerosol
transmission, high virulence and high mortality of consequent infec-
tion with F. tularensis subsp. tularensis mean that this organism is a
potential bioweapon.17
The virulence of F. tularensis is based on its ability to survive and
replicate within the cytosol of infected cells, including macrophages,
hepatocytes and endothelial cells.18 After entering the body, the organ-
isms spread to the regional lymph nodes, from where they may dis-
seminate via the lymphatic system or bloodstream to involve multiple
organs. There is probably a transient bacteremia at this early stage.
Intracellular survival is enabled by a number of virulence genes,
including the ‘macrophage growth locus’ (mgl) A and B and the ‘Fran-
cisella pathogenicity island’. Organisms are protected from humoral
antibodies, which are directed against carbohydrate antigens. Recovery
from tularemia depends largely on cell-mediated immunity, which is
directed against the protein antigens of the organism.
Prevention
Prevention of tularemia is best achieved by avoiding exposure to the
organism. Gloves, masks and goggles should be worn when skinning
or eviscerating animals. Animals that look sick should be left intact.
Game meat should be thoroughly cooked and fresh water that is pos-
sibly contaminated should not be drunk. Ticks should be promptly
removed and chemical insect repellants may be used.
There is no licensed vaccine available. To be effective a vaccine
would need to elicit a T-cell memory response. The live attenuated
vaccine (LVS) given to laboratory staff working with F. tularensis in
the USA was withdrawn because of variable immunogenicity and risk
of reversion to virulence. Killed and subunit vaccines have not been
effective. A vaccine using an attenuated strain of Type A tularemia,
SchuS4, appears to provide protection against parenteral and intrana-
sal challenge with a fully virulent SchuS4 strain.19
Diagnostic Microbiology
F. tularensis is a Hazard Group 3 pathogen, so it is imperative that
the laboratory is notified if tularemia is suspected so appropriate described in Brazil in the 1980s and was caused by a single clone of
containment precautions can be taken. The detection of F. tularensis
in a Gram-stained smear of aspirates or other samples is rarely
successful.
Specialized culture media such as cysteine–glucose–blood agar or
cysteine-enriched media, such as buffered charcoal yeast extract agar
(BCYE), have been developed for F. tularensis. It also grows on choco-
late blood agar supplemented with cysteine (e.g. IsoVitaleX™). Plates
should be incubated at 98.6°F (37°F) with carbon dioxide-enriched air.
F. tularensis grows slowly, taking 2–4 days to produce visible colonies,
and incubation should be extended for 3 weeks. Colonies appear blue-
gray, round, smooth and somewhat mucoid. They are β-hemolytic on
blood-containing media. Identification can be confirmed by slide
agglutination or fluorescent antibody staining. Alternatively, PCR tar-
geting the fopA or tul4 gene may be used.20 Real-time PCR for F.
tularensis is based on four target genes, ISFtu2, 23kDA, tul4 and fopA21
and real-time PCR has been used to differentiate subspecies tularensis
and holarctica.
Antibody titers reach detectable levels 10–20 days post-infection. A
fourfold rise in antibody titer or a single titer greater than 1:160 is
considered diagnostic. ELISA is more sensitive than agglutination
assays. IgG and IgM antibodies can persist for months or years and it
may be difficult to distinguish past from current infection. Infections
with Brucella spp. can give rise to antibodies that cross-react with
F. tularensis.
Chapter 183  Gram-Negative Coccobacilli 1617
Clinical Manifestations (see also Chapter 127)
The clinical manifestations of F. tularensis infection depend on the
portal of entry, the virulence of the infecting strain, infecting dose and
host immune status. Up to 30% of untreated infections can be lethal.
Management
Fluoroquinolones, such as ciprofloxacin, are effective treatment for
uncomplicated tularemia. For the treatment of more severe cases,
including pneumonic tularemia, aminoglycosides are indicated. A
high rate of relapse has been associated with the use of tetracyclines.
For postexposure prophylaxis, oral doxycycline or ciprofloxacin for 14
days should be considered.
Haemophilus spp.
Nature
Haemophilus spp. are small, pleomorphic, nonmotile, nonsporing
gram-negative rods or GNCB. They are aerobic and facultatively
anaerobic, and addition of 5–10% carbon dioxide to the incubation
atmosphere may enhance growth. The oxidase and catalase reactions
vary. The differential requirements for X and V factors are important
criteria for defining species of Haemophilus. X factor can be provided
by hemin, protoporphyrin IX or other iron-containing porphyrins.
X-dependent Haemophilus spp. cannot synthesize protoporphyrin
from δ-aminolevulinic acid, a process involving several enzyme-
mediated steps, some or all of which may be defective. V factor is nico-
tinamide adenine dinucleotide (NAD) or NAD phosphate or certain
unidentified precursors of these compounds. It is essential for the
oxidation–reduction processes.
The species of Haemophilus associated with human infections are
shown in Table 183-4. Haemophilus influenzae is the major human
pathogen in the group. There are six distinct antigenic types of encap-
sulated H. influenzae, designated a–f. H. influenzae type b (Hib) has a
polyribosyl-ribitol-phosphate (PRP) capsule and before the introduc-
tion of Hib conjugate vaccines, was associated with most invasive
infections. There is considerable debate as to whether H. aegyptius
(which is associated with purulent conjunctivitis) is a species distinct
from H. influenzae. The syndrome of epidemic purpura fulminans and
purulent conjunctivitis, known as Brazilian purpuric fever, was first
Haemophilus (H. influenzae biogroup aegyptius).22
The name Haemophilus quentini has been proposed for variant
strains of H. influenzae isolated from the genitourinary tract which
may be associated with maternal-neonatal infections.23 These strains
are distinguishable from typical strains of nontypeable Haemophilus
influenzae (NTHi) by multilocus enzyme electrophoresis, 16S rRNA
gene sequence and DNA hybridization. Most of these strains were
reported to be biotype IV and were described as a ‘cryptic genospecies
of Haemophilus biotype IV’. Subsequent studies, however, have
reported more diverse H. influenzae biotypes causing such infections.
H. haemolyticus is a pharyngeal commensal of low pathogenicity,
which can be hemolytic or nonhemolytic, and may be misidentified as
H. influenzae.24 V-factor requiring species of Haemophilus include H.
parainfluenzae, H. parahaemolyticus, H. paraphrohaemolyticus, H. pitt­
maniae and H. sputorum.23
H. ducreyi, the cause of chancroid, is not closely related to the other
Haemophilus spp.
H. aphrophilus and H. paraphrophilus have recently been combined
as a single species and reassigned to the genus Aggregatibacter as Aggre-
gatibacter aphrophilus.23 This species forms part of the HACEK group
(see below). H. segnis (Aggregatibacter segnis) is occasionally associated
with acute appendicitis and bacteremia.
DNA sequencing is increasingly being used for identification and
typing of Haemophilus spp. and MALDI-TOF is a valuable tool for
1618 SECTION 8  Clinical Microbiology: Bacteria

TABLE
183-4  Species of Haemophilus and Aggregatibacter Associated with Human Infection
REQUIREMENT FOR ACID FROM IgA1 protease
(probable
Species X factor V factor CO2 Hemolysis Sucrose Mannose Lactose virulence factor)

H. influenzae + + − − − − − +

H. aegyptius + + − − − − − +

H. haemolyticus + + − +§ − − − −

H. parainfluenzae − + − − + + − −

H. parahaemolyticus − + − + + − − +

H. paraphrohaemolyticus − + − + + − − −

H. sputorum − + − + + − − −

H. pittmaniae − + − + + + − −

H. ducreyi + − − v − − − −

Aggregatibacter aphrophilus* h v + − + + + −

A. segnis †
+ − − w w − −
A. actinomycetemcomitans − − − − + − −

h, A. aphrophilus requires hemin for primary isolation; w, weak reaction; v, variable.

*H. aphrophilus and H. paraphrophilus have been reassigned to the genus Aggregatibacter as Aggregatibacter aphrophilus sp. nov. comb.
†
H. segnis has been reassigned to the genus Aggregatibacter as Aggregatibacter segnis.
§
H. haemolyticus strains can be hemolytic or nonhemolytic.

identification in a clinical microbiology laboratory. Of note, the
genome of Haemophilus influenzae strain Rd was the first bacterial
genome to be fully sequenced.25
Epidemiology
Haemophilus influenzae is only found in humans and colonizes the
throat and nasopharynx, and, to a lesser extent, the conjunctivae and
genital tract. The respiratory tract is mainly colonized by H. parainflu-
enzae and nontypeable (noncapsulate) H. influenzae (NTHi). Carriage
rates for Hib are low (3–5%).
The epidemiology of invasive infections caused by H. influenzae is
distinct from noninvasive infections. NTHi is a major cause of mucosal
infections such as otitis media, sinusitis, conjunctivitis and exacerba-
tions of chronic obstructive pulmonary disease, associated with the
formation of biofilms.26
Pathogenicity
H. influenzae is transmitted by aerosols of respiratory secretions or by
direct contact with contaminated material. The primary event is colo-
nization of the nasopharynx. Prior infection with respiratory viruses
(e.g. influenza) predisposes to nasopharyngeal colonization by mecha-
nisms including obstruction to the outflow of respiratory secretions,
depression of local immunity and suppression of mucociliary clear-
ance. Contiguous spread (usually of NTHi) may result in noninvasive
mucosal infections including acute sinusitis and otitis media.
Colonization may be promoted by microbial factors such as fim-
briae and other adhesins, lipo-oligosaccharide (LOS) and IgA1 prote-
ase.26 LOS consists of two covalently linked moieties, lipid A (which
mediates endotoxic effects) and core oligosaccharide. LOS is a cilio-
toxin and is important for colonization, persistence and survival of the
bacteria in the human host.
IgA1 protease inactivates secretory IgA, facilitating bacterial access
to the mucosal surface. Fimbriae and the autotransporter proteins
Hap, HMW1/HMW2 and Hia/Hsf are associated with binding to
respiratory tract epithelium. Hap promotes bacterial entry into epithe-
lial cells. HMW1/HMW2 proteins are expressed by the majority of
NTHi but are not found in capsulate strains. Hia is found in nearly all
NTHi. Its analog, Hif, is expressed in almost all capsulate strains. Pos-
session of the outer membrane protein P2 is another important
virulence factor for NTHi. Lipoprotein D (LPD) impairs respiratory
ciliary function.
In the pre-Hib vaccine era, invasive infections, notably meningitis
and epiglottitis, were mainly caused by Hib and resulted from invasion
of the bloodstream. The capsule of type b H. influenzae is the single
most important virulence determinant for invasion because it protects
the organism from phagocytosis and complement-mediated lysis. The
rarity of infections in the first 2 months of life correlates with the pres-
ence of maternal antibodies to PRP, and the occurrence of infection
in early infancy with the absence of antibodies having such specificity.
As the mean level of PRP antibodies in the population rises, so Hib
infections become less common. It is unclear whether natural antibody
production is stimulated by exposure to Hib or to some other organ-
ism (e.g. E. coli K100) that possesses cross-reacting antigens.
With the widespread use of Hib conjugate vaccines, invasive Hib
infections have become uncommon and the majority of invasive infec-
tions are caused by NTHi or other capsulated serotypes. Invasive NTHi
infections mainly occur in very young infants and in the elderly. Preg-
nant women have an increased susceptibility to invasive NTHi disease,
which is associated with miscarriage, premature labor and post-partum
infection in the mother and the neonate.27
Prevention
VACCINATION
H. influenzae type b conjugate vaccines consist of the PRP covalently
linked to a protein carrier, such as tetanus toxoid, diphtheria toxoid,
a non-toxic mutant diphtheria and an outer membrane protein
complex from Neisseria meningitidis group B. These vaccines produce
a lasting anamnestic response, which is not age related. They can be
given to infants as young as 2 months of age and are also effective in
high-risk patients who have a poor response to polysaccharide vac-
cines. In the UK Hib vaccine is administered at 2, 3 and 4 months as
a component of a pentavalent vaccine (diphtheria, tetanus, acellular
pertussis, Hib and inactivated polio vaccine), with a booster dose (Hib
combined with meningococcus C vaccine) at 12–13 months of age.
Countries where Hib vaccine is routinely offered have witnessed a
dramatic decline in the occurrence of Hib infections (Figure 183-4).
Immunization of infants with conjugate Hib vaccine results in a reduc-
tion in the rate of nasopharyngeal colonization by Hib and a marked
 Chapter 183  Gram-Negative Coccobacilli 1619

Incidence of Haemophilus influenzae type b disease in England 1990–2012

Number of laboratory 350

reports Hib vaccine introduced All ages

<5 years
300

250

200

Hib catch-up
150 campaign

100
12-month Hib
booster introduced

50

0
90
90
91
92
93
93
94
95
96
96
97
98
99
99
00
01
02
02
03
04
05
05
06
07
08
08
09
10
11
11
12
19
19
19
19
19
19
19
19
19
19
19
19
19
19
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
Year (by quarter)

Figure 183-4  The incidence of Haemophilus influenzae type b disease in England, 1990–2012. Routine laboratory data combined with reference laboratory data. Data
supplied by Public Health England.

herd immunity effect. Hib vaccine is recommended post splenectomy

and for patients with functional asplenia, together with pneumococcal TABLE Candidate Vaccine Antigens of Nontypeable
183-5  Haemophilus influenzae (NThi)
and meningococcal vaccines.
There is currently considerable interest in developing vaccines
Protective In
for NTHi.28 A 10-valent pneumococcal conjugate vaccine, using H. Antigen Animal Model Comments
influenzae-derived protein D as a carrier protein for the pneumococcal
polysaccharide, showed protection against both pneumococcal and Lipo-oligosaccharide + Common epitopes among
(LOS) NTHi strains
noncapsulate H. influenzae otitis media.29 Outer membrane vesicles
(OMVs) present a combination of multiple heterologous antigens to P6 + Highly conserved lipoprotein
the immune system which may increase vaccine efficacy against
OMP26 + Highly conserved
hetero­­logous strains.30 Orally administered killed vaccines which aim
to prevent acute exacerbations of chronic obstructive pulmonary OMP P5 + Highly conserved
disease are under investigation. Several other candidate antigens are Lipoprotein D (LPD) + Recombinant deacylated form
being investigated (Table 183-5).31 (protein D) used as carrier
protein for pneumococcal
CHEMOPROPHYLAXIS conjugate vaccine
Oral rifampin for 4 days is recommended for eradicating carriage of OMP P2 >50% of total OMP of NTHi
H. influenzae type b and has been used to prevent secondary infection Heterogeneous and
in household and nursery contacts. Its effectiveness is unproven. conserved regions

Diagnostic Microbiology HtrA ±

HMW1/HMW2 + Found in >75% NTHi strains

Gram-stained smears of clinical samples such as cerebrospinal fluid or
pus may aid rapid diagnosis, although H. influenzae tends to stain P4 lipoprotein + Highly conserved
poorly. Dilute carbol fuchsin is a better counterstain than neutral red OMVs + Present a combination of
or safranin. Rapid tests for Hib capsular antigens, such as latex agglu- multiple heterologous
tination or a PCR-based method, can be applied to clinical material. antigens
False-positive latex agglutination results have been reported in cere-
brospinal fluid samples collected from children who had recently
received Hib vaccine.
1620 SECTION 8  Clinical Microbiology: Bacteria

Chocolate agar is used most commonly to culture Haemophilus
spp. The addition of bacitracin suppresses growth of other respiratory
organisms, thus assisting the recovery of H. influenzae from respiratory
samples. Cultures should be incubated at 98.6°F (37°C) in an aerobic
atmosphere enriched with 5–10% carbon dioxide. After 24 hours of
incubation, colonies of nontypeable H. influenzae (NTHi) are small,
circular, smooth and pale gray. Capsulated strains produce somewhat
larger, mucoid colonies, with a characteristic seminal odor. Phenotypic
identification depends on growth factor requirement. To perform the
‘disk test’, the culture is plated onto nutrient agar with paper disks
containing X factor alone, V factor alone, and both X and V factors.
After overnight incubation, growth is observed around the disks sup-
plying the required growth factor(s) (Figure 183-5). Besides H. influ-
enzae, a second X- and V-factor dependent species, H. haemolyticus, is
frequently found in the upper respiratory tract, and has long been
regarded as a commensal. Until recently, microbiologists relied on the
production of zones of β-hemolysis on horse blood agar to differenti-
ate H. haemolyticus from NTHi. However, 10-40% of H. haemolyticus
strains are nonhemolytic,24 so a scheme including hpd- and iga-based
PCR assays has been recommended to improve the identification of
H. haemolyticus.31
The capsular serotype of H. influenzae is normally determined by
slide agglutination using type-specific antisera. This method is prone
to misinterpretation, with problems of autoagglutination, cross-
reacting antisera and observer error. There are eight biotypes of H.
influenzae (I-VIII) and eight biotypes of H. parainfluenzae, based on
indole production, ornithine decarboxylase and urease activity. In
clinical practice the designation of biotype is of little value. The defini-
tive method of typing H. influenzae is capsular genotyping using a
PCR-based method.

Clinical Manifestations
Haemophilus influenzae is associated with two distinct types of infec-
tion: invasive and noninvasive infections (Table 183-6).

Figure 183-5  Growth factor requirement of Haemophilus influenzae. Strain of
H. influenzae sown on Columbia agar plate. Filter paper disks containing X factor,
V factor and both X and V factors were placed on the surface of the inoculated
plate, but colonies of H. influenzae grow only around the disk with both X and V
factors.
INVASIVE INFECTIONS
Introduction of the Hib conjugate vaccine has resulted in a dramatic
decline in the incidence of invasive H. influenzae infections. In coun-
tries with established Hib vaccination programmes, most invasive H.
influenzae infections are caused by NTHi with a median age of onset
of approximately 60 years. Disease in neonates is almost invariably due
to NTHi. Epiglottitis is almost always caused by Hib, and has a peak
incidence in children aged 2–3 years (see also Chapter 25). Most inva-
sive NTHi cases in children (40–70%) and adults (60–80%) occur in
individuals with underlying co-morbidities, particularly chronic respi-
ratory disease and impaired immunity. H. influenzae type b (Hib)
childhood meningitis and bacteremia remain common where the Hib
vaccine is not used.
Other less common invasive H. influenzae infections include pneu-
monia, endocarditis, periorbital cellulitis, osteomyelitis, septic arthritis
and a bacteremia with no obvious focus of infection. H. influenzae
pneumonia is a major cause of mortality in young children in LMIC.
H. parainfluenzae is the most common cause of non-H. influenzae
bacteremia and infective endocarditis. Aggregatibacter aphrophilus may
cause infective endocarditis and cerebral abscesses, Aggregatibacter
actinomycetemcomitans also causes infective endocarditis and is associ-
ated with periodontal disease. Aggregatibacter segnis is a rare cause of
endocarditis, acute appendicitis and bacteremia.

TABLE
183-6  Spectrum of Infections Caused by Haemophilus influenzae
Infection Age Group Affected Details Strains

Invasive infections: 90% children <4 years old* Hib meningitis principally affects children ~90% type b (Hib)
Meningitis 10% older children and adults aged 2 months–2 years. Epiglottitis occurs ~10% NTHi
Epiglottitis in slightly older children (peak incidence 1% types e and f
Pneumonia 2–3 years). Risk factors for invasive disease
Septic arthritis include overcrowding, attendance at
Osteomyelitis daycare centers, chronic illness and poor
Cellulitis access to healthcare facilities
Bacteremia

Neonatal and maternal sepsis Neonates >90% NTHi

Parturient mothers
Noninvasive respiratory infections: Children and adults >90% NTHi
Otitis media
Sinusitis
Conjunctivitis
Acute exacerbations of chronic bronchitis

Hib, Haemophilus influenzae type b.

NTHi, nontypeable (noncapsulated) Haemophilus influenzae.
*Figures reflect prevaccine incidence and age distribution.

NONINVASIVE INFECTIONS
Respiratory tract infections, acute exacerbations of chronic bronchitis,
otitis media, sinusitis and conjunctivitis are usually caused by NTHi
(see Chapter 27).
H. influenzae biogroup aegyptius is a distinct strain of nontypeable
H. influenzae. For more than a century this organism was known to
cause seasonal epidemics of acute purulent conjunctivitis, especially in
warmer climates. In 1984, a virulent clone of H. influenzae biogroup
aegyptius, emerged in Brazil as the cause of an acute febrile illness in
children with a high mortality rate.22 This infection, subsequently
known as Brazilian purpuric fever, caused a series of outbreaks and
sporadic cases almost exclusively in Brazil over the next decade but has
virtually disappeared since then.
Unusual infections due to H. influenzae include urinary tract infec-
tions, cholecystitis, salpingitis and epididymo-orchitis. These may be
associated with pre-existing damage, e.g. the presence of urinary stones
or gallstones. Haemophilus pittmaniae is part of the normal oropha-
ryngeal flora and may be isolated from various sites of infection,
including blood and bile. It may cause respiratory tract infections in
patients with chronic lung disease.
Management
Third-generation cephalosporins are the treatment of choice for Hae-
mophilus meningitis. They are bactericidal for H. influenzae, achieve
high concentrations in the meninges and cerebral tissues, and have
proved highly effective in clinical practice. Ampicillin is also effective
but resistance due to a β-lactamase is now high (for example, 13% of
Hib strains in the UK are β-lactamase-producers). The β-lactamase is
usually TEM-1 and, rarely, ROB-1. No TEM-type extended-spectrum
β-lactamases (ESBLs) have yet been detected in H. influenzae. In addi-
tion to β-lactamase production, BLNAR (β-lactamase negative ampi-
cillin resistant) strains occur, especially in Japan, South East Asia,
Spain and France. BLNAR strains have mutations in the ftsI gene,
which encodes penicillin-binding protein 3 (PBP3) involved in septum
formation of dividing cells. BLNAR are subdivided into genotypes I,
II (low gBLNAR) and III (high gBLNAR) linked to different minimum
inhibitory concentration (MIC) values: genotype I/II strains usually
have ampicillin MIC values between 0.5–2 µg/mL and genotype III
strains have MICs ranging between 1.0–16 µg/mL, often combined
with reduced susceptibility to cephalosporins. There are also BLPACR,
which are β-lactamase positive amoxicillin–clavulanic acid-resistant
strains. These strains combine β-lactamase production with the pres-
ence of PBP3 mutations. Compared to BLNAR with the same PBP3
mutations but without a β-lactamase, these BLPACR strains have
higher ampicillin MICs and similar amoxicillin–clavulanic acid and
cephalosporin MICs. BLPACR can also be subdivided based on the
specific ftsI mutations.26,32
For the treatment of less serious respiratory infections, oral antibi-
otics including amoxicillin (if β-lactamase negative), amoxicillin–
clavulanate, tetracycline or macrolides are usually effective.
Haemophilus ducreyi
Epidemiology
Haemophilus ducreyi (see Chapter 64) which causes chancroid, or soft
sore, has declined in importance as a sexually transmitted pathogen in
most countries where it was previously endemic (Central and South-
ern Africa, South East Asia, India, and the Caribbean). The epidemiol-
ogy of chancroid is not well characterized because of syndromic
management and inadequate diagnostic laboratories and surveillance
systems. However, it still occurs sporadically in other countries, and is
more common in non-white, uncircumcised males in low socioeco-
nomic groups. Genital ulcers, including chancroid, are associated with
increased transmission of HIV. Co-infection with Treponema pallidum
or herpes simplex virus (HSV) is common. There have been recent
Chapter 183  Gram-Negative Coccobacilli 1621
reports of H. ducreyi causing chronic skin ulceration in Papua New
Guinea.
Pathogenicity
The pathogenesis of chancroid is poorly understood. Haemophilus
ducreyi gains access via a break in the epithelium and establishes infec-
tion in a focal area of mucosa or skin in the genital tract. Hemolysin
is important in epithelial cell invasion and ulcer formation. Superoxide
dismutase enzymes contribute to survival of the bacteria within the
host. Potential virulence factors include fimbriae, pili, LPS, cytotoxins
and outer membrane proteins (OMP).
Prevention
The use of condoms dramatically reduces the transmission of H.
ducreyi. All sexual partners should be identified by contact tracing
and treated. Education is vitally important. H. ducreyi hemolysin or
hemoglobin receptor (HgbA) are possible candidates for vaccine
development.
Diagnostic Microbiology
Gram-stained examination of material from ulcers or buboes may
reveal pleomorphic GNCB or short rods. The organisms characteristi-
cally resemble ‘shoals of fish’. However, microscopy is not recom-
mended as a diagnostic test, as ulcer material is generally contaminated
and direct Gram staining identifies only 10% of culture-positive
cases.33 Specimens collected from the base and margins of ulcers or by
aspirating a bubo should be plated directly onto culture media and
incubated in a humid atmosphere with 5% carbon dioxide at 91.4°F
(33°C) for 2–3 days.
H. ducreyi is a fastidious organism that grows slowly on chocolate
agar. Alternatives include GC agar supplemented with 1–2% bovine
hemoglobin plus 5% fetal calf serum (or 0.2% activated charcoal) or
Mueller–Hinton agar enriched with 5% chocolatized horse blood.
Both of these media should be supplemented with 1% IsoVitaleX
and 3 mg/L vancomycin to prevent overgrowth by gram-positive
organisms.
The appearance of colonies of H. ducreyi varies with the medium
used. Typically they are pinpoint, yellow-gray and translucent or semi-
opaque. Cultures often appear mixed, with a variety of colonial forms.
The colonies are very cohesive and can be pushed across the agar
surface with an inoculating loop.
H. ducreyi requires X factor but not V factor for growth. It can be
identified using a number of rapid test systems (for example API-ZYM
and RapIDNH) and MALDI-TOF (which can also identify strain-
specific biomarkers from different patients). PCR is the most sensitive
diagnostic technique and has been shown to be 95% sensitive com-
pared to culture. Multiplex PCR tests that also amplify Treponema
pallidum and HSV have been developed. PCR may be negative in some
culture-positive cases due to the presence of Taq polymerase inhibitors
in DNA extracts from genital ulcers. Antigen detection and serological
methods have been described but are not in common use.
Sequencing of 16S rRNA and nucleic acid hybridization studies
suggest H. ducreyi is a valid member of the family Pasteurellaceae,
but is more similar to the Actinobacillus rather than the genus
Haemophilus.
Clinical Manifestations
These are described in Chapter 64.
Management
The susceptibility of H. ducreyi to antimicrobial agents varies from one
geographic area to another. Azithromycin, ceftriaxone or ciprofloxacin
may be used, but strains with intermediate resistance to ciprofloxacin
have been reported. HIV-infected patients require careful follow-up
due to reports of treatment failure with single dose regimens. Buboes
should be drained to prevent sinus formation.
1622 SECTION 8  Clinical Microbiology: Bacteria

HACEK Group TABLE Legionella Species Associated With

183-7  Human Disease
Nature
Species Number of Serogroups
The HACEK group comprises the Haemophilus species, Aggregati-
bacter actinomycetemcomitans (formerly Actinobacillus actinomy­ Legionella pneumophila 16
cetemcomitans), Aggregatibacter aphrophilus (formerly Haemophilus Legionella anisa 1
aphrophilus and Haemophilus paraphrophilus), Aggregatibacter segnis
(formerly H. segnis), Cardiobacterium hominis, Cardiobacterium val- Legionella bozemanii 2
varum, Eikenella corrodens and Kingella species (Kingella kingae, Kin- Legionella cincinnatiensis 1
gella denitrificans and Kingella oralis). This group of fastidious GNCB
Legionella dumoffii 1
account for 1–3% of cases of infective endocarditis.34
All of the HACEK group are small, fastidious, pleomorphic GNCB Legionella feeleii 2
and are commensals of the oropharyngeal/respiratory tract. They cause Legionella gormanii 1
endocarditis and a range of other infections.35-38 K. kingae has recently
emerged as a major cause of septic arthritis in children less than 2 years Legionella hackeliae 2
of age39 and has been associated with outbreaks of invasive infection Legionella jordanis 1
in daycare facilities.40 K. kingae has a polysaccharide capsule and viru-
lence factors include type IV pili and an RTX toxin which is cytotoxic Legionella longbeachae 2
to synovial cells, respiratory epithelium, and macrophage-like cells.41 Legionella lansingensis 1

Legionella lytica* 1
Diagnostic Microbiology
Legionella maceachernii 1
The slow growing HACEK group requires enriched culture media and
an increased carbon dioxide tension. Cardiobacterium hominis and Legionella micdadei 1
Eikenella corrodens usually require hemin (X factor) and carbon Legionella oakridgensis 1
dioxide for primary isolation and may be misidentified as Haemophilus
spp.; these requirements are lost on subculture. The colonies of E. Legionella parisiensis 1
corrodens have a characteristic ‘bleach-like’ odor and often ‘pit’ the Legionella sainthelensi 2
surface of the agar. Older cultures of E. corrodens may appear yellow.
Legionella tucsonensis 1
Colonies of K. kingae produce slight β-hemolysis on blood agar and
may be misidentified as pathogenic Neisseria species as they grow on Legionella wadsworthii 1
Neisseria selective agar. A. actinomycetemcomitans colonies may be
*Formerly called Legionella-like amebal pathogen (LLAP).
firm, adherent, star-shaped and difficult to remove from the agar
surface. When Aggregatibacter spp. produce extracellular slime, cul-
tures appear sticky on primary isolation. MALDI-TOF is a reliable tool
for identification of HACEK organisms.42 ordinary media. They generally grow well on BCYE supplemented with
L-cysteine, α-ketoglutarate and ferric ions, adjusted to pH 6.9. Some
Clinical Manifestations species of Legionella grow poorly on BCYE but can be isolated by
These organisms cause infective endocarditis and a variety of other co-cultivation with amebae. Originally called Legionella-like amebal
infections (see Chapter 51). pathogens (LLAP), these have been renamed L. lytica, L. drozanskii, L.
falloni and L. rowbothamii.44
Prevention There are more than 50 species of Legionella and human infections
have been documented for 19 of these species (Table 183-7). Infections
Approximately 60% of cases of endocarditis caused by HACEK organ- due to species other than L. pneumophila are rare and usually occur in
isms are associated with poor oral hygiene, bad dentition or recent immunocompromised hosts. The exception is Legionella longbeachae
dental work.43 Prevention of endocarditis should therefore be based on serogroup 1 which causes community-acquired pneumonia in Western
good dental and oral hygiene in patients at risk of endocarditis. Australia, possibly associated with handling compost and soil.45

Management
The treatment of endocarditis caused by HACEK organisms should Legionella spp. are distributed worldwide, in fresh water streams,
be based on antimicrobial susceptibility tests, including tests for
β-lactamase activity. Currently, third-generation cephalosporins are
the agents of choice. While Eikenella corrodens is generally susceptible
to penicillin, broad-spectrum antibiotics are usually used to treat
mixed infections, such as human bites.
Legionella spp.
Nature
Legionella spp. are slender, aerobic, noncapsulated, nonspore-forming,
pleomorphic gram-negative coccobacilli. They may stain poorly in
clinical material but can be filamentous in older cultures. Basic fuchsin
may be a better counterstain. Legionella are motile with a single polar
flagellum.
Legionella can grow in the temperature range 68–107.6°F (20–
42°C). They are nutritionally fastidious and cannot be cultured on
Epidemiology
rivers, ponds, lakes and mud. The organisms can survive in moist
environments for long periods and can withstand temperatures
of 32–154°F (0–68°C) and chlorination. They proliferate in air-
conditioning cooling towers, hot water systems, shower heads, taps,
whirlpool spas and respiratory ventilators, and form biofilms which
render them less susceptible to biocides and chlorine. The growth of
Legionella spp. is aided by coexisting bacteria and algae, which provide
nutrients, and protozoa, in which the Legionella spp. can live and
multiply.
The exact incidence of Legionella infections is unknown. Exposure
to Legionella spp. occurs fairly frequently and serological surveys
suggest that asymptomatic infection and seroconversion are common.
Large-scale surveys of pneumonia suggest that Legionella spp. cause
2–5% of community-acquired pneumonia and up to 30% of nosoco-
mial pneumonia.
Cases may be sporadic or occur as part of an outbreak. The original
description of Legionnaires’ disease was of an outbreak of a febrile

respiratory illness in delegates at an American Legion convention in
Philadelphia in 1976. A total of 221 people developed pneumonia and
34 died.46 Retrospective studies have revealed that the first proven case
occurred in 1947 and the first known epidemic was in 1965 in Wash-
ington, DC.
Legionnaire’s disease usually occurs in the middle-aged and heavy colonization with Legionella spp., avoiding ‘dead spaces’, stagna-
elderly, especially in smokers, the immunocompromised and people
with impaired respiratory and cardiac function. Cases have been build-up of sediment. Regular monitoring for Legionella spp. is advis-
documented in children. Person-to-person spread has not been
demonstrated.
Legionella is a good example of an organism that has been present
in the environment for a long time but has been brought into close
contact with humans as a result of technical developments. Organisms
are disseminated via contaminated water droplets from nebulizers and
humidifiers and in aerosols from cooling towers, whirlpools and evap-
orative condensers. Infection arises from inhalation of contaminated
aerosols, direct instillation during surgery or possibly by ingestion of
contaminated water. Hospital equipment that has been rinsed in tap
water prior to use may be a source of infection.
L. pneumophila contains 16 different serogroups. More than 80%
of human infections are caused by Legionella pneumophila serogroup
1. This serogroup can be divided into subtypes, on the basis of mono-
clonal antibody typing or genotyping, which is important during out-
break investigation.
Pathogenicity
Legionella spp. are transmitted to humans via aerosols. Droplet nuclei
of less than 5 µm reach the alveoli, where alveolar macrophages ingest
the organisms. Legionella is a facultative intracellular parasite and mul-
tiplies freely within the macrophages. The bacteria bind to alveolar
macrophages via complement receptors and are engulfed in phago-
somes. The bacteria resides in a specialized ‘Legionella-containing
vacuole’ (LCV), and phagolysosome fusion is inhibited. LCVs provide
an intracellular niche for replication and help prevent release of bacte-
rial components into the cytoplasm. The Dot/Icm type IVB secretion
system effector SdhA maintains LCV integrity (see below). The bacte-
ria respond to depletion of nutrients by expressing virulence proteins,
which cause host cell lysis and release of bacteria. These bacteria are
then taken up by other macrophages and the infection cycle is
repeated.47 This process produces chemotactic substances that attract
polymorphonuclear leukocytes and monocytes, and the inflammatory
response results in a destructive pneumonia.
There are two phases in the life cycle of Legionella pneumophila – an
intracellular replicative phase (RP) and an infectious nonreplicating
transmissive phase (TP). Bacteria in the RP are avirulent, non-
flagellated and sodium resistant, whereas those in the TP are virulent,
flagellated and motile. Complex regulatory systems govern the dif-
ferentiation between these two phases.48
Multiple virulence factors are involved in initial attachment and
early intracellular infection, including type IV pili, Hsp60, the pore-
formation protein RtxA, the macrophage infectivity potentiator Mip
and the macrophage-specific infectivity protein MilA. The icm (intra-
cellular multiplication) and dot (defect in organelle trafficking) groups
of genes form a type IV secretion system and enable bacterial survival
and intracellular replication. Other effectors (including RalF LidA;
LepA/B), regulators (RpoS; LetA), a type II protein secretion system,
iron-uptake systems and iron-acquisition genes (feoB; iraA; ccmC)
have been described.
Prevention
Prevention of legionellosis depends upon identifying the environmen-
tal source and reducing colonization. The simple preventive measure
of keeping water from hot taps ‘hot’ and water from cold taps ‘cold’
should always be observed. Periodic superheating and flushing of
water supplies may be useful during an outbreak. The continuous
chlorination of hot and cold water service systems, after initial disin-
fection, is not recommended because chlorine has a limited ability to
Chapter 183  Gram-Negative Coccobacilli 1623
penetrate biofilms and inactivate sessile micro-organisms. Treatment
using copper/silver ionization or chlorine dioxide can be used. Despite
numerous costly measures it is often impossible to eliminate Legionella
spp. from water supplies.49
New water systems should be designed to minimize the risk of
tion, materials that support the growth of Legionella spp. and the
able, particularly in high-risk areas.
Vaccination may be a future option for highly susceptible patients.
Diagnostic Microbiology
Legionella spp. can be cultured from sputum, endotracheal aspirates,
bronchoalveolar lavages, lung biopsies and pleural fluid. Sputum
samples should be diluted in distilled water because saline can inhibit
some Legionella spp. Gram-stained smears may reveal poorly staining
gram-negative rods or coccobacilli. Direct immunofluorescence using
a monoclonal antibody to a major outer membrane protein common
to all serogroups of L. pneumophila gives a rapid diagnosis, but is less
sensitive than culture and only detects L. pneumophila.
Cultures should therefore also be carried out, using a selective and
non-selective agar incubated at 98.6°F (37°C) in 2–5% carbon dioxide
for up to 14 days. Legionella species will grow on Legionella agar base
(BCYE) supplemented with L-cysteine. Those colonies that fail to grow
on the BCYE without L-cysteine, are presumptive Legionella. Growth
on both plates indicates it is not Legionella.
Colonies of Legionella may take 3–5 days to appear, and have a
ground-glass appearance, which may be white, gray, pale blue or
purple-tinged (Figure 183-6). Some species, other than L. pneumoph-
ila, fluoresce blue-white under long-wave UV light while others fluo-
resce dull yellow or brick red. Legionella lytica (and other LLAPs) can
be isolated on BCYE if the plates are incubated at 86°F (30°C).
Colonies that grow on L-cysteine-containing BCYE, but not on
BCYE lacking L-cysteine, which are catalase-positive, oxidase-negative
and have characteristic Gram stain, should be regarded as presumptive
Legionella spp. Biochemical tests contribute little to identification.
Serologic typing using direct or indirect fluorescent antibody tests
with polyclonal or monoclonal antibodies will confirm the identity.
Sequence analysis of the mip gene can be used to differentiate most
Legionella spp.50 and real-time PCR-based on the mip gene can provide
a rapid diagnosis.
Urine samples can be examined for Legionella soluble antigen using
either an enzyme immunoassay or a radioimmunoassay kit, or com-
mercially available immunochromatographic tests. These rapid tests
are specific for L. pneumophila serogroup 1, which is most common,
Figure 183-6  Colonies of Legionella pneumophila on BCYE agar, showing the
typical ground-glass appearance. (With permission from Harrison T.G., Taylor
A.G., eds. A laboratory manual for Legionella. Chichester: John Wiley; 1998.)
1624 SECTION 8  Clinical Microbiology: Bacteria

but will not detect other species. The antigen persists in urine for 1–2
weeks, and occasionally for months. TABLE Candidate Vaccine Antigens of Moraxella
183-8  catarrhalis
The most commonly used serological technique is an indirect fluo-
rescent antibody test (IFAT), but ELISA or rapid microagglutination
Antigen Action Comments
tests are also available. A fourfold or greater rise in antibody titer in a
patient who has had clinical pneumonia indicates Legionella infection. UspA1, A2 and Adhesin, interfere Highly immunogenic
Serology is useful in epidemiological studies, rather than diagnosis of A2H with complement- Contains both conserved
mediated killing and variable regions
acute diseases, as it may take weeks or months for the antibody titer
to rise, and antibodies can persist for years. The only validated sero- MID/Hag IgD binding Highly immunogenic
logic test is for L. pneumophila serogroup 1, and patients with Campy- Adhesin Contains both conserved
lobacter infections may have cross-reacting antibodies. Hemagglutinin and variable regions

McaP Adhesin, transporter Highly conserved

Clinical Manifestations OMP CD Porin, mucin-binding Highly conserved
Asymptomatic Legionella infections are relatively common. Symptom-
OMP M35 ? porin Highly conserved
atic Legionella infection can present as a severe pneumonia (Legion-
naires’ disease) or an acute influenza-like illness (Pontiac fever). See MhaB1, MhaB2, Filamentous –
Chapter 28. MhaC haemagglutinin,
adhesion

Management OlpA ? Highly conserved

Immunogenic
For an acutely ill patient with Legionnaires’ disease, quinolones are
the treatment of choice; macrolides are an alternative. The co- TbpA, TbpB Transferrin-binding Immunogenic
proteins
administration of rifampin does not appear to give any additional
benefit. Cases of Pontiac fever usually resolve spontaneously. LbpA, LbpB Lactoferrin-binding Immunogenic
proteins

OMP B2/CopB Iron acquisition Immunogenic

Moraxella spp.
OMP E ? porin Immunogenic

Nature LOS A, LOS B,

LOS C
Lipo-oligosaccharide,?
adhesin
Immunogenic

Moraxella spp. are gram-negative short rods, coccobacilli or, in the

case of M.catarrhalis, diplococci (GNDC) that phenotypically resemble
Neisseria spp. diplococci (GNDC) that phenotypically resemble Neis-
seria spp. They are strictly aerobic, oxidase-positive, catalase-positive,
DNAse-positive, non-encapsulated and asaccharolytic. Moraxella
catarrhalis causes upper and lower respiratory tract infections in chil-
dren and adults.
Moraxella lacunata is associated with conjunctivitis. Moraxella non-
liquefaciens is an upper respiratory tract commensal which may be a
secondary invader in respiratory infections. Moraxella osloensis is a
genital tract commensal which may be misidentified as Neisseria gonor-
rhoeae. M. osloensis has also been reported in cases of septic arthritis,
osteomyelitis and bacteremia. Moraxella atlantae can cause bacteremia
in immunocompromised patients.
Epidemiology
Moraxella catarrhalis is exclusively found in humans. Up to 75% of
children are colonized within the first year of life. Adults with chronic
lung disease are more likely to be carriers than healthy adults. Naso-
pharyngeal carriage rates are significantly higher in autumn and
winter.51
Pathogenicity
M. catarrhalis is a mucosal pathogen, causing infections of the upper
respiratory tract in young children and the lower respiratory tract in
adults with chronic lung disease. M. catarrhalis is the third most
common cause of otitis media in children (after Haemophilus influen-
zae and Streptococcus pneumoniae). Binding of M. catarrhalis to host
epithelial cells and extracellular matrix (ECM) is multifactorial. Adhes-
ins include ubiquitous surface proteins (Usp) A1, A2 and A2H, M.
catarrhalis IgD-binding protein hemagglutinin (MID/Hag), OMP CD
and OMP M35, Mha B1 and B2 and C, OlpA, McaP and LOS.52 Fol-
lowing colonization of the host epithelium and ECM, M. catarrhalis
invade host cells, enabling evasion of both humoral and cellular immu-
nity and antimicrobial therapy. The organisms persist within the enzymes (BRO-1 and BRO-2) are unique to this genus and are
cytoplasm in vacuoles. M. catarrhalis can form biofilms, which
are important in the development of acute and chronic respiratory
mucosal infections.
Prevention
A number of vaccine candidates are under development or clinical
testing. However the phase-variable expression of several important
virulence factors (e.g. UspA1, UspA2, UspA2H, MID/Hag) and high
genotypic heterogeneity of M. catarrhalis suggest that a multicompo-
nent vaccine will be required to prevent colonization and pathogenesis
(Table 183-8).53
Diagnostic Microbiology
M. catarrhalis grows well on blood and chocolate agar, producing
small, nonhemolytic, grayish-white colonies that slide across the agar
surface, like a hockey puck, when pushed with a bacteriologic loop.
Gram-stained films reveal GNDC but M. catarrhalis often resists decol-
orization and thus may appear gram-positive. M. catarrhalis is DNAse
positive, reduces nitrate and hydrolyses tributyrin (enabling differen-
tiation from most Neisseria spp.). PCR (multiplexed with other respi-
ratory pathogens, or bacteria causing middle ear infections) and
MALDI-TOF are useful diagnostic tools.
Clinical Manifestations (see also Chapter 26)
M. catarrhalis causes upper respiratory tract infections, otitis media
and sinusitis in children. In adults M. catarrhalis is associated with
acute exacerbations of chronic bronchitis, pneumonia or invasive
infections, including bacteremia, meningitis, septic arthritis and
endocarditis.
Management
Most strains of M. catarrhalis are β-lactamase-positive. The β-lactamase
inducible; therefore ampicillin therapy should be avoided.54 Many
infections due to M. catarrhalis can be treated with oral antibiotics,
such as amoxicillin–clavulanate. Other options include tetracyclines,

macrolides and fluoroquinolones, although antibiotic resistance to
these agents has been reported.
Pasteurella spp.
Nature
Pasteurella spp. are very small, nonmotile, nonspore-forming gram-
negative bacteria that are coccoid, oval or rod-shaped. Pasteurella spp.
grow on ordinary laboratory media at 98.6°F (37°C), and most species
are catalase-positive and oxidase-positive. There are 15–20 species cur-
rently included in the genus Pasteurella, but some are more closely
related genotypically to Actinobacillus. The type species is P. multocida,
which is subdivided into four subspecies – multocida, septica, gallicida
and tigris.
Epidemiology
Pasteurella spp. are distributed worldwide. They are commensals or
parasitic organisms in the upper respiratory tract and gastrointestinal
tracts of many domestic and wild animals and birds.
Many Pasteurella spp. are opportunistic pathogens that can cause
endemic disease and are associated increasingly with epizootic out-
breaks. Pasteurella multocida is found in the oropharynx of 50–90% of
domestic cats, 50–70% of dogs and 50% of pigs. It causes hemorrhagic
sepsis and bronchopneumonia in cattle and water buffalo, pneumonia
in pigs and sheep, swine atrophic rhinitis, snuffles in rabbits and fowl,
and fowl cholera in chickens, ducks and turkeys. It is generally carried
asymptomatically by cats and dogs, and humans become infected
through contact with infected animals.
Pasteurella multocida can remain viable in soil and water for up to
4 weeks and may survive in animal carcasses for up to 3 months. Most
human infections are caused by P. multocida subsp. multocida and P.
multocida subsp. septica. Occasionally, Pasteurella canis, Pasteurella
dagmatis or Pasteurella stomatis may be implicated.
Pathogenicity
Pasteurella multocida is transmitted to humans by contact with infected
animals, usually following bites or scratches from cats or dogs. Respi-
ratory tract infections may occur through airborne transmission of the Enterobacteriaceae.
(see Chapter 73). Occasionally, an animal source of infection is not
documented.
Virulence in P. multocida is associated with the degree of encapsula-
tion, which prevents phagocytosis. There are five serogroups based on
capsular antigens – A, B, D, E and F – and 16 serovars (1–16) based
on lipopolysaccharide (LPS) antigens. Capsular types A and D produce
a dermonecrotic toxin (PMT), encoded by the toxA gene, which mod-
ulates the immune response. Enhanced survival in the host environ-
ment also depends on membrane lipopolysaccharide that confers
serum resistance, iron-acquisition mechanisms that enable growth,
surface components such as fimbriae, which provide adherence prop-
erties, extracellular matrix-degrading enzymes, such as neuramini-
dases, hyaluronidases and proteases that facilitate colonization and/or
dissemination.55
Prevention
The only way of effectively preventing Pasteurella infections is avoiding
contact with domestic or wild animals. Prophylactic antibiotics may
be indicated after cat and dog bites, particularly if the patient is immu-
nocompromised or diabetic or the wound affects the hands or face.
Vaccine research is focused on controlling animal disease, as the inci-
dence of human infections is relatively low.
Diagnostic Microbiology
A clinical history of animal bites should alert the laboratory to the
possibility of Pasteurella spp. After 24 hours of culture on blood agar
Chapter 183  Gram-Negative Coccobacilli 1625
plates at 98.6°F (37°C) P. multocida appear as small, gray, nonhemo-
lytic colonies, with a strong odor of indole. P. multocida does not grow
on MacConkey agar, but can grow poorly on some cystine lactose
electrolyte deficient (CLED) agars. The organisms often show bipolar
staining in methylene blue preparations. Identification can be con-
firmed by biochemical tests, serology or MALDI-TOF.
Clinical Manifestations
Pasteurella multocida causes opportunistic soft tissue infections in
humans, especially the elderly and immunocompromised. Up to 75%
of cat bites and 50% of dog bites are contaminated with P. multocida.
The infection may spread rapidly along fascial planes. Patients
with underlying respiratory disease or cirrhosis are more susceptible
to respiratory infections and systemic infections respectively (see
Chapter 73).
Management
Penicillin is the treatment of choice for Pasteurella infections.
Other agents with good activity include ampicillin, amoxicillin–
clavulanate, cefuroxime and ciprofloxacin. Broad-spectrum antibiotics
are usually recommended for infected animal bites, which are often
polymicrobial.
Yersinia spp.
Nature
Yersinia spp. are members of the Enterobacteriaceae. They are short,
pleomorphic gram-negative rods or GNCB, which often exhibit
bipolar staining. Yersinia pestis is nonmotile. Other species are non-
motile at 98.6°F (37°C) but motile at temperatures less than
86°F (30°C) by means of peritrichous flagella. They are nonlactose
fermenters, oxidase-negative and catalase-positive. Yersinia pestis is
urease-negative; Y. enterocolitica and Y. pseudotuberculosis are both
urease-positive. Yersinia spp. grow on simple laboratory media and are
tolerant of bile salts. The optimal temperature for growth is 82–86°F
(27.8–30°C). They do not form spores or capsules, but Y. pestis pro-
duces a capsule-like envelope. They share antigens with other members
In total, there are 11 species of Yersinia, three of which are impor-
tant human pathogens associated with zoonotic infections – Y. pestis
causes plague and Y. pseudotuberculosis and Y. enterocolitica give rise
to yersiniosis. These two diseases are very different so are described
separately below. Genomic studies suggest that Y. pseudotuberculosis
(serotype O:1b) is the direct evolutionary ancestor of Y. pestis.56 This
occurred 15–20 000 years ago by lateral gene transfer and gene inacti-
vation. The acquisition of the pFra plasmid by Y. pestis, plus the ability
to express chromosomally-encoded proteins not expressed in Y. pseu-
dotuberculosis, conferred transmissibility from one mammalian host to
another via a vector flea.
Yersiniosis
The clinical features of yersiniosis range from gastroenteritis, entero-
colitis, mesenteric adenitis, reactive arthritis to sepsis (see Chapters
43 and 47). Recipients of blood transfusions, or patients with iron
overload, are at particular risk of infection with this siderophilic
bacterium.
EPIDEMIOLOGY
Yersinia enterocolitica is harbored in the gastrointestinal tract of a range
of mammals, including rodents, cattle, sheep, pigs, cats and dogs.
Infected animals tend to become chronic carriers and excrete large
numbers of bacteria, which may contaminate water and dairy prod-
ucts. Humans are infected by eating inadequately cooked meat (espe-
cially pork) or other contaminated food, or through contact with an
1626 SECTION 8  Clinical Microbiology: Bacteria

infected domestic animal. In the USA, outbreaks associated with dairy
products, chocolate and milk have been reported. The ability of Y.
enterocolitica to grow at 39°F (4°C) means that refrigerated meat and
meat products can become a potent source of infection. Yersinia
enterocolitica sepsis has been reported following transfusion of blood
stored for more than 3 weeks at 39°F (4°C).
Yersinia enterocolitica are classified phenotypically into six bio-
groups, five of which (1B and 2–5) are regarded as pathogens. There
are more than 57 ‘O’ serogroups, based on the lipopolysaccharide
surface antigen, only a few of which have been associated with human
or animal disease. In Europe, serogroup O:3 is most important fol-
lowed by O:9, whereas in the USA, where yersiniosis is less common,
serogroup O:8 predominates.
Yersinia pseudotuberculosis infection is less common. The organism
is harbored in the gastrointestinal tract of rodents, farm animals and
birds. Human infections have been reported worldwide, but as with Y.
enterocolitica, it is most common in northern Europe.
PATHOGENICITY
Yersinia enterocolitica and Y. pseudotuberculosis usually enter the body
via the gastrointestinal tract. Organisms invade the ileal mucosa via
specialized follicle-associated epithelial cells (M cells) involved in
antigen uptake. Adherence and invasion of epithelial cells requires two
chromosomally-encoded proteins, Invasin and Ail.57 Following inva-
sion the bacteria enter Peyer’s patches and macrophages, and can
survive intracellularly and multiply. In Peyer’s patches the organisms
express Yad A, which protects them against phagocytosis. Yad A and
Ail facilitate dissemination of bacteria to regional lymph nodes. Yad A
also helps adhesion to collagen, which is crucial in the development of
reactive arthritis in Y. pseudotuberculosis infection.
Y. enterocolitica usually causes acute gastroenteritis, while Y. pseu-
dotuberculosis infection causes mesenteric adenitis, which can mimic
acute appendicitis. Systemic dissemination may cause sepsis and
splenic/hepatic abscesses. A reactive polyarthritis can occur, particu-
larly in HLA-B27 positive patients. Erythema nodosum is also a re­­
cognized post-infectious complication. A clone of Y. pseudotuberculosis
which produces a superantigenic exotoxin mitogen (YPM) causes a
toxic-shock like syndrome called Far East scarlet-like fever (or Izumi
fever in Japan).58
PREVENTION
Prevention of yersiniosis depends on good animal husbandry and
careful slaughtering techniques. Meat should not be consumed raw
and should not be stored at 39°F (4°C) for prolonged periods before
consumption. There is no effective vaccine.
DIAGNOSTIC MICROBIOLOGY
Material for culture includes stool, blood, lymph nodes, abscesses and
food samples. Specimens should be cultured on blood and MacConkey
agars at 80°F (26.7°C) for 24 hours. A selective medium, CIN (cefsu-
lodin, irgasan, novobiocin), is available.

TABLE
183-9  Key Virulence Factors of Yersinia species
Location Y. pestis Y. enterocolitica Y. pseudotuberculosis Action

Phospholipase D pFra plasmid + – – Promotes survival of Y. pestis in flea

vector

F1 antigen pFra plasmid + – – Fibrillar capsule inhibits macrophage

activity

Plasminogen activator (Pla) pPCP1 plasmid + – – Activates mammalian plasminogen

and anticomplementary serum
resistance
Promotes dissemination from
subcutaneous inoculation of
Y. pestis

Yersinia outer protein (YOPs) Plasmid encoded - + + Invasion epithelial cells

Yad A

Lipopolysaccharide Chromosomal + + + Endotoxin

Yersiniabactin Chromosomal + + + Siderophore-essential for full

virulence of Y. pestis by
subcutaneous inoculation

YOPs Chromosomal + + +

Yad BC + + + Adhesion

YopH + + + Antiphagocytic

YopE + + + Antiphagocytic

YopT + + + Antiphagocytic

YpKA/YopO Antiphagocytic

pH 6 antigen Chromosomal + + + Fimbrial adhesin

Binds host lipoprotein
Antiphagocytic

Invasin Chromosomal – + + Invasion of epithelial cells

Ail Chromosomal – + + Host cell attachment and serum

resistance
HPI (high pathogenicity Chromosomal + (pgm locus)* Biotype 1B Serotype O1 Iron uptake
island)

*pgm locus includes (i) hms (hemin storage); (ii) irp1 and irp2 (iron-repressible high molecular weight proteins HNWP1 and HMWP2) and (iii) fyuA or psn gene (ferric
yersiniabactin uptake or pesticin sensitivity).
 Chapter 183  Gram-Negative Coccobacilli 1627

Cold enrichment of heavily contaminated samples such as feces
involves inoculation into phosphate-buffered saline and incubation at
39°F (4°C) for at least 3 weeks. To reduce contamination the broth can
be treated with potassium hydroxide. The broth is subcultured at
weekly intervals on to MacConkey or CIN agar. Y. enterocolitica colo-
nies give a ‘bulls-eye’ appearance on CIN agar, while Y. pseudotuber-
culosis colonies are smaller on CIN, with a deep-red center and a sharp
border surrounded by a translucent zone. Confirmation of identifica-
tion is usually by biochemical or molecular tests or MALDI-TOF. The
serotype of Yersinia can be determined using slide agglutination tests.
A serologic diagnosis depends on demonstrating a significant rise of
serotype-specific antibodies. Yersinia pseudotuberculosis is rarely iso-
lated from feces.
CLINICAL MANIFESTATIONS
Yersiniosis encompasses a variety of clinical presentations including
gastroenteritis, enterocolitis, mesenteric adenitis, and reactive
arthritis.
MANAGEMENT
Yersinia enteritis and mesenteric adenitis do not generally require anti-
microbial chemotherapy. Sepsis, extra-intestinal foci of infection and
enteritis in immunocompromised patients should be treated with pestis tends to form chains.
antimicrobials, such as aminoglycosides, quinolones, tetracyclines or
third-generation cephalosporins. Yersinia pseudotuberculosis is usually
sensitive to benzylpenicillin and ampicillin.
Plague
Plague is a zoonotic infection caused by Y. pestis, and has caused dev-
astating pandemics such as the ‘Plague of Justinian’ in 541–3ce and
the Black Death of the Middle Ages. Plague is still responsible for many
thousands of human infections annually.59 The clinical features, patho-
genesis, epidemiology and management of plague are discussed in
detail in Chapter 126.
Y. pestis possesses unique proteins associated with virulence,
including Pla, Yad A, and Yad C (Table 183-9).60,61 The key step in
evolution of virulence appears to be the acquisition of the Fra plasmid,
which together with the expression of chromosomal proteins not
expressed in Y. pseudotuberculosis conferred the transmissibility from
mammalian host to mammalian host via vector fleas.56
DIAGNOSTIC MICROBIOLOGY
Plague should be suspected in febrile patients exposed to flea bites or
rodents in endemic areas. There is a considerable risk of laboratory-
acquired infection, so high-risk samples must be handled in a contain-
ment level 3 laboratory by experienced trained personnel. Gram stain
of smears taken from bubo aspirates, blood, sputum or cerebrospinal
fluid may reveal pleomorphic GNCB with rounded ends. Wright–
Giemsa or Wayson staining may demonstrate bipolar staining. Yersinia
pestis appear light blue with dark blue polar bodies. Direct immuno-
fluorescence of smears may be beneficial.
Culture of material on blood or MacConkey agars or in broth
cultures, at 80°F (26.7°C), may reveal tiny, translucent, nonhemolytic
colonies after 24 hours. After further incubation the colonies enlarge
and become opaque. Yersinia pestis grows poorly on MacConkey agar
and the colonies tend to autolyse after 2–3 days. In fluid culture Y.
Typically, Y. pestis is oxidase-negative, catalase-positive, urea-
negative and indole-negative. Strains may be misidentified as Y. pseu-
dotuberculosis or other Enterobacteriaceae. Definitive identification
can be confirmed by immunofluorescence of F1 antigen or molecular
techniques, such as real-time PCR for Y. pestis pla gene.62 A serologic
diagnosis can be made by a passive hemagglutination test using tanned
sheep red cells to which F1 antigen has been adsorbed, or a comple-
ment fixation test. ELISA tests for antibodies to F1 antigen are useful
in large-scale seroepidemiologic surveys.
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