244   MEDICAL MICROBIOLOGY
T he four most important genera in the family Pasteurel-
laceae are Haemophilus, Actinobacillus, Aggregati-
bacter, and Pasteurella (Table 24-1), and they are responsible
for a broad spectrum of diseases (Box 24-1). The members
of this family are small (0.2 to 0.3 × 1.0 to 2.0 µm), facultative
anaerobic, gram-negative rods. Most have fastidious proper-
ties, requiring enriched media for isolation. Members of the
genus Haemophilus, particularly H. influenzae, are the most
common pathogens in this family and will be the main focus
of this chapter (Table 24-2).
• Haemophilus
Haemophilae are small, sometimes pleomorphic, gram-
negative rods present on the mucous membranes of humans
(Figure 24-1). Haemophilus influenzae is the species most
commonly associated with disease, although introduction of
the H. influenzae type b vaccine has dramatically reduced the
incidence of disease, particularly in the pediatric population.
Haemophilus aegyptius is an important cause of acute puru-
lent conjunctivitis. Haemophilus ducreyi is well recognized
as the etiologic agent of the sexually transmitted disease soft
chancre or chancroid. The other members of the genus are
commonly isolated in clinical specimens (e.g., Haemophilus
parainfluenzae is the most common species in the mouth)
but are rarely pathogenic, being responsible primarily for
opportunistic infections.
Table 24-1 Important Pasteurellaceae
Organism Historical Derivation
Haemophilus haemo, blood; hilos, lover (“blood lover”;
requires blood for growth on agar media)
H. influenzae Originally thought to be the cause of influenza
H. aegyptius aegyptius, Egyptian (observed by Robert
Koch in 1883 in exudates from Egyptians
with conjunctivitis)
H. ducreyi Named after the bacteriologist Ducrey, who
first isolated this organism
Actinobacillus actinis, ray; bacillus, small staff or rod (“ray
bacillus”; refers to the growth of
filamentous forms [rays])
Aggregatibacter aggregare, to come together; bacter,
bacterial rod; rod-shaped bacteria that
aggregate or clump together
A. actinomycetemcomitans comitans, accompanying (“accompanying
an actinomycete”; isolates are frequently
associated with Actinomyces)
A. aphrophilus aphros, foam; philos, loving (“foam loving”)
Pasteurella Named after Louis Pasteur
P. multocida multus, many; cidus, to kill (“many-killing”;
pathogenic for many species of animals)
P. canis canis, dogs (isolated from the mouths of
dogs)
Physiology and Structure
The growth of most species of Haemophilus requires supple-
mentation of media with one or both of the following
growth-stimulating factors: (1) hemin (also called X factor
for “unknown factor”) and (2) nicotinamide adenine dinu-
cleotide (NAD; also called V factor for “vitamin”). Although
both factors are present in blood-enriched media, sheep
blood agar must be gently heated to destroy the inhibitors of
V factor. For this reason, heated blood (“chocolate”) agar is
used for the isolation of Haemophilus in culture.
The cell wall structure of Haemophilus is typical of other
gram-negative rods. Lipopolysaccharide with endotoxin
activity is present in the cell wall, and strain-specific and
species-specific proteins are found in the outer membrane.
Analysis of these strain-specific proteins is valuable in epi-
demiologic investigations. The surface of many, but not all,
strains of H. influenzae is covered with a polysaccharide
capsule, and six antigenic serotypes (a through f) have been
identified. Before the introduction of the H. influenzae type
b vaccine, H. influenzae serotype b was responsible for more
than 95% of all invasive Haemophilus infections. After intro-
duction of the vaccine, most disease caused by this serotype
disappeared in vaccinated populations, and more than half
of all invasive disease is now caused by nonencapsulated
(nontypeable) strains. A 2014 study reported that serogroup
A was responsible for an increase in H. influenzae invasive
disease, so a reemergence of this pathogen may be observed
in the future.
Box 24-1 Pasteurellaceae: Clinical Summaries
Haemophilus influenzae
Meningitis: a disease primarily of unimmunized children characterized
by fever, severe headache, and systemic signs
Epiglottitis: a disease primarily of unimmunized children characterized
by initial pharyngitis, fever, and difficulty breathing, and progressing to
cellulitis and swelling of the supraglottic tissues, with obstruction of
the airways possible
Pneumonia: inflammation and consolidation of the lungs observed
primarily in the elderly with underlying chronic pulmonary disease;
typically caused by nontypeable strains
Haemophilus aegyptius
Conjunctivitis: an acute purulent conjunctivitis (“pink eye”)
Haemophilus ducreyi
Chancroid: sexually transmitted disease characterized by a tender papule
with an erythematous base that progresses to painful ulceration with
associated lymphadenopathy
Aggregatibacter actinomycetemcomitans
Endocarditis: responsible for subacute form of endocarditis in patients
with underlying damage to the heart valve
Aggregatibacter aphrophilus
Endocarditis: as with A. actinomycetemcomitans
Pasteurella multocida
Bite wound: most common manifestation is infected cat or dog bite
wound; particularly common with cat bites because the wounds are
deep and difficult to disinfect
 CHAPTER 24 HAEMOPHILUS AND RELATED BACTERIA   245

Table 24-2 Haemophilus Species Associated with Human Disease

Species Primary Diseases Frequency
H. influenzae Pneumonia, sinusitis, otitis, meningitis, epiglottitis, cellulitis, bacteremia Common worldwide; uncommon in United States
H. aegyptius Conjunctivitis Uncommon
H. ducreyi Chancroid Uncommon in United States
H. parainfluenzae Bacteremia, endocarditis, opportunistic infections Rare
H. haemolyticus Opportunistic infections Rare
H. parahaemolyticus Opportunistic infections Rare

Pathogenesis and Immunity

A leading to damage of the respiratory epithelium. The bacteria
B anti-PRP antibodies, those with depletion of complement,
FIGURE 24-1 Gram stains of Haemophilus influenzae. A, Small
coccobacilli forms seen in sputum from patient with pneumonia.
B, Thin pleomorphic forms seen in a 1-year-old unvaccinated child
in Africa with overwhelming meningitis.
In addition to the serologic differentiation of H. influen-
zae, the species is subdivided into eight biotypes (I through
VIII) as determined by three biochemical reactions: indole
production, urease activity, and ornithine decarboxylase
activity. The separation of these biotypes is useful for epide-
miologic purposes.
Haemophilus species, particularly H. parainfluenzae and
nonencapsulated H. influenzae, colonize the upper respira-
tory tract in virtually all people within the first few months
of life. These organisms can spread locally and cause disease
in the ears (otitis media), sinuses (sinusitis), and lower respi-
ratory tract (bronchitis, pneumonia). Disseminated disease,
however, is relatively uncommon. In contrast, encapsulated
H. influenzae (particularly serotype b [biotype I]) is uncom-
mon in the upper respiratory tract or is present in only very
small numbers but is a common cause of disease in unvac-
cinated children (i.e., meningitis, epiglottitis [obstructive
laryngitis], cellulitis). Pili and nonpilus adhesins mediate
colonization of the oropharynx with H. influenzae. Cell wall
components of the bacteria (e.g., lipopolysaccharide and a
low-molecular-weight glycopeptide) impair ciliary function,
can then be translocated across both epithelial and endothe-
lial cells and can enter the blood. In the absence of specific
opsonic antibodies directed against the polysaccharide
capsule, high-grade bacteremia can develop, with dissemina-
tion to the meninges or other distal foci.
The major virulence factor in H. influenzae type b is
the antiphagocytic polysaccharide capsule, which contains
ribose, ribitol, and phosphate (commonly referred to as
polyribitol phosphate [PRP]). Antibodies directed against
the capsule greatly stimulate bacterial phagocytosis and
complement-mediated bactericidal activity. These antibodies
develop because of natural infection, vaccination with puri-
fied PRP, or the passive transfer of maternal antibodies. The
severity of systemic disease is inversely related to the rate of
clearance of bacteria from the blood. The risk of meningitis
and epiglottitis is significantly greater in patients with no
and those who have undergone splenectomy. The lipopoly-
saccharide lipid A component induces meningeal inflamma-
tion in an animal model and may be responsible for initiating
this response in humans. Immunoglobulin IgA1 proteases
are produced by H. influenzae (both encapsulated and non-
encapsulated strains) and may facilitate colonization of the
organisms on mucosal surfaces by interfering with humoral
immunity.
Epidemiology
Haemophilus species are present in almost all individuals,
primarily colonizing the mucosal membranes of the respira-
tory tract. H. parainfluenzae is the predominant Haemophi-
lus species in the mouth. Nonencapsulated strains of
246   MEDICAL MICROBIOLOGY

H. influenzae are also commonly found in the upper respira- Meningitis

tory tract; however, encapsulated strains are detectable only CSF 50%-95% culture positive
in small numbers and only when highly selective culture Blood 50%-95% culture positive
methods are used. Before the introduction of the H. influen- Conjunctivitis
zae vaccine, even though H. influenzae type b was the most Eye 50%-75% culture positive
Blood <10% culture positive
common serotype that caused systemic disease, it was rarely
Sinusitis
isolated in healthy children (a fact that emphasizes the viru- Sinus aspirate
lence of this bacterium). 50%-75% culture positive
The epidemiology of Haemophilus disease has changed Cellulitis
dramatically. Before the introduction of conjugated Skin 75%-90% culture positive
H. influenzae type b vaccines, an estimated 20,000 cases of Blood 50%-75% culture positive
invasive H. influenzae type b disease occurred annually in Otitis media
children younger than age 5 years in the United States. The Tympanocentesis
50%-70% culture positive
first polysaccharide vaccines for H. influenzae type b were
not protective for children younger than 18 months (the Epiglottitis
Blood 90%-95% culture positive
population at greatest risk for disease) because there is a Epiglottitis culture contraindicated
natural delay in the maturation of the immune response to
polysaccharide antigens. When vaccines containing purified Pneumonia, bronchitis
Sputum 25%-75% culture positive
PRP antigens conjugated to protein carriers (i.e., diphtheria Blood 10%-30% culture positive
toxoid, tetanus toxoid, meningococcal outer membrane
protein) were introduced in December 1987, a protective
antibody response in infants aged 2 months and older was
produced, and systemic disease in children younger than age
Arthritis
5 was virtually eliminated in the United States, with only 14 Synovial fluid
cases reported in 2011. Most of the H. influenzae type b 70%-90% culture positive
infections now occur in children who are not immune Blood 50%-80% culture positive
(because of incomplete vaccination or a poor response to the
vaccine) and in elderly adults with waning immunity. In
addition, invasive H. influenzae disease caused by other sero-
types of encapsulated bacteria and by nonencapsulated
strains has now become proportionally more common than
that resulting from serotype b. It should be noted that suc-
cessful elimination of H. influenzae type b disease in the
United States has not been seen in many developing coun-
tries where vaccination programs have been difficult to
implement. Thus H. influenzae type b remains the most sig-
nificant pediatric pathogen in many countries of the world.
It is estimated that 3 million cases of serious disease and up
to 700,000 fatalities occur in children each year worldwide,
a tragedy considering that vaccination could eliminate virtu-
ally all disease. The epidemiology of disease caused by non- FIGURE 24-2 Infections caused by Haemophilus influenzae. With
encapsulated H. influenzae and other Haemophilus species is the advent of the conjugated vaccine, most infections in adults
distinct. Ear and sinus infections caused by these organisms involve areas contiguous with the oropharynx (i.e., lower
are primarily pediatric diseases but can occur in adults. respiratory tract, sinuses, ears). Serious systemic infections (e.g.,
Pulmonary disease most commonly affects elderly people, meningitis, epiglottitis) can occur in nonimmune patients. CSF,
particularly those with a history of underlying chronic Cerebrospinal fluid.
obstructive pulmonary disease (COPD) or conditions
predisposing to aspiration (e.g., alcoholism, altered mental
state). by all Haemophilus species are described in the following
H. ducreyi is an important cause of genital ulcers (chan- sections.
croid) in Africa and Asia but is less common in Europe and
North America. The incidence of disease in the United States Meningitis
is cyclic. A peak incidence of more than 5000 cases was H. influenzae type b was the most common cause of pediatric
reported in 1988, which decreased to 8 cases in 2011. Despite meningitis, but this situation changed rapidly when the con-
this favorable trend, the Centers for Disease Control and jugated vaccines became widely used. Disease in nonim-
Prevention has documented that the disease is significantly mune patients results from bacteremic spread of the
unrecognized and underreported, making the true incidence organisms from the nasopharynx and cannot be differenti-
unknown. ated clinically from other causes of bacterial meningitis. The
initial presentation is a 1- to 3-day history of mild upper
Clinical Diseases (see Table 24-2) respiratory disease, after which the typical signs and symp-
The clinical syndromes seen in patients with H. influenzae toms of meningitis appear. Mortality is less than 10% in
infections are represented in Figure 24-2. The diseases caused patients who receive prompt therapy, and carefully designed
 CHAPTER 24
studies have documented a low incidence of serious neuro-
logic sequelae (in contrast with the 50% incidence of severe
residual damage reported in nonimmune children seen in
early studies). Person-to-person spread in a nonimmune
population is well documented, so appropriate epidemio-
logic precautions must be used.
Epiglottitis
Epiglottitis, characterized by cellulitis and the swelling of the
supraglottic tissues, represents a life-threatening emergency.
Although epiglottitis is a pediatric disease, the peak inci-
dence of this disease during the prevaccine era occurred in
children 2 to 4 years of age; in contrast, the peak incidence
of meningitis was seen in children 3 to 18 months of age.
Children with epiglottitis have pharyngitis, fever, and diffi-
culty breathing, which can progress rapidly to obstruction of
the airway and death. Since the introduction of the vaccine,
the incidence of this disease has also decreased dramatically
in children and remains relatively rare in adults.
Cellulitis
Like meningitis and epiglottitis, cellulitis is a pediatric H.
influenzae disease that has largely been eliminated by vac-
cination. When it is observed, patients have fever and cel-
lulitis characterized by the development of reddish-blue
patches on the cheeks or periorbital areas. The diagnosis is
strongly suggested by the typical clinical presentation, cel-
lulitis proximal to the oral mucosa, and lack of documented
vaccination in the child.
Arthritis
Before the advent of conjugated vaccines, the most common
form of arthritis in children younger than 2 years was an
infection of a single, large joint secondary to bacteremic
spread of H. influenzae type b. Disease does occur in older
children and adults, but it is very uncommon and generally
affects immunocompromised patients and patients with pre-
viously damaged joints.
Otitis, Sinusitis, and Lower Respiratory Tract
Disease (Clinical Case 24-1)
Nonencapsulated strains of H. influenzae are opportunistic
pathogens that can cause infections of the upper and lower
airways. Most studies have shown that H. influenzae and
Streptococcus pneumoniae are the two most common causes
of acute and chronic otitis and sinusitis. Primary pneumonia
Clinical Case 24-1 Pneumonia Caused by
Haemophilus influenzae
Holmes and Kozinn (J Clin Microbiol 18:730–732, 1983) described a
61-year-old woman with pneumonia caused by Haemophilus influenzae
serotype d. The patient had a long history of smoking, chronic obstructive
lung disease, diabetes mellitus, and congestive heart failure. She pre-
sented with left upper lobe pneumonia, producing purulent sputum with
many gram-negative coccobacilli. Both sputum and blood cultures were
positive for H. influenzae serotype d. The organism was susceptible to
ampicillin, to which the patient responded. This case illustrates the sus-
ceptibility of patients with chronic underlying pulmonary disease to infec-
tions with non–serotype b strains of H. influenzae.
HAEMOPHILUS AND RELATED BACTERIA   247
is uncommon in children and adults who have normal pul-
monary function. These organisms commonly colonize
patients who have chronic pulmonary disease (including
cystic fibrosis), and frequently are associated with exacerba-
tion of bronchitis and frank pneumonia.
Conjunctivitis
H. aegyptius, also called the Koch-Weeks bacillus, causes an
acute purulent conjunctivitis. This contagious organism is
associated with epidemics, particularly during the warm
months of the year.
Chancroid
Chancroid is a sexually transmitted disease that is most com-
monly diagnosed in men, presumably because women can
have asymptomatic or inapparent disease. Approximately 5
to 7 days after exposure, a tender papule with an erythema-
tous base develops on the genitalia or perianal area. Within
2 days the lesion ulcerates and becomes painful, and ingui-
nal lymphadenopathy is commonly present. Other causes
of genital ulcers, such as syphilis and herpes simplex disease,
must be excluded to confirm the diagnosis of chancroid.
Other Infections
Other species of Haemophilus can cause opportunistic infec-
tions such as otitis media, conjunctivitis, sinusitis, endocar-
ditis, meningitis, and dental abscesses.
Laboratory Diagnosis
Specimen Collection and Transport
Because most Haemophilus infections in vaccinated indi-
viduals originate from the oropharynx and are restricted to
the upper and lower respiratory tract, contamination of the
specimen with oral secretions should be avoided. Direct
needle aspiration should be used for the microbiological
diagnosis of sinusitis or otitis, and sputum produced from
the lower airways is used for the diagnosis of pneumonia.
Culture of blood for patients with pneumonia may be useful
but would be predictably negative in patients with upper
respiratory infections. Both blood and cerebrospinal fluid
(CSF) should be collected from patients with the diagnosis
of meningitis. Because there are approximately 107 bacteria
per ml of CSF in patients with untreated meningitis, 1 to
2 ml of fluid is generally adequate for microscopy, culture,
and antigen-detection tests. Microscopy and culture are less
sensitive if the patient has been exposed to antibiotics before
the CSF is collected. Blood cultures should also be collected
for the diagnosis of epiglottitis, cellulitis, and arthritis. Speci-
mens should not be collected from the posterior pharynx in
patients with suspected epiglottitis because the procedure
may stimulate coughing and obstruct the airway. Specimens
for the detection of H. ducreyi should be collected with
a moistened swab from the base or margin of the ulcer.
Culture of pus collected by aspiration from an enlarged
lymph node can be performed but is generally less sensitive
than culture of the ulcer. The laboratory should be notified
that H. ducreyi is suspected, because special culture tech-
niques must be used for recovery of the organism.
Microscopy
If microscopy is performed carefully, the detection of Hae-
mophilus species in clinical specimens is both sensitive and
248   MEDICAL MICROBIOLOGY
specific. Gram-negative rods ranging in shape from cocco-
bacilli to long, pleomorphic filaments can be detected in
more than 80% of CSF specimens from patients with
untreated Haemophilus meningitis (see Figure 24-1). Micro-
scopic examination of Gram-stained specimens is also useful
for the rapid diagnosis of the organism in arthritis and lower
respiratory tract disease.
Antigen Detection
The immunologic detection of H. influenzae antigen, specifi-
cally the PRP capsular antigen, is a rapid and sensitive way
to diagnose H. influenzae type b disease. PRP can be detected
with the particle agglutination test, which can detect less
than 1 ng/ml of PRP in a clinical specimen. In this test,
antibody-coated latex particles are mixed with the clinical
specimen; agglutination occurs if PRP is present. Antigen
can be detected in CSF and urine (in which the antigen is
eliminated intact). However, this test has limited use because
it can detect only H. influenzae type b, which is now uncom-
mon in the United States and other countries with an estab-
lished vaccine program. Other capsular serotypes and
nonencapsulated strains do not give a positive reaction.
Culture
It is relatively easy to isolate H. influenzae from clinical speci-
mens inoculated onto media supplemented with the appro-
priate growth factors. Chocolate agar is used in most
laboratories. However, if chocolate agar is overheated during
preparation, V factor is destroyed, and Haemophilus species
requiring this growth factor (e.g., H. influenzae, H. aegyptius,
H. parainfluenzae) will not grow. The bacteria appear as 1- to
2-mm, smooth, opaque colonies after 24 hours of incuba-
tion. They can also be detected growing around colonies of
Staphylococcus aureus on unheated blood agar (satellite phe-
nomenon [Figure 24-3]). The staphylococci provide the
requisite growth factors by lysing the erythrocytes in the
medium and releasing intracellular heme (X factor) and
excreting NAD (V factor). The colonies of H. influenzae in
these cultures are much smaller than they are on chocolate
FIGURE 24-3 Satellite phenomenon. Staphylococcus aureus excretes
nicotinamide adenine dinucleotide (NAD, or V factor) into the
medium, providing a growth factor required for Haemophilus influ-
enzae (small colonies surrounding S. aureus colonies [arrow]).
agar because the V factor inhibitors present in blood are not
inactivated.
The growth of Haemophilus in blood cultures is generally
delayed because most commercially prepared blood culture
broths are not supplemented with optimum concentrations
of X and V factors and inhibitors of V factor. Furthermore,
the growth factors are released only when the blood
cells lyse. Isolates of H. influenzae often grow better in
anaerobically incubated blood cultures because, under these
conditions, the organisms do not require X factor for
growth.
H. aegyptius and H. ducreyi are fastidious and require
specialized growth conditions. H. aegyptius grows best on
chocolate agar supplemented with 1% IsoVitaleX (mixture
of chemically defined supplements), with growth detected
after incubation in a carbon dioxide atmosphere for 2 to 4
days. Culture for H. ducreyi is relatively insensitive (<85% of
cultures yield organisms under optimal conditions) but
reportedly is best on gonococcal (GC) agar supplemented
with 1% to 2% hemoglobin, 5% fetal bovine serum,
IsoVitaleX enrichment, and vancomycin (3 µg/ml). Cultures
should be incubated at 33° C in 5% to 10% carbon dioxide
for 7 days or more. Because the media and incubation condi-
tions are not used for other bacterial cultures, success in
recovering H. ducreyi requires that the microbiologist look
specifically for this organism.
Identification
A presumptive identification of H. influenzae can be made
by the Gram stain morphology and demonstration of a
requirement for both X and V factors. Further subgrouping
of H. influenzae can be done with biotyping, electrophoretic
characterization of the membrane protein antigens, and
analysis of the strain-specific nucleic acid sequences. Bio-
chemical tests, nucleic acid analysis, or mass spectrometry is
used to identify other species in this genus.
Treatment, Prevention, and Control
Patients with systemic H. influenzae infections require
prompt antimicrobial therapy because the mortality rate in
patients with untreated meningitis or epiglottitis approaches
100%. Serious infections are treated with broad-spectrum
cephalosporins. Less severe infections, such as sinusitis and
otitis, can be treated with amoxicillin (if susceptible; approx-
imately 30% of strains are resistant), an active cephalosporin,
azithromycin, doxycycline, or a fluoroquinolone. Most iso-
lates of H. ducreyi are susceptible to erythromycin, the drug
recommended for treatment.
The primary approach to preventing H. influenzae type b
disease is through active immunization with purified capsu-
lar PRP. As discussed previously, the use of conjugated
vaccines has been remarkably successful in reducing the
incidences of H. influenzae type b disease and colonization.
Currently, it is recommended that children receive two or
three doses of vaccine against H. influenzae type b disease
before the age of 6 months, followed by a booster dose at age
12 to 15 months.
Antibiotic chemoprophylaxis is used to eliminate the car-
riage of H. influenzae type b in children at high risk for
disease (e.g., children <2 years in a family or day-care center
where systemic disease is documented). Rifampin prophy-
laxis has been used in these settings.
 CHAPTER 21
Treatment, Prevention, and Control
Erysipelothrix is susceptible to penicillin, which is the anti-
biotic of choice for both localized and systemic diseases.
Cephalosporins, carbapenems, fluoroquinolones, and
clindamycin are also active in vitro, but the organism has
variable susceptibility to macrolides, sulfonamides, and ami-
noglycosides and is resistant to vancomycin. For patients
allergic to penicillin, ciprofloxacin or clindamycin can be
used for localized cutaneous infections, and ceftriaxone or
imipenem can be considered for disseminated infections.
Infections in people at a higher occupational risk are pre-
vented by the use of gloves and other appropriate coverings
on exposed skin. Vaccination is used to control disease in
swine.
• Corynebacterium diphtheriae
The genus Corynebacterium is a large, heterogeneous collec-
tion of more than 100 species and subspecies that have a cell
wall with arabinose, galactose, meso-diaminopimelic acid
(meso-DAP), and (in most species) short-chain mycolic
acids (22 to 36 carbon atoms). Although organisms with
medium- and long-chain mycolic acids stain with acid-fast
stains (see Chapter 22), Corynebacterium organisms are not
acid-fast. Gram stains of these bacteria reveal clumps and
short chains of irregularly shaped (club-shaped) rods (Figure
21-4). Corynebacteria are aerobic or facultatively anaerobic,
nonmotile, and catalase positive. Most (but not all) species
ferment carbohydrates, producing lactic acid as a byproduct.
Many species grow well on common laboratory media;
however, some species form small colonies because they
require lipid supplemented media for good growth (lipo-
philic strains).
Corynebacteria are ubiquitous in plants and animals, and
they normally colonize the skin, upper respiratory tract, gas-
trointestinal tract, and urogenital tract in humans. Although
all species of corynebacteria can function as opportunistic
pathogens, relatively few are associated with human disease
FIGURE 21-4 Gram stain of Corynebacterium species in sputum
specimen.
LISTERIA AND RELATED GRAM-POSITIVE BACTERIA   215
(see Table 21-2). The most famous of these is C. diphtheriae,
the etiologic agent of diphtheria. A number of other genera
of coryneform bacteria have been characterized. Three
genera associated with human disease (Arcanobacterium,
Rothia, Tropheryma) are listed in Table 21-2 but will not be
discussed further.
Physiology and Structure
C. diphtheriae is an irregularly staining, pleomorphic rod
(0.3 to 0.8 × 1.0 to 8.0 µm). After overnight incubation, large
1- to 3-mm colonies are observed on blood agar medium.
More selective, differential media can be used to recover this
pathogen from specimens with other organisms present,
such as pharyngeal specimens. This species is subdivided
into four biotypes based on their colonial morphology and
biochemical properties: belfanti, gravis, intermedius, and
mitis, with most disease caused by biotype mitis.
Pathogenesis and Immunity
Diphtheria toxin is the major virulence factor of C. diphthe-
riae. The tox gene that codes for the exotoxin is introduced
into strains of C. diphtheriae by a lysogenic bacteriophage,
β-phage. Two processing steps are necessary for the active
gene product to be secreted: (1) proteolytic cleavage of the
leader sequence from the Tox protein during secretion from
the bacterial cell and (2) cleavage of the toxin molecule into
two polypeptides (A and B) that remain attached by a disul-
fide bond. This 58,300-Da protein is an example of the classic
A-B exotoxin.
Three functional regions exist on the toxin molecule: a
catalytic region on the A subunit and a receptor-binding
region and a translocation region on the B subunit. The
receptor for the toxin is heparin-binding epidermal growth
factor, which is present on the surface of many eukaryotic
cells, particularly heart and nerve cells; its presence explains
the cardiac and neurologic symptoms observed in patients
with severe diphtheria. After the toxin becomes attached to
the host cell, the translocation region is inserted into the
endosomal membrane, facilitating the movement of the cata-
lytic region into the cell cytosol. The A subunit then termi-
nates host cell protein synthesis by inactivating elongation
factor-2 (EF-2), a factor required for the movement of
nascent peptide chains on ribosomes. Because the turnover
of EF-2 is very slow and approximately only one molecule
per ribosome is present in a cell, it has been estimated that
one exotoxin molecule can inactivate the entire EF-2 content
in a cell, completely terminating host cell protein synthesis.
Toxin synthesis is regulated by a chromosomally encoded
element, diphtheria toxin repressor (DTxR). This protein,
activated in the presence of high iron concentrations, can
bind to the toxin gene operator and prevent toxin
production.
Epidemiology
Diphtheria is a disease found worldwide, particularly in poor
urban areas where there is crowding and the protective level
of vaccine-induced immunity is low. The largest outbreak in
the latter part of the 20th century occurred in the former
Soviet Union, where in 1994 almost 48,000 cases were docu-
mented, with 1746 deaths. C. diphtheriae is maintained in
the population by asymptomatic carriage in the oropharynx
or on the skin of immune people. Respiratory droplets or
216   MEDICAL MICROBIOLOGY
skin contact transmits it from person to person. Humans are
the only known reservoir for this organism.
Diphtheria has become uncommon in the United States
because of an active immunization program, as shown by the
fact that more than 200,000 cases were reported in 1921 but
no cases have been reported since 2003. An analysis of C.
diphtheriae infections in the United Kingdom between 1986
and 2008 identified that the major risk factor for infection
was travel of nonimmune individuals to countries with
endemic disease (e.g., Indian subcontinent, Africa, Southeast
Asia). Diphtheria is primarily a pediatric disease, but the
highest incidence has shifted toward older age groups in
areas where there are active immunization programs for
children. Skin infection with toxigenic C. diphtheriae (cuta-
neous diphtheria) also occurs, but it is not a reportable
disease in the United States, so its incidence is unknown.
Clinical Diseases
The clinical presentation of diphtheria is determined by the
(1) site of infection, (2) immune status of the patient, and (3)
virulence of the organism. Exposure to C. diphtheriae may
result in asymptomatic colonization in fully immune people,
mild respiratory disease in partially immune patients, or a
fulminant, sometimes fatal, disease in nonimmune patients.
Diphtheria toxin is produced at the site of the infection and
then disseminates through the blood to produce the systemic
signs of diphtheria. The organism does not need to enter the
blood to produce disease.
Respiratory Diphtheria (Clinical Case 21-3)
The symptoms of diphtheria involving the respiratory tract
develop after a 2- to 4-day incubation period. Organisms
multiply locally on epithelial cells in the pharynx or adjacent
Clinical Case 21-3 Respiratory Diphtheria
Lurie and associates (JAMA 291:937–938, 2004) reported the last patient
with respiratory diphtheria seen in the United States. An unvaccinated
63-year-old man developed a sore throat while on a week-long trip in rural
Haiti. Two days after he returned home to Pennsylvania, he visited a local
hospital with complaints of a sore throat and difficulties in swallowing. He
was treated with oral antibiotics but returned 2 days later with chills,
sweating, difficulty swallowing and breathing, nausea, and vomiting. He
had diminished breath sounds in the left lung, and radiographs confirmed
pulmonary infiltrates as well as enlargement of the epiglottis. Laryngos-
copy revealed yellow exudates on the tonsils, posterior pharynx, and soft
palate. He was admitted to the intensive care unit and treated with azithro-
mycin, ceftriaxone, nafcillin, and steroids, but over the next 4 days he
became hypotensive with a low-grade fever. Cultures were negative for
Corynebacterium diphtheriae. By the eighth day of illness, a chest radio-
graph showed infiltrates in the right and left lung bases, and a white
exudate consistent with C. diphtheriae pseudomembrane was observed
over the supraglottic structures. Cultures at this time remained negative
for C. diphtheriae, but polymerase chain reaction testing for the exotoxin
gene was positive. Despite aggressive therapy the patient continued to
deteriorate, and on the seventeenth day of hospitalization he developed
cardiac complications and died. This patient illustrates (1) the risk factor
of an unimmunized patient traveling to an endemic area, (2) the classic
presentation of severe respiratory diphtheria, (3) delays associated with
diagnosis of an uncommon disease, and (4) the difficulties most labora-
tories would now have isolating the organism in culture.
surfaces and initially cause localized damage as a result of
exotoxin activity. The onset is sudden, with malaise, sore
throat, exudative pharyngitis, and a low-grade fever. The
exudate evolves into a thick pseudomembrane composed of
bacteria, lymphocytes, plasma cells, fibrin, and dead cells
that can cover the tonsils, uvula, and palate and can extend
up into the nasopharynx or down into the larynx (Figure
21-5). The pseudomembrane firmly adheres to the underly-
ing tissue and is difficult to dislodge without making the
tissue bleed (unique to diphtheria). As the patient recovers
after the approximately 1-week course of the disease, the
membrane dislodges and is expectorated. Systemic compli-
cations in patients with severe disease primarily involve the
heart and nervous system. Evidence of myocarditis can be
detected in the majority of patients with diphtheria, typically
developing 1 to 2 weeks into the illness and at a time when
the pharyngeal symptoms are improving. Symptoms can
present acutely or gradually, progressing in severe disease to
congestive heart failure, cardiac arrhythmias, and death.
Neurotoxicity is proportional to the severity of the primary
disease, which is influenced by the patient’s immunity. The
majority of patients with severe primary disease develop
neuropathy, initially localized to the soft palate and pharynx,
later involving oculomotor and ciliary paralysis, with pro-
gression to peripheral neuritis.
Cutaneous Diphtheria
Cutaneous diphtheria is acquired through skin contact with
other infected persons. The organism colonizes the skin and
gains entry into the subcutaneous tissue through breaks in
the skin. A papule develops first and then evolves into a
chronic, nonhealing ulcer, sometimes covered with a
grayish membrane. Staphylococcus aureus or Streptococcus
pyogenes is also frequently present in the wound.
Laboratory Diagnosis
The initial treatment of a patient with diphtheria is instituted
on the basis of the clinical diagnosis, not laboratory results,
because definitive results are not available for at least a week.
FIGURE 21-5 Pharynx of a 39-year-old woman with bacteriologi-
cally confirmed diphtheria. The photograph was taken 4 days after
the onset of fever, malaise, and sore throat. Hemorrhage caused by
removal of the membrane by swabbing appears as a dark area on
the left. (From Mandell G, Bennett J, Dolin R: Principles and prac-
tice of infectious diseases, ed 8, Philadelphia, 2015, Elsevier.)
 CHAPTER 21
Microscopy
The results of microscopic examination of clinical material
are unreliable. Metachromatic granules in bacteria stained
with methylene blue have been described, but this appear-
ance is not specific to C. diphtheriae.
Culture
Specimens for the recovery of C. diphtheriae should be col-
lected from both the nasopharynx and throat and should be
inoculated onto a nonselective, enriched blood agar plate
and a selective medium (e.g., cysteine-tellurite blood agar
[CTBA], Tinsdale medium, colistin-nalidixic agar [CNA]).
Tellurite inhibits the growth of most upper respiratory tract
bacteria and gram-negative rods and is reduced by C. diph-
theriae, producing a characteristic gray to black color on
agar. Degradation of cysteine by C. diphtheriae cysteinase
activity produces a brown halo around the colonies. CTBA
has a long shelf life (practical for cultures that are infre-
quently performed) but inhibits some strains of C. diphthe-
riae. Tinsdale medium is the best medium for recovering C.
diphtheriae in clinical specimens, but it has a short shelf life
and requires addition of horse serum. Because infections
caused by C. diphtheriae are rarely seen or suspected in non-
endemic areas, CTBA and Tinsdale medium are not com-
monly available in most laboratories. CNA is commonly
used for the selective recovery of gram-positive bacteria;
therefore this is a practical alternative medium. Regardless
of the media used, all isolates resembling C. diphtheriae must
be identified by biochemical testing and the presence of the
diphtheria exotoxin confirmed because nontoxigenic strains
occur.
Identification
The presumptive identification of C. diphtheriae can be based
on the presence of cystinase and absence of pyrazinamidase
(two enzyme reactions that can be rapidly determined).
More extensive biochemical tests or nucleic acid sequencing
of species-specific genes is required for identification at the
species level.
Toxigenicity Testing
All isolates of C. diphtheriae should be tested for the produc-
tion of exotoxin. The gold standard for detection of diphthe-
ria toxin is an in vitro immunodiffusion assay (Elek test).
An alternative method is detection of the exotoxin gene
using a polymerase chain reaction (PCR)–based nucleic
acid amplification method. This test can detect the tox gene
in clinical isolates and directly in clinical specimens (e.g.,
swabs from the diphtheritic membrane or biopsy material).
Although this test is rapid and specific, strains in which the
tox gene is not expressed (presumably because the diphthe-
ria toxin repressor is expressed) can give a positive signal.
Nontoxigenic strains of C. diphtheriae do not produce classic
diphtheria; however, they should not be ignored, because
these strains have been associated with other significant dis-
eases, including septicemia, endocarditis, septic arthritis,
osteomyelitis, and abscess formation.
Treatment, Prevention, and Control
The most important aspect of the treatment for diphtheria is
early administration of diphtheria antitoxin to specifically
LISTERIA AND RELATED GRAM-POSITIVE BACTERIA   217
neutralize the exotoxin before it is bound by the host cell.
Once the cell internalizes the toxin, cell death is inevitable.
Unfortunately, because diphtheria may not be suspected ini-
tially, significant progression of disease can occur before the
antitoxin is administered. Antibiotic therapy with penicillin
or erythromycin is also used to eliminate C. diphtheriae and
terminate toxin production. Bed rest, isolation to prevent
secondary spread, and maintenance of an open airway in
patients with respiratory diphtheria are all important. After
the patient has recovered, immunization with toxoid is
required because most patients fail to develop protective
antibodies after a natural infection.
Symptomatic diphtheria can be prevented by actively
immunizing people with diphtheria toxoid. The nontoxic,
immunogenic toxoid is prepared by formalin treatment of
the toxin. Initially, children are given five injections of this
preparation with pertussis and tetanus antigens (DPT
vaccine) at ages 2, 4, 6, 15 to 18 months, and 4 to 6 years.
After that time, it is recommended that booster vaccinations
with diphtheria toxoid combined with tetanus toxoid be
given every 10 years. The effectiveness of immunization is
well documented, with disease restricted to nonimmune or
incompletely immunized individuals.
People in close contact with patients who have docu-
mented diphtheria are at risk for acquiring the disease. Naso-
pharyngeal specimens for culture should be collected from
all close contacts and antimicrobial prophylaxis with eryth-
romycin or penicillin started immediately. Any contact who
has not completed the series of diphtheria immunizations or
who has not received a booster dose within the previous 5
years should receive a booster dose of toxoid. People exposed
to cutaneous diphtheria should be managed in the same
manner because it is reported that they are more contagious
than patients with respiratory diphtheria. If the respiratory
or cutaneous infection is caused by a nontoxigenic strain, it
is unnecessary to institute prophylaxis in contacts.
Bibliography
Allerberger F, Wagner M: Listeriosis: a resurgent foodborne infection, Clin
Microbiol Infect 16:16–23, 2010.
Fenollar F, Puechal X, Raoult D: Whipple’s disease, N Engl J Med 356:55–66,
2007.
Freitag N, Port G, Miner M: Listeria monocytogenes—from saprophyte to
intracellular pathogen, Nat Rev Microbiol 7:623–628, 2009.
Funke G, von Graevenitz A, Clarridge JE 3rd, et al: Clinical microbiology
of coryneform bacteria, Clin Microbiol Rev 10:125–159, 1997.
Gorby GL, Peacock JE Jr: Erysipelothrix rhusiopathiae endocarditis: micro-
biologic, epidemiologic, and clinical features of an occupational disease,
Rev Infect Dis 10:317–325, 1988.
Gray MJ, Freitag NE, Boor KJ: How the bacterial pathogen Listeria mono-
cytogenes mediates the switch from environmental Dr. Jekyll to patho-
genic Mr. Hyde, Infect Immun 74:2505–2512, 2006.
McCollum JT, Cronquist AB, Silk BJ, et al: Multistate outbreak of listeriosis
associated with cantaloupe, N Engl J Med 369:944–953, 2013.
Pamer EG: Immune responses to Listeria monocytogenes, Nat Rev Immunol
4:812–823, 2004.
Popovic T, Kombarova SY, Reeves MW, et al: Molecular epidemiology of
diphtheria in Russia, 1985-1994, J Infect Dis 174:1064–1072, 1996.
Wagner KS, White JM, Crowcroft NS, et al: Diphtheria in the United
Kingdom, 1986-2008: the increasing role of Corynebacterium ulcerans,
Epidemiol Infect 138:1519–1530, 2010.
Wang Q, Chang BJ, Riley TV: Erysipelothrix rhusiopathiae, Vet Microbiol
140:405–417, 2010.
Wing E, Gregory S: Listeria monocytogenes: clinical and experimental
update, J Infect Dis 185(Suppl 1):S18–S24, 2002.