MICROBIOLOGY:
Haemophilus influenzae
Haemophilus and Other Fastidious Gram-Negative Bacilli
Haemophilus
• Consists of gram-negative, pleomorphic coccobacilli or rods that can
vary microscopically from small coccobacilli to long filaments.
• Nonmotile and facultatively anaerobic
• Ferment carbohydrates
• Generally oxidase- and catalase-positive
• Reduce nitrates to nitrites
• Obligate parasites on the mucous membranes of humans and animals
• Most members of the genus Haemophilus are nonpathogenic or
produce opportunistic infection.
• The emphasis of this section is on the major pathogenic species: H.
influenzae, H. aegyptius, and H. ducreyi.
• Require preformed growth factors present in blood: X factor (hemin
or hematin) (“X for unknown”) or V factor (nicotinamide adenine
dinucleotide [NAD]) (“V for vitamin”), or both.
• The production of hemolysis on 5% horse or rabbit blood agar is an
important differential characteristic (SRBCs contain enzymes;
NADases that hydrolyze V factor).
• A phenomenon that helps in the recognition of Haemophilus
spp. that require V factor is satellitism.
A.
Virulence Factors:
• Capsule
• Immunoglobulin A (IgA) proteases
• Adherence by fimbriae and other structures
• Outer membrane proteins and lipopolysaccharide (LPS)
Clinical Infections:
• Two patterns of disease
✓ Invasive disease caused by encapsulated strains, in which
bacteremia plays a significant role. Examples of invasive
disease include septicemia, meningitis, arthritis, epiglottitis,
tracheitis, and pneumonia.
✓ Localized infection caused by the contiguous spread of NTHi
strains and occurs within or in close proximity to the
respiratory tract. Examples of localized infection include
conjunctivitis, sinusitis, and otitis media with effusion (middle
ear infections).
• NTHi strains are also occasionally associated with invasive diseases
such as bronchitis and pneumonia in older patients.
• NTHi strains can enter the central nervous system (CNS) by direct
extension through infected sinuses, otitis media, and head trauma.
• NTHi strains can cause meningitis in adults, especially in
immunocompromised or debilitated patients, and they can cause
neonatal sepsis.
 • Meningitis
✓ Virtually all cases were caused by serotype b (before the Hib
vaccine).
✓ Bloodstream invasion and bacteremic spread follow
colonization, invasion, and replication of this organism in the
respiratory mucous membranes.
✓ Headache, stiff neck, and other meningeal signs are usually
preceded by mild respiratory disease.
• Epiglottis
✓ Manifestations include rapid onset, acute inflammation, and
intense edema of the epiglottis that may cause complete airway
obstruction, requiring an emergency tracheostomy.
✓ To avoid causing further possible damage, the area is not
swabbed for culture, but is treated empirically based on signs
and symptoms
✓ Maintenance of a secure airway is the most important aspect of
treatment.
• Bacterial Tracheitis
✓ Can arise after an acute, viral respiratory infection and initially
presents with a mild to moderate illness that progresses rapidly.
✓ Use of broad-spectrum antimicrobial agents during the early
stages of the disease is imperative because thick secretions can
occlude the trachea.
B. Haemophilus aegyptius (Koch-Weeks bacillus)
Clinical Infections:
• Genetically related to H. influenzae.
• Difficult to differentiate H. influenzae from H. aegyptius and H.
influenzae biogroup aegyptius in the clinical laboratory.
• Observed in conjunctivitis exudates from Egyptians by Koch.
• Associated with an acute, contagious conjunctivitis, commonly
referred to as “pinkeye”.
C. Haemophilus influenzae Biogroup aegyptius
Clinical Infections:
• Can cause conjunctivitis.
• Despite being nonencapsulated, a clone of this organism first
caused a severe systemic disease known as Brazilian
purpuric fever (BPF).
• BPF is characterized by recurrent or concurrent
conjunctivitis, high fever, vomiting, petechial/purpural rash,
septicemia, shock, and vascular collapse.
• Mortality rate for BPF may reach 70% within 48 hours after
onset.
D. Haemophilus ducreyi
Clinical Infections:
• Strict human pathogen.
• Infects mucosal epithelium, genital and nongenital skin, and
regional lymph nodes.
• Causative agent of chancroid, a highly communicable
sexually transmitted genital ulcer disease (GUD).
• Chancroid is commonly referred to as soft chancre, in
contrast to the hard chancre of syphilis.
• Organism is not part of the normal microbiota.
• Men have symptoms related to the inguinal tenderness and
genital lesions, whereas most women are asymptomatic.
 E. Haemophilus parainfluenzae • The use of GC agar (Gibco Laboratories) containing 1% hemoglobin,
Clinical Infections: 5% fetal calf serum, 1% IsoVitaleX, and 3 mg/L of vancomycin is also
reliable.
• H. parainfluenzae and the other species found in the oral
• The use of vancomycin (most H. ducreyi are resistant) in the media
cavity have a very low incidence of pathogenicity and have
helps reduce the growth of commensal biota from genital specimens
been rarely implicated as causative agents of endocarditis.
and improves detection.
• The mitral valve is the primary site of infection.
• The plates for the recovery of this organism should be incubated in a
• In the absence of other pathogens, H. parahaemolyticus may
5% to 10% CO2 atmosphere containing high humidity.
be a cause of some cases of pharyngitis.
• In contrast to the optimal growth temperature (35° C to 37° C) of the
LABORATORY DIAGNOSIS other Haemophilus spp., H. ducreyi grows best at 33° C.
• Specimens submitted for H. aegyptius should be held for at least 4
a. Specimen Processing and Isolation:
days and specimens for H. ducreyi should be held for at least 7 days
• Common sources include blood, CSF, middle ear exudate, joint
before reporting a negative result.
fluids, upper and lower respiratory tract specimens, swabs from
conjunctivae, vaginal swabs, and abscess drainage.
b. Colony Morphology:
• For culture of the lower respiratory tract, bronchial washing is
• Colonies of H. influenzae on CA appear translucent, tannish, moist,
recommended.
smooth, and convex, with a distinct “mousy” or bleachlike odor.
• Haemophilus spp. die rapidly in clinical specimens, and prompt
• Encapsulated strains of H. influenzae grow larger and more mucoid
transportation and processing are vital for their isolation.
than the NTHi strains.
• When attempting to isolate H. influenzae, CA is a commonly used
• Haemophilus spp. are MAC negative.
medium incubated between 33° C and 37° C in an atmosphere of 5%
• Haemophilus spp. sometimes grow on SBA plates around colonies of
to 10% carbon dioxide (CO2).
other bacterial species—a phenomenon known as satellitism.
• CA supplemented with bacitracin (300 mg/L) is an excellent medium
for the isolation of Haemophilus spp. from respiratory specimens.
• For H. aegyptius, enriched CHOC agar supplemented with 1% Iso-
VitaleX (BD) or Vitox (Oxoid) is required.
• Enriched CHOC medium is commonly used in most clinical
microbiology laboratories.
• H. ducreyi also grows on enriched CHOC medium, or Nairobi biplate
can be used.
• Nairobi biplate consists of GC agar base with 2% bovine hemoglobin
and 5% fetal calf serum on one half and Mueller Hinton agar with 5%
chocolatized horse blood on the other. Both sides contain 3 mg/L of
vancomycin.
 • H. parainfluenzae colonies appear tannish and drier with a medium
to large size compared with H. influenzae on CA.
• H. parahaemolyticus resembles H. parainfluenzae on CA; however,
when grown on horse or rabbit blood agar, it is β-hemolytic, whereas
H. influenzae is nonhemolytic.
• H. ducreyi appears as small, flat, smooth, nonmucoid, transparent to
opaque colonies or appears tan or yellow on CA and individual
colonies can be pushed intact using a loop across the agar plate
surface. They are difficult to pick up and produce a “clumpy”
nonhomogeneous appearance when suspended in saline.
c. Microscopic Morphology:
• Microscopic morphology varies from small, gram-negative
coccobacilli to long filaments.
• Coccobacillary morphology is the more predominant form found
in clinical specimens.
• Capsules of H. influenzae may be observed in Gram-stained direct
smears as clear, nonstaining areas (“halos”) surrounding the
organisms in purulent secretions.
• Organism is small and pleomorphic and often stains a faint pink, it
can resemble the amorphous serous material (serumlike or
proteinaceous background material).
• Because of the low specificity and sensitivity of Gram stains, an
acridine orange or methylene blue stain of the specimen may help in
detecting Haemophilus.
• Gram stains for microscopic morphology of genital lesions or colonies
for H. ducreyi may show pale staining gram-negative coccobacilli
arranged singly or in groups (clusters) commonly referred to as
“school of fish” or “railroad tracks” that are loosely coiled clusters of
organisms lined up in parallel, or appearing as “fingerprints”.
d. Laboratory identification:
• X Factor and V Factor Requirement
✓ Using impregnated strips or disks (traditional
approach) for identification of Haemophilus spp.
✓ When Haemophilus spp. are grown anaerobically,
they do not require heme but still require NAD.
✓ Haemophilus Quad Plate contains four zones: media
with X factor only, with V factor only, with X and V
factors, and with X and V factors with horse red
blood cells.
✓ The Haemophilus isolate may be identified based on
the factors required for growth and the presence of
hemolysis.
✓ H. haemolyticus is generally β-hemolytic on horse
blood while H. influenzae is negative.
 ✓ Species that are
porphyrin-negative
cannot synthesize
heme and are X factor-
positive (require
hemin) when the
impregnated strip is
used.
✓ The main advantage of the porphyrin test is that X factor is
not required, and the problem of carryover is eliminated.
✓ Disadvantage is that primary identification is based on a
negative test result.
• Biochemical Test
✓ Biochemical tests, such as carbohydrate fermentation, can
help further differentiate Haemophilus spp.
✓ In addition, indole, urease, and ornithine decarboxylase
tests are used to biotype some Haemophilus spp.
• Porphyrin Test
✓ For differentiating the heme-producing species of
Haemophilus.
✓ Can be performed in agar, in broth, or on a disk.
✓ Test principle is based on the ability of the organism to
convert the substrate δ-aminolevulinic acid (ALA) into
porphyrins or porphobilinogen, which are intermediates in
the synthesis of X factor.
✓ After incubation at 35° C for 4 hours, porphobilinogen is
detected by the addition of p-dimethylaminobenzaldehyde
(Kovacs’ reagent).
✓ After the addition of Kovacs’ reagent, a red color forms in
the lower aqueous phase if porphobilinogen is present.
✓ Porphyrins can be detected using an ultraviolet light with a
wavelength of about 360 nm (Wood’s lamp).
✓ Porphyrins fluoresce reddish orange under ultraviolet light.
RESISTANCE • In contrast to Haemophilus spp., the latter four members of the
HACEK group are considered to be more dysgonic (slower or poorer
• Increased resistance to ampicillin by Haemophilus spp. owing to β-
growing). Their predilection for attachment to heart valves, usually
lactamase or, to a lesser extent, altered penicillin-binding proteins.
damaged or prosthetic, makes many of them an important cause of
• Several rapid tests to detect β-lactamase production are available,
endocarditis.
including the chromogenic cephalosporin test (Cefinase; BD) and
• Members of the HACEK group include both fermentative and
acidometric tests.
nonfermentative, gram-negative bacilli.
• A positive β-lactamase test means that the microorganism is
• All members can be normal biota of the oral cavity, allowing for their
resistant to ampicillin and amoxicillin.
introduction in the bloodstream and resultant infections.
• Antimicrobial susceptibility testing of H. influenzae should be limited
• All HACEK organisms are opportunists and generally require a
only to isolates known to be clinically significant.
compromised host.
TREATMENT • Risk factors for infective (bacterial) endocarditis include tooth
extraction, history of endocarditis, gingival surgery, heart valve
• Current recommended treatment of life-threatening illness caused
surgery, and mitral valve prolapse.
by H. influenzae is cefotaxime or ceftriaxone.
• Because of increased resistance to ampicillin, this drug should not be
used alone for initial therapy.
• Non–life-threatening H. influenzae infection may be treated with
amoxicillin-clavulanate, an oral second-generation or third-
generation cephalosporin, or trimethoprimsulfamethoxazole.
• For the treatment of H. ducreyi, azithromycin, ceftriaxone,
ciprofloxacin, or erythromycin is recommended.

HACEK Group
• Haemophilus spp. (e.g. H. paraphrophilus)
• Aggregatibacter actinomycetemcomitans (formerly Actinobacillus
actinomycetemcomitans) and Aggregatibacter aphrophilus (formerly
H. aphrophilus) A. Aggregatibacter aphrophilus
• Cardiobacterium hominis • Most prevalent species in the HACEK group involved in
• Eikenella corrodens endocarditis.
• Kingella spp. • Does not require CO2 but grows better in its presence.
• Found in dental plaque and gingival scrapings.
 • Patients with infections present commonly with clinical features
of fever, heart, murmur, congestive heart failure, and embolism.
• H. aphrophilus and H. paraphrophilus have been reclassified into
the single species, A. aphrophilus.
• This species contains X factor–dependent and X factor–
independent strains.
• Colonies are convex, granular, and yellow with an opaque zone
near the center on CA.
B. Aggregatibacter actinomycetemcomitans
• Formerly in the genus Actinobacillus.
• Found as normal oral microbiota in humans.
• Divided into six serotypes (a through f) based on a surface
polysaccharides.
• Has been clinically isolated from blood, lung tissue, abscesses of the
mouth and brain, and sinuses.
• Recognized etiologic pathogen in the development of periodontitis
and can cause destruction of the alveolar bone that supports the
teeth.
• Major virulence factors include collagenase and a leukotoxin that is
toxic to polymorphonuclear cells and monocytes.
• Produce small bacilli to coccoid gram-negative bacilli that are
nonmotile.
• Grows better with increased CO2.
• Isolates may require more than 24 hours for visible growth; a
distinctive “star shape with four to six points” in the center of the
colonies is often seen at 48 hours.
• In broth, the organism is granular and may adhere to the sides of the
tube.
• Catalase-positive and oxidase variable.
• Do not grow on MAC agar.
• Negative for X and V growth factors, urease, indole, esculin, and
citrate.
• Glucose fermentation is positive (with or without gas), although the
addition of serum to the carbohydrate-containing medium is often
necessary to demonstrate fermentation.
• Xylose, mannitol, and maltose fermentation are variable.
• Do not ferment lactose or sucrose.
• Demonstrates sensitivity to penicillin in vitro, although this agent is
not always successful clinically, and resistance to ampicillin is
common.
 • Typically susceptible to aminoglycosides, third-generation
cephalosporins, quinolones, chloramphenicol, and tetracycline.
• Resistance to vancomycin and erythromycin is common.
• Usual treatment for endocarditis is with penicillin and an
aminoglycoside.
C. Cardiobacterium hominis
• Pleomorphic, nonmotile, fastidious, gram-negative bacillus.
• Normal microbiota of the nose, mouth, and throat and may be
present in the gastrointestinal tract.
• Oral infections or dental procedures usually precede endocarditis.
• Usual clinical manifestation is endocarditis, often manifesting with
very large vegetations and no demonstrable fever.
• Infects the aortic valve more frequently than the other HACEK
organisms.
• Rarely associated with meningitis
• Gram stains of the bacilli often show false gram-positive reactions in
parts of the cells.
• Organisms tend to form rosettes, swellings, long filaments, or
sticklike structures in yeast extract.
• Grow slowly on BA and CA agar and do not grow at all on MAC agar.
• Incubation in a humid atmosphere (either aerobic or anaerobic) with
5% CO2 is required for growth.
• On agar, “pitting” may be produced.
• Is a fermenter but reactions may be weak, and serum might be
needed.
• Ferments glucose, mannitol, sucrose, and maltose.
• Oxidase-positive, catalase-negative, and indole-positive.
• Negative for urease, nitrate, gelatin, and esculin.
• Sensitivity can be seen to β-lactams, chloramphenicol, and
tetracycline with variable response to aminoglycosides,
erythromycin, clindamycin, and vancomycin.
• Usual therapy includes penicillin and an aminoglycoside.
D. Eikenella corrodens
• Member of the normal biota of the oral and bowel cavities.
• Most infections associated with this organism have been mixed and
often occur as a result of trauma, especially after human bites or
fights (i.e., “clenched fist wounds,” or after the skin has been broken
by human teeth).
• An opportunistic pathogen, especially in immunocompromised
individuals.
• Reported as the cause of adult periodontitis, meningitis, empyema,
pneumonia, osteomyelitis, arthritis, and postoperative tissue
infections.
• Shows a predilection for attachment to heart valves and causes
endocarditis, although it is the least common isolate of the HACEK
group in adult infectious endocarditis.
• Fastidious, gram-negative coccobacilli that grow best under
conditions of increased CO2 with hemin.
• Nonmotile, oxidase-positive, and asaccharolytic.
• Catalase-negative and often produce a yellow pigment.
• 45% of the isolates of E. corrodens “pit” (make a depression) or
corrode the surface of the agar.
• Although they are nonhemolytic on SBA, a slight greening effect
secondary to growth may occur around the colonies.
• A chlorine bleach–like odor from the agar surface may be obvious.
 • Do not usually grow on MAC agar or eosin-methylene blue (EMB)
agar.
• In broth media, E. corrodens may adhere to the sides of the tube and
produce granules.
• Resistant to clindamycin and narrow-spectrum cephalosporins.
• Demonstrate sensitivity to penicillin, ampicillin, cefoxitin,
chloramphenicol, carbenicillin, and imipenem.
E. Kingella
• Members of the genus Kingella are coccobacillary to short bacilli with
squared ends that occur in pairs or short chains and tend to resist
decolorization in Gram stain.
• Typically nonmotile.
• Nutritionally fastidious, oxidase-positive, catalase-negative
fermenters of glucose and other sugars but with no gas.
• Colonize the upper respiratory tract, especially the tonsils.
• Viral infections can be a precursor to infections by these organisms.
• Poor dental hygiene or oral surgery is associated with infection.
• Genus consists of four species: Kingella kingae, K. denitrificans,
K. oralis, and K. potus.
• Can grow on Neisseria selective agar (e.g., modified ThayerMartin
medium) and can resemble Neisseria gonorrhoeae if the isolate does
not pit the agar as many strains do.
• Gram stain morphology of a rod with square ends and in chains should
aid in distinguishing Kingella spp. from N. gonorrhoeae.
• Kingella spp., especially K. kingae, are recognized as an important
pathogen in the pediatric population with a predilection for bones and
joints.
• Usually susceptible to most agents, including penicillin.
a. Kingella kingae
• Also been associated with HACEK endocarditis, particularly in
immunocompromised patients.
• Gram-negative short coccoid bacilli • Forms large white to beige
β-hemolytic colonies on SBA, no growth on MAC agar, catalase-
negative, and oxidase-positive.
• Weakly ferments glucose and maltose but is negative for sucrose
• May produce a yellow-brown pigment.
• Two types of colony morphologies: a spreading, corroding
colony or a smooth, convex, and β-hemolytic colony.
b. Kingella denitrificans • Fastidious, facultatively anaerobic, gram-negative bacilli and require
• Rarely isolated as a pathogen but has been associated with increased CO2 for growth.
bacteremia and abscesses. • Thin and often fusiform (pointed ends) resembling Fusobacterium
• Positive for glucose fermentation, catalase-negative and spp., spindle-shaped, coccoid, and curved filaments may be also
superoxol-negative and nitrate reduction. seen.
• Negative for urease, indole, esculin, gelatin, and citrate and • Flagella are usually absent but can produce gliding motility on solid
does not grow on MAC surfaces.
agar. • Colonies are often adherent, produce a yellow-orange pigment and
• Might grow at 42° C. can resemble colonies of E. corrodens.
• Two types of colonies: • Most isolates are nonhemolytic except for C. haemolytica
smooth, convex and a (βhemolytic).

spreading corroding type.

Capnocytophaga
• Genus consists of nine species, five of which
are normal microbiota of the oral cavity of
humans and have been associated with
septicemias and other human infections.
• Not as commonly involved in endocarditis
as they are in septicemia.
• Common sites of clinical isolation include
blood cultures from neutropenic patients
who have oral ulcers, juvenile periodontal • Ferment sucrose, glucose, maltose, and lactose, although triple sugar
disease, and endocarditis. iron agar (TSI) may be negative without enrichment.
• The five normal inhabitants of the human • Negative for most biochemical reactions, including indole, they may
oral cavity— C. ochracea, C. gingivalis, C. reduce nitrates and hydrolyze esculin.
sputigena, C. haemolyticus, and C. • Susceptible to imipenem, erythromycin, clindamycin, tetracycline,
granulosa—all are oxidase and catalase chloramphenicol, quinolones, and β-lactams.
negative.
• Resistant to the aminoglycosides.
• C. ochracea is the most common clinical isolate.
• For C. canimorsus and C. cynodegmi infections, penicillin is the drug
• C. canimorsus and C. cynodegmi are normal inhabitants of the oral of choice.
cavity of dogs and cats and are oxidase- and catalase-positive.C.
• C. canimorsus can cause a fulminant, life-threatening infection in
humans after a dog or cat bite or through continuous contact.
Pasteurella
• Pasteurellosis (infection with Pasteurella spp.) is a zoonotic disease
that humans acquire from exposure to infected animals or products
made from infected animals.
• Although systemic and pneumonic forms are possible, cutaneous
infection, frequently resulting from animal bites, is the most common
presentation.
• Wounds can become infected because these organisms often reside
in the respiratory tract and oral cavity of birds and mammals; and
often occur as the result of feline bites.
• At least 17 species of Pasteurella have been identified.
• P. multocida is the most frequently isolated species and includes three
subspecies: multocida, septica, and gallicida and consists of five
Brucella
serogroups (A, B, D, E, and F) defined by capsular antigens.
• Brucellosis (infection with genus Brucella) is an important zoonotic
• P. canis: associated with dogs.
disease that is found worldwide.
• P. stomatis and P. dagmatis: both associated with dogs and cats (also
• Considered category B select biological agents by the CDC because of
isolated from humans).
their potential application in bioterrorism.
• Gram-negative, nonmotile, facultative, anaerobic coccobacilli that
• Brucellosis is acquired through aerosol, percutaneous, and oral
appear ovoid, filamentous, or as bacilli.
routes of exposure.
• Bipolar staining (safety pin appearance when the poles of the cells are
• Although direct person-to-person transmission is considered rare,
more intensely stained) is frequently observed.
cases resulting from sexual contact and breast-feeding have been
• Catalase and oxidase (most isolates) positive.
reported.
• Ferment glucose with weak to moderate acid production without gas
• The three clinical stages of brucellosis are acute, subchronic, and
(TSI: weak glucose fermentation).
chronic.
• All Pasteurella spp. grow on BA and CA, producing grayish colonies.
• Symptoms of acute infection are nonspecific and occurs 1 to 4 weeks
• MC agar does not support the growth of most Pasteurella spp.
of exposure.
• P. multocida produces nonhemolytic colonies on SBA that may appear
• The subchronic or undulant form of the disease typically occurs
mucoid after 24 hours of incubation at 37° C followed by the
within a year of exposure and is characterized by undulating fevers,
production of a narrow green-to-brown halo around the colony after
arthritis, and epididymoorchitis.
48 hours.
• Chronic form commonly manifests 1 year after exposure with
symptoms such as depression, arthritis, and chronic fatigue
syndrome.
• The four species that are most commonly associated with human
illness are B. melitensis, B. abortus, B. suis, and B. canis.
• The bacteria are facultative intracellular pathogens that can reside
within phagocytic cells.
• It can be difficult to diagnose brucellosis through direct examination
of a clinical sample, most often blood or bone marrow, and the ability
for direct isolation and culture.
• Serologic tests are frequently used in conjunction with patient history
and disease status for diagnosis.
• Brucellae are small gram-negative, aerobic, nonmotile, Francisella
unencapsulated bacteria that do not form spores and may appear as • There are at least four species in the genus Francisella.
coccobacilli or bacilli. • tularensis has been implicated in most human infections and has
• Colonies appear as smooth, three subspecies or biovars: subsp. tularensis (type A), subsp.
raised, and translucent. holarctica (type B), subsp. Mediasiatica.
• Grows on SBA and CA and can be • tularensis subsp. tularensis causes the most severe disease.
isolated on modified Thayer • Tularemia (infection with genus Francisella) is a zoonotic disease and
Martin or Martin-Lewis media has many other names, including rabbit fever, deerfly fever, lemming
from contaminated specimens. fever, and water rat trappers’ disease.
• Oxidase and catalase-positive and • Tularemia can be contracted through ingestion, inhalation,
are urease-positive within 2 arthropod bite (e.g., ticks, biting flies), or contact with infected
hours. tissues.
• Brucella spp. are usually subtyped into biovars using molecular • The most common clinical form is ulceroglandular, in which an ulcer
biology technics. forms at the site of inoculation and is followed by an enlargement of
• Because of the aerosol mode of transmission, Brucella spp. should be the regional lymph nodes.
handled under biosafety level 3 conditions. • F. tularensis is a CDC category A select biological agent.
• Clinical symptoms, patient exposure to geographic locations where
tularemia is endemic, and serology are used in the diagnosis of F.
tularemia infections.
• Are facultative intracellular pathogens that appear as small,
nonmotile, non–sporeforming, gram-negative bacilli or coccoid
bacteria and are strictly aerobic.
• Fastidious and require supplementation with cysteine, cystine, or
thiosulfate.
• CA, modified Thayer-Martin, and buffered charcoal yeast extract Clinical Significance:
(BCYE) agars and Mueller-Hinton and tryptic soy broths may be used
• Infections produce a spectrum of symptoms ranging from mild upper
• MAC and EMB agars do not support F. tularensis growth.
respiratory tract infections to pneumonia.
• Because of slow growth rates, F. tularensis colonies may not be visible
• Responsible for 2% to 15% of community-acquired pneumonia.
before 48 hours of incubation at 37° C; once visible, gray-white,
• Also associated with nosocomial infections.
raised colonies with a smooth appearance are seen.
• Most human cases of legionellosis are caused by Legionella
• Oxidase, urease, and satellite or X and V test-negative and weakly
pneumophila.
positive for catalase and β-lactamase activity.
• Confirmation requires culture identification or a fourfold increase in Virulence Factors:
antibody titer.
• Organism’s ability to enter, survive, and multiply within the host’s
• F. tularensis is a highly infectious
cells, especially bronchoalveolar macrophages, and the ability to
agent with 50 organisms causing
produce proteolytic enzymes.
an infection through the
• Cause intracellular infections in humans but also survive in an
cutaneous or inhalational routes.
extracellular environment.
• Biosafety level 3 conditions should
be implemented when working Clinical Infections:
with suspected F. tularensis
• Febrile disease with pneumonia (legionnaires’ disease).
samples.
• Febrile disease without pulmonary involvement (Pontiac fever).
Legionella • Asymptomatic infection
• Ubiquitous gram-negative bacilli acquired primarily through • Mode of transmission and the number of infecting organisms in the
inhalation. inoculum play a role in the clinical features
• Patients ca present with a wide variety of conditions ranging from • Host factors, such as a suppressed immune system, chronic lung
asymptomatic infection to life-threatening disease. disease, alcoholism, and heavy smoking, predispose individuals to
legionnaires’ disease.
• Laboratory diagnosis depends on one or more of the following
methods:
✓ Isolation using special • Legionnaires’ Disease
media ✓ Manifests in three major patterns:
✓ Urine antigen 1. Sporadic cases (most common)
detection 2. Epidemic outbreaks (short duration and low attack rates)
✓ Direct fluorescent 3. Nosocomial clusters (compromised patient populations)
antibody (DFA) ✓ Pneumonia is the predominant manifestation of legionellosis
✓ Serology ✓ Incubation period: 2-10 days
✓ Typically present with a nonproductive cough, fever, headache,
and myalgia.
 ✓ Later, as pulmonary infiltrates develop, sputum may be bloody
or purulent. Rales, dyspnea, and shaking chills are clinical
manifestations of progressing disease.
✓ Dissemination via the circulatory system can lead to
extrapulmonary infections with or without pneumonia.
✓ Infections of the kidneys, liver, heart, CNS, lymph nodes, spleen,
and bone marrow and cutaneous abscesses have been
described.
✓ Bacteremia, renal failure, liver function abnormalities, watery
diarrhea, nausea, vomiting, headache, confusion, lethargy, and
other CNS abnormalities have been associated with these
infections.
• Pontiac Fever
✓ Nonpneumonic form of legionellosis.
✓ Incubation period: 2 days
✓ Flulike symptoms of fever, headache, and myalgia that last 2
to 5 days and then subside without medical intervention.
✓ L. pneumophila is responsible for most cases
EPIDEMIOLOGY
• Found worldwide, occurring naturally in aquatic sources such as
lakes, rivers, hot springs, and mud.
• Can tolerate chlorine concentrations of 3 mg/L.
• Resist water treatment and subsequently gain entry into and colonize
human-made water supplies and distribution systems.
• Legionella spp. are transmitted to human hosts from environmental
sources and transmission between humans has not been
demonstrated.
LABORATORY DIAGNOSIS
a. Specimen Collection and Handling
• Specimens commonly include sputum, bronchoalveolar lavage,
and bronchial washings.
• Saline or buffer should not be used (inhibitory effects of sodium
on Legionella spp.).
• Specimens should be refrigerated to avoid overgrowth of
contaminating microbiota (delay > 2 hours).
• Freeze specimens at −70° C; urine -20°C (delay > several days)
• Urine is an important specimen to be collected for antigen
detection (most useful for L. pneumophila serogroup 1).
b. Microscopic Examination
• Pleomorphic, weakly staining, gram-negative bacilli.
• Extending the safranin counterstaining time to at least 10
minutes can enhance the staining intensity of the organisms.
• L. micdadei is weakly acid-fast in tissue and stains best with the
modified Kinyoun procedure.
c. Isolation Methods
• Most important test for legionnaires’ disease is culture of the
organism and should always be attempted even when
employing a rapid test such as urine antigen detection.
• Acid treatment of specimens contaminated with other
bacteria, such as sputum, before inoculation enhances
isolation of Legionella spp.
• Legionella spp. are fastidious, aerobic bacteria that do not
grow on SBA and require L-cysteine for growth.
• Tiny colonies may appear on CHOC agar that contains
Lcysteine
• BCYE agar with L-cysteine is best for Legionella isolation.
d. Colony Morphology ✓ Biochemical testing has limited value in the further identification
• On BCYE agar, colonies appear as grayish white or blue-green, to the species level.
convex, and glistening, measuring approximately 2 to 4 mm ✓ Protocol can be used to evaluate suspected colonies:
in diameter. 1. Gram stain any suspicious colony growing on BCYE agar.
• When these colonies are viewed with a dissecting microscope Legionella spp. are thin, gram-negative bacilli that may
illuminated from above, the central portion of young colonies show size variation from 2 to 20 μm in length.
has a “ground-glass” 2. Subculture the isolate to BCYE agar with L-cysteine and
appearance, light gray and to either SBA or BCYE agar without L-cysteine. Legionella
granular, whereas the spp. grow only on BCYE agar supplemented with L-
periphery of the colony has cysteine.
pink or light blue or bottle 3. Prepare smears from colonies that require L-cysteine
green bands with a and test with polyvalent and monovalent conjugates to
furrowed appearance. determine specific species and serogroup.
✓ Definitive identification of less common species is usually
e. Identification Methods performed at reference or public health laboratories, frequently
• Conventional using 16S ribosomal RNA sequencing methodology.
✓ Slow growth (3 to 5 days) • Rapid
✓ Characteristic “ground-glass” colony morphology. ➢ Urine Antigen Test
✓ Lightly staining, gram-negative bacillus ✓ Includes radioimmunoassay, microplate enzyme
✓ Requires L-cysteine for primary isolation immunoassay, and rapid immunochromatographic
✓ No growth on unsupplemented sheep blood agar assay for Legionella antigen detection in urine.
(SBA) Asaccharolytic ✓ Antigen can be detected by day 3 of the infection and
✓ Catalase or oxidase: weakly positive can persist for 1 year (prolonged secretion has been
associated with immunosuppression renal failure, and
chronic alcoholism).
✓ Early antimicrobial intervention with macrolides may
decrease antigen excretion in some patients.
➢ Direct Fluorescent Antibody Test
✓ For detection of the more common species of Legionella
found in clinical samples from the lower respiratory
tract.
✓ Fluorescein isothiocyanate–labeled conjugates are
available that detect all known serogroups of L.
pneumophila, L. bozemanii, L. dumoffii, L. gormanii, L.
longbeachae groups 1 and 2, L. micdadei, and L. jordanis.
 ✓ Conjugate binds to antigens on the cell surface, and the antigen-
antibody complexes are detected using a fluorescence
microscope.
✓ The organisms appear as bright yellow to green, short or
coccobacillary bacilli with intense peripheral staining.
➢ DNA Detection
✓ Nucleic acid amplification tests such as PCR and strand
displacement amplification have demonstrated potential;
however, testing is not commonly available for routine clinical
use.
➢ Indirect Fluroescent Antibody (IFA) Assay
✓ Most common method employed for serologic diagnosis of
legionellosis.
✓ Heat-killed or formalin-killed bacteria are fixed to a microscope
slide.
✓ A fourfold increase in IFA titer to at least 1 : 128 from the acute
serum phase (obtained within 1 week of onset of symptoms)
to the convalescent serum phase (3 to 6 weeks later) is
evidence of recent infection.
ANTIMICROBIAL SUSCEPTIBILITY TESTING
• Testing is not standardized or routinely performed.
• When infections are diagnosed early, they usually can be treated
successfully with a macrolide such as azithromycin or a
fluoroquinolone.
• If the disease often takes an aggressive course, an alternative drug
(doxycycline) is used.
Bordetella
• Small gram-negative bacilli or coccobacilli
• All are obligate aerobic bacteria
• Grow best at 35° C to 37° C
• Do not ferment carbohydrates
• Do not ferment carbohydrates, oxidize amino acids, are relatively
inactive in biochemical test systems, and produce catalase (variable in
B. pertussis)
• Both Bordetella pertussis and B. parapertussis are primary human
pathogens of the respiratory tract, causing whooping cough or
pertussis.
• B. pertussis is fastidious and requires special collection and transport
systems and culture media, inhibited by fatty acids, metal ions,
sulfides, and peroxides, constituents found in many media.
• Media for the isolation of B. pertussis require protective substances,
such as charcoal, blood, or starch.
• At least six other species are recognized: B. bronchiseptica, B. avium,
B. hinzii, B. holmesii, B. petrii, B. trematum.
• The other Bordetella spp. except for B. pertussis are less fastidious and
grow on MAC agar or media containing blood.
Bordetella pertussis and Bordetella parapertussis
Virulence Factors:
• Filamentous hemagglutinin (FHA) and pertactin (a 69-kDa outer
membrane protein)
• Pertussis toxin (PT)
• Adenylate cyclase toxin
• Tracheal cytotoxin
Clinical Manifestations:
• Incubation period: 1-3 weeks (usually 7-10 days)
• Classic pertussis or whooping cough resulting from B. pertussis
infection occurs after exposure to the organism through the
respiratory tract.
 1. Catarrhal phase
✓ Symptoms are insidious and nonspecific and include
sneezing, mild cough, runny nose, and perhaps conjunctivitis.
✓ The infection is highly communicable because of the large
number of organisms in the respiratory tract.
2. Paroxysmal phase
✓ Sudden onset of severe, repetitive coughing followed by the
LABORATORY DIAGNOSIS
characteristic “whoop” at the end of the coughing spell.
✓ Whooping sound is caused by the rapid gasp for air following
the prolonged bout of coughing.
3. Convalescent phase
✓ Generally begins within 4 weeks of onset with a decrease in
frequency and severity of the coughing spells.
EPIDEMIOLOGY
• Pertussis is a human disease.
• No animal reservoir or vector has been found .
• Infections caused by Bordetella spp. are acquired through the
respiratory tract via respiratory droplets or direct contact with
infectious secretions.
• Organisms are uniquely adapted to adhere to and replicate on
ciliated respiratory epithelial cells.
• The organisms remain localized to the respiratory tract, but toxins
and other virulence factors are produced and have systemic effects.
• Pertussis is one of the most highly communicable diseases of
childhood.
• Vaccine is available, however, Immunity is short-lived.
• Infants younger than 6 months old, who are too young to be fully
vaccinated, continue to be at highest risk for severe disease
MISCELLANEOUS SPECIES
• The remaining Bordetella spp. are either opportunists or not primary
human pathogens.
• B. bronchiseptica is a respiratory tract pathogen of numerous animals
including dogs.
• Symptomatic B. bronchiseptica infections in humans generally
manifest with a nonspecific cough or bronchitis, and often patients
have underlying conditions, such as immunosuppression or contact
with animals.
a. Specimen Collection and Handling
• Nasopharyngeal aspirates or swabs (calcium alginate or
Dacron polyester with a flexible wire shaft) constitute the
specimen of choice for culture, DFA, and PCR testing.
• Specimens should be transported at room temperature and
transferred to culture media as soon as they arrive at the
laboratory.
• In situations requiring transport overnight or over several
days, half-strength charcoal agar containing 10% horse blood
and 40 mg/L cephalexin (Regan-Lowe transport medium)
should be used.
b. Nucleic Acid Detection
• Detection by PCR from nasopharyngeal swabs is a primary
rapid diagnostic strategy.
• Laboratories that offer PCR must conduct in-house
verification and validation studies because FDA approval of
products is limited to analyte-specific reagents.
c. Microscopic Examination
• Clinical specimens can be examined for Bordetella spp.
microscopically only using DFA staining.
• DFA test should be used only along with culture.
• Slides for DFA testing may be prepared directly from swab
specimens or after expression of the material from the swab.
• On microscopic examination, the organisms appear as small,
fat bacilli or coccobacilli with intense peripheral yellow-green
fluorescence and darker centers.
d. Isolation Method • Suspicious colonies should be screened further for B.
• Most successful is charcoal agar supplemented with 10% pertussis and B. parapertussis using agglutinating or
horse blood and 40 mg/L cephalexin. fluorescein-labeled antisera.
• Medium is identical in composition to the transport medium • When the results of fluorescent staining or
of Regan and Lowe except that it contains agar at full agglutination are clear, confirmatory testing is not
strength. required.
• Plates for the recovery of Bordetella spp. should be
incubated at 35° C in ambient air for a
minimum of 7 days.
• A stereomicroscope should be used to
detect the colonies before they become
visible to the unaided eye.

e. Colony Morphology
• On charcoal–horse blood and Regan-Lowe media, young
colonies are smooth, glistening, and silver, resembling
mercury droplets.
• Colonies turn whitish gray as they age
• Bordet-Gengou agar, colonies of B. pertussis and B.
parapertussis are hemolytic.

f. Identification Methods
• On Gram stain of culture isolates, the organisms stain as tiny
gram-negative coccobacilli and may become elongated if
recovered from media containing cephalexin.
• Increasing the safranin counterstaining time to 2 minutes may be
necessary to see typical morphology.
 g. Serologic Testing Friendly Reminder Don’t forget to pray.
• Tests for routine purposes are not approved for diagnostic use.
• If serologic assays are to be performed, reference sera available
from the Laboratory of Pertussis (FDA, Bethesda, MD) should be
used for quality control.
• Serology also tends to be retrospective; several weeks are often
required to demonstrate a diagnostic response.

ANTIMICROBIAL SUSCEPTIBILITY

• Erythromycin is the drug of choice for treatment and prophylaxis of

pertussis.
• Routine antimicrobial susceptibility testing of B. pertussis or B.
parapertussis is unnecessary because of predictable sensitivity to
macrolides.
• Susceptibility of B. bronchiseptica is unpredictable, and tests should
be performed, although the organism is usually susceptible to the
aminoglycosides.

-END-

Kapoy dzae? Taas kayo noh?

“It’s okay to feel tired, it’s okay to feel stressed, It’s okay to even cry.
What isn’t okay is giving up. It isn’t okay to stop trying.”
Prepared by:
-Amira Askira
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