Haemophilus and Other Fastidious GramNegative Rods
Case Study
 A 2-year-old unvaccinated child was seen in the
emergency room because of complaints of headache
and fever
 A cerebrospinal fluid(CSF) sample was obtained and
sent to the laboratory for culture
 The Gram stain showed many white blood cells and
many gram-negative, small bacilli
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Points to Consider
 What clinical findings led the physician to request a
CSF culture?
 Based on the patient’s age, which organism would be
suspected?
 What growth requirements must be met to recover
the suspected agent?
 Which other fastidious organisms would be
considered?
 Other points to consider
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Haemophilus and Other Fastidious Gramnegative Rods
 The fastidious group of gram-negative bacilli
include:
 Haemophilus
 HACEK
 Legionella
 Bordetella
 Pasteurella
 Brucella
 Francisella
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Haemophilus Influenzae: Clinical Infections
 Infections caused by typable (encapsulated) strains
 Acute epiglottis or laryngotracheal infection in small children
 Cellulitis/arthritis
 Meningitis
 Pneumonia/septicemia (in children)
 Conjunctivitis
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Haemophilus Influenzae: Clinical Infections
 Infections caused by Nontypable strains
 Otitis media
 Sinusitis
 Pneumonia, bronchitis (in adults)
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Haemophilus Species
H. influenzae
satellitism around
and between the
large, white,
hemolytic
staphylococci
Haemophilus species require hemoglobin for growth:
X-factor ( hemin): Heat-stable substance
V-factor (NAD): Heat- labile, coenzyme I, nicotinamide
adenine dinucleotide, found in blood or secreted by
certain organisms
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Haemophilus Species
Direct smear of H. influenzae in CSF in a
case of meningitis. Note the intracellular
and extracellular pleomorphic gramnegative bacilli.
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Haemophilus species
H.influenzae growing on
chocolate agar. Notice the
semi-opaque, gray-white,
mucoid colonies characteristic
of encapsulated strains.
Gram stain of H. influenzae
from colony
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Haemophilus Species: Identification
This organism would be identified as H. influenzae
because it is using both X and V factors.
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Haemophilus Species: Identification
This organism would be identified as H.
parainfluenzae because it is using V factor only.
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Haemophilus Species: Identification
This organism would be identified as H. aphrophilus because it is
using X factor only.
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Haemophilus Species: Identification
Under ultraviolet light, the organism on the bottom
is showing a positive porphyrin reaction, whereas
the organism on the top is demonstrating a
negative porphyrin reaction.
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HACEK Group and Capnocytophaga
 HACEK is an acronym of the first initial of each genus
that belong in the group:
 Haemophilus aphrophilus
 Actinobacillus actinomycetemcomitans
 Cardiobacterium hominis
 Eikenella corrodens
 Kingella species
 Capnocytophaga sp.
 Has similar requirements as the HACEK group
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HACEK Group and Capnocytophaga: General
Characteristics
 Gram-negative bacilli
 Require an increased CO2 (5%-10%) environment
 Significant cause of endocarditis
 Usual flora of the oral cavity
 Opportunists in immunocompromised hosts
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HACEK Group
H. aphrophilus growing
on blood agar
Gram stain morphology of H.
aphrophilus
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HACEK Group
A. actinomycetemcomitans on
blood agar. Note the starshaped centers of the
colonies.
Microscopic morphology of A.
actinomycetemcomitans (Gram
stain).
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HACEK Group
The 48-hour growth of
colonies of C. hominis
Gram stain of C. hominis
showing typical “rosettes”
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HACEK Group
E. corrodens on
chocolate agar
Gram stain morphology
of E. corrodens
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HACEK Group and Capnocytophaga
Growth of Capnocytophaga
colonies on chocolate agar.
Compare this growth with
Eikenella
Gram stain of
Capnocytophaga
species
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HACEK group
Gram stain of Kingella kingae showing plump
rods in chains. Compare this morphology with
other members of the HACEK group.
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Pasteurella species
 General characteristics
 Colonizes mucous membranes of the upper respiratory tract
and gastrointestinal tracts of mammals and birds
 Human infections occur from bites and scratches inflicted by
animals
 Most common isolated species is Pasteurella multocida
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Pasteurella species
 P. multocida: Clinical manifestations
 Localized infection after a bite or scratch
 Respiratory tract infection
 Life-threatening systemic diseases (e.g., meningitis,
bacteremia)
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Pasteurella Multocida
 Culture characteristics
 Growth on 5% blood or chocolate shows small, smooth, convex
colonies
 “Musty” odor
 No growth on MacConkey agar; oxidase positive
 Microscopic examination
 Very small gram-negative rods
 Bipolar staining with Giemsa or methylene blue
 “Safety-pin” appearance
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Pasteurella Multocida
P. multocida growing on blood
agar and chocolate agar. No
growth on MacConkey agar
plate.
Gram stain morphology of P.
multocida showing bipolar staining
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Case Study
 Ten members of a group of 2 dozen retirees from the
tobacco industry became acutely ill with pneumonia
during a 2-week cruise
 The group was staying in a block of rooms together
and spent time socializing over drinks and cigarettes
in the cabins and saunas and at the poolside
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Case Study
 On the fifth day of the cruise, several members went
to see the ship’s doctor because of a worsening
cough
 Chest x-rays revealed patchy lobar pneumonia in all
affected individuals
 The condition improved with erythromycin therapy
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Legionella Species
 General characteristics
 Ubiquitous gram-negative rods
 Acquired by humans primarily through inhalation of aerosols
 Clinical infections
 Febrile disease with pneumonia (Legionnaire’s disease) and
extrapulmonary involvement
 Pontiac fever (without pulmonary involvement)
 Asymptomatic infection
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Legionella pneumophila
Gram stain of specimen showing
intracellular and extracellular
Legionella pneumophila
L. pneumophila in specimen stained by direct fluorescent antibody technique.
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Legionella pneumophila
A
B
(A) Nonselective buffered charcoal yeast extract (BCYE) plate
inoculated with sputum specimen.
(B) Selective BCYE inoculated with the same specimen but
treated before inoculation. Legionella colonies are the smallest
visible colonies.
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Legionella pneumophila
L. pneumophila colony on BCYE agar after 3, 4, 5, and
7 days of incubation
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Legionella pneumophila: Identification
Schema for identification of L. species
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Points to Remember
 Clinical manifestations presented by the patient
 The types of infections these organisms produce
 The risk factors that predispose susceptible
individuals
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Points to Remember
 What special growth supplements are required for
isolation
 Where these groups of organisms are usually found
 Characteristic features of the organisms for
identification and differentiation among closelyrelated species
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