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GROUP 2
GENUS HAEMOPHILUS
Submitted by:
Chua, Albert Y.
Cielo, Angela V.
De Guzman, Masao E.
Genus Haemophilus
General Characteristics
Colony characteristics
Haemophilus grow in small, round, convex colonies, which may be iridescent, develop in
24 hours on chocolate blood agar. Encapsulated strains appear small, semi-opaque, gray-white,
and mucoid on chocolate agar.
Microscopic appearance
Virulence factors
Capsule
The capsule is the most significant virulence factor present in Haemophilus organisms.
H. influenzae, though not all, can express one of six antigenically distinct polysacchaide
capsules, designated a through f. The most invasive infections were caused by encapsulated
strains of Haemophilus influenza belonging to serotype b (Hib) and occurred primarily in
systemic infections in children, whereas most respiratory tract isolates are uncapsulated,
referred to as non-typeable. In unvaccinated children, type b is the leading cause of meningitis.
The serotype b capsule is a unique polymer composed of ribose, ribitol, and phosphate
(polyribitol phosphate [PRP]). The antiphagocytic property and anticomplementary activity of the
type b are important factors in virulence.
IgA proteases
Most nonencapsulated strains are adherent to human epithelial cells, while most
serotype b strains are not. The lack of this adherent capability of type b strains may explain the
tendency of type b strains to cause systemic infections. One example of adhesion protein is the
haemagglutinating pili, also called fimbriae, which promote adherence to respiratory mucus and
human oropharyngeal epithelial cells, as well as promote interaction with heparin-binding
extracellular matrix proteins. Others are Hap (promote adherence and invasion), Hia/Hsf (high-
affinity adhesive activity and mediates interaction with a broad array of respiratory epithelial cell
types), HMW1/HMW2 (mediate attachment to human epithelial cells found in non-typeable
strains), and OapA (required for efficient colonization of the nasopharynx and may play a minor
role in adherence).
The LPS comprises two distinct, covalently linked regions, the lipid A and the core
oligosaccharide. LPS is an important virulence determinant of H. influenzae, playing a key role
in the processes of bacterial colonization, persistence and survival in the human host, but also
represents an ideal target for bactericidal as well as endotoxin-neutralizing antibodies. The lipid
A portion of H. influenzae LPS is embedded in the outer membrane and mediates the endotoxic
effects of the LPS molecule responsible for some of the pathophysiology associated with severe
infection. LOS phosphorylcholine (ChoP) may influence invasion via interaction with PAF
receptor and stimulates inflammatory response. P2 protein is the most abundant major outer
membrane protein of non-typeable H. influenza. It allows the organism to evade clearance by
potentially protective antibodies and contributes to the development of chronic infection. P5
protein interacts with a glycoprotein expressed by respiratory epithelial cell.
Cultivation
Haemophilus isolation agar with bacitracin is a primary plating medium used for the
selective isolation of Haemophilus species. Haemophilus Isolation Agar is formulated with Fildes
Enrichment and BBL IsoVitaleX enrichment to supply the essential X and/or V growth factors.
Horse blood provides appropriate hemolytic reactions to facilitate the differentiation of
Haemophilus species. The antimicrobial agent bacitracin is incorporated to inhibit the growth of
bacteria that could mask the presence of Haemophilus species. Haemophilus isolation agar with
bacitracin consists of Brain Heart Infusion Agar supplemented with Fildes enrichment,
IsoVitaleX enrichment and horse blood. Fildes enrichment is a peptic digest of sheep blood that
supplies both X and V factors. IsoVitaleX enrichment is a chemically-defined supplement that
provides V factor and other nutrients, such as thiamine and cysteine, to stimulate the growth of
Haemophilus species.
Haemophilus Test Medium Agar (HTM Agar) is intended for use in the antimicrobial disc
diffusion susceptibility procedure for Haemophilus spp. This medium is Mueller Hinton agar or
broth supplemented with X factor (hemin or hematin), V factor (nicotinamide adenine
dinucleotide, NAD) and yeast extract. A major advantage of HTM Agar compared with Mueller
Hinton Chocolate Agar is optical clarity, permitting zone diameter measurements from the
bottom of the dish as is the standard test procedure for nonfastidious organisms on Mueller
Hinton Agar. Furthermore, HTM Agar contains low levels of thymidine and is, therefore, suitable
for testing sulfamethoxazole/trimethoprim (SXT).
X & V test
Members of the Haemophilus genus are typically cultured on blood agar plates as all
species require at least one of the following blood factors for growth: hemin (X factor) and/or
nicotinamide adenine dinucleotide (V factor). Chocolate agar is an excellent Haemophilus
growth medium as it allows for increased accessibility to these factors.
X & V Strips
Satellite Test
ALA-Porphyrin test
A better test for X factor requirement is based on the inability of H. influenzae (and a few
other Haemophilus species) to synthesize heme from δ-aminolevulinic acid. The organism is
inoculated into 0.5 ml of 0.1 M sodium phosphate buffer, pH 6.9, containing 0.08 mM
magnesium sulfate and 2 mM δ-aminolevulinic acid and then incubated at 37°C for 4 hours. .
Factor X-independent bacteria incorporate the aminolevulinic acid into the heme biosynthesis
pathway and excrete pathway intermediates, such as porphobilinogen and porphyrins.
Porphobilinogen is detected by the addition of p-dimethylaminobenzaldehyde (Kovac’s reagent);
a red color forms in the lower aqueous phase if porphobilinogen is present. The presence of red
fluorescence under a Wood’s light in a dark room (approximately 360 nm) indicates the
presence of porphyrins and a positive test; a bluish fluorescence is negative. Haemophilus
species that synthesize porphyrins (and thus heme) are not H. influenzae.
Carbohydrate fermentation
Haemophilus species are facultative anaerobes and have both a respiratory and a
fermentative type of metabolism. Biochemical test such as carbohydrate fermentation can help
furher differentiate the Haemophilus spp. They are capable of fermenting glucose and other
carbohydrates, producing acids and sometimes gas.
Table 2. Carbohydrate Fermentation of Haemophilus spp.
Indole test is a biochemical test performed on bacterial species to determine the ability
of the organism to split indole from the amino acid tryptophan. Haemophilus influenzae will yield
a positive result. Reagents include 0.05 M phosphate buffer (pH 8.0) containing 0.1%
tryptophan; after 4 hours incubation at 35 °C, Kovac’s reagent is added and a red color
indicates a positive result. Yellow color indicates negative result.
Urease test is a test performed on bacterial species to determine if the bacteria can
produce carbon dioxide from urea. A balanced salts solution (0.1% KH2PO4, 0.1% K2HPO4,
0.5% NaCl, and 0.5 ml of a 2% solution phenol red), pH 7.0, is prepared and autoclaved.
Development of red color indicates positive result. Christensen’s urea agar slants may also be
used.
Ornithine decarboxylase test is used to determine the ability of the bacteria to catalyze
the decarboxylation of ornithine. Standard Moeller’s ornithine decarboxylase broth is used.
Heavy inoculum is used and each tube is overlaid with sterile mineral oil.
Pathogenesis and Clinical Manifestations
Haemophilus influenzae
Otitis media – inflammation of the middle ear, located between the tympanic membrane
and the inner ear, including a duct known as the Eustachian tube; treatment with five day
treatment with amoxicillin or amoxicillin-clavulanate
Haemophilus aegyptius
Brazilian purpuric fever (BPF) – preceded by conjunctivitis, prostrating high fevers and
chills, vomiting and gastrointestinal complaints progress rapidly to extensive purpura
(red or purple discolorations of the skin), septic shock and acidosis; treated with
ampicillin plus chloramphenicol but the mortality rate is around 70% even with therapy
Haemophilus ducreyi
Other species
From books:
Adelberg, Jawetz, & Melnick. (2007). Medical Microbiology, 24th Edition. The McGraw-Hill
Companies: USA.
Dworkin, M. (ed.) The Prokaryotes: A handbook on the biology of bacteria, 3d ed., Vol. 6:
Gillespie, S. H. & Hawkey, P. M. (ed.). (2006). Principles and practice of clinical bacteriology.
State Key Laboratory for Moleclular Virology and Genetic Engineering. (2003). Virulence factors
bin/VFs/vfs.cgi?VFID=VF0040#VF0040
St. Louis Community College website. (2008). Biochemical tests. Retrieved on September 11,