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Haemophilus
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Taxonomic Placement
Phylum Class
: Proteobacteria,
:

Gammaproteobacteria, Order
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Pasteurellales,

General Characters

Minute to medium-sized coccobacilli or rods, Less than 1 m in width. sometimes forming threads or laments and showing marked pleomorphism. Gram ve, Nonmotile, Aerobic or facultatively anaerobic. Require pre-formed growth factors present in blood,
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Optimum temperature, 3537 C,

Cell Morphology

Cells of Haemophilus species occur as individual short rods or coccobacilli. Filament formation is environmentally inuenced. Capsules are present in some species, notably H. inuenzae, H. paragallinarum, and H. parasuis. Capsules play an important part in pathogenesis, determination of type specicity, and induction of anti-infective immunity in H. inuenzae and H. paragallinarum. Occasional capsulate strains of H. parainuenzae have been described.
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Cell wall composition: The cell walls resemble those of other gram-negative bacteria in structure, composition, and endotoxic activity. Unlike the lipopolysaccharide (LPS) of Enterobacteriaceae, H. inuenzae LPS lacks long polysaccharide O chains and, thus, may be described more accurately as a lipooligosaccharide (LOS). Fresh clinical isolates of H. inuenzae usually express peritrichous pili with hemagglutinating properties. However, after serial subcultivations most isolates, either capsular or noncapsular, do not express 4/12/12 such pili.

Colonial Characters

Surface colonies of Haemophilus species on sufficiently rich media are usually nonpigmented or slightly yellowish, flat, and convex. Diameter of 0.52.0 mm within 48 h at 37C. H. influenzae undergoes variation in colony opacity. spontaneous phase

Opacity variation and is associated with the ability to colonize the nasopharynx (Weiser et al. 1995). Most species produce smooth colonies, but some variation is seen, particularly in H. parainfluenzaeand H. aphrophilus.
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Some species show haemolysis on blood agar.

The optimum growth temperature is 3537C and the minimum temperature is 2025C. Most Haemophilus strains are killed by heating at 55C for 30 min. H. inuenzae grows better under aerobic than anaerobic conditions. The most universally satisfactory propagative media are chocolate agar and Levinthal media (agar and broth). The former has the virtue of relative ease of preparation, the latter that of transparency, which facilitates the recognition of colonial phases and dissociation phenomena.

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X & V Factor

The most critical ingredients of any medium for Haemophilus spp., whether for propagation or for characterization, are the growth factors X and/or V (vitamin-like ). All members of the genus require one or both of these growth factors. X factor is usually protoporphyrin IX but, under conditions where iron cannot be obtained from another source, the iron-containing protohaeme is required. Blood 4/12/12 or blood derivatives, including haemin, are

The V factor is minimally nicotinamide mononucleotide nicotinamide riboside (NR),

equivalent (NMN)

to or

but is usually described as nicotinamide adenine dinucleotide (NAD). None of the members of the family Pasteurellaceae are capable of de novo synthesis of NAD. The V factor is inactivated by autoclaving and is therefore absent in traditional agar or broth media. Traditional sources of V factor, apart from blood, have been yeast derivatives which must be added after 4/12/12 autoclaving of the medium.

Satellitism

When inoculated on media deficient in V factor but containing the X factor (e.g., blood agar) Haemophilus colonies cluster around contaminant colonies of other bacteria which produce the critical factors in excess. This phenomenon is called satellitism. It may be demonstrated by inoculating a medium such as blood agar with a strain requiring the V factor. The inoculated area is crossed with a single streak of Staphylococcus aureus. Early 4/12/12 growth of the Haemophilus strain is

It gradually spreads peripherally but remains heaviest in the area nearest the feeder streak, reecting the diffusion gradient of the limiting growth factor. Satellitism on blood agar is strongly suggestive of a Vfactor requirement.

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Pathogenicity

Young or previously susceptible.

unexposed

animals

are

Although virulence factors have not been fully identified, endotoxins are thought to play important role. Haemophilus somnus can adhere firmly to several host cell types, including endothelial and vaginal epithelium. Organism is reported to cause degeneration of macrophages and suppression of neutrophils. Certain outer membrane proteins which confer virulence allow widespread dissemination of the 4/12/12

Diagnostic Procedures

Specimens for lab examination depend on clinical condition and type of lesions. Haemophilus species are fragile and neither refrigeration nor transport media maintain viability. Ideally clinical specimens should be frozen in dry ice and delivered to lab within 24 hours. Satellism test : Either chocolate or blood agar, inoculated with streak of S. aureus, 4/12/12 incubated at 37C under 5-10% CO2 for 2-

Identification isolates:

criteria

for

Small, dewdrop like colonies after 1-2 days, Enhancement of growth under CO2 , Requirement of X & V factors, Biochemical Profile.

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Prepared by : SANDEEP SHARMA