Haemophilus influenzae: Habitat, Cell Morphology, Cultural Characteristic, Biochemical Tests, Pathogenicity, Diagnosis and Treatment

Habitat

• Mucous membrane of the upper respiratory tract of humans.
• Found in the nasopharynx of approx. 75% of healthy peoples.

Cell Morphology

• Slender, short, gram negative rods or coccobacillus
• 3-0.5 um X 0.5-1 um with rounded ends.
• Prolonged counterstaining for 5-15 minutes with very dilute carbol fuschin often gives satisfactory results.
• Non-Motile, No flagella or pilli
• Capsules are present and important in pathogenicity.
• Encapsulated form are coccobacilli, CSF Sample
• Non-Capsulated are pleomorphic, long, thread, filamentous
• Fastidious Microorganism
• It is the first free living organisms to have its entire genome sequenced; 1,830,140 bp of DNA and 1740 Genes.

Cultural Characteristic

• On Blood Agar: translucent, low, convex or flat pin point colonies, Satellitism.
• On Chocolate Agar: Grayish, Transparent, smooth, low, convex or flat with a slightly splayed out, entire edge, mucoid, pale
• In liquid medium like Levinthal’s Broth or Filde’s Broth: Uniform Turbidity.
• 5 to 0.8 mm after incubation at 37˚C for 24 hours, can enlarge upto 1-1.5 mm by 48 hrs.
• Capsulated strains form larger, more opaque, smooth, mucoid colonies, 3-4 mm in diameter.
• Fastidious and Facultative anaerobes and aerobic, grow also in 5% CO2.
• Optimum Temperature: 35-37˚C, Min: 20-25˚C, Optimum pH: 7.6
• Killed at 55˚C for 30 minutes of heating
• Requires 2 accessory growth factors, a head stable growth promoting substances present in Red Blood Cells (X-Factor) and a heat labile vitamin like substances (V-Factor).

X-Factor (Hemin)

• Hemin, plays a role as an enzyme in respiration.
• Use human transferrin as a sole source of iron
• Provide protophyrin IX, iron can be obtained from another source.

V-Factor (NAD)

• Present in the tissue of plants and animals.
• Heating blood agar to about 75˚C, until it acquires a chocolate agar, releases V-Factor from Red cells and also inactivates NADase activity.
• aureus release V-Factor during growth, which defuses into the surrounding medium, enhancing the growth of H. influenzae.
• influenzae will show Satellitism.
• V-Factor is involved in oxidation-reduction process in the growing bacterial cell.
• influenzae requires pantothenic acid, thiamine and uracil.

Biochemical Tests

Pathogenicity

• Haemophilus influenza b (Hib) is the major cause of childhood infectious disease.
• It causes meningitis, primary bacteremia, pneumonia, epiglottitis and arthritis.
• Non-Capsulated influenzae (Nc-hi) causes otitis media, conjunctivitis and sinusitis
• Causes chronic bronchitis in adults

Mechanism
Encapsulated organisms –> penetrate the epithelium of the nasopharynx –> invade the blood capillaries –> infection.

• Capsule resists phagocytosis and complement mediated lysis.
• May be opportunistic
• Transmitted by direct contact, inhalation of the respiratory tract droplets.

Diagnosis

Specimen: CSF, Sterile site aspirates, fluids, urine, sputum, Respiratory tract specimens.

1. Direct Detection Method

– Gram Staining –>Gram –ve, coccobacilli, small rods

2. Antigen Detection

–  Agglutination Assays (Latex Agglutination Test)

– For H. influenzae type b

3. Cultivation

– On Blood Agar: translucent, low, convex or flat pin point colonies, Satellitism.

– On Chocolate Agar: Grayish, Transparent, smooth, low, convex or flat with a slightly splayed out, entire edge, mucoid, pale.

4. Polymerase Chain Reaction (PCR)

– More sensitive

– CSF is the sample,

– Test in case of meningitis.

Treatment

• First line of treatment: 3^rd Generation Cephalosporin with good CSF penetration.
• Ceftriaxone and Cefotaxime is used in case of Meningitis and Septicemia.
• In poor countries, Chloramphenicol alone or in combination with Ampicillin.
• For non-invasive infections: otitis media, sinusitis, bronchitis: Amoxicillin is used orally.

Prevention and Control

• PRP-conjugate vaccines (capsular antigen)
• PRP-T, PRP-OMP, PRP-CRM, HbOC.

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