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Introduction of Haemophilus influenzae

Haemophilus influenzae colonies on chocolate agar after overnight incubation in a carbon dioxide atmosphere as shown above image. It must contain haemin or other iron-containing porphyrin and nicotinamide adenine dinucleotide  (NAD) or its phosphate (NADP). The porphyrin requirement is referred to as growth factor X and the NAD or NADP requirement as growth factor V. It was a causative agent of the devastating 1918 pandemic of influenza to now from local to systemic infections.

Classification of Haemophilus

• Domain: Bacteria
• Kingdom: Eubacteria
• Phylum: Proteobacteria
• Class: Gammaproteobacteria
• Order: Pasteurellales
• Family: Pasteurellaceae
• Genus: Haemophilus
• Species: H. influenzae

History of Haemophilus

1. First described in 1892 by Richard Pfeiffer during an influenza pandemic
2. Isolated by Smith, Andrewes, and Laidlaw in 1933
3. Need one or both of accessory growth factors X and V present in the Blood

General Features of Haemophilus

• Small
• Non-motile
• Non-sporing
• Oxidase test positive
• Pleomorphic Gram-negative rods
• Transmitted via respiratory droplets, or direct contact with contaminated secretions
• Normal flora of the human respiratory tract and oral cavity

Clinical Importance of Hamophilus

1. H. influenzae type b is an important human pathogen
2. H. ducreyi is a sexually transmitted pathogen causing chancroid.
3. Other Haemophilus are normal flora like H. parainfluenzae – pneumonia and endocarditis,  H. aphrophilus causes pneumonia and endocarditis and  H. aegyptius causes pink eye (purulent conjunctivitis).

 Haemophilus Influenzae

• Aerobic gram-negative bacteria
• Polysaccharide capsule
• Six different serotypes (a-f) of the polysaccharide capsule
• 95% of invasive diseases caused by type b (Hib) Haemophilus Influenzae (formerly called Pfeiffer’s bacillus or Bacillus influenzae )

Morphology of  Haemophilus Influenzae

It is small having a size of  1.5 x 0.3 µm, Gram-negative, non -motile rods showing considerable pleomorphism. It is non-sporing and non-acid fast. The cells are usually cocobacilliary in young cultures ( 18-24 hours), while in older cultures, long filamentous forms may be seen. Pleomorphic appear as clusters or coccobacillus forms in sputum sample while infected CSF ( meningitis) long and filamentous form predominates.

Cultural Characteristics of  Haemophilus Influenzae

H. influenzae needs fastidious growth requirements. Factors X and V are essential for growth.  X is hemin heat stable and porphyrins for the synthesis of cytochromes. V factor is coenzyme nicotinamide adenine dinucleotide (NAD) phosphate acts as a hydrogen acceptor. It grows well on chocolate agar and it is a non-selective, enriched growth medium that is the lysed blood agar. The name of agar is for its color when the red blood cells (RBCs) lysis gives the medium a chocolate-brown color without having chocolate products. It uses for the isolation of fastidious bacteria, such as Haemophilus influenzae, when incubated at 35-37°C in a 5% CO₂   incubator. Haemophilus influenzae shows opaque cream to gray colonies.

H. influenzae in a Gram stain of a sputum sample appears as Gram-negative coccobacilli. Haemophilus influenzae requires X and V factors for growth and therefore, it grows only around the paper disk that has been impregnated with X and V factors on non-selective and non-enriched media like nutrient agar or MHA. There is no bacterial growth around the disk that only contains either X or V factor but may be in between X and V disks as shown above picture.  It also shows satellitism. When Staphylococcus aureus is streaked across a plate of blood agar with a species containing H. Influenzae the colonies which are large development along with the streak of Staphylococcus and are small further away as shown above image and below video.

Biochemical Reactions of  Haemophilus Influenzae

H. influenzae is catalase and oxidase test positive ferments glucose and galactose, reduces nitrate to nitrite. It does fement lactose, mannitol and sucrose. On the basis of production of urease, indole, and ornithine decarboxylase, it is divided into 8 biotypes.

Biotypes  Urease  Indole  Ornithine decarboxylase

• I       +         +                           +
• II       +        +                           –
• III      +        –                           –
• IV     +         –                           +
• V       –         +                          +
• VI      –         –                         +
• VII     –         –                         –
• VIII    –         –                        –

Resistance

It is a delicate organism and it is readily killed by heating at 55°C for 30 minutes. Refrigeration, drying, and disinfectants destroy the organisms. For long-term preservation, the culture may be lyophilized.

Antigenic Structures of  Haemophilus Influenzae

It contains 3 major surface antigens and they are-

1. Capsular polysaccharide
2. Outer membrane proteins (OMP)
3. Lipopolysaccharides ( LPS )

Pitman Classification– The major antigenic determinant of capsulated strains into six capsular types type a to f. Typing by agglutination reaction using type-specific antisera. Other methods include-

• Quelling reactions
• Coagglutination
• Countercurrent electrophoresis ( CIEP)
• ELISA

Type b characteristics

Hib has unique characters contains pentose sugars, ribose ribitol, instead of hexose in others, and hexosamines. The capsular polyribosyl ribitol phosphate ( PRP ) of Hib induces IgG,  IgM, and IgA antibodies which are bactericidal, opsonic, and protective. So Hib PRP ( polyribosyl ribitol phosphate) was employed for Immunization.

 Mode of Transmission

Droplet infection and discharge from the upper respiratory tract (URT) during the infectious period. The incubation period is unknown, probably short, 2-4 days. Infectious Period – As long as the organism is present, even in the absence of nasal discharge. Non-infectious within 24 to 48 hours after the start of effective antibiotics.

Haemophilus influenzae type b Clinical Features

1. Epiglottitis
2. Meningitis
3. Pneumonia
4. Osteomyelitis
5. Arthritis
6. Cellulitis
7. Bacteremia

Non -typable Strains

H. Influenzae lacking capsules that are nontypable are most relevant in clinical infections. Outer membrane proteins (OMP) of Hib are classified into 13 subtypes.  H. Influenzae lipopolysaccharides are more complex. The genome of the organism is sequenced.

Pathogenicity

A human pathogen can produce invasive and non-invasive lesions. The prominent organism is producing meningitis. It can produce laryngoepiglottitis, conjunctivitis, bacteremia, pneumonia, arthritis,  endocarditis and pericarditis. Most strains are opportunistic pathogens. Most strains of H. influenzae are opportunistic pathogens; that is, they usually live in their host without causing disease, but cause problems only when other factors (such as a viral infection or reduced immune function) create an opportunity.

Most Important Clinical Illness associated with H. influenzae 

Pneumonia: Severe shortness of breath, rapid heart rate, fever, cough, and evidence of pneumonia by chest radiograph.

Septic Arthritis: Swelling, warmth, pain with movement and decreased mobility of a single large weight-bearing joint.

Haemophilus Influenza

Mode of Transmission

Droplet infection and discharge from the upper respiratory tract during the infectious period. Incubation Period Unknown, probably short, 2-4 days. The infectious period is as long as the organism is present, even in the absence of nasal discharge. Non-infectious within 24 to 48 hours after the start of effective antibiotics.

Haemophilus influenzae type b meningitis

Accounted for approximately 50%-65% of cases in the prevaccine era. Hearing impairment or neurologic sequelae in 15%-30%. The case-fatality rate is 2%-5% despite effective antimicrobial therapy.

Secondary Infections:  Respiratory tract infections, otitis media,  Sinusitis, chronic bronchitis

Haemophilus Meningitis:  It carries high mortality of 90% if not treated. The Bacteria reach meninges from the nasopharynx.

Laryngol epiglottitis: Causes epiglottis, obstructive laryngitis, > 2 years children are vulnerable which may be fatal in 2 hours.

Pneumonia:  Pneumonia along with Meningitis,  Lobar Pneumonia, bronchopneumonia. It can present with empyema ( a collection of pus).

Suppurative Lesions: Arthritis, endocarditis, pericarditis, hematogenous dissemination, otitis media, cellulitis

Laboratory Diagnosis of  Haemophilus Influenzae

Specimens: It depends on the site of infection, the following specimens may be collected-

• Sputum
• CSF
• Throat Swab
• Pus
• Aspirates from joints, middle ears, or sinuses

Collection and Transport of specimens

Specimens should be collected in sterile conditions and under all aseptic conditions.

Microscopy

Gram stain: Gram-stained smear of CSF in meningitis shows pleomorphic long and filamentous form predominate while in sputum coccobacillary forms.

Immunofluorescence and quellung reaction: For a direct demonstration of H. influenzae after mixing with specific tube b antiserum.

Antigen detection:  Type b capsular antigen can also be detected in patient serum, CSF, urine, or pus by following methods-

1. Latex agglutination
2. Coagglutination
3. Counter immunoelectrophoresis (CIE)

Culture

CSF culture- Inoculate CSF on chocolate age and incubate the plate at 35-37 °C, aerobically with 5-10 % CO₂, overnight. After that, identify on the basis of colony morphology, gram staining, oxidase test, satellitism, serotyping, etc.

Blood culture: It is useful in cases of pneumonia and epiglottitis.

Sputum culture

Colony Morphology and Staining

Serotyping: It may be performed with type-specific antisera as shown above picture.

Treatment

Following antimicrobial drugs are useful to treat and they are-

• Cefotaxime
• Ceftazidime
• Ampicillin
• Contrimixazole
• Amoxycillin with Clavulanate
• Clarithromycin

Epidemiology and Prevention

1. Similar to Pneumococci
2. The infection enters through the respiratory tract
3. Immunity is type-specific
4. HIB is protected by the PRP vaccine
5. Poorly immunogenic in children below 2 years
6. Rifampicin can be given for 4 days and prevents secondary infection and eradicates carrier state.

Public Health Action

Current Vaccines:  Haemophilus B conjugate vaccine is widely using Haemophilus influenzae type b vaccine has reduced H. influenzae type b meningitis in children by 95%.

Newer vaccines: The previous vaccines’ PRP is immunogenic in older children. PRP is poorly immunogenic in children below two years and Immunogenicity can be improved when coupled with protein carriers like diphtheria and tetanus toxoid that should be used in young children.

Key Notes on Haemophilus

• Virulent strains possess capsules while the avirulent strains and older cultures are non-capsulated.
• 95% of H. Influenzae isolates belong to type b in the Pitman classification.
• Plasmid born resistance set in Ampicillin
• H. ducreyi a causative agent of Chancroid in bipolar stain shows bacilli in small groups appear as parallel chains giving school of fish appearance.
• Haemophilus aegypticus is also called Koch – Weeks Bacillus. Identified as Biotype of H. influenzae that produces pink eye infection for which sulphonamides and gentamycin are effective antimicrobial agents.
• H. aphrophilus requires X and V factors that produce bacterial endocarditis, brain abscess, sinusitis, and abscess.
• H. influenzae biogroup aegyptius causes Brazilian Purpuric Fever.
• Haemophilus influenzae type b medical management requires hospitalization and treatment with an effective 3rd generation cephalosporin, or chloramphenicol plus ampicillin.
• Ampicillin-resistant strains of HIB are now common throughout the United States.
• As you know, H. influenzae is very sensitive to low temperature and thus, clinical samples should never be refrigerated, instead, samples should be transported to the laboratory without delay and inoculated on culture media immediately.

Related Text and Video

Various Haemophilus species Identification using X, V, and XV test

Haemophilus spp. have varying requirements for X, V, and XV growth factors. Consequently, the significant differences in growth factor requirements of Haemophilus spp. allows for their differentiation. Differentiation is based on the presence or absence of growth around and/or between discs impregnated with factors X, V, and XV.

Each X-Factor Disk is impregnated with hemin. Each V-Factor Disc is impregnated with NAD (nicotinamide adenine dinucleotide). Each XV-Factor Disc is impregnated with a combination of hemin and NAD.

Further Reading

1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
2. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
3. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
4. https://www.thermofisher. com/order/catalog/product/R01293#/R01293
5. https://www.sciencedirect.com/topics/immunology-and-microbiology/chocolate-agar
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC271442/
7. https://www.slideshare.net/doctorrao/hinfluenza
8. https://jcm.asm.org/content/jcm/20/4/822.full.pdf
9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533390/
10. https://anaerobesystems.com/products/plated-media/chocolate-agar-choc
11. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
12.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
13.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
14. A textbook of Microbiology, 2nd ed, Prof. C.P. Baveja
15. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042752/
16. https://www.webmd.com/children/meningococcal-meningitis-symptoms-causes-treatments-and-vaccines