Haemophilus influenzae: An Overview

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The History of Discovery


  • Hemophilic bacteria were primarily discovered in the early 1880s by R. Koch, who detected them in conjunctival exudate of patient with purulent conjunctivitis.
  • Some time later, M. Afanassiev in 1891 and R. Pfeiffer in 1892 isolated similar bacteria from patients in the course of influenza epidemic.
  • As the result, for a long time these pathogens were regarded as the causative agents of influenza and therefore, acquired their own species name Haemophilus influenzae.

Classification


  • Hemophilic bacteria pertain to the family Pasteurellaceae and genus Haemophilus. This genus comprises more than 10 species of bacteria; some of them are seriously pathogenic for humans.
  • Haemophilus influenzae species is the dominant human pathogen. The members of this species commonly cause respiratory infections, but in certain cases they may trigger severe invasive disorders, such as meningitis or septicemia.
  • Similar pathogenic activity is sporadically demonstrated by H. parainfluenzae, H. haemolyticus and H. parahaemolyticus species.
  • H. ducreyi causes chancroid or soft chancre – one of the bacterial sexually transmitted diseases (STD).
  • Genera Aggregatibacter and Pasteurella that pertain to the same microbial family also harbor human pathogenic representatives.
  • For instance, Аggregatibacter aprophilus can be isolated in patients with bacterial endocarditis; one more agent Аggregatibacter actinomycetemcomitans is an agressive oral pathogen that participates in progression of periodontitis.

Structure and Properties of Haemophilus influenzae


Morphology

These organisms are small 0.3-1.0 μm gram-negative polymorphic coccobacteria. They are non-sporeforming, but produce capsule.

Cultivation

  • Hemophilic bacteria are rather difficult for culture. As fastidious microorganisms, they need a number of auxiliary factors for efficient growth.
  • For instance, they require factor V (nicotinamide adenine dinucleotide or NAD) and factor X (hemin), which are commonly present in red blood cells.
  • Thus, the optimal medium for them is chocolate agar, where erythrocytes are lysed by heating. The bacteria grow better in presence of elevated concentrations of CO2.
  • Also H. influenzae can be cultured on blood agar, but only nearby paper disc impregnated with V and X factors. Likewise, H. influenzae may grow together with satellite hemolytic bacteria (e.g., S. aureus) that liberate factors V and X from red blood cells.
  • The colonies of bacteria are small, convex, and glistening. Pathogenic H. influenzae render S- or M (mucous) forms of colonies. Non-pathogenic strains usually produce R-forms.

Biochemical properties

  • The bacteria are facultative anaerobes with mixed type of metabolism. They produce catalase and oxidase.
  • H. influenzae ferment glucose. Some strains produce indole and metabolize urea. The latter reactions are used for biotyping of H. influenzae.
  • Biochemical differentiation of hemophils and related bacteria is based on a number of tests, presented in table

Antigenic structure

  • The bacteria possess thermostable somatic O-antigen made of lipooligosaccharide (or LOS) and superficial capsular polysaccharide K-antigen. Six basic serovars or types (a, b, c, d, e, and f) are recognized by capsular K-Ag. Non-capsulated strains are referred to as nontypable. Protein M-antigen is present in non-pathogenic strains.

Virulence factors

  • The major factor of bacterial virulence is capsule. It shows adhesive properties and prevents bacteria from phagocytosis and complement activity. Most of severe invasive infections are caused by capsular strains of Н. influenzae type b (or Hib).
  • Unlike other types, Hibs are covered with the capsule that contains polyribosil ribitol phosphate (PRP). All other capsulated Н. influenzae have a hexose instead of pentose (ribose) in the structure of PRP.
  • PRP is a strong T-independent antigen.The lipooligosaccharide (LOS) shows endotoxin activity.
  • Multiple pili play a role of the adhesins. They promote microbial attachment to epithelial cells.
  • IgA proteases of hemophilic bacteria destroy human IgAs thereby downgrading mucosal immunity.
  • Synthesis of beta-lactamases confers microbial resistance to certain β-lactam antibiotics.

Resistance

  • Н. influenzae is markedly sensitive to environmental factors being rapidly inactivated outside the body. However, in sputum and mucus it stays viable up to 18 h, on plastic surfaces – for 12 h.
  • Microbial cells are readily inactivated by all standard disinfectants (e.g., sodium hypochlorite, phenol, or formaldehyde).

Pathogenesis and Clinical Findings of Infections, Caused by Haemophilus influenzae


  • Н. influenzae is solely human pathogen engendering various kinds of anthroponotic infections. Nevertheless, hemophilic bacteria especially their nontypable strains are normal inhabitants of human respiratory tract.
  • The decline of mucosal immunity of respiratory tract as well as airway damages predispose to active propagation of Н. influenzae. They replicate extra- and intracellularly and may enter the bloodstream.
  • Sick persons and carriers of Н. influenzae are the major sources of infection.
  • The diseases are transmitted by air droplet route and, to lesser extent, by contact route.
  • Children under the age of 4-5 years are the most susceptible to Н. influenzae.
  • The infections caused by Н. influenzae are divided into two main groups – non-invasive and invasive.
  • Non-invasive diseases affect the epithelium of respiratory tract. Among them are acute sinusitis, acute otitis media and exacerbations of chronic bronchitis.
  • They result from the colonization of bronchial mucosa by Н. influenzae after the impairment of mucociliary clearance. In most of the cases they occur as the complications of primary respiratory infections, e.g., caused by viruses.
  • Invasive diseases are predominantly associated with Н. influenzae of Hib type. They comprise the severe disorders with hematogenous spread meningitis, epiglottitis (acute inflammation of epiglottis), pneumonia, and septicemia (sepsis).
  • The leading clinical forms of infections, caused by various types of Н. influenzae are presented in table

  • The association of the most severe infections with Н. influenzae type b (Hib) seems to be related with the expression of PRP capsule by these bacteria. It protects Hib from phagocytosis, opsonization, and complement lysis, ensuring microbial survival in the bloodstream.
  • Hib-associated meningitis has the mortality rate of 3-6% in affected children. The bacteria cause acute pyogenic damage of brain tissues resulted from the inflammatory action of microbial endotoxin.
  • About 10-20% of children recovered from meningitis retain long-term and stable neurological complications, e.g., hearing loss.
  • Local invasive disease caused by Н. influenzae is acute epiglottitis that may result in purulent necrosis of epiglottis with asphyxia of infant.
  • Newborns and postpartum women are under the risk of development of Н. influenzae septicemia, largely mediated by nontypable bacterial serovars.
  • In adults Н. influenzae may cause pneumonia or bronchitis mainly as a complication of primary viral or bacterial respiratory infection.
  • Post-infectious immunity is stable. It is maintained predominantly by antibacterial antibodies. Babies and infants are passively protected with maternal antibodies for 2-3 months after birth.

Laboratory Diagnosis of Haemophilus influenzae Infections


  • Specimen collection for laboratory examination depends on the clinical form of the diasease. Initially sterile media are of the most clinical value – cerebrospinal fluid (CSF), blood, pleural exudate, pericardial or synovial fluids.
  • Microscopy with Gram stain of the sediments of centrifuged CSF reveals small gram-negative non-sporeforming coccobacteria.
  • Rapid detection of antigens of Н. influenzae type b (Hib) in cerebrospinal fluid, blood or pleural exudate is achieved by latex agglutination or ELISA test.
  • Molecular typing of DNA of Н. influenzae in clinical samples is performed by genetic tests (PCR).
  • For culture of CSF or other biological fluids the clinical materials should be managed immediately, or stored maximum 30 minutes at room temperature before culturing to prevent microbial autolysis.
  • CSF is primarily centrifuged before inoculation. Microbial antigens are detemined in supernatants by ELISA.
  • Sediments of CSF are cultured on chocolate agar, or blood agar with factors V and X in aerobic atmosphere with 5-10% CO2.
  • The test of satellite cultures on blood agar can be applied as well. Here Н. influenzae is inoculated along the streaks of satellite hemolytic bacteria (e.g., S. aureus) that release factors V and X from red blood cells. The growth of Н. influenzae is possible only in close proximity to hemolytic satellite culture.
  • After assessment of microbial growth, the bacteria are further identified by the number of biochemical and serological tests. Identification of specific antigens allows to determine antigenic type of Н. influenzae.

Treatment and Prophylaxis of Infections, Caused by Haemophilus influenzae


  • Invasive infections, associated with Н. influenzae (meningitis, acute epiglottitis, or septicemia) require urgent antibiotic treatment.
  • Taking into account possible resistance of Н. influenzae to penicillins, mediated by production of beta-lactamases, third-generation cephalosporins (cefotaxime or ceftriaxone) are the drugs of choice for treatment of these diseases. Fluoroqunolones and macrolides (clarithromycin) can be administered as well.
  • For specific prophylaxis highly efficient polysaccharide chemical vaccines based on capsular antigen of Н. influenzae type b (Hib) were developed. They are commonly used in combination with DPT vaccine (diphtheria, pertussis, tetanus vaccine) and vaccine against hepatitis B.
  • Infants are vaccinated four times in 3, 4, 5 and 18 months after birth.
  • Non-specific prophylaxis includes isolation and successful treatment of patients, prevention of carriage, improvement of sanitary conditions, proper disinfection.