Haemophilus influenzae

Bacteriology at UW- Madison

The Microbial World

University of Wisconsin - Madison

Haemophilus influenzae

Introduction

Haemophilus influenzae is a small, nonmotile Gram-negative bacterium in the family Pasteurellaceae, on the level with the Vibrionaceae and the Enterobacteriaceae. The family also includes Pasteurella and Actinobacillus, two other genera of bacteria that are parasites of animals. Encapsulated strains of Haemophilus influenzae isolated from cerebrospinal fluid are coccobacilli, 0.2 to 0.3 to 0.5 to 0.8 um, similar in morphology to Bordetella pertussis, the agent of whooping cough. Non encapsulated organisms from sputum are pleomorphic and often exhibit long threads and filaments. The organism may appear Gram-positive unless the Gram stain procedure is very carefully carried out. Furthermore, elongated forms from sputum may exhibit bipolar staining, leading to an erroneous diagnosis of Streptococcus pneumoniae.

Figure 1. Gram stain of Haemophilus influenzae from sputum

H. influenzae is highly adapted to its human host. It is present in the nasopharynx of approximately 75 percent of healthy children and adults. It is rarely encountered in the oral cavity and it has not been detected in any other animal species. It is usually the non encapsulated strains that are harbored as normal flora, but a minority of healthy individuals (3-7 percent) intermittently harbor H. influenzae type b (Hib) encapsulated strains in the upper respiratory tract. Pharyngeal carriage of Hib is important in the transmission of the bacterium. The success of current vaccination programs against Hib is due in part to the effect of vaccination on decreasing carriage of the organism.

What's in a name?

Haemophilus influenzae is widespread in its distribution among the human population. It was first isolated by Pfeiffer during the influenza pandemic of 1890. It was mistakenly thought to be the cause of the disease influenza, and it was named accordingly. Probably, H. influenzae was an important secondary invader to the influenza virus in the 1890 pandemic, as it has been during many subsequent influenza epidemics. In pigs, a synergistic association between swine influenza virus and Haemophilus. suis is necessary for swine influenza. Similar situations between human influenza virus and H. influenzae have been observed in chick embryos and infant rats.

Haemophilus "loves heme", more specifically it requires a precursor of heme in order to grow. Nutritionally, H.aemophilus influenzae prefers a complex medium and requires preformed growth factors that are present in blood, specifically X factor (i.e., hemin) and V factor (NAD or NADP). In the laboratory it is usually grown on chocolate blood agar which is prepared by adding blood to an agar base at 80 degrees. The heat releases X and V factors from the RBCs and turns the medium a chocolate brown color. The bacterium grows best at 35-37 degrees and has an optimal pH of 7.6. Haemophilus influenzaeis generally grown in the laboratory under aerobic conditions or under slight CO₂ tension (5% CO₂), although it is capable of glycolytic growth and of respiratory growth using nitrate as a final electron acceptor.

In 1995, Haemophilus influenzae was the first free-living organism to have its entire chromosome sequenced, sneaking in just ahead of Escherichia coliin that race, mainly because its genome is smaller in size than E. coli's. For a relatively obscure bacterium, there was already a good understanding of its genetic processes, especially transformation.

Figure 2. A map of the circular chromosome of Haemophilus influenzae illustrating the location of known genes and predicted coding regions

Observations of genetic transformation in Haemophilus have included drug resistance and synthesis of specific capsular antigens. The latter is thought to be the main determinant of H. influenzae.

Transformation in Haemophilus influenzae occurs by several different mechanisms and is more efficient than in enteric bacteria. When developing competence, the bacterium develops membranous "blebs" in the outer membrane that contain a specific DNA-binding protein. This outer membrane protein recognizes a specific 11-base pair sequence of DNA nucleotides that appears in Haemophilus DNA with much higher frequency than in other genera of bacteria. There is some evidence that Haemophilus is able to undergo both interspecies and intraspecies transformation in vivo (in host tissues). The restriction endonucleases from Haemophilus, e.g. Hind III, are widely used in biotechnology and in the analysis and cloning of DNA.

Pathogenesis

The pathogenesis of H. influenzae infections is not completely understood, although the presence of the type b polysaccharide capsule is known to be the major factor in virulence. Encapsulated organisms can penetrate the epithelium of the nasopharynx and invade the blood capillaries directly. Their capsule allows them to resist phagocytosis and complement-mediated lysis in the the nonimmune host. Nontypable (non encapsulated) strains are less invasive, but they are apparently able to induce an inflammatory response that causes disease. Outbreaks of H. influenzae type b infection may occur in nurseries and child care centers, and prophylactic administration of antibiotics is warranted. Vaccination with type b polysaccharide (in the form of Hib conjgate vaccines) is effective in preventing infection, and several vaccines are now available for routine use.

Naturally-acquired disease caused by H. influenzae seems to occur in humans only. In infants and young children (under 5 years of age), H. influenzae type b causes bacteremia and acute bacterial meningitis. Occasionally, it causes epiglottitis (obstructive laryngitis), cellulitis, osteomyelitis, and joint infections. Nontypable H. influenzae causes ear infections (otitis media) and sinusitis in children, and is associated with respiratory tract infections (pneumonia) in infants, children and adults.

Figure 3. Tissues infected by type b and nontypable strains of Haemophilus influenzae

Seven serotypes of the bacterium have been identified on the basis of capsular polysaccharides. Until the implementation of widespread vaccination programs, type b H. influenzae was the most common cause of meningitis in children between the ages of 6 months and 2 years (see Figure 4 below), resulting in 12,000 to 20,000 cases annually in the U.S. It would be interesting to view comparative data since the era of vaccination against H. influenzae meningitis, which began in 1985. Certainly, there are fewer than 100 cases annually of bacterial meningitis caused by H. influenzae type b.

Figure 4. Age-specific incidence of bacterial meningitis caused by Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae prior to 1985

Disease caused by H. influenzae usually begins in the upper respiratory tract as nasopharyngitis and may be followed by sinusitis and otitis, possibly leading to pneumonia. In severe cases, bacteremia may occur which frequently results in joint infections or meningitis.

Virulence

H. influenzae does not produce any demonstrable exotoxins The direct role of endotoxin in meningitis or bacteremia is unclear, although the Gram-negative bacterium's outer membrane lipooligosaccharide is thought to play a role in inflammation associated with otitis media. All virulent strains produce neuraminidase and an IgA protease, but the role of these extracellular enzymes in invasion is unproven. Fimbriae increase the adherence of bacteria to human mucosal cells in vitro, and they are required for successful colonization of the nasopharynx. The Anton antigen, as defined in red blood cells, appears to be the receptor.

Virulence, at least in the case of bacteremia and meningitis, is directly related to capsule formation. Virtually all of these infections are caused by the type b serotype, and its capsular polysaccharide, containing ribose, ribitol and phosphate, is the proven determinant of virulence. The capsule material is antiphagocytic, and it is ineffective in inducing the alternative complement pathway, so that the bacterium can invade the blood or cerebrospinal fluid without attracting phagocytes or provoking an inflammatory response and complement-mediated bacteriolysis. For this reason, anticapsular antibody, which promotes both phagocytosis and bacteriolysis, is the main factor in immune defense against H. influenzae infections (below).

The polyribosyl ribitol phosphate (PRP) capsule is the most important virulence factor because it renders type b H. influenzae resistant to phagocytosis by polymorphonuclear leukocytes in the absence of specific anticapsular antibody, and it reduces the bacterum's susceptibility to the bactericidal effect of serum. However, susceptibility to the bactericidal effect of serum depends on the presence of antibodies to a number of other antigenic sites, including the lipooligosaccharide and outer membrane proteins designated as P1 and P2.

Type b H. influenzae is plainly the most virulent of the Haemophilus species; 95 percent of bloodstream and meningeal Haemophilus infections in children are due to this bacterium. In contrast, in adults, nontypable strains of H. influenzae are the most common cause of Haemophilus infection, presumably because most adults have naturally acquired antibody to PRP.

Immunity

The age incidence of H. influenzae meningitis is inversely proportional to the titer of bactericidal antibody in the blood, whether passively acquired from the mother or actively formed (see Figure 5 below). Without artificial immunization, in children aged 2 months to 3 years, antibody levels are minimal; thereafter antibody levels increase and the disease becomes much less common. From this curve, it is obvious that artificial active immunization should begin at 2 months of age, when nearly all passive immunity has waned, and the child enters a vulnerable non immune period of life.

Figure 5. Relation of the age incidence of bacterial meningitis caused by Haemophilus influenzae to bactericidal antibody titers in the blood (data pre 1985)

H. influenzae is susceptible to lysis by antibody and complement. Furthermore, anticapsular antibodies promote phagocytosis, as well as bacteriolysis. Thus, serum antibody, complement, lysozyme and phagocytes can work in concert during a bacteremia. During meningitis, phagocytosis is probably the main host defense mechanism since complement rarely occurs in the cerebrospinal fluid.

For many years it was believed that bactericidal antibody directed against PRP capsule ofH. influenzae type b was entirely responsible for host resistance to infection. However, some recent studies have stressed a role for antibody to somatic (cell wall) antigens as well. For example, antibody to PRP can often be detected in the sera of children on admission to the hospital with sepsis due to H. influenzae type b. Adsorption of immune serum with PRP alone does not remove its protective capabilities, whereas adsorption with whole organisms does. Separation of the outer membrane of type b H. influenzae into its many protein constituents reveals several individual membrane proteins that may be associated with immunity. Bactericidal antibodies that react with individual outer membrane proteins or with lipooligosaccharide constituents have been identified. These findings support indicate the potential importance of antibody to noncapsular antigens in immunity to H. influenzae type b infection. In addition, opsonizing antibodies, which also play a role in protection, may be directed against PRP or somatic constituents (see Figure 6 below).

Figure 6. Phagocytic engulfment of H. influenzae bacterium opsonized by antibodies specific for the capsule and somatic (cell wall) antigen

Recent studies of nontypable H. influenzae have shown that bactericidal antibody to outer membrane proteins develop in infants in response to otitis media caused by the organism. Normal adults generally have both bactericidal and opsonizing antibodies directed against nontypable H. influenzae.

Treatment and Prevention

Virtually all patients treated early in the course of H. influenzae meningitis are cured. The mortality rate of treated infections is less than 10 percent, but nearly 30 percent of the children who recover have residual neurologic effects. Ampicillin has been the drug of choice, but presently over 20 percent of all strains of H. influenzae are resistant to ampicillin because of plasmid-mediated ß-lactamase production.

The recommended treatment for H. influenzae meningitis is ampicillin for strains of the bacterium that do not make ß-lactamase, and a third-generation cephalosporin or chloramphenicol for strains that do. Amoxicillin, together with a substance such as clavulanic acid, that blocks the activity of ß-lactamase, has been unreliable in treatment of meningitis, although it is effective in treatment of sinusitis, otitis media and respiratory infections. Chloramphenicol was long considered the drug of choice for meningitis caused by penicillin-resistant H. influenzae, and it is still highly effective, but not without potential toxic side effects. Third-generation cephalosporins, such as ceftriaxone or cefotaxime, are effective against H. influenzae and penetrate the meninges well. Tetracyclines and sulfa drugs remain effective in treating sinusitis or respiratory infection caused by nontypable H. influenzae. Amoxicillin plus clavulanic acid (Augmentin) is effective against ß-lactamase producing strains. Erythromycin is ineffective in treatment ofH. influenzae infections.

The use of polyribosyl ribitol phosphate (PRP) vaccine and, more recently, protein-conjugated PRP, has vastly reduced the frequency of infection due to type b H. influenzae. The PRP vaccine consists of the type b capsular polysaccharide. Like most bacterial polysaccharides, it elicits a strong primary antibody response, but with little induction of memory. H. influenzae type b Hib conjugate vaccines, which couple the polysaccharide to a protein, induce memory type antibody responses in children and are effective in younger infants who are at higher risk for the disease.

There are several types of Hib conjugate vaccines available for use. All of the vaccines are approved for use in children 15 months of age and older and some are approved for use in children beginning at 2 months of age. All of the vaccines are considered effective. The vaccines are given by injections. More than 90% of infants obtain long term immunity with 2-3 doses of the vaccine.

All children should have a vaccine approved for infants beginning at 2 months. Depending on the type used, the recommended schedule for infants will vary. All unvaccinated children 15 - 59 months old should receive a single dose of conjugate vaccine. Children 60 months of age or older and adults normally do not need to be immunized.

Whether the vaccine provides protection against ear infections is not known. It also does not protect against diseases caused by other types of Haemophilus. nor does it protect against meningitis caused by other types of bacteria.

Specific characteristics of the four conjugate vaccines available for infants and children vary based on the type of protein carrier, the size of the polysaccharide, and the chemical linkage between the polysaccharide and carrier (see Table 1 below).

Current recommendations for vaccination of infants require parenteral administration of three different vaccines, diphtheria-tetanus-pertussis (DTP), Hib conjugate, and hepatitis B, during two or three different visits to a health-care provider. TETRAMUNE (see table footnote below) is the first licensed combination vaccine that provides protection against diphtheria, tetanus, pertussis, and Hib disease.

Table 1. Hib conjugate vaccines licensed for use among children

Vaccine	Trade name (manufacturer)	Polysaccharide	Linkage	Protein carrier
PRP-D	ProHIBiT (Connaught)	Medium	6-carbon	Diphtheria toxoid
HbOC*	HibTITER (Lederle-Praxis)	Small	None	CRM197 mutant Corynebacterium diphtheriae toxin protein
PRP-OMP	PedvaxHIB (Merck Sharp and Dohme)	Medium	Thioether	Neisseria meningitidis outer membrane protein complex
PRP-T	ActHIB OmniHIB (Pasteur Merieux Vaccins)	Large	6-carbon	Tetanus toxoid

* TETRAMUNE consists of HbOC and DTP vaccine (TRI-IMMUNOL), also manufactured by Lederle-Praxis.

Tailpiece

Before 1985, Haemophilus influenzae type b (Hib) was the most common cause of bacterial meningitis in children under 5 years of age (approximately 12,000 cases per year, most in children younger than 18 months). Approximately 5% of affected children died, and neurologic sequelae developed in 15% to 30% of the surviving children. An additional estimated 7,500 cases of other invasive Hib infections also occurred annually in young children. The cumulative risk for Hib invasive disease before the age of 5 was one in 200 children, similar to the risk for poliomyelitis during the 1950s.

In 1985, the first Hib polysaccharide vaccines were licensed for use in the United States. These vaccines contained purified polyribosylribitol phosphate (PRP) capsular material from the type b serovar. Antibody against PRP was shown to be the primary component of serum bactericidal activity against the organism. PRP vaccines were ineffective in children less than 18 months of age because of the T-cell-independent nature of the immune response to PRP polysaccharide.

Conjugation of the PRP polysaccharide with protein carriers confers T-cell-dependent characteristics to the vaccine and substantially enhances the immunologic response to the PRP antigen. In 1989, the first Hib conjugate vaccines were licensed for use among children 15 months of age or older. In 1990, two new vaccines were approved for use among infants.

The incidence of Hib invasive disease among children aged 4 years or younger has declined by 98% since the introduction of Hib conjugate vaccines. One goal of the Childhood Immunization Initiative was to eliminate invasive Hib disease among children aged 4 years or younger by 1996. However, approximately 300 cases of Haemophilus influenzae invasive disease per year continue to be reported in the U.S., mainly in non immunized children. Most cases are caused by nontypable Haemophilus influenzae. The bar graph below (Figure 7) shows the age distribution of cases in 1996 and is comparable to Figure 5, which displays results from the pre-imuunization era.

Figure 7. Age-specific incidence of bacterial meningitis in children caused by Haemophilus influenzae in 1996