Haemophilus Species, Including H. influenzae and H. ducreyi (Chancroid)

Distinguishing Haemophilus influenzae from Haemophilus haemolyticus

Strains of H. haemolyticus are frequently misidentified as H. influenzae in clinical microbiology laboratories and in published studies. The confusion results from the observation that many strains of H. haemolyticus are not hemolytic, and this is the sole characteristic used routinely to distinguish H. haemolyticus from H. influenzae in commercial kits and in clinical microbiology laboratories. H. influenzae and H. haemolyticus both have growth requirements for X and V factors. Analysis of 500 strains originally identified as nontypeable H. influenzae revealed that 27% of nasopharyngeal isolates from children and 40% of sputum isolates from adults were in fact H. haemolyticus. H. haemolyticus is a commensal but does cause disease rarely. Strains of H. haemolyticus and H. influenzae can be distinguished from one another through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or differences in various genetic markers, including 16S ribosomal RNA (rRNA), superoxide dismutase C, outer membrane protein (OMP) P6, protein D, fuculose kinase, and others. Because H. influenzae and H. haemolyticus share the human upper airway as their ecologic niche and are competent for DNA exchange, there appears to be an evolutionary continuum between the two species.

Biotypes

Viability of H. influenzae is lost rapidly, so clinical specimens should be inoculated onto appropriate media without delay. A biotyping scheme devised by Kilian (based on indole production, urease, and ornithine decarboxylase activity) may be used to characterize individual isolates. Biotype III includes Haemophilus aegyptius, the “Koch-Weeks bacillus.” A clone of biotype IV strains is associated with neonatal and postpartum infections.

Serotypes

Colonies of H. influenzae are usually granular, transparent (or slightly opaque), circular, and dome shaped. On chocolate agar, most colonies attain a size of about 0.5 to 0.8 mm during the first 24 hours of growth at 37°C, enlarging to 1.0 to 1.5 mm by 48 hours. Six serotypes, designated a to f, are based on antigenically distinct capsular polysaccharide types. Colonies of encapsulated strains are mucoid (iridescent when grown on transparent media and examined with an indirect source of light) and may attain a size of 3 to 4 mm. Capsular type b strains are important invasive pathogens in humans. Strains of H. influenzae that lack a polysaccharide capsule are generally referred to as nontypeable because they are nonreactive with typing antisera raised against each of the six capsules. The population structure of H. influenzae type b is clonal, whereas nontypeable strains demonstrate substantial genetic diversity. Most unencapsulated isolates are not capsule-deficient variants of extant capsule clones; they are genetically distinct from encapsulated strains of H. influenzae.

Colonization in Adults With Chronic Obstructive Pulmonary Disease

Nontypeable H. influenzae frequently colonizes the lower respiratory tract in the setting of chronic obstructive pulmonary disease (COPD) and cystic fibrosis; multiple strains colonize the respiratory tract of these patients simultaneously. Acquisition of new strains of nontypeable H. influenzae is associated with an increased risk of exacerbations of COPD. Use of selective media improves the recovery rate of H. influenzae from the sputum of patients with cystic fibrosis.

Based on its binding to mucin and adherence to epithelial cells, H. influenzae has long been considered an extracellular pathogen. Several lines of evidence, however, have established that H. influenzae has both an extracellular and an intracellular niche in the human respiratory tract. Therefore, H. influenzae is present in the airway lumen, bound to mucin, adherent to respiratory cells, found within the interstitium of the submucosa, and found within cells of the respiratory tract. This observation has important implications for understanding the dynamics of colonization of the human respiratory tract and the human immune response to the bacterium.

Colonization and Conjugate Vaccines

Before the widespread use of conjugate vaccines, type b strains colonized the nasopharynx of children at a rate of 2% to 4%. The rate of nasopharyngeal colonization by type b strains has decreased substantially with the use of conjugate vaccines to prevent invasive infections caused by H. influenzae type b. Table 225.1 summarizes several features of nontypeable and type b strains.

Otitis Media

The first step in the pathogenesis of infection is colonization of the upper respiratory tract. H. influenzae expresses a variety of adhesin molecules ( Table 225.2 ), each of which has its own specificity for host receptors. The prevalence and distribution of adhesins vary among nontypeable strains. In contrast to type b strains, which gain access to the bloodstream, nontypeable strains cause disease by local invasion of mucosal surfaces. The pathogenesis of otitis media involves direct extension of bacteria from the nasopharynx to the middle ear via the eustachian tube. Release of lipooligosaccharide, lipoproteins, peptidoglycan fragments, and other antigens induces host inflammation.

Strains of nontypeable H. influenzae show differences in their pathogenic potential. A subset of strains that colonize the nasopharynx is capable of causing otitis media and has different sets of genes compared with strains that cause asymptomatic colonization. For example, otitis media strains are more likely to have the lipooligosaccharide synthesis gene lic2B, the histidine operon, and the urease operon than are asymptomatic colonizing strains. Nontypeable H. influenzae rapidly and reversibly regulates the expression of many virulence genes through phase variation of methyltransferase proteins, which methylate DNA at sequence-specific sites, leading to changes in expression of many genes. This phasevarion switching plays an important role in the pathogenesis of nontypeable H. influenzae otitis media in the chinchilla model.

Otitis Media With Effusion

Nontypeable H. influenzae has also been implicated as a cause of otitis media with effusion, a term that refers to the presence of middle ear fluid in the absence of clinical signs of acute otitis media. In addition to positive cultures of some middle ear fluids, analysis with PCR reveals the presence of microbial DNA and messenger RNA (mRNA), suggesting that H. influenzae is present in a viable but nonculturable form in some cases of otitis media with effusion.

Biofilms

H. influenzae in the form of biofilms is present in the middle ear in animal models and in the middle ears of children with otitis media. A biofilm is a community of bacteria encased in a matrix and attached to a surface. Bacteria in biofilms are more resistant to host clearance mechanisms and more resistant to antibiotics compared with planktonic bacteria. Nontypeable H. influenzae biofilms are associated with recurrent and chronic otitis media.

Exacerbations of Chronic Obstructive Pulmonary Disease

The lower respiratory tract of adults with COPD is chronically colonized by nontypeable H. influenzae. The course of COPD is characterized by intermittent exacerbations of the disease. Several lines of evidence implicate H. influenzae as the most common bacterial cause of exacerbations, including bronchoscopic sampling of the lower respiratory tract during exacerbations, analysis of immune responses to H. influenzae isolated from patients experiencing exacerbations, correlation of airway inflammation with sputum bacteriology, and molecular analysis of prospectively collected isolates. Differences in pathogenic potential among strains in the setting of COPD are based on genome content. A complex host-pathogen interaction most likely determines the outcome of the acquisition of a new strain of nontypeable H. influenzae ; the determinants include virulence of the strain, degree of host impairment of innate immunity and pulmonary function, host inflammatory response, preexisting immunity, perception of symptoms, and other factors. Strains of nontypeable H. influenzae that persist in COPD airways regulate expression of critical virulence functions with slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes as a major mechanism for survival in the hostile environment of the human airways.

Invasive Infections Caused by Haemophilus influenzae Type b

The importance of the type b capsule as a critical virulence factor in the pathogenesis of invasive disease has been well established with the use of genetic techniques and an infant rat model of bacteremia and meningitis. Mutants lacking the polyribitol ribose phosphate (PRP) capsule do not cause invasive disease, whereas the isogenic parent strains are highly virulent in the infant rat model. The type b capsular polysaccharide is composed of PRP. The capsule enables the organism to invade the bloodstream after colonization of the respiratory tract (see later).

Nontypeable Haemophilus influenzae

Immunity to infection by nontypeable H. influenzae is complex and not completely understood. A hallmark of noninvasive infections caused by nontypeable H. influenzae is their propensity for recurrence. The immune response to surface antigens of nontypeable strains is intimately involved in the pathogenesis of recurrent infection. Studies in animal models, in adults with COPD, and in children with otitis media have all demonstrated that the most prominent antibody response is directed at strain-specific determinants. The clinical observation of recurrent infections in immunocompetent hosts (recurrent otitis media in children and recurrent exacerbations in COPD) suggests that strain-specific immune responses leave the host susceptible to recurrent infections by different strains of H. influenzae. A variety of membrane-associated surface-exposed determinants are immunogenic and potential targets of protective host immune responses. For example, OMP P2, the major porin protein, contains immunodominant strain-specific determinants on the bacterial surface. Adults with COPD make potentially protective antibodies to strain-specific determinants on P2 after infection. Patients remain susceptible to recurrent infections by other strains. Furthermore, the P2 genes of strains that colonize adults with COPD undergo point mutations in the human respiratory tract. The mutations result in amino-acid changes in the surface-exposed loops of the P2 molecule. A similar phenomenon has been observed with OMP P5. These variants have a selective advantage and are able to evade the host response and cause recurrent or persistent infection.

The presence of serum bactericidal antibody is associated with protection from otitis media caused by nontypeable H. influenzae, as is serum antibody to protein D . Because nontypeable H. influenzae causes mucosal infection, mucosal immunity likely plays a role in host defense; however, the mucosal immune response to H. influenzae is poorly understood. Finally, observations have suggested that cell-mediated immune responses, including Th17 responses, play a role in protection against infection.

Haemophilus influenzae Type b

Protection against invasive H. influenzae type b infections is mediated by antibodies to the type b capsular polysaccharide PRP. Serum anti-PRP antibodies activate complement-mediated bactericidal and opsonic activity in vitro and mediate protective immunity against systemic infections in humans. The level of maternally acquired serum antibody to PRP declines after birth and reaches a nadir at approximately 18 to 24 months of age, the peak age incidence of meningitis caused by H. influenzae type b in an unimmunized child. The level of antibody to PRP then gradually rises, apparently as a result of exposure to H. influenzae type b or cross-reacting antigens. Systemic disease is unusual after the age of 6 years even in the absence of immunization, because of, at least in part, naturally acquired antibody to PRP.

Immunization with vaccines that are composed of PRP conjugated to carrier proteins affords protection by inducing antibodies to PRP. These vaccines are now widely used and are highly effective in preventing invasive disease caused by H. influenzae type b in infants and children. In addition, these vaccines prevent colonization of the nasopharynx; this effect accounts for herd immunity by reducing the circulation of type b strains.