Haemophilus influenzae is both a human respiratory pathogen and pharyngeal commensal, while H. haemolyticus, the closest phylogenetic relative of H. influenzae, is arguably a strict pharyngeal commensal. A hemolytic phenotype has historically differentiated H. haemolyticus from H. influenzae, but the recent recognition of significant nonhemolytic H. haemolyticus colonization has decreased this trait's resolvability. Given this and the potential of recombination between the species, we examined the distribution of microbiologic and molecular traits between collections of H. influenzae and H. haemolyticus strains separated within a dendrogram obtained by multilocus sequence analysis (MLSA). All strains hybridizing with a probe to iga, a gene encoding an immunoglobulin A protease of H. influenzae, clustered apart from strains that did not hybridize with the probe. Other traits also segregated significantly along this division, suggesting a separation of the species. Of note, the LOS genes licA, lic2A, and lgtC of H. influenzae were approximately 2, 6, and 54 times, respectively, more prevalent in H. influenzae than in H. haemolyticus. In contrast to species separation, interspecies recombination was evidenced by the inability of single gene sequences to phylogenetically separate the species and by the "fuzzy" distribution of some species-specific traits across the species dividing line. Together, these data support the historically accurate and pragmatic division of these species while recognizing their potential for recombination. Future comparative genomic studies identifying common and distinctive genes could be useful in evaluating their role in the commensal or virulent growth, respectively, of H. influenzae.Haemophilus influenzae is one of eight Haemophilus species that reside as commensal organisms in the pharyngeal cavity of humans. These other commensal Haemophilus species include H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, H. paraphrohaemolyticus, H. segnis, H. aphrophilus, and H. paraphrophilus. H. influenzae is by far the most pathogenic member of the genus in humans. Strains possessing a type b capsule are often associated with invasive diseases, such as meningitis, sepsis, and pneumonia; and strains lacking a capsule (referred to as nontypeable [NT]) are associated with localized mucosal diseases, such as otitis media, sinusitis, and bronchitis. Other haemophili are considered to be rarely associated with disease, and H. haemolyticus has never been implicated as an infectious disease agent (1).In contrast to other haemophili, H. haemolyticus and H. influenzae both depend on X (hemin) and V (NAD) growth factors, both lack the ability to ferment sucrose, both share similar GϩC contents, and both are nearly indistinguishable by their colony and cellular morphologies (25). Initial phylogenetic studies performed by using DNA-DNA hybridization revealed that H. haemolyticus associated with H. influenzae in a cluster of the family Pasteurellaceae termed Haemophilus sensu stricto (6,41,47), and t...

Summary New methods to distinguish between nontypeable Haemophilus influenzae and nonhemolytic Haemophilus haemolyticus were compared. The results of iga variable region hybridization to dotblots and library-on-a-slide microarrays were more similar to a “gold standard” multigene phylogenetic tree than iga conserved region hybridization or P6 7F3 epitope immunoblots.

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA-and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA-and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o ؊ and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o ؊ cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o ؊ and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA-and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.Nontypeable Haemophilus influenzae (NTHI; i.e., strains lacking a polysaccharide capsule) strains colonize the human pharynx and nasopharynx and may cause respiratory infections such as otitis media, bronchitis, sinusitis, and pneumonia...

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