Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing.

The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.

Chloramphenicol is a substrate for a novel nitroreductase pathway in Haemophilus influenzae.

The p-nitroaromatic antibiotic chloramphenicol has been used extensively to treat life-threatening infections due to Haemophilus influenzae and Neisseria meningitidis; its mechanism of action is the inhibition of protein synthesis. We found that during incubation with H. influenzae cells and lysates, chloramphenicol is converted to a 4-aminophenyl allylic alcohol that lacks antibacterial activity. The allylic alcohol moiety undergoes facile re-addition of water to restore the 1,3-diol, as well as further dehydration driven by the aromatic amine to form the iminoquinone. Several Neisseria species and most chloramphenicol-susceptible Haemophilus species, but not Escherichia coli or other gram-negative or gram-positive bacteria we examined, were also found to metabolize chloramphenicol. The products of chloramphenicol metabolism by species other than H. influenzae have not yet been characterized. The strains reducing the antibiotic were chloramphenicol susceptible, indicating that the pathway does not appear to mediate chloramphenicol resistance. The role of this novel nitroreductase pathway in the physiology of H. influenzae and Neisseria species is unknown. Further understanding of the H. influenzae chloramphenicol reduction pathway will contribute to our knowledge of the diversity of prokaryotic nitroreductase mechanisms.

Role of lgtC in resistance of nontypeable Haemophilus influenzae strain R2866 to human serum.

We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

Characterization of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae.

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.