Analysis of Genetic Relatedness of Haemophilus influenzae Isolates by Multilocus Sequence Typing

Abstract

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.Haemophilus influenzae is a small (1 to 2 m in length) gram-negative bacterium that is found only in humans. The polysaccharide-protein conjugate vaccines against serotype b H. influenzae have almost eliminated H. influenzae as a cause of pediatric meningitis in the western world. However, unencapsulated (nontypeable) H. influenzae (NTHI) remains an important pathogen, particularly in children and the elderly (5,8,23). NTHI infections are usually limited to respiratory mucosal sites such as the middle ear or bronchi but are occasionally systemic. It is not known whether NTHI isolates associated with localized or systemic disease are genetically distinct from each other or distinct from isolates associated with asymptomatic colonization of the nasopharynx.Efforts to understand and control NTHI disease have been hampered by the diversity of these bacteria. Many of the surface antigens that have been studied display interstrain and intrastrain heterogeneity as a result of both sequence divergence and phase variation. It is increasingly recognized that NTHI isolates also vary in genetic content. We use the term island to refer to a genetic locus (one or more genes) that occurs in some but not all strains. As used here, the term does not imply that the locus is known to be readily transferred between strains or is thought to have been recently acquired. Islands whose functi...

Phylogenetic relatedness and diversity of non-typable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media

“…Previously, a study by Meats et al (2003) has shown phylogenetic diversity between all the strains of H. influenzae using the MLST approach. Our results are consistent with the observations of Erwin et al (2008), who showed considerable diversity within NTHi strains. Erwin et al (2008) found 13 clades or 10 eBURST groups from the H. influenzae MLST database.…”

“…Our results are consistent with the observations of Erwin et al (2008), who showed considerable diversity within NTHi strains. Erwin et al (2008) found 13 clades or 10 eBURST groups from the H. influenzae MLST database. Eighty per cent of their NTHi strains were in group 2.…”

“…With more strains added to the database, we found new eBURST groups (29 eBURST groups compared to 10 groups in their study using the same group definition). Specifically, 41.4 % of the NTHi STs found in our study were distributed in 10 eBURST groups from the Erwin et al (2008) study but 27.1 % of the STs from our study were found in new eBURST groups, and 31.5 % of the NTHi STs we evaluated did not correspond to any of the 29 eBURST groups.…”

Population subdivision and the detection of recombination in non-typable Haemophilus influenzae

“…However, later work that specifically compared NT isolates with encapsulated ones using the same approach found a lower degree of congruence between housekeeping loci in NT isolates (Meats et al, 2003). NT strains are more diverse (both genetically and phenotypically) than their encapsulated relatives (Erwin et al, 2008;Musser et al, 1986), and it is not clear whether this reflects differences in the ability or opportunity for NT and typable strains to take up DNA (Meats et al, 2003). , and a better understanding of the flow of genetic material between bacterial lineages is hence important for public health.…”

“…NTHi is also a leading cause of economically significant, but rarely fatal, infections such as otitis media (Klein, 1997;Talbird et al, 2010). Phylogenetically, typable Hi have been found to group into lineages generally concordant with their serotype (Meats et al, 2003;Musser et al, 1988aMusser et al, , b, 1990, while NTHi is more phylogenetically diverse (Erwin et al, 2008;Meats et al, 2003;Quentin et al, 1990). Hi is naturally transformable, taking up DNA from its environment by the recognition of specific Hi uptake sequences in free DNA (Danner et al, 1980) and integrating it into its chromosome (Goodgal & Herriott, 1961;Smith et al, 1981).…”