Characterization of Encapsulated and Noncapsulated Haemophilus influenzae and Determination of Phylogenetic Relationships by Multilocus Sequence Typing

Abstract

A multilocus sequence typing (MLST) scheme has been developed for the unambiguous characterization of encapsulated and noncapsulated Haemophilus influenzae isolates. The sequences of internal fragments of seven housekeeping genes were determined for 131 isolates, comprising a diverse set of 104 serotype a, b, c, d, e, and f isolates and 27 noncapsulated isolates. Many of the encapsulated isolates had previously been characterized by multilocus enzyme electrophoresis (MLEE), and the validity of the MLST scheme was established by the very similar clustering of isolates obtained by these methods. Isolates of serotypes c, d, e, and f formed monophyletic groups on a dendrogram constructed from the differences in the allelic profiles of the isolates, whereas there were highly divergent lineages of both serotype a and b isolates. Noncapsulated isolates were distinct from encapsulated isolates and, with one exception, were within two highly divergent clusters. The relationships between the major lineages of encapsulated H. influenzae inferred from MLEE data could not be discerned on a dendrogram constructed from differences in the allelic profiles, but were apparent on a tree reconstructed from the concatenated nucleotide sequences. Recombination has not therefore completely eliminated phylogenetic signal, and in support of this, for encapsulated isolates, there was significant congruence between many of the trees reconstructed from the sequences of the seven individual loci. Congruence was less apparent for noncapsulated isolates, suggesting that the impact of recombination is greater among noncapsulated than encapsulated isolates. The H. influenzae MLST scheme is available at www.mlst.net, it allows any isolate to be compared with those in the MLST database, and (for encapsulated isolates) it assigns isolates to their phylogenetic lineage, via the Internet.

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

“…We also explored the possibility that strains expressing a b-lactamase enzyme (which confers penicillin resistance) might cluster phylogenetically, and found they did not. 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.…”

“…Bacterial genomic DNA was extracted from pure cultures of NTHi isolated from NP, TS or MEF samples (if children had AOM). The internal fragments of seven housekeeping genes of NTHi were amplified by PCR, using a PCR Master Mix (Promega) as described previously (Meats et al, 2003). The PCR products were purified using the ExoSAP kit (USB) and identified by DNA sequencing.…”

Genotyping ofMycoplasma mycoidessubsp.mycoidesSC by multilocus sequence analysis allows molecular epidemiology of contagious bovine pleuropneumonia

“…The rationale for this is that a single genetic event resulting in a new allele can occur by a point mutation, altering only a single nucleotide site, or by a recombinational replacement that will often result in the modification of multiple sites. This typing scheme has been applied successfully to a wide variety of bacterial pathogens such as Staphylococcus aureus [14], Campylobacter jejuni [12], and Haemophilus influenzae [26], to cite only a few. This approach has multiple applications such as phylogeny [18], molecular structure analysis [42], and molecular epidemiology.…”

Identification and interrogation of highly informative single nucleotide polymorphism sets defined by bacterial multilocus sequence typing databases

“…Therefore, MLST databases for a number of different species were searched for high-D SNP sets. Species subject to this analysis were Burkholderia pseudomallei/ Burkholderia mallei, Helicobacter pylori, Campylobacter jejuni, Streptococcus pneumoniae, Streptococcus pyogenes and Enterococcus faecium (Feil et al, 2000;Enright et al, 2001;Sa-Leao et al, 2001;Dingle et al, 2002;Homan et al, 2002;Godoy et al, 2003;Meats et al, 2003). Ten different sets of SNPs were identified for each species.…”