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.

FIG. 1.

Clustering of encapsulated and noncapsulated H. influenzae isolates constructed using UPGMA from the matrix of pairwise differences in the allelic profiles of all 131 isolates. The prefix to the isolate name indicates the serotype of the isolate or whether it is noncapsulated (NT-). The relationship between STs and clonal complexes defined by MLST and the MLEE lineages defined by Musser et al. (28) are indicated alongside the branches of the dendrogram. The STs marked with an asterisk (encapsulated isolates) or a plus sign (noncapsulated isolates) were used in the statistical analysis of congruence between loci.

FIG. 2.

Phylogenetic relationships among diverse STs. A UPGMA tree was constructed from the differences in the allelic profiles of the 12 diverse STs of encapsulated (A) and noncapsulated (C) H. influenzae isolates (these STs are identified in Fig. 1). All isolates differed from each other at a genetic distance of ≥0.55 (N.B.: the tree is truncated at a genetic distance of 0.5). The concatenated sequences were used to construct a minimum evolution tree from the same sets of diverse encapsulated (B) and noncapsulated (D) STs. The percentage of recovery of the nodes in 1,000 replicate trees using the bootstrap test are shown on the minimum evolution trees.

FIG. 3.

Phylogenetic relationships among STs of encapsulated and noncapsulated H. influenzae. A minimum evolution tree was reconstructed using the concatenated sequences from the seven MLST loci (3,057 bp) for each of the 68 STs. The percent recoveries of the nodes in 1,000 bootstrap replicates are shown where these are ≥50%. The serotypes and MLEE lineages of the STs and the major phylogenetic division of isolates into group I and group II are shown. The tree is rooted at the midpoint of the longest distance between the STs. The box (inset) shows a simplified tree illustrating the relationships between major lineages inferred from the pairwise differences in the electrophoretic profiles of isolates obtained using MLEE. Tree shown in inset adapted from reference with permission of the publisher.