Using multilocus sequence data to define the pneumococcus

Abstract

We investigated the genetic relationships between serotypeable pneumococci and nonserotypeable presumptive pneumococci using multilocus sequence typing (MLST) and partial sequencing of the pneumolysin gene (ply). Among 121 nonserotypeable presumptive pneumococci from Finland, we identified isolates of three classes: those with sequence types (STs) identical to those of serotypeable pneumococci, suggesting authentic pneumococci in which capsular expression had been downregulated or lost; isolates that clustered among serotypeable pneumococci on a tree based on the concatenated sequences of the MLST loci but which had STs that differed from those of serotypeable pneumococci in the MLST database; and a more diverse collection of isolates that did not cluster with serotypeable pneumococci. The latter isolates typically had sequences at all seven MLST loci that were 5 to 10% divergent from those of authentic pneumococci and also had distinct and divergent ply alleles. These isolates are proposed to be distinct from pneumococci but cannot be resolved from them by optochin susceptibility, bile solubility, or the presence of the ply gene. Complete resolution of pneumococci from the related but distinct population is problematic, as recombination between them was evident, and a few isolates of each population possessed alleles at one or occasionally more MLST loci from the other population. However, a tree based on the concatenated sequences of the MLST loci in most cases unambiguously distinguished whether a nonserotypeable isolate was or was not a pneumococcus, and the sequence of the ply gene fragment was found to be useful to resolve difficult cases.

FIG. 1.

Phylogenetic tree from concatenated sequences of MLST loci excepting ddl. The tree shows the relationships between the pneumococcal reference set (indicated by open circles) and nontypeable isolates (closed circles). Nontypeable isolates that had the same ST as members of the reference set are shown in gray. Group 1 contains the reference set of pneumococci and all nontypeable strains clustering with them. Group 1b is defined in the text. Group 2 contains the remaining nontypeable isolates, indicated with the prefix NT in Table 1. Trees were generated using MrBayes as described in Materials and Methods, using all nucleotide sites. The scale bar indicates substitutions per site. The asterisk marks the node that is considered to separate authentic pneumococci from group 2 isolates.

FIG.2.

Minimum evolution trees for the seven MLST loci. Trees were constructed using the sequences of all alleles from the pneumococcal MLST database and those from the nontypeable isolates. The latter are indicated by red markers. (a) aroE; (b) gdh; (c) gki; (d) recP; (e) spi; (f) xpt; (g) ddl. Trees were generated using MEGA 2.1. All nucleotide differences were used in the analysis, and distances were corrected using the K2P model. The scale bar indicates substitutions per site.

FIG. 3.

Minimum evolution tree for the ply alleles. All alleles found in the pneumococcal reference set or other group 1 isolates (shown in boldface) were found to descend from a single node. The remainder were alleles from nontypeable isolates that clustered apart from group 1 in Fig. 1. The tree was generated using MEGA 2.1. All nucleotide differences were used in the analysis, and distances were corrected using the K2P model. The scale bar indicates substitutions per site.