Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococcal strains are classified according to their capsular serotype and through a Multi-Locus Sequence Typing schema (MLST) based on the sequencing of seven housekeeping genes. However, strains with a defined allelic profile (Sequence Type, ST) can have different serotypes, suggesting that the micro-evolution of the MLST lineages leads to a considerable degree of phenotypic variability. To better investigate the genetic diversity within these lineages, we set-up and then validated an extended molecular typing schema (96-MLST) based on the sequencing of ninety-six genomic loci. 96-MLST loci were designed within core-genes in a collection of 39 complete genomes of S. pneumoniae. None of the capsular genes was included in the schema. When tested on a collection of 69 isolates, 96-MLST was able to partition strains with the same ST and diverse serotypes into groups that were homogenous for capsular serotype, improving our understanding of the evolution of epidemiologically relevant lineages. Phylogenetic sequence analysis showed that the capsular heterogeneity of three STs that were sampled more extensively could be traced back to a limited number of capsular switch events, indicating that changes of serotype occur occasionally during the short term expansion of clones. Moreover, a geographical structure of ST156 was identified, suggesting that the resolution guaranteed by this method is sufficient for phylogeographic studies. In conclusion, we showed that an extended typing schema was able to characterize the expansion of individual lineages in a complex species such as S. pneumoniae.
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