Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Monica Moschioni,Morena Lo Sapio,Alessandro Muzzi,Silvia Guidotti,Giulia Torricelli,Giovanni Crisafulli,Michèle A Barocchi,Claudio Donati

doi:10.1371/journal.pone.0061003

Abstract

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.

Highlights

Disease-causing pneumococci represent a phenotypically and genotypically diverse population of strains that cause bacteraemia, meningitis, pneumonia, sinusitis, and acute otitis media in children [1,2,3,4]
As detailed elsewhere [25,27,43], CC162 strains were associated with serotypes 9V and 14 and were Pilus Islet-1 (PI-1) positive (PI-1 clade I), CC124 and CC392 strains were associated with serotypes 14 and 17F and were PI-1 negative, CC146 strains were associated with serotype 6B and PI-1 positive and CC176 strains were generally associated with serotype 6B and 23F and heterogeneous for the PI-1 presence
The implementation of increased regional surveillance combined with molecular typing methods has allowed for the identification of several successful S. pneumoniae clones with a higher invasive disease potential and an ability to spread across different geographical regions

Summary

Introduction

Disease-causing pneumococci represent a phenotypically and genotypically diverse population of strains that cause bacteraemia, meningitis, pneumonia, sinusitis, and acute otitis media in children [1,2,3,4]. Along with the characterization and the classification of the circulating strains, are important tools to understand the evolution and the population dynamics that support the success of specific pneumococcal lineages [5,6,7,8,9]. The accessibility of sequence analysis tools has increased the diffusion of Streptococcus pneumoniae molecular genotyping methods such as Multi Locus Sequence Typing (MLST) (http://spneumoniae.mlst.net/) [15,16]. The MLST classification reveals important insights into the geographic spread of successful pathogenic clones and the emergence of associations between STs and serotypes, combinations that can be traced back to serotype switching events [18,19,20,21,22]

Methods

Results

Conclusion