CC156 strain panel used in this study.

<p>For each strain name, ST, serotype/serogroup, country of isolation, number of MLST alleles in common with ST156 and ST4945, data source, strain source and lineage (as identified by 96-MLST hierarchical clustering, <i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061003#pone-0061003-g002" target="_blank">Figure 2</a>) are indicated.</p

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

<div><p>Multi-Locus Sequence Typing (MLST) of <i>Streptococcus pneumoniae</i> 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 <i>S. pneumoniae</i> 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.</p> </div

Minimum Spanning Tree analysis based on 96-MLST allelic profiles identifies seven distinct lineages by imposing a maximum threshold of 75 different loci.

<p>The Minimum Spanning Tree analysis was performed by using PHYLOVIZ on the 96-MLST alleles of the 66 strains considered in this study. The lineages identified by applying the threshold of 75/96 different loci are highlighted with shadowed shapes and named according to the lineage identification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061003#pone-0061003-g002" target="_blank">Figure 2</a>.</p

Classification of the transposable elements identified in 96 sequenced non- encapsulated strains.

<p>Classification of the transposable elements identified in 96 sequenced non- encapsulated strains.</p

GBS SNP-based Neighbor-joining phylogenetic trees highlighting GBS hosts and NT strains.

<p>(A) Phylogenetic tree where non-typeable or capsulated phenotypes are indicated by colored dots. (B) Phylogenetic tree where strain host origin is indicated by colored dots.</p

Analysis of the influence of genetic alterations on the transcription of the <i>cps</i> operon in NT isolates.

<p>(A) Mutations in the <i>cps</i> promoter or <i>cpsA-D</i> detected in 18 NT strains. Their positions are indicated as +/- numbers in relation to the first base pair of the <i>cpsA</i> coding sequence. ISs are represented by colored triangles; point mutations in the -10 sequence are marked in bold; deleted regions are represented by dotted lines and stop codons by crosses. Numbers within parentheses identify the strains reported in the x-axis of panel B. (B) Transcription of the <i>cps</i> operon in the 18 NT strains measured with primers cpsAup-F/R (filled bars) and to cpsE-F/R (hatched bars). The relative fold expression for each strain was estimated in comparison to the expression of <i>cpsA</i> in strain 515. For strains 25, 26, 52 and 53 the transcript in <i>cpsE</i> gene was not determined.</p

Relative frequency of the different types of mutations possibly responsible for the lack of capsule expression in GBS.

<p>(A) Distribution of 126 genetic alteration events detected in 89 isolates. (B) Number of strains bearing each of the <i>cps</i> mutation types among to the five selected capsular genotypes.</p

NT GBS strains presenting missense mutations and the results of <i>cpsE</i> complementation in same strains.

<p>The nucleotide and amino acid changes in the isolates were based on the encapsulated 2603 V/R reference genome sequence. AA, single letter amino acid designation; del, deletion of the nucleotide; n.a. not applicable.</p><p>NT GBS strains presenting missense mutations and the results of <i>cpsE</i> complementation in same strains.</p

GBS SNP-based and MST-based phylogenetic trees.

<p>(A) SNP-based Neighbor-joining phylogenetic tree. The tree was generated using 14,092 polymorphic sites extracted from the alignment of 0.42 Mbp non-duplicated core regions shared by all 373 strains aligned to the reference strain 2603 V/R. CCs assigned to the 12 major clusters are indicated in colored ribbons. Dots represent single strains and are colored according to their capsular genotype. Asterisks indicate strains where the CC assigned by MLST (CC-1 or CC-6-8-10) differs from that assigned to strains belonging to the same SNP clade. (B) Minimum Multilocus Sequence Typing spanning tree of GBS strains. Each node represents one ST and STs differing by only one allele are connected by a line. Node dimensions refer to the relative number of strains belonging to each ST. Colored dots represent the assigned 17 CCs after refinement based on the SNP analysis. CCs included in the dotted line circle are linked by transition STs.</p

Complementation of <i>cpsE</i> missense mutations in selected NT GBS strains.

<p>Flow cytometry analysis of NT GBS strains carrying missense mutations in <i>cpsE</i> and of their counterpart after transformation with pAM-<i>cpsE</i>. Bacteria were incubated with mouse monoclonal antibodies specific for the five capsular polysaccharides, and then treated with labeled secondary antibodies. Fluorescence after incubation with the secondary antibody alone is indicated by empty histograms, while staining after treatment with type the specific antibodies is shown by the colored histograms.</p