Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl trasferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity

Genomic analysis reveals multi-drug resistance clusters in Group B Streptococcus CC17 hypervirulent isolates causing neonatal invasive disease in southern mainland China

Neonatal invasive disease caused by group B Streptococcus (GBS) represents a significant public health care concern globally. However, data related to disease burden, serotype distribution and molecular epidemiology in China and other Asian countries are very few and specifically relative to confined regions. The aim of this study was to investigate the genetic characteristics of GBS isolates recovered from neonates with invasive disease during 2013-2014 at Guangzhou and Changsha hospitals in southern mainland China. We assessed the capsular polysaccharide (CPS) type, pilus islands (PIs) distribution and hvgA gene presence in a panel of 26 neonatal clinical isolates, of which 8 were recovered from Early Onset Disease (EOD) and 18 from Late Onset Disease (LOD). Among 26 isolates examined, five serotypes were identified. Type III was the most represented (15 cases), particularly among LOD strains (n=11), followed by types Ib (n=5), V (n=3), Ia (n=2) and II (n=1). We performed whole-genome sequencing (WGS) analysis and antimicrobial susceptibility testing on the 14 serotype III isolates belonging to the hypervirulent Clonal Complex 17 (serotype III-CC17).The presence of PI-2b alone was associated with 13 out of 14 serotype III-CC17 strains. Genome analysis led us to identify two multi-drug resistance gene clusters harbored in two new versions of integrative and conjugative elements (ICEs), carrying five or eight antibiotic resistance genes, respectively. These ICEs replaced the 16 kb-locus that normally contains the PI-1 operon. All isolates harboring the identified ICEs showed multiple resistances to aminoglycoside, macrolide and tetracycline antibiotic classes. In conclusion, we report the first whole-genome sequence analysis of 14 GBS serotype III-CC17 strains isolated in China, representing the most prevalent lineage causing neonatal invasive disease. The acquisition of newly identified ICEs conferring multiple antibiotic resistances could in part explain the spread of this specific clone among Chinese neonatal isolates and underlines the need for a constant epidemiological surveillance

Genomic Analysis Reveals the Molecular Basis for Capsule Loss in the Group B <i>Streptococcus</i> Population

<div><p>The human and bovine bacterial pathogen <i>Streptococcus agalactiae</i> (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the <i>cps</i> operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the <i>cps</i> genes, and the main target was found to be <i>cpsE</i> encoding the portal glycosyl trasferase of capsule biosynthesis. Complementation of strains carrying missense mutations in <i>cpsE</i> with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.</p></div

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

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

Different kinds of genetic alterations detected in the GBS <i>cps</i> operon.

<p>(A) Distribution of 37 transposable elements (blue), 24 insertion or deletion (indels) targeting a single gene (light green) and 54 point mutations (dark green) scattered across the <i>cps</i> operon. Genes that are missing in one or more capsular types are shown with red font color in the operon diagram. (B) Deletions comprising more than a single gene are indicated with dotted lines; empty spaces represent the absence of <i>cps</i> genes in a specific capsular genotype. (C) Gene targets of capsule inactivation mutations that appeared as single events (blue arrows); the figures below the target genes indicate the number of strains presenting the individual mutation.</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

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