Whole-Genome Comparison Uncovers Genomic Mutations between Group B Streptococci Sampled from Infected Newborns and Their Mothers

Abstract

Streptococcus agalactiae (group B Streptococcus or GBS), a commensal of the human gut and genitourinary tract, is a leading cause of neonatal infections, in which vertical transmission from mother to child remains the most frequent route of contamination. Here, we investigated whether the progression of GBS from carriage to disease is associated with genomic adaptation. Whole-genome comparison of 47 GBS samples from 19 mother-child pairs uncovered 21 single nucleotide polymorphisms (SNPs) and seven insertions/deletions. Of the SNPs detected, 16 appear to have been fixed in the population sampled whereas five mutations were found to be polymorphic. In the infant strains, 14 mutations were detected, including two independently fixed variants affecting the covRS locus, which is known to encode a major regulatory system of virulence. A one-nucleotide insertion was also identified in the promoter region of the highly immunogenic surface protein Rib gene. Gene expression analysis after incubation in human blood showed that these mutations influenced the expression of virulence-associated genes. Additional identification of three mutated strains in the mothers' milk raised the possibility of the newborns also being a source of contamination for their mothers. Overall, our work showed that GBS strains in carriage and disease scenarios might undergo adaptive changes following colonization. The types and locations of the mutations found, together with the experimental results showing their phenotypic impact, suggest that those in a context of infection were positively selected during the transition of GBS from commensal to pathogen, contributing to an increased capacity to cause disease.

Importance: Group B Streptococcus (GBS) is a major pathogen responsible for neonatal infections. Considering that its colonization of healthy adults is mostly asymptomatic, the mechanisms behind its switch from a commensal to an invasive state are largely unknown. In this work, we compared the genomic profile of GBS samples causing infections in newborns with that of the GBS colonizing their mothers. Multiple mutations were detected, namely, within key virulence factors, including the response regulator CovR and surface protein Rib, potentially affecting the pathogenesis of GBS. Their overall impact was supported by differences in the expression of virulence-associated genes in human blood. Our results suggest that during GBS's progression to disease, particular variants are positively selected, contributing to the ability of this bacterium to infect its host.

FIG 1

Whole-genome phylogeny of mother-child GBS pairs. Maximum-likelihood phylogenetic tree built with snpTree (49) based on genomic SNPs. The unrooted tree depicts the phylogenetic relationship of each strain in our study with the published complete GBS genomes in the NCBI database (highlighted in blue), i.e., 09mas018883 (GenBank accession no. HF952104), 2603V/R (GenBank accession no. AE009948), A909 (GenBank accession no. CP000114), COH1 (GenBank accession no. HG939456), GD201008-001 (GenBank accession no. CP003810), and NEM316 (GenBank accession no. AL732656). Strain names are as indicated in Table 1 (see also Table S1 in the supplemental material). Strains from pairs 7 and 11 are highlighted in red and green, respectively.

FIG 2

Characteristics of the mutations identified. (A) Classification of the SNPs identified in 10 of the 19 pairs of GBS samples analyzed by comparative genomics. (B) Functional category of each mutated region based on homology search of the flanking gene of each site, as annotated in the SagaList database of GBS NEM316. Both the type of mutation and the functional category are represented by different colors, as shown in the key. Each value depicted is the number of mutations identified belonging to each category.

FIG 3

Relative expression of mutated genes (pair 12). RT-qPCR results obtained with GBS strains from pair 12 in TH medium (A) and after incubation for 1 h in human blood (B). The mother 12 isolate was mutated in the 5′ UTR of gbs1946 (ptsG), and both child strains were mutated in gbs0668 (d-lactate dehydrogenase) and gbs1038 (permease). The isolate from child 12-1 tested presented additional mutations affecting gbs1946 (ptsG), gbs1377 (homocysteine S-methyltransferase), and a phage terminase, while the child 12-2 strain specifically was mutated in the 5′ UTR of gbs1672 (covR) and gbs0231 (putative transporter). Gene expression is represented after normalization to the housekeeping gene gyrA. Gene names are indicated below each graph. Experiments were performed in triplicate with three independent cultures. Error bars represent standard deviations. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 4

Relative expression of mutated genes (pair 19). RT-qPCR results obtained with GBS strains from pair 19 in TH medium (A) and after incubation for 1 h in human blood (B). The mother 19 isolate tested was mutated in gbs2047 (recA), and the child strain was mutated in gbs1159 (pta), gbs1672 (covR), and the promoter of GBSCOH1_0416 (rib). Gene expression is represented after normalization to the housekeeping gene gyrA. Gene names are indicated below each graph. Experiments were performed in triplicate with three independent cultures. Error bars represent standard deviations. ***, P < 0.001; *, P < 0.05.

FIG 5

Transmission pathways between GBS samples. Graphical representation of the most likely transmission route hypothesized for each pair of GBS samples. The colonization site and whether it corresponds to a situation of carriage or disease are represented by different shapes and colors according to the key. The arrows indicate the presumed direction of transfer inferred from the clinical information available together with the number of mutations determined in the mother and child GBS samples. Polymorphic variants, present in <97% of the population sampled, are indicated by asterisks. When the child was diagnosed with EOD and sampled at the time of birth, even if the mutant strain was retrieved from the mother, we assumed mother-to-child transmission. Ambiguous transmission direction is indicated by a double-headed arrow. D. L. indicates that the two strains belong to distinct lineages, so the origin of the newborn GBS clone is unknown.