Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Abstract

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

Figure 1. Population structure of human GBS is driven by tetracycline resistance acquisition

(a) Whole-genome-based phylogeny of 229 sequenced GBS isolates and strain SS1219 isolated from fish. Maximum Likelihood (ML) using MEGA was used to infer phylogenetic relationships. The major clonal complexes (CC) 1, 10, 17, 19, 23 and 26 as defined on the GBS MLST web site (http://pubmlst.org/sagalactiae/) correspond to well-defined branches. Isolates are indicated by dots coloured according to their geographical origin. Flanking the whole-genome phylogeny, are four Bayesian maximum clade credibility phylogenies (b–e) based on the non-recombinogenic genome for the GBS CC17 (b), CC23 (c), CC19 (d) and CC1 strains (e). Divergence dates (median estimates with 95% highest posterior density dates in brackets) are provided in blue for the major nodes. Coloured branches relate to the major tetracycline-resistant clones. Arrows indicate the predicted time of insertion of the ICE carrying the tet(M) resistance determinant within the major clones. Capsular serotypes are indicated on the right of each tree according to the indicated colour code.

Figure 2. Distribution of SNPs and recombination across all GBS isolates from the six major CCs

The maps were generated by using the SyntView software. Isolates were ordered according to the distance from the reference genome depicted at the inner circle. CC numbers are indicated in the centre. Recombined regions compared with the reference genome correspond to regions with a higher density of SNPs indicated by short lines on each circle corresponding to one strain. Around the outside circle are the relative positions of selected antigenic loci. The reference genomes were BG-NI-011 for CC1, DK-NI-008 for CC10, COH1 for CC17, RBH11 for CC19, CCH210801006 for CC23 and Bangui-IP-105 for CC26.

Figure 3. Phylogeny of the ‘hypervirulent’ CC17 lineage

(a) ML phylogeny based on the alignment of 3,922 polymorphic positions. Six independent ICE insertions (indicated on the right and by blue arrows) corresponding to six different lineages (indicated by different colours) were identified and are numbered from 11 to 16 (Table 4). A star indicates that Tn5801 has been lost by this isolate. Following the loss of Tn5801, strains CCH210160764 and CCH207800974 have acquired unrelated ICE expressing tet(M) and erm(B), and tet(O) and erm(B), respectively. Nodes with >90% bootstrap support are indicated by black dots. (b) Genetic maps and alignment of Tn916 and Tn5801. Comparisons were performed by BLASTn. The tet(M) gene is coloured in yellow, genes encoding type 4 secretion system components are in blue and the integrase and excisionase genes which are not conserved between the two transposons in red. Percentages of identities are shown in blue scale and range between 68 and 98% for the tet(M) region.

Figure 4. Phylogeny of clonal complex CC1

(a) ML phylogeny from the alignment of pseudosequences of the 1,244 polymorphic positions in 914 interrogated kbases. The five independent Tn916 or Tn5801 insertions are indicated in blue and numbers from 1 to 5 refer to their description in Table 4. The two TcR lineages with more than one isolate are coloured in blue and red. Three sub-lineages have acquired an erm resistance gene. Within lineage Tn916-1, 40% of the isolates (12) carry Tn3872 (dark-blue branch and strain Bangui-IP-30). The four observed serotypes (cps) II, IV, V and VI are indicated in violet. A star indicates that Tn916 has been lost by the isolate. Antibiotic resistance genes other than tet(M) are indicated in red. Nodes with >90% bootstrap support are indicated by black dots. (b) Genetic map of Tn3872. Tn917 carrying the erm(B) gene is in grey the erm(B) gene being in orange, Tn916 genes are coloured as in Fig. 3. The location of 13 out of the 15 SNPs between strain CZ-NI-006 Tn916 and strain DE-NI-001 Tn3872, indicated by black bars are located between positions 10,407 and 13,659 (Tn916 coordinates) and the 76 SNPs between strain Bangui-IP-50 and strain CZ-NI-006, all located between position 10,407 and 13,497 are in blue.

Figure 5. Correlation of isolation date with maximum likelihood root-to-tip branch length for the five major TcR lineages calculated with Path-O-Gen

These analyses predict the origin of these clones in agreement with the BEAST analysis except for the CC23 lineage where there was a lack of temporal sampling to support tree root estimates. X axis, time in years; Y axis root-to-tip branch length in SNP per Mb. (a) CC1 lineage Tn916-1; (b) CC19 lineage Tn916-17; (c) CC17 lineage Tn5801-11; (d) CC17 lineage Tn916-12; (e) CC23 lineage Tn5801-23.