Acquisition and evolution of SXT-R391 integrative conjugative elements in the seventh-pandemic Vibrio cholerae lineage

Abstract

SXT-R391 Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements able to confer multidrug resistance and other adaptive features to bacterial hosts, including Vibrio cholerae, the causative agent of cholera. ICEs are arranged in a mosaic genetic structure composed of a conserved backbone interspersed with variable DNA clusters located in conserved hot spots. In this study, we investigated ICE acquisition and subsequent microevolution in pandemic V. cholerae. Ninety-six ICEs were retrieved from publicly available sequence databases from V. cholerae clinical strains and were compared to a set of reference ICEs. Comparative genomics highlighted the existence of five main ICE groups with a distinct genetic makeup, exemplified by ICEVchInd5, ICEVchMoz10, SXT, ICEVchInd6, and ICEVchBan11. ICEVchInd5 (the most frequent element, represented by 70 of 96 elements analyzed) displayed no sequence rearrangements and was characterized by 46 single nucleotide polymorphisms (SNPs). SNP analysis revealed that recent inter-ICE homologous recombination between ICEVchInd5 and other ICEs circulating in gammaproteobacteria generated ICEVchMoz10, ICEVchInd6, and ICEVchBan11. Bayesian phylogenetic analyses indicated that ICEVchInd5 and SXT were independently acquired by the current pandemic V. cholerae O1 and O139 lineages, respectively, within a period of only a few years.

Importance: SXT-R391 ICEs have been recognized as key vectors of antibiotic resistance in the seventh-pandemic lineage of V. cholerae, which remains a major cause of mortality and morbidity on a global scale. ICEs were acquired only recently in this clade and are acknowledged to be major contributors to horizontal gene transfer and the acquisition of new traits in bacterial species. We have reconstructed the temporal dynamics of SXT-R391 ICE acquisition and spread and have identified subsequent recombination events generating significant diversity in ICEs currently circulating among V. cholerae clinical strains. Our results showed that acquisition of SXT-R391 ICEs provided the V. cholerae seventh-pandemic lineage not only with a multidrug resistance phenotype but also with a powerful molecular tool for rapidly accessing the pan-genome of a large number of gammaproteobacteria.

FIG 1

BLAST atlas using ICEVchInd5 showing the genetic rearrangements in the ICE data set. Contigs from each de novo assembly of 96 V. cholerae genomes were mapped against the sequence of ICEVchInd5 (outer circle, with annotations). ICE profiles are shown with different colors according to their variable levels of genetic content within the hot spots. Green, ICEVchInd5 (74 ICEs); orange, ICEVchMoz10 (6 ICEs); red, ICEVchInd6 (13 ICEs); purple, ICEVchBan11 (1 ICE); blue, SXT (2 ICEs). Absence of color represents missing genetic material compared to ICEVchInd5. Gray bars represent deletion regions gap 1 and gap 2 in the antibiotic resistance gene cluster and hotspot 5, respectively. Color gradients are proportional to the BLAST percent identity (90% to 100%).

FIG 2

Genetic organization of the five ICE profiles detected in V. cholerae clinical strains. Backbone genes are depicted in black. Different hot spots and variable regions are highlighted in gray. Variable genes shared between ICEVchInd5 and other ICEs are represented in green. Genes present in both SXT and ICEVchMoz10 are depicted in blue. Genes present in both ICEVchInd6 and ICEVchMoz10 are depicted in red. Genes unique to ICEVchBan11 and ICEVchMoz10 are represented in purple and orange, respectively.

FIG 3

Signatures of recombination between ICEs in V. cholerae. The distribution of SNPs is shown for each ICE profile with respect to the 52 core genes of different reference ICEs. The y axis data give the number of SNPs within a 100-bp window. (A) ICEVchBan11 reads (accession number ERS016137) are mapped against ICEPmiUSA1 (purple) and ICEVchInd5 (green). (B) ICEVchInd6 reads (accession number ERS013257) are mapped against ICEVflInd1 (orange) and ICEVchInd5 (green). (C) ICEVchMoz10 reads (accession number ERS013126) are mapped against ICEVflInd1 (orange) and ICEVchInd5 (green). (D) SXT reads (accession number ERS013124) are mapped against ICEVchInd5. Genes with high (>98%) sequence similarity to the respective reference ICE are represented in the same color. No recent recombination signatures were detected in dark-gray genes. Red bars depict low coverage and intergenic regions where SNPs could not be called (<5× coverage, >25 bp).

FIG 4

Estimated date for the acquisition of ICEVchInd5 and SXT in V. cholerae. Data represent the posterior-probability distributions (red dashed lines) for the time to most recent common ancestor (TMRCA) of ICEVchInd5 (A) and SXT (B). Median values and 95% confidence intervals are reported in the legend beside the graph. The x axis shows the age of the root in years. Flat priors (blue dashed line) were applied in BEAST, as the tree was calibrated using tip dates only (see Materials and Methods for details).

FIG 5

Schematic representation of the acquisition of SXT-R391 ICEs in seventh-pandemic V. cholerae. The three overlapping pandemic waves of global transmission are depicted in blue (first wave), green (second wave), and red (third wave), according to the color scheme and the inferred dates from Mutreja et al. (8). The O139 serogroup is shown as a subset of the second pandemic wave, in light green. Vertical arrows depict the acquisition events from the environment and subsequent rearrangements by recombination with other SXT-R391 ICEs acquired by conjugation (hypothetical bacterial donors are depicted using a purple bar). The likely acquisition dates of ICEVchInd5 and SXT are shown by green and blue arrows, respectively, with color bars spanning the 95% confidence intervals estimated by our analysis of the ICE sequences (see text and Fig. 4B). Subsequent inter-ICE recombination events giving rise to new hybrid ICEs are depicted as green dashed double arrows, with corresponding dates associated with the oldest isolate showing the recombinant ICE. Env., environmental.