Recombining population structure of Plesiomonas shigelloides (Enterobacteriaceae) revealed by multilocus sequence typing

Abstract

Plesiomonas shigelloides is an emerging pathogen that is widespread in the aquatic environment and is responsible for intestinal diseases and extraintestinal infections in humans and other animals. Virtually nothing is known about its genetic diversity, population structure, and evolution, which severely limits epidemiological control. We addressed these questions by developing a multilocus sequence typing (MLST) system based on five genes (fusA, leuS, pyrG, recG, and rpoB) and analyzing 77 epidemiologically unrelated strains from several countries and several ecological sources. The phylogenetic position of P. shigelloides within family Enterobacteriaceae was precisely defined by phylogenetic analysis of the same gene portions in other family members. Within P. shigelloides, high levels of nucleotide diversity (average percentage of nucleotide differences between strains, 1.49%) and genotypic diversity (64 distinct sequence types; Simpson's index, 99.7%) were found, with no salient internal phylogenetic structure. We estimated that homologous recombination in housekeeping genes affects P. shigelloides alleles and nucleotides 7 and 77 times more frequently than mutation, respectively. These ratios are similar to those observed in the naturally transformable species Streptococcus pneumoniae with a high rate of recombination. In contrast, recombination within Salmonella enterica, Escherichia coli, and Yersinia enterocolitica was much less frequent. P. shigelloides thus stands out among members of the Enterobacteriaceae. Its high rate of recombination results in a lack of association between genomic background and O and H antigenic factors, as observed for the 51 serotypes found in our sample. Given its robustness and discriminatory power, we recommend MLST as a reference method for population biology studies and epidemiological tracking of P. shigelloides strains.

FIG. 1.

Allelic profiles, STs, O and H serotypes, sources, and countries of isolation of the 77 strains of P. shigelloides. The unweighted-pair group method using average linkage dendrogram was constructed from a distance matrix consisting of pairwise distances between allelic profiles.

FIG. 2.

Unrooted neighbor-joining tree of 31 Enterobacteriaceae species (type strains were sequenced) and three other Gammaproteobacteria constructed using the concatenated sequences of four loci (fusA, leuS, pyrG, and rpoB). Sequences of the following strains were retrieved from public databases: Y. pestis CO92 (accession no. NC_003143), P. multocida subsp. multocida Pm70 (NC_002663), S. oneidensis MR-1 (NC_004347), and H. influenzae Rd KW20 (NC_000907). All P. shigelloides strains were placed on the same branch as the type strain. The numbers at the nodes correspond to bootstrap values obtained with 1,000 replicates that were more than 80%.

FIG. 3.

Minimum spanning tree analysis of the 77 strains of P. shigelloides based on the number of allelic mismatches among MLST profiles. The colors of the circles represent the serotypes of the strains. (A) O antigen. (B) H antigen. Each circle corresponds to one ST; the size of the circle indicates the number of strains in the ST. The numbers on the lines between circles are the numbers of allelic differences between the corresponding profiles.

FIG. 4.

Relationship between allelic profile distance (x axis) and average number of nucleotide differences (y axis) in the distinct alleles. The error bars indicate the standard deviations for N. meningitidis and P. shigelloides. A positive trend was observed for S. aureus, E. coli, and Y. pseudotuberculosis, three species that are generally considered clonal. In contrast, P. shigelloides did not show a positive trend, similar to the recombining bacteria N. meningitidis and S. pneumoniae.