Genomic portrait of the evolution and epidemic spread of a recently emerged multidrug-resistant Shigella flexneri clone in China

Abstract

Shigella flexneri is the major cause of shigellosis in developing countries. A new S. flexneri serotype, Xv, appeared in 2000 and replaced serotype 2a as the most prevalent serotype in China. Serotype Xv is a variant of serotype X, with phosphoethanolamine modification of its O antigen mediated by a plasmid that contained the opt gene. Serotype Xv isolates belong to sequence type 91 (ST91). In this study, whole-genome sequencing of 59 S. flexneri isolates of 14 serotypes (serotypes 1 to 4, Y, Yv, X, and Xv) indicated that ST91 arose around 1993 by acquiring multidrug resistance (MDR) and spread across China within a decade. A comparative analysis of the chromosome and opt-carrying plasmid pSFXv_2 revealed independent origins of 3 serotype Xv clusters in China, with different divergence times. Using 18 cluster-dividing single-nucleotide polymorphisms (SNPs), SNP typing divided 380 isolates from 3 provinces (Henan, Gansu, and Anhui) into 5 SNP genotypes (SGs). One SG predominated in each province, but substantial interregional spread of SGs was also evident. These findings suggest that MDR is the key selective pressure for the emergence of the S. flexneri epidemic clone and that Shigella epidemics in China were caused by a combination of local expansion and interregional spread of serotype Xv.

FIG 1

Genomic relationships of S. flexneri isolates. (A) Phylogenetic tree of 64 S. flexneri isolates based on 1,790 SNPs constructed by the maximum likelihood method. Maximum likelihood bootstrap values that supported major lineages are indicated below the branches. The 6 lineages are marked on the right. The distribution of antibiotic resistance genomic contents is shown for the Shigella resistance locus (SRL) island, Tn7, gyrA mutations, and dfrA5. +, presence of a given gene or mutation; −, absence of a given gene or mutation. Isolate details (year, region, serotype, ST, and opt type) are shown. opt* indicates that the isolate carries a defective optII. The median and estimated 95% highest posterior density (HPD) of divergence time are given for the major lineages. Blue, cluster 1; purple, cluster 2; red, cluster 3. TET, tetracycline; STR, streptomycin; AMP, ampicillin; CHL, chloramphenicol; TMP, trimethoprim; NAL, nalidixic acid. (B) Phylogenetic tree of lineage I, to show opt gene variations. The phylogenetic tree was from A. The 3 clusters of Xv isolates are marked with boxed cluster names and divergence times. Blue, cluster 1; purple, cluster 2; red, cluster 3. The number above the branch that defines a cluster is the number of cluster-specific SNPs. ns, nonsynonymous SNP (nsSNP); s, synonymous SNP (sSNP); nc, noncoding SNP (ncSNP). Isolate details (year, region, serotype, ST, and opt type) are shown. opt* indicates that the isolate carries a defective optII. The opt SNPs are listed on the right, with the consensus bases in the top row. Dots, bases identical to the consensus bases. −, single-base deletion, resulting in a stop codon at the position shown.

FIG 2

Frequency and geographic distribution of the SNP genotypes of the 380 S. flexneri isolates in China, typed using cluster-specific SNPs. (A) Frequencies of the 5 SGs in different provinces, shown in pie charts. The numbers in parentheses after the province names are the numbers of isolates from the given province. SG3 and SG4 were grouped together to be consistent with genome clusters. (B) Geographic distribution of different SGs (genome clusters). The frequencies of the 5 SGs in different geographic regions (provinces) are shown in pie charts. For consistency with genome clusters, SG3 and SG4 are combined in one group. The genome clusters are indicated in parentheses after the SGs. The numbers in parentheses are the numbers of isolates from the given SG(s).