Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of Shigella flexneri

Abstract

Shigella spp. are the causative agent of shigellosis with Shigella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.

FIG. 1.

Temporal trends of S. flexneri serotypes in Henan Province, China. Only the top four predominant serotypes for each year are shown. The frequencies were based on 1,890 isolates from Henan Province, and the number of isolates per year varies.

FIG. 2.

Overall genome features of the serotype X variant isolate 2002017 in comparison with other S. flexneri genome-sequenced strains (2457T, Sf301, and Sf8401). The locations of SRLs and SHI-O are indicated. From the outer ring to the innermost in order are 2002017, 2457T, Sf301, and Sf8401. The inset depicts the structures of the SHI-Os in the 4 genomes. For details of 2002017 SHI-O see Fig. S1 in the supplemental material.

FIG. 3.

The evolutionary relationship of the four genome-sequenced strains of S. flexneri. The root of the tree was determined using genome sequences of E. coli K-12 (GenBank accession no. NC_000913) and EDL933 (GenBank accession no. NC_002655) as an outgroup. The number of genes gained (+) or lost (−) was marked on each branch. The number of mutational and recombinational changes was marked on 2002017 and 2457T only. rec, recombination; sSNP, synonymous SNPs; nsSNP, nonsynonymous SNPs. Sequence types are shown in parentheses beside the strain names. For a detailed comparison, see Fig. S3 in the supplemental material.

FIG. 4.

Minimum spanning tree of S. flexneri isolates based on PFGE data. The tree was constructed based on the presence or absence of PFGE bands. Pulse types with a band difference of one are connected by a thick line while those with a difference of two are connected by a thin line and those with a difference greater than two are connected by a dashed line. Clonal complexes are shaded. Each pulse type is color coded by serotype(s), and the pulse type is labeled alongside the circle. The circle size is proportional to the number of serotypes in the given pulse type.