Genomic Portrait Of The Evolution And Epidemic Spread Of A Recently Emerged Multidrug-Resistant Shigella Flexneri Clone In China

Shigella includes four species, in which, Shigella flexneri is the major cause of bacillary dysentery in developing countries. S. flexneri is divided into at least19serotypes, based on the combinations of antigenic determinants present on the O-antigen. The addition of glucosyl and/or acetyl groups and/or a phosphoethanolamine （PEtN） group to different sugars of the tetrasaccharide unit results in different serotypes. O-antigen glucosylation and acetylation are mediated by temperate bacteriophages, and PEtN modification is medicated by op?-carrying plasmid pSFXv₂. The opt gene, encodes the PEtN transferase, which is responsible for attaching a PEtN at the second （RhaⅡ） or third （RhaⅢ） rhamnose, or both, of the tetrasaccharide repeat unit on the O-antigen. There are2known opt alleles, optll and optâ…¢, which are carried by plasmids pSFXv₂and pSFyv₂, respectively, and preferentially mediate modifications of Rhaâ…¡ and Rhaâ…¢, to form Xv and Yv （4av） respectively. A new S. flexneri serotype, Xv, appeared in2000and subsequently replaced serotype2a as the most prevalent serotype in Henan and other provinces in China. Serotype Xv is a variant of serotype X, with PEtN modification on its O-antigen. Serotype Xv isolates belong to sequence type91（ST）, and ST91is predominant ST in China. Genome sequencing of a representative serotype Xv strain2002017（ST91）, revealed that it gained2unique multidrug-resistance （MDR） islands, i.e., the Shigella resistance locus （SRL） and Tn7, which carry multidrug-resistant genes. The SRL was first identified in the Japanese S. flexneri2a strain YSH6000. The SRL located in2002017carries an additional set of tetracycline resistance genes, but does not contain the ferric transport system present in YSH6000. Tn7is a composite transposon. The sequence of2002017Tn7is similar with strain2457T （serotype2a, ST86）, Sf301（2a, ST18）, Sf8401（5b, ST93） and YSH6000（2a）.To elucidate evolution and epidemic spread of the multidrug-resistant clone, and the temporal and geographic dynamics of S. flexneri epidemics in China, we sequenced59S. flexneri isolates of14serotypes （Xv [25isolates], X [9isolates],1a [7isolates], lb [1isolate],1d [1isolate],2a [5isolates],2b [1isolate],3a [3isolates],3b [1isolate],4a [1isolate],4av [1isolate],4b [1isolate], Y [2isolates] and Yv [1isolate]） and7STs （ST91[50isolates], ST109[1isolate], ST18[3isolates], ST142[1isolate], ST143[lisolate], ST103[2isolates] and ST16[lisolate]）. Five published complete S. flexneri genomes （2002017, Sf301,2457T, Sf8401and M90T） were also included for comparison. We searched for the antibiotic resistant islands and genes on the draft genome, and found that by acquiring2MDR islands （SRL and Tn7） and two gyrA mutations related to quinolone resistance, ST91arose around1993, rapidly expanded, and spread across China within a decade, to become predominant clone. Phylogenetic tree based on1,790SNPs （single-nucleotide polymorphisms） in the core genome regions constructed by BEAST analysis indicated that Xv isolates form distinctive and extremely tight3genome clusters with different divergence times on branch, suggesting independent origins of the Xv serotype. The3main clusters of serotype Xv isolates, which were localized in their respective regions, further supported multiple and local origins of serotype Xv. In particular, cluster1, which was predominant in Henan, emerged earliest （1999）, spread to other regions with much greater proportions. Subsequently, cluster3（Anhui predominant cluster） and cluster2（Gansu predominant cluster） diverged in succession. Thus, there seems to be a regional prevalence of different serotype Xv clusters.We determined the presence of opt by mapping Illumina reads to the plasmid sequence and further confirmed the sequence by sequencing the amplified opt PCR products. All25Xv strains carried optâ…¡, and3variants, differing from the optâ…¡ of2002017by1or2bases, were observed. A single Yv strain carried an optâ…¢ form with a single base change from the optâ…¢ of HN006strain. Three nonfunctional opt alleles were also observed, with one each in3serotype X strains. A single-base deletion rendered the opt gene nonfunctional in all3cases. We further extended our SNP analysis to the whole pSFXv₂plasmid. By mapping raw reads to the reference pSFXv₂plasmid, we found that all serotype Xv isolates and3serotype X isolates carried the pSFXv₂, while all other strains did not contain the plasmid. The pSFXv₂plasmids of cluster1and cluster2are highly homologous, however cluster3was dissimilar from the other2clusters with15unique base changes. There was good concordance between plasmid variants and chromosomal clusters, consistent with coevolution of the plasmid and chromosome. A comparative analysis of the chromosome and opt-carrying plasmid pSFXv₂revealed a possible common origin of cluster1and2, and cluster3seems to have arisen independently as an Xv serotype.The overwhelming regional clustering of serotype Xv isolates as3genome clusters based on the whole-genome sequences prompted us to determine the regional dynamics of the3clusters using a much larger sample, by SNP typing of cluster-specific SNPs. There are18cluster-specific SNPs, including4SNPs from cluster1,12from cluster2, and2from cluster3. We typed all380serotype Xv isolates （177isolated from Henan,125from Gansu and78from Anhui） into5SNP genotypes （SGs）. SG1grouped together all isolates that did not belong to any of the3 clusters. SG2and SG5corresponded to genome clusters1and3, respectively. Cluster2was divided into SG3and SG4, because one of the cluster2-specific SNPs （SNP14） varied within cluster2and divided cluster2into2SGs. SG2and SG5are predominantly Henan and Anhui SGs, while SG3and SG4are predominantly Gansu SGs. Overall SG2is predominant of all isolates. Thus, the regional clustering seen for the genome-sequenced strains is confirmed with this large set of isolates. However, there is also a substantial interregional flow of strains. One SG predominated in each province, but substantial interregional spread of SGs was also evident. These findings suggest that Shigella epidemics in China were caused by a combination of local expansion and interregional spread of serotype Xv.The SRL and Tn7sequences of59S.flexneri isolates were compared with published genomes of2002017and YSH6000. All50ST91isolates,1ST109and1ST142isolate each carried the SRL and Tn7islands. Insertion or deletions on SRL islands were observed in9ST91isolates, as compared with2002017genome. Of which,7ST91isolates deleted tetracycline resistance tet genes;1ST91isolate deleted (3-lactamases oxa-1gene and chloramphenicol acetyltransferase cat gene and1ST91isolate had an insertion of β-lactamase bla TEM-1gene. No sequence variation was observed for Tn7islands for all draft genomes analyzed in this study.1ST18isolate carried only3resistant genes from both islands.1ST16isoalte does not contain the two MDR islands, but carried the fee gene, encoding ferric transport system present in YSH6000.In conclusion, whole-genome sequencing of59S. flexneri isolates revealed that, by acquiring MDR islands （SRL and Tn7） and quinolone resistance gyrA mutations, ST91arose in the early1990s, rapidly expanded, and spread across China within a decade. These findings suggest that MDR is the key selective pressure for the emergence of the S. flexneri epidemic clone. The acquisition of a novel plasmid by S. flexneri, leading to the emergence of a new serotype, occurred more than once. Both the pSFXv₂plasmid and the opt gene variation suggest independent gain of the plasmid by different clusters, with clear evidence that cluster3arose independently as an Xv serotype. There were3main clusters of serotype Xv isolates, which were localized in their respective regions, further supporting local origins of serotype Xv. However, there was also a substantial interregional flow of the clusters. In particular, cluster1, which was predominant in Henan and emerged earliest, spread to other regions with much greater proportions. Therefore, S, flexneri epidemics represent a dynamic play of local strains and interregional spread of different strains. All of S. flexneri ST91isolates analyzed in this study carried SRL and Tn7islands. Moreover, genetic variations such as deletions and insertion were observed on SRL island. The ST18might play an important role in acquiring and spreading of the two MDR islands, which was phylogenetically ancient as compared with ST91. Our results provide significant insights into the evolution of S. flexneri.