A novel Escherichia coli O157:H7 clone causing a major hemolytic uremic syndrome outbreak in China

Abstract

An Escherichia coli O157:H7 outbreak in China in 1999 caused 177 deaths due to hemolytic uremic syndrome. Sixteen outbreak associated isolates were found to belong to a new clone, sequence type 96 (ST96), based on multilocus sequence typing of 15 housekeeping genes. Whole genome sequencing of an outbreak isolate, Xuzhou21, showed that the isolate is phylogenetically closely related to the Japan 1996 outbreak isolate Sakai, both of which share the most recent common ancestor with the US outbreak isolate EDL933. The levels of IL-6 and IL-8 of peripheral blood mononuclear cells induced by Xuzhou21 and Sakai were significantly higher than that induced by EDL933. Xuzhou21 also induced a significantly higher level of IL-8 than Sakai while both induced similar levels of IL-6. The expression level of Shiga toxin 2 in Xuzhou21 induced by mitomycin C was 68.6 times of that under non-inducing conditions, twice of that induced in Sakai (32.7 times) and 15 times higher than that induced in EDL933 (4.5 times). Our study shows that ST96 is a novel clone and provided significant new insights into the evolution of virulence of E. coli O157:H7.

Figure 1. Geographic distribution of HUS cases of the 1999 outbreak in Jiangsu and neighbouring Anhui province.

Figure 2. Genetic relationships of O157:H7 isolates.

(A): eBURST analysis of the O157:H7 sequence types. Majority of the STs belong to two clonal complexes which was defined based on one out 15 genes being different. Names after ST in brackets are strain names. The recently sequenced strains and their ST assignments are as follows: TW14588: ST23; EC4009, EC4127, EC4191, EC4401, EC4045, EC4076, EC869, 1125, FRIK2000, FRIK966: ST24; EC536: ST97; EC4486: ST110; EC4042: ST111; and LSU-61: ST112. (B). Genomic relationships of Xuzhou21 with other O157:H7 strains. The evolutionary relationship of the four genome sequenced strains of O157:H7 were determined using mutational SNPs. Allocation of SNPs to specific lineages were done using E. coli O55:H7 CB9615 as an outgroup . Changes were shown in the boxes along the branches. Rec: recombination; sSNP: synonymous SNPs; nsSNP: non-synonymous SNPs. The lineages were defined based on the LSPA6 lineages , and clades were based on Manning et al. SNP typing . (C): Genetic changes and relationships of the pO157 plasmids from the four strains. The bar at the top indicates coding genes in alternative colour with those transcribed in reverse direction pointing downwards. The key genes are marked above the bar. Changes in each plasmid are marked on branches on the left. Ins, del, nc, s, ns and large del denote single base insertion, single base deletion, non- coding SNP, sSNP, nsSNP, and large deletion respectively. The colours for the vertical lines on the grey strips are red for insertion, green for deletion and blue for other single base changes with shorter line for synonymous changes. The scale is in kb.

Figure 3. Comparison of the expressions of cytokines and stx2 among Xuzhou21, Sakai and EDL933.

(A): The levels of cytokines (IL-6 and IL-8) of peripheral blood mononuclear cells response to 10⁷ heat-inactivated O157:H7 after four-hour incubation. All data were from three independent experiments. Differences were analyzed for significance by using the least significant difference (LSD) test following a significant F-test. Significant values relative to EDL933 (P<0.01) are indicated by a *. (B): Expression of stx2. The relative levels of expression under non-induction and induction conditions were relative to the expression level in EDL933 before induction by mitomycin C which was arbitrary set at 1.0. The relative value was averaged from three experiments. Error bars represent the standard errors. Differences in induction conditions were analyzed for significance by using the least significant difference (LSD) test following a significant F-test. Statistical significance for comparison of a given value with the value for EDL933 (non-induction condition) (P<0.01) is indicated by a *.