Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany

Abstract

Background: A large outbreak of diarrhea and the hemolytic-uremic syndrome caused by an unusual serotype of Shiga-toxin-producing Escherichia coli (O104:H4) began in Germany in May 2011. As of July 22, a large number of cases of diarrhea caused by Shiga-toxin-producing E. coli have been reported--3167 without the hemolytic-uremic syndrome (16 deaths) and 908 with the hemolytic-uremic syndrome (34 deaths)--indicating that this strain is notably more virulent than most of the Shiga-toxin-producing E. coli strains. Preliminary genetic characterization of the outbreak strain suggested that, unlike most of these strains, it should be classified within the enteroaggregative pathotype of E. coli.

Methods: We used third-generation, single-molecule, real-time DNA sequencing to determine the complete genome sequence of the German outbreak strain, as well as the genome sequences of seven diarrhea-associated enteroaggregative E. coli serotype O104:H4 strains from Africa and four enteroaggregative E. coli reference strains belonging to other serotypes. Genomewide comparisons were performed with the use of these enteroaggregative E. coli genomes, as well as those of 40 previously sequenced E. coli isolates.

Results: The enteroaggregative E. coli O104:H4 strains are closely related and form a distinct clade among E. coli and enteroaggregative E. coli strains. However, the genome of the German outbreak strain can be distinguished from those of other O104:H4 strains because it contains a prophage encoding Shiga toxin 2 and a distinct set of additional virulence and antibiotic-resistance factors.

Conclusions: Our findings suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak. More broadly, these findings highlight the way in which the plasticity of bacterial genomes facilitates the emergence of new pathogens.

Figure 1. Comparison of Genomes of Eight Enteroaggregative Escherichia coli O104:H4 Isolates

In Panel A, the eight circular bands represent the tracks for the different enteroaggregative E. coli (EAEC) O104:H4 isolates sequenced in our study (from inner to outer tracks: 55989, C227-11, C734-09, C35-10, C682-09, C760-09, C754-09, and C777-09). These different bands represent coverage plots mapped against the reference genome TY2482, with blue or green indicating a high degree of coverage, yellow moderate coverage, and orange or red little or no coverage. The C227-11 coverage plot indicates near-complete sequence identity with the TY2482 genome. The annotations on the outside of the chromosome plot highlight positions of known virulence-factor genes and genes previously characterized on the AA plasmid in the E. coli O42 strain. The band ordering and color coding for the plasmids are the same as those described for the chromosome plot. The thin red lines in the pAA plot show internal repeats between 55989 and C227-11 that highlight shuffling of these elements around the plasmid sequence. In Panel B, the reference genome is 55989. Panel C shows a comparison between the genome of the outbreak strain C227-11 (left semicircle) and the 55989 genome (right semicircle), mapped against the 55989 genome and the genome of the outbreak strain TY2482, respectively. The colored ribbons inside the innermost track (blue, pink, and purple) represent homologous mappings between the two genomes, C227-11 and 55989, indicating a high degree of similarity between these genomes. The red lines show the chromosomal positioning of repeat elements, such as insertion sequences and other mobile elements, which reveal more heterogeneity between the genomes than is evident from mapping regions of homology.

Figure 2. Phylogenetic Comparisons of 53 Escherichia coli and Shigella Isolates

Genomic sequences were compared with the use of 100 bootstrap calculations, as described by Sahl et al. The species-based phylogeny was inferred with the use of 2.56 Mbp of the conserved core genome. The O104:H4 isolates are shown in orange, the reference enteroaggregative E. coli (EAEC) isolates in blue, and the enterohemorrhagic E. coli isolates in green. (The classification of the other strains is shown in Fig. 4 and Table 4 in the Supplementary Appendix.) The O104:H4 isolates cluster into a single clade (dark gray); in contrast, the reference EAEC isolates are extremely divergent and are represented throughout the phylogeny.