The two sporadic STEC O104:H4 strains 11–06681 and 11–07153 and the outbreak strain isolate 11–02027 show comparable levels of toxicity towards Vero cells.

<p>EHEC EDL933 served as a positive control and <i>E</i>. <i>coli</i> K12 C600 as a negative control. Toxicity of strain EDL933 as a quantitative reference was set to 100%. Shown are mean values of three independent experiments, each performed in triplicates. Bars represent means and standard deviation of three experiments. No significant difference in cytotoxicity of the STEC strains was observed.</p

Phylogeny of the 13 STEC O104:H4 isolates under investigation and of ten more STEC O104:H4 strains based on selected SNPs in their published genome sequences assignes the two German sporadic isolates 11–06681 and 11–07153 to a clade clearly separate from the one containing the 2011 outbreak strain isolates.

<p>The tree was calculated using the MrBayes tool from the GENEIOUS software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122074#pone.0122074.ref026" target="_blank">26</a>] and routed to the German historical isolate 01–09591 which served as outgroup. The sequences for EL-2071 and EL-2050, two sporadic isolates from cases in Georgia in 2009 and C-3493, an isolate obtained in the USA from a person with travelling history to Germany in May 2011 were published by Ahmed et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122074#pone.0122074.ref021" target="_blank">21</a>]. The sequences for Ec04-8351 and Ec09-7901, two historical strains from France and the sequences of Ec11-9450, Ec11-9941, Ec11-9990, Ec12-0456, Ec12-0466, five more recent sporadic isolates from France were published by Grad et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122074#pone.0122074.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122074#pone.0122074.ref023" target="_blank">23</a>]. The selected SNPs used for tree calculation are given in supplementary files <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122074#pone.0122074.s001" target="_blank">S1 Dataset</a> (SNP positions) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122074#pone.0122074.s002" target="_blank">S2 Dataset</a> (sequences).</p

Comparative Genomic Analysis of Two Novel Sporadic Shiga Toxin-Producing <i>Escherichia coli</i> O104:H4 Strains Isolated 2011 in Germany

<div><p>A large outbreak of gastrointestinal disease occurred in 2011 in Germany which resulted in almost 4000 patients with acute gastroenteritis or hemorrhagic colitis, 855 cases of a hemolytic uremic syndrome and 53 deaths. The pathogen was an uncommon, multiresistant <i>Escherichia coli</i> strain of serotype O104:H4 which expressed a Shiga toxin characteristic of enterohemorrhagic <i>E</i>. <i>coli</i> and in addition virulence factors common to enteroaggregative <i>E</i>. <i>coli</i>. During post-epidemic surveillance of Shiga toxin-producing <i>E</i>. <i>coli</i> (STEC) all but two of O104:H4 isolates were indistinguishable from the epidemic strain. Here we describe two novel STEC O104:H4 strains isolated in close spatiotemporal proximity to the outbreak which show a virulence gene panel, a Shiga toxin-mediated cytotoxicity towards Vero cells and aggregative adherence to Hep-2 cells comparable to the outbreak strain. They differ however both from the epidemic strain and from each other, by their antibiotic resistance phenotypes and some other features as determined by routine epidemiological subtyping methods. Whole genome sequencing of these two strains, of ten outbreak strain isolates originating from different time points of the outbreak and of one historical sporadic EHEC O104:H4 isolate was performed. Sequence analysis revealed a clear phylogenetic distance between the two variant strains and the outbreak strain finally identifying them as epidemiologically unrelated isolates from sporadic cases. These findings add to the knowledge about this emerging pathogen, illustrating a certain diversity within the bacterial core genome as well as loss and gain of accessory elements. Our results do also support the view that distinct new variants of STEC O104:H4 repeatedly might originate from yet unknown reservoirs, rather than that there would be a continuous diversification of a single epidemic strain established and circulating in Germany after the large outbreak in 2011.</p></div

The <i>Xba</i>I macrorestriction patterns of the two sporadic STEC O104:H4 strains 11–06681 and 11–07153 are distinct from outbreak strain isolates and from the historical strain.

<p>Lanes 1, 6, 9: molecular weight standard <i>Salmonella</i> serotype Braenderup strain H9812; Lane 2: historical O104:H4 isolate 01–09591; Lanes 3, 4, 5: independent isolates of the epidemic strain (11–02027, 11–02135, 11–06811); Lanes 7, 8: variant isolates 11–07153 and 11–06681.</p

The two sporadic STEC O104:H4 strains 11–06681 and 11–07153 and the outbreak strain isolate 11–02027 show aggregative adherence as characteristic for EAEC.

<p>Assay of mannose-resistant adherence to Hep-2 cells included the EHEC O157:H7 strain EDL933 for comparison. Images were taken at 600-fold magnification.</p