Characteristics of the novel EHEC/EAEC strains analyzed in the study compared to the O59:H⁻ EAEC and the O104:H4 2011 outbreak strain.

<p>Characteristics of the novel EHEC/EAEC strains analyzed in the study compared to the O59:H⁻ EAEC and the O104:H4 2011 outbreak strain.</p

Plasmid profile of the two novel EHEC/EAEC strains and identification of the pAA plasmid.

<p>(<b>A</b>) EHEC/EAEC 12-05829 (Orough:H⁻), EAEC 11-08343 (O59:H⁻), EHEC/EAEC 10-06235 (O59:H⁻), EHEC/EAEC 11-02027 (O104:H4 outbreak 2011), and EHEC EDL933 (O157:H7) were analyzed for their plasmid profile. <i>E. coli</i> reference strain 39R861 plasmids served as molecular mass standard. (<b>B</b>) Southern hybridization with plasmid DNA of the same strains as mentioned in (A) was performed using a digoxigenin-labelled <i>aatA</i> gene probe. *EHEC/EAEC; ?pAA plasmid.</p

Identification of two novel EHEC/EAEC strains.

<p>(<b>A</b>) Duplex <i>stx1/2</i> PCR analysis (left side of panel) and single <i>aatA</i> PCR analysis (right side of panel) of EHEC EDL933, known EHEC/EAEC 11-02027 (O104:H4 outbreak 2011), and the two novel EHEC/EAEC: 10-06235 (O59:H⁻) and 12-05829 (Orough:H⁻). (<b>B</b>) Novel Triplex PCR analysis for simultaneous detection of <i>stx1/2</i> and <i>aatA</i> in different EHEC, EHEC/EAEC, and EAEC strains. S = bp standard; *EHEC/EAEC.</p

Toxicity of the two novel EHEC/EAEC strains towards Vero cells.

<p>As a positive control EHEC EDL933 and as negative controls <i>E. coli</i> K12c600 and EAEC 042 were incubated with Vero cells. Further EHEC/EAEC 11-02027 (O104:H4 outbreak 2011), EHEC/EAEC 10-06235 (O59:H⁻), EHEC/EAEC 12-05829 (Orough:H⁻), and EAEC 11-08343 (O59:H⁻) were analyzed. Toxicity of EHEC EDL933 served as a quantitative reference and was set to 100%. Bars represent means and standard deviations of triplicate samples and 256-fold diluted culture preparations. Asterisks indicate significantly lower cytotoxicity compared to the EDL933 reference strain, however all EHEC/EAEC strains were significantly more cytotoxic then the <i>E. coli</i> K12 control, the EAEC 042, as well as EAEC 11-08343 (O59:H⁻) strains (two tailed student’s t-test type 1, p<0.01; p<0.03 for EHEC/EAEC 12-05829 compared to <i>E. coli</i> K12).</p

Pulsed-field gel analysis after XbaI macrorestriction shows that the EHEC/EAEC O59:H⁻ (upper lane) and EAEC O59:H⁻ (lower lane) are not closely related.

<p>Pulsed-field gel analysis after XbaI macrorestriction shows that the EHEC/EAEC O59:H⁻ (upper lane) and EAEC O59:H⁻ (lower lane) are not closely related.</p

The novel O59:H⁻ EHEC/EAEC codes for AAF/IV fimbriae and both novel EHEC/EAEC show aggregative adherence.

<p>(<b>A</b>) PCR-based determination of AAF type I, III, and IV genes of the EHEC/EAEC O59:H⁻ strain (3) compared to two known EHEC/EAEC O104:H4 (1 - outbreak 2011 and 2 - HUSEC 041) strains. (<b>B</b>) PCR-based determination of AAF/V-coding gene <i>aaf5A</i> of different EHEC/EAEC strains and an O111:H21 EAEC strain as positive control. (<b>C</b>) Aggregative adherence of the two novel EHEC/EAEC strains to Hep-2 cells. The controls EHEC EDL933 (O157:H7) and <i>E. coli</i> K12c600 without aggregative adherence and the aggregative adherence reference strains EAEC 042 (O44:H18), EHEC/EAEC 11-02027 (O104:H4 outbreak 2011) were analyzed in addition to the EHEC/EAEC 10-06235 (O59:H⁻), EHEC/EAEC 12-05829 (Orough:H⁻), and the EAEC 11-08343 (O59:H⁻) strains. Images were taken at 600-fold magnification. S = bp standard; *EHEC/EAEC.</p

Characteristics of EHEC/EAEC, EAEC, and EHEC strains analyzed in the study.

<p>MLST sequence types were assigned or cited according to the MLST database at the University College Cork, Ireland <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095379#pone.0095379-Wirth1" target="_blank">[46]</a>. Abbreviations: Bd = bloody diarrhea, Hc = hemorrhagic colitis, n.d. = not determined, n.a. = not applicable, AMP = ampicillin, CTM = Cefotiam, MEZ = mezlocillin, MSU = mezlocillin/sulbactam, OTE = oxytetracyclin, SMZ = sulfamethoxazol, STR = streptomycin, SXT = trimethoprim/sulfamethoxazol, CAZ = ceftazidim, CTX = cefotaxim, and NAL = nalidixic acid.</p><p>*<b>-</b>pAA plasmid.</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

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

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