Presence of OI 122 among EHEC isolates from HUS patients.

a<p><i>pagC</i> was present in one strain.</p>b<p>C, complete OI 122 (all genes tested present); I, incomplete OI 122 (<i>pagC</i> absent).</p

Phylogeny of EHEC associated with HUS in the Czech Republic.

<p>Minimum-spanning tree illustrating the clonal relationship between HUS-associated EHEC from the Czech Republic (green) and the HUSEC collection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073927#pone.0073927-Mellmann1" target="_blank">[3]</a> (red) based on MLST allelic profiles. Each MLST sequence type (ST) is represented by a node named with its ST. The size of the node is proportional to the number of isolates reported in this study sharing the same ST. The number on the connecting lines indicates the number of alleles that were different between the two connected nodes. In addition, for the major serogroups (e.g. O157, O26) the STs and their corresponding clonal complexes (CC) were given and shaded in grey.</p

Phylogeny of EHEC isolated from HUS patients in the Czech Republic determined by MLST.

<p>ST, sequence type; CC, clonal complex; n.a., not assigned.</p>a<p>ST29 strains belong to the new EHEC O26 clone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073927#pone.0073927-Bielaszewska1" target="_blank">[7]</a>.</p

Phenotypes of EHEC strains isolated from patients with HUS in the Czech Republic.

a<p>EHEC-Hly, EHEC hemolysin production; α-Hly, α hemolysin production; growth on CT-SMAC, indicator of tellurite resistance; urease, urease production; SMAC, utilization of sorbitol on sorbitol MacConkey agar; SOR, utilization of sorbitol (API 20E); RHA, utilization of rhamnose (API 20E); LDC, production of lysine decarboxylase; GLR, production of ß-D-glucuronidase.</p>b<p>The highest dilution of culture supernatant which caused cytotoxicity in 50% Vero cells after 3 days.</p>c<p>Production of Stx1 and Stx2 tested using the VTEC - RPLA kit.</p>d<p>−, the phenotype was absent; +, the phenotype was present (the numbers in parentheses indicated numbers of positive strains in the case that not all strains expressed the respective phenotype).</p>e<p>one O26:H11 and O172:NM and Orough:NM strains did not express EHEC-<i>hlyA</i> gene.</p

Seasonal distribution of EHEC strains of different serotypes isolated from patients with HUS in the Czech Republic, 1998–2012.

<p>Seasonal distribution of EHEC strains of different serotypes isolated from patients with HUS in the Czech Republic, 1998–2012.</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>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

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

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

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