Enterohemorrhagic Escherichia coli as Causes of Hemolytic Uremic Syndrome in the Czech Republic

Background: Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea-associated hemolytic uremic syndrome (D+ HUS) worldwide, but no systematic study of EHEC as the causative agents of HUS was performed in the Czech Republic. We analyzed stools of all patients with D+ HUS in the Czech Republic between 1998 and 2012 for evidence of EHEC infection. We determined virulence profiles, phenotypes, antimicrobial susceptibilities and phylogeny of the EHEC isolates. Methodology/Principal Findings: Virulence loci were identified using PCR, phenotypes and antimicrobial susceptibilities were determined using standard procedures, and phylogeny was assessed using multilocus sequence typing. During the 15-year period, EHEC were isolated from stools of 39 (69.4%) of 56 patients. The strains belonged to serotypes [fliC types] O157:H7/NM[fliCH7] (50% of which were sorbitol-fermenting; SF), O26:H11/NM[fliCH11], O55:NM[fliCH7], O111:NM[fliCH8], O145:H28[fliCH28], O172:NM[fliCH25], and Orough:NM[fliCH25]. O26:H11/NM[fliCH11] was the most common serotype associated with HUS (41% isolates). Five stx genotypes were identified, the most frequent being stx2a (71.1% isolates). Most strains contained EHEC-hlyA encoding EHEC hemolysin, and a subset (all SF O157:NM and one O157:H7) harbored cdt-V encoding cytolethal distending toxin. espPα encoding serine protease EspPα was found in EHEC O157:H7, O26:H11/NM, and O145:H28, whereas O172:NM and Orough:NM strains contained espPγ. All isolates contained eae encoding adhesin intimin, which belonged to subtypes β (O26), γ (O55, O145, O157), γ2/θ (O111), and ε (O172, Orough). Loci encoding other adhesins (efa1, lpfAO26, lpfAO157OI-141, lpfAO157OI-154, iha) were usually associated with particular serotypes. Phylogenetic analysis demonstrated nine sequence types (STs) which correlated with serotypes. Of these, two STs (ST660 and ST1595) were not found in HUS-associated EHEC before. Conclusions/Significance: EHEC strains, including O157:H7 and non-O157:H7, are frequent causes of D+ HUS in the Czech Republic. Identification of unusual EHEC serotypes/STs causing HUS calls for establishment of an European collection of HUS-associated EHEC, enabling to study properties and evolution of these important pathogens

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

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 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

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

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

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