Fach P. Specific detection of enteroaggregative hemorrhagic Escherichia coli O104:H4 strains by use of the CRISPR locus as a target for a diagnostic real-time PCR

In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an en-teroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coliO104:H4 (CRISPRO104:H4) for specific detection of EAEC STECO104:H4 strains from different geographical locations and time periods. The specificity of the CRISPRO104:H4 PCR was investigated using 1,321 E. coli strains, including reference strains for E. coliO serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting dif-ferent O104 antigen-encoding genes (wbwCO104,wzxO104, andwzyO104). The PCR assays reacted with all types of E. coliO104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coliO8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STECO104:H4 type. A single PCR developed for the CRISPRO104:H4 target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coliO104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPRO104:H4 locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174: H2) reacted positive in the CRISPRO104:H4 PCR (99.06 % specificity). More than 400 serotypes of Shiga toxin (Stx)-producingEscherichia coli (STEC) strains have been described as agents of disease in humans, and some of these have bee