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 enteroaggregativeEscherichia coli (EAEC) and a Shiga toxin-producingE. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting thec lusteredr egularlyi nterspaceds hortp alindromicr epeats locus ofE. coli O104:H4 (CRISPRO104:H4 ) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPRO104:H4 PCR was investigated using 1,321E. coli strains, including reference strains forE. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC O104 ,wzx O104 , andwzy O104 ). The PCR assays reacted with all types ofE. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and withE. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104: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 35E. coli O104 strains expressing H types other than H4 as well as 8E. 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:H4E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPRO104:H4 PCR (99.06% specificity).