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 enteroaggregative 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. coli O104:H4 (CRISPR(O104:H4)) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR(O104:H4) PCR was investigated using 1,321 E. coli strains, including reference strains for E. 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), and wzy(O104)). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. 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 CRISPR(O104:H4) target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR(O104: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 CRISPR(O104:H4) PCR (99.06% specificity).
French Agency for Food, Environmental and Occupational Health and Safety, Food Safety Laboratory, Maisons-Alfort, France.
Sabine
S
Delannoy
Lothar
L
Beutin
Ylanna
Y
Burgos
Patrick
P
Fach
D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis*; D005060:Europe; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial