Use of Clustered Regularly Interspaced Short Palindromic Repeat Sequence Polymorphisms for Specific Detection of Enterohemorrhagic Escherichia coli Strains of Serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by Real-Time PCR

Abstract

bWe explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of enterohemorrhagic Escherichia coli (EHEC) to design simplex real-time PCR assays for each of the seven most important EHEC serotypes worldwide. A panel of 958 E. coli strains investigated for their CRISPR loci by high-throughput real-time PCR showed that CRISPR polymorphisms in E. coli strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx and eae genes). The CRISPR sequences chosen for simplex real-time PCR amplification of EHEC strains belonging to the top 7 EHEC serogroups differentiated clearly between EHEC and non-EHEC strains. Specificity estimates for the CRISPR PCR assays varied from 97.5% to 100%. Sensitivity estimates for the assays ranged from 95.7% to 100%. The assays targeting EHEC O145: H28, O103:H2, and O45:H2 displayed 100% sensitivity. The combined usage of two simplex PCR assays targeting different sequences of the O26 CRISPR locus allowed detection of EHEC O26:H11 with 100% sensitivity. By combining two simplex PCR assays targeting different sequences of the EHEC O157 CRISPR locus, EHEC O157:H7 was detected with 99.56% sensitivity. EHEC O111:H8 and EHEC O121:H19 were detected with 95.9% and 95.7% sensitivity, respectively. This study demonstrates that the identification of EHEC serotype-specific CRISPR sequences is more specific than the mere identification of O-antigen gene sequences, as is used in current PCR protocols for detection of EHEC strains. Shiga toxin-producing Escherichia coli (STEC) strains are a diverse group of E. coli strains belonging to over 400 E. coli O:H serotypes, some of which cause outbreaks and sporadic cases of food-borne illnesses ranging from diarrhea to hemorrhagic colitis (HC) and the hemolytic-uremic syndrome (HUS) (11,15,16). According to their pathogenicity for humans, the latter strains were also designated enterohemorrhagic E. coli (EHEC) (17, 18). Numerous cases of HC and HUS have been attributed to EHEC O157:H7 strains (25), but it has now been recognized that other STEC serotypes belong to the EHEC group (8, 24). Cumulative evidence from numerous countries indicates that up to 30 to 60% of human EHEC infections are caused by non-O157 EHEC strains (7). There are seven "priority" EHEC serotypes most frequently implicated in outbreaks and sporadic cases of HC and HUS (8, 24). These comprise serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 and their nonmotile derivatives.Although regulations are disparate throughout the world, many food inspection programs aim at detecting STEC strains that pose a significant threat to human health in foods that are the most likely to disseminate EHEC and to be consumed raw or undercooked. Some beef products are thus of particular interest in that aspect. The U.S. regulations have precisely been revised to add 6 additional serogroups (O26, O103, O45, O111, O121, and O145) to the existing O157:H7 regulation (22,23). This regulation imposes testin...

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

“…Interestingly, the CRISPR polymorphisms were found to provide a more specific typing profile than the established techniques, which were based on stx and eae gene polymorphisms alone or together with O:H serotypes (52). This suggests that a significant correlation exists between CRISPR genotypes and an isolate's virulence.…”

“…For enterohemorrhagic E. coli (EHEC) bacteria, (type I-E/I-F) CRISPR polymorphisms were found to correlate with the presence of two EHEC virulence genes, stx and eae, encoding the phage-delivered Shiga toxin and the intimin virulence factor, respectively (52). Interestingly, the CRISPR polymorphisms were found to provide a more specific typing profile than the established techniques, which were based on stx and eae gene polymorphisms alone or together with O:H serotypes (52).…”

CRISPR– cas Loci Profiling of Cronobacter Sakazakii Pathovars

“…Subtyping protocols based on CRISPR-cas systems have been proposed for Salmonella and these have included combined analysis with multi-virulence-locus sequence typing and PFGE [28][29][30]. In contrast, CRISPR typing cannot be used for all E. coli strains as it is absent from the extra intestinal phylogenetic group B2 but can be used for specific identification of enterohemorrhagic and Shiga toxin producing E. coli serotypes [27,31,32]. This could be linked to the absence of evidence for the type I-E CRISPR-cas system having a role in adaptive immunity in E. coli, and instead is proposed to be involved in different functions such as the regulation of endogenous gene expression and possible links to virulence [33,34].…”

Subtyping of Salmonella enterica Serovar Newport Outbreak Isolates by CRISPR-MVLST and Determination of the Relationship between CRISPR-MVLST and PFGE Results

“…Due to both acquisition and loss of these spacer elements, CRISPRs arguably represent the most rapidly evolving prokaryotic loci (19)(20)(21). Although originally used for spacer oligonucleotide typing, or spoligotyping, for Mycobacterium tuberculosis (22,23), we and others have successfully exploited CRISPR spacer sequence differences for subtyping several different pathogens, including group A Streptococcus (24), Campylobacter species (25), Salmonella (15,(26)(27)(28), and Shiga toxin-producing Escherichia coli (29,30). In Salmonella, CRISPR spacer compositions are highly conserved at the serovar level, suggesting that CRISPR sequence typing alone may be sufficient to detect, identify, and distinguish serotypes (27).…”