Improved traceability of Shiga-toxin-producing Escherichia coli using CRISPRs for detection and typing

Abstract

Among strains of Shiga-toxin-producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are frequently associated with severe clinical illness in humans. The development of methods for their reliable detection from complex samples such as food has been challenging thus far, and is currently based on the PCR detection of the major virulence genes stx1, stx2, and eae, and O-serogroup-specific genes. However, this approach lacks resolution. Moreover, new STEC serotypes are continuously emerging worldwide. For example, in May 2011, strains belonging to the hitherto rarely detected STEC serotype O104:H4 were identified as causative agents of one of the world's largest outbreak of disease with a high incidence of hemorrhagic colitis and hemolytic uremic syndrome in the infected patients. Discriminant typing of pathogens is crucial for epidemiological surveillance and investigations of outbreaks, and especially for tracking and tracing in case of accidental and deliberate contamination of food and water samples. Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of short, highly conserved DNA repeats separated by unique sequences of similar length. This distinctive sequence signature of CRISPRs can be used for strain typing in several bacterial species including STEC. This review discusses how CRISPRs have recently been used for STEC identification and typing.

Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection

“… Delannoy et al (2013a , b , 2016b ) have already demonstrated that individual combinations of an extended set of known markers beyond the classical STEC marker genes allow the identification of STEC serotypes (O157:H7, O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and their non-motile derivatives), which are most frequently implicated in outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Additionally they showed that clustered regularly interspaced short palindromic repeat (CRISPR) regions can be used to discriminate the same serotypes as well as O104:H4 ( Delannoy et al, 2012a , b , 2016a ). Furthermore, Wong et al (2014) identified a novel O157:H7-specific marker in a genome wide insertion/deletion-based approach.…”

CRISPR Visualizer: rapid identification and visualization of CRISPR loci via an automated high-throughput processing pipeline

“…The industrial relevance of CRISPR-based genotyping has been demonstrated in several bacterial species widely found in the food supply chain, encompassing starter cultures and food spoilage organisms such as Streptococcus thermophilus [14], Bifidobacterium longum [15], and Lactobacillus buchneri [16]. CRISPR-based genotyping also exhibits significant medical relevance through its usage in identification of pathogens such as Mycobacterium tuberculosis [17,18], Salmonella enterica [19,20], Clostridium difficile [21], Escherichia coli and many others [22,23], including food-borne pathogens. Characterization of CRISPR-Cas systems has also revealed alternative functions such as roles in virulence regulation and DNA repair [24].…”