Methods for Genotyping Verotoxin‐Producing Escherichia coli

Abstract

Verotoxin-producing Escherichia coli (VTEC) is annually incriminated in more than 100,000 cases of enteric foodborne human disease and in losses amounting to $US 2.5 billion every year. A number of genotyping methods have been developed to track VTEC infections and determine diversity and evolutionary relationships among these microorganisms. These methods have facilitated monitoring and surveillance of foodborne VTEC outbreaks and early identification of outbreaks or clusters of outbreaks. Pulsed-field gel electrophoresis (PFGE) has been used extensively to track and differentiate VTEC because of its high discriminatory power, reproducibility and ease of standardization. Multiple-locus variable-number tandem-repeats analysis (MLVA) and microarrays are the latest genotyping methods that have been applied to discriminate VTEC. MLVA, a simpler and less expensive method, is proving to have a discriminatory power comparable to that of PFGE. Microarrays are successfully being applied to differentiate VTEC and make inferences on genome diversification. Novel methods that are being evaluated for subtyping VTEC include the detection of single nucleotide polymorphisms and optical mapping. This review discusses the principles, applications, advantages and disadvantages of genotyping methods that have been used to differentiate VTEC strains. These methods have been mainly used to differentiate strains of O157:H7 VTEC and to a lesser extent non-O157 VTEC.

Molecular Risk Assessment and Epidemiological Typing of Shiga Toxin-Producing Escherichia coli by Using a Novel PCR Binary Typing System

“…Phenotypic methods for typing STEC, such as serotyping and phage typing, offer limited applicability, as few laboratories are equipped to carry out the full range of O-and H-antigen serotyping and phage typing has not yet been developed for non-O157 STEC (28). Octamer-based genome scanning (OBGS), lineagespecific polymorphism assay (LSPA), single nucleotide polymorphisms (SNPs), and microarray analysis are molecular methods with the potential for high discriminatory power; however, protocols have not been developed for application of these methods to non-O157 STEC, and expensive sequencing or microarray scanning equipment is required (28).…”

“…Octamer-based genome scanning (OBGS), lineagespecific polymorphism assay (LSPA), single nucleotide polymorphisms (SNPs), and microarray analysis are molecular methods with the potential for high discriminatory power; however, protocols have not been developed for application of these methods to non-O157 STEC, and expensive sequencing or microarray scanning equipment is required (28). Random amplification of polymorphic DNA (RAPD) typing is a simple and inexpensive PCR-based method that has been developed for both O157 and (28).…”

“…Octamer-based genome scanning (OBGS), lineagespecific polymorphism assay (LSPA), single nucleotide polymorphisms (SNPs), and microarray analysis are molecular methods with the potential for high discriminatory power; however, protocols have not been developed for application of these methods to non-O157 STEC, and expensive sequencing or microarray scanning equipment is required (28). Random amplification of polymorphic DNA (RAPD) typing is a simple and inexpensive PCR-based method that has been developed for both O157 and (28). Pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) are highly discriminatory typing methods that are suitable for subtyping O157 and non-O157 STEC; however, these methods are labor-intensive and require expensive equipment and software to facilitate comparisons of strain types (28).…”

A precise reconstruction of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone concatenomic analysis

“…Pulsed field gel electrophoresis, the standard technique (20), is hindered by genomic alterations (11,21), and theoretic and technical challenges (22). Known SNPs can differentiate pathogens, but confidence that 2 isolates of the same SNP type are from the same source is directly proportional to the amount of identical de novo-generated sequence between them.…”

Precise Dissection of an Escherichia coli O157:H7 Outbreak by Single Nucleotide Polymorphism Analysis

“…The typing is usually performed in public health laboratories where the endonuclease-cleaved bacterial DNA is separated using pulsed-field gel electrophoresis (PFGE) (2) to produce isolate-specific patterns. The retrospective determination that Escherichia coli O157:H7 isolates from different individuals in a 1993 outbreak had identical PFGE patterns (3) prompted adoption of this technique in North America and elsewhere (2)(3)(4)(5). While PFGE is widely used as a tool in outbreak management, its limitations are emerging.…”