Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

Murase, Toshiyuki; Yamai, Shiro; Watanabe, Haruo

doi:10.1007/pl00006771

Cited by 44 publications

(40 citation statements)

References 20 publications

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“…However, challenges in using PFGE include the fact that it is a labor-intensive and time-consuming technique that requires expensive instrumentation and software to interpret the complex electrophoretic patterns generated, and there are several factors inherent to the protocol itself that influence the patterns generated and lead to misclassification of strains (12,15,23). Some of the factors influencing the electrophoretic patterns of DNA in PFGE are improper storage and repeated subculturing of the isolates used to prepare the genomic DNA, which can cause spontaneous loss of plasmids or other recombinational events that alter DNA profiles; nuclease-related or electrophoresis-related degradation of DNA; methylation of DNA, causing incomplete restriction digestion of DNA; inactivation of restriction enzymes by reagents used to prepare DNA; improper resolution of smaller or larger DNA fragments, depending on the size limits of gel conditions; comigration of similar-sized DNA fragments; and nonhomologous DNA migrating as same-size bands, resulting in untypeable or incorrect profiles (3,6,23). In fact, PFGE fails to resolve all fragments generated after restriction digestion of the DNA on the gel under a single set of conditions, and the number of bands visualized does not match the number of sites for the restriction enzyme used to digest the DNA (3,4,6,12).…”

Section: Discussionmentioning

confidence: 99%

“…However, over time, certain drawbacks of PFGE have become more evident, requiring researchers to implement several modifications of the technique in order to increase sample turnaround, overcome inhibition of restriction enzymes or degradation of DNA, and minimize changes in electrophoretic patterns of DNA between gels to avoid reliance on sophisticated pattern recognition computer software to interpret the patterns generated (3,12,15,23,29). The ability of PFGE to accurately assess the genetic relatedness of O157 isolates in outbreaks was recently assessed (6).…”

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confidence: 99%

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Insertions, Deletions, and Single-Nucleotide Polymorphisms at Rare Restriction Enzyme Sites Enhance Discriminatory Power of Polymorphic Amplified Typing Sequences, a Novel Strain Typing System for Escherichia coli O157:H7

et al. 2004

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Polymorphic amplified typing sequences (PATS) for Escherichia coli O157:H7 (O157) was previously based on indels containing XbaI restriction enzyme sites occurring in O-island sequences of the O157 genome. This strain-typing system, referred to as XbaI-based PATS, typed every O157 isolate tested in a reproducible, rapid, straightforward, and easy-to-interpret manner and had technical advantages over pulsed-field gel electrophoresis (PFGE). However, the system was less discriminatory than PFGE and was unable to differentiate fully between unrelated isolates. To overcome this drawback, we enhanced PATS by using another infrequently cutting restriction enzyme, AvrII (also known as BlnI), to identify additional polymorphic regions that could increase the discriminatory ability of PATS typing. Referred to as AvrII-based PATS, the system identified seven new polymorphic regions in the O157 genome. Unlike XbaI, polymorphisms involving AvrII sites were caused by both indels and single-nucleotide polymorphisms occurring in O-island and backbone sequences of the O157 genome. AvrII-based PATS by itself provided poor discrimination of the O157 isolates tested. However, when primer pairs amplifying the seven polymorphic AvrII sites were combined with those amplifying the eight polymorphic XbaI sites (combined PATS), the discriminatory power of PATS was enhanced. Combined PATS matched related O157 isolates better than PFGE while differentiating between unrelated isolates. PATS typed every O157 isolate tested and directly targeted polymorphic sequences responsible for differences in the restriction digest patterns of O157 genomic DNA, utilizing PCR rather than relying on gel electrophoresis. This enabled PATS to resolve the ambiguity in PFGE typing, including that arising from the "more distantly related" and "untypeable" profiles.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Insertions, Deletions, and Single-Nucleotide Polymorphisms at Rare Restriction Enzyme Sites Enhance Discriminatory Power of Polymorphic Amplified Typing Sequences, a Novel Strain Typing System for Escherichia coli O157:H7

et al. 2004

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“…For these two reasons, isolates might appear identical when they are not. Conversely, PFGE patterns can change during subculturing, presumably because of the mobilization of elements of the genome such as bacteriophages (7,8). Such alterations can also occur in the course of human infections (9,10).…”

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

et al. 2013

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The current pathogen-typing methods have suboptimal sensitivities and specificities. DNA sequencing offers an opportunity to type pathogens with greater degrees of discrimination using single nucleotide polymorphisms (SNPs) than with pulsed-field gel electrophoresis (PFGE) and other methodologies. In a recent cluster of Escherichia coli O157:H7 infections attributed to salad bar exposures and romaine lettuce, a subset of cases denied exposure to either source, although PFGE and multiple-locus variable-number tandem-repeat analysis (MLVA) suggested that all isolates had the same recent progenitor. Interrogation of a preselected set of 3,442,673 nucleotides in backbone open reading frames (ORFs) identified only 1 or 2 single nucleotide differences in 3 of 12 isolates from the cases who denied exposure. The backbone DNAs of 9 of 9 and 3 of 3 cases who reported or were unsure about exposure, respectively, were isogenic. Backbone ORF SNP set sequencing offers pathogen differentiation capabilities that exceed those of PFGE and MLVA.

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“…The distinctive profiles generated enable differentiation of strains in a reproducible manner. Not all strains are typeable by PFGE, however, because of methylation of restriction sites, degradation of DNA in agarose plugs, or other technical problems (7,8,11). The most important drawback of PFGE, however, is that the comparison of results for isolates analyzed at different locations or times (and hence on different gels) requires sophisticated pattern-recognition computer software (12).…”

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Polymorphic Amplified Typing Sequences Provide a Novel Approach to Escherichia coli O157:H7 Strain Typing

et al. 2002

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Escherichia coli O157:H7 (O157) strains are commonly typed by pulsed-field gel electrophoresis (PFGE) following digestion of genomic DNA with the restriction enzyme XbaI. We have shown that O157 strains differ from each other by a series of discrete insertions or deletions, some of which contain recognition sites for XbaI, suggesting that these insertions and deletions are responsible for the differences in PFGE patterns. We have devised a new O157 strain typing protocol, polymorphic amplified typing sequences (PATS), based on this information. We designed PCR primer pairs to amplify genomic DNA flanking each of 40 individual XbaI sites in the genomes of two O157 reference strains. These primer pairs were tested with 44 O157 isolates, 2 each from 22 different outbreaks of infection. Thirty-two primer pairs amplified identical fragments from all 44 isolates, while eight primer pairs amplified regions that were polymorphic between isolates. The isolates could be differentiated solely on the basis of which of the eight polymorphic amplicons was detected. PATS correctly identified 21 of 22 outbreak pairs as being identical or highly related, whereas PFGE correctly identified 14 of the 22 outbreak pairs as being identical or highly related; PATS was also able to type isolates from three outbreaks that were untypeable by PFGE. However, PATS was less sensitive than PFGE in discriminating between outbreaks. These data suggest that typing by PATS may provide a simple procedure for strain typing of O157 and other bacteria and that further evaluation of the utility of this method for epidemiologic investigations is warranted.

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Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

Cited by 44 publications

References 20 publications

Insertions, Deletions, and Single-Nucleotide Polymorphisms at Rare Restriction Enzyme Sites Enhance Discriminatory Power of Polymorphic Amplified Typing Sequences, a Novel Strain Typing System for Escherichia coli O157:H7

Insertions, Deletions, and Single-Nucleotide Polymorphisms at Rare Restriction Enzyme Sites Enhance Discriminatory Power of Polymorphic Amplified Typing Sequences, a Novel Strain Typing System for Escherichia coli O157:H7

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

Polymorphic Amplified Typing Sequences Provide a Novel Approach to Escherichia coli O157:H7 Strain Typing

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