Molecular characterisation of Escherichia coli O157: H7 isolates by pulsed-field gel electrophoresis and plasmid DNA analysis

Meng, Jianghong; Zhao, Shixiang; Zhao, Tong; Doyle, Michael P.

doi:10.1099/00222615-42-4-258

Cited by 55 publications

(38 citation statements)

References 6 publications

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“…is method has widely been used as a molecular subtyping method of STEC strains, including O157, due to its high discriminatory power and good reproducibility (Bohm and Karch, 1992;Meng et al, 1995). In the present study, it was shown that epidemiologically unrelated O157 E. coli isolates produced very similar XbaI restriction profiles.…”

Section: Discussionsupporting

confidence: 51%

“…coli strains of the O157 serogroup show a clonal nature and highly sensitive molecular biology-based subtyping methods are needed to compare and to differentiate unrelated strains isolated from different sources (Whittam et al, 1988;Feng et al, 1998). Several methods have been used for genetic analysis of STEC O157 and among them differentiation of chromosomal DNA based on restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) appear to be the most powerful techniques that provide the greatest discrimination and reproducibility (Bohm and Karch, 1992;Meng et al, 1995). e aim of this study was to analyse, using polymerase chain reaction (PCR), the presence of the STEC-associated virulence genes stx1, stx2, eaeA, ehly, and tir, in E. coli strains of the O157 serogroup isolated from cattle and from pigs.…”

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confidence: 99%

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Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

Osek¹,

Gallien²

2002

Vet. Med. - Czech

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ABSTRACT:Fourteen Escherichia coli O157 strains isolated from cattle and pigs in Poland and in Germany were investigated, using PCR, for the genetic markers associated with Shiga toxin-producing E. coli (STEC). Only two strains, both of cattle origin, were positive for the fliC (H7) gene and could be classified as O157 : H7. Nine isolates had stx shiga toxin genes, either stx1 (1 strain), stx2 (4 isolates) or both (4 strains). e stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that all but one stx2-positive bacteria possessed the stx2c Shiga toxin gene type and one stx2 STEC isolate had the stx2d virulence factor subtype. e eaeA (intimin) gene was found in 9 strains (8 isolates from cattle and one strain from pigs); all of them harboured the genetic marker characteristic of the gamma intimin variant. e translocated intimin receptor (tir) gene was detected in 7 isolates tested and among them only one tir-positive strain was recovered from pigs. e ehly E. coli enterohemolysin gene was amplified in all but one strains obtained from cattle and only in one isolate of porcine origin. e genetic relatedness of the analysed E. coli O157 strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with XbaI. Two distinct but related RFLP pattern clusters were observed: one with 9 strains (8 isolates of bovine origin and one strain obtained from pigs) and the other one comprises the remaining 5 E. coli isolates (4 of porcine origin and one strain recovered from cattle). e results suggest that pigs, besides cattle, may be a reservoir of E. coli O157 strains potentially pathogenic to humans. Moreover, epidemiologically unrelated isolates of the O157 serogroup, recovered from different animal species, showed a clonal relationship as demonstrated by the RFLP analysis.

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Section: Discussionsupporting

confidence: 51%

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confidence: 99%

Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

Osek¹,

Gallien²

2002

Vet. Med. - Czech

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“…Since the XbaI restriction enzyme site occurs infrequently in the O157 genome, it is frequently used with PFGE for typing of this organism (3,4,7). Although PFGE has successfully been used to support outbreak investigations, it may be impossible to fully resolve all bands on a gel under a single set of conditions, making interpretation and comparisons difficult (7,8,10).…”

Section: Discussionmentioning

confidence: 99%

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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“…PFGE analysis represents a "gold standard" in epidemiological studies of diverse bacterial pathogens including other E. coli groups (2,18,19,26). In the present study, XbaI-generated DNA fingerprints proved to be more sensitive than RAPD Table 1 and are indicated at the top.…”

Section: Discussionmentioning

confidence: 99%

Beyond Serotypes and Virulence-Associated Factors: Detection of Genetic Diversity among O153:H45 CFA/I Heat-Stable Enterotoxigenic Escherichia coli Strains

et al. 2001

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Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heatstable (ST) toxins and colonization factors (CFs).In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like São Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.Enterotoxigenic Escherichia coli (ETEC) represents one of the main etiologic agents of diarrhea in infants and travelers in developing countries (5). ETEC strains are identified by the ability to produce enterotoxins, either heat-labile toxin or heatstable toxin (ST), or both, and surface adhesins known as colonization factors (CFs). Also, characterization of ETEC strains has relied on the serological determination of a number of different combinations of O (lypopolyssacharide) and H (flagellar) serogroups (4,7,11,13,28). Antigen heterogeneity is a striking feature among ETEC strains, as demonstrated in a survey that evaluated the diversity, distribution, and association of ETEC phenotypes in epidemiological studies carried out in different parts of the world (31).The application of DNA-based typing methods to investigate the genetic relationship among ETEC strains isolated from humans has been rare (16,19). Previous studies, based on the use of randomly amplified polymorphic DNA (RAPD) analyses, indicated that, in contrast to other pathogenic E. coli groups (8, 30), ETEC strains that share an O:H serotype but not necessarily the same virulence-associated factors belong to clonal clusters (21,22,23). Moreover, RAPD analysis can reveal variant genotypes among ETEC strains that share the same O:H serotype and that belong to the same clonal cluster (22, ...

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Molecular characterisation of Escherichia coli O157: H7 isolates by pulsed-field gel electrophoresis and plasmid DNA analysis

Cited by 55 publications

References 6 publications

Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

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

Beyond Serotypes and Virulence-Associated Factors: Detection of Genetic Diversity among O153:H45 CFA/I Heat-Stable Enterotoxigenic Escherichia coli Strains

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