Development of a PCR-Restriction Fragment Length Polymorphism Assay for the Epidemiological Analysis of Shiga Toxin-Producing
            <i>Escherichia coli</i>

Shima, Kensuke; Terajima, Jun; Sato, Toshio; Nishimura, Kazuhiko; Tamura, Kazumichi; Watanabe, Haruo; Takeda, Yoshifumi; Yamasaki, Shinji

doi:10.1128/jcm.42.11.5205-5213.2004

Cited by 18 publications

(31 citation statements)

References 58 publications

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“…PCR/RFLP is a simple and reproducible method of bacterial differentiation that does not require special equipment and is performed in many laboratories (Shima et al, 2004;Ciantar et al, 2005). Moreover, by using PCR the limitations of traditional culturedependent technique, which requires the isolation and culture of bacteria, is overcome (Ranjard et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Comparative microbiota of Rickettsia felis-uninfected and -infected colonized cat fleas, Ctenocephalides felis

et al. 2007

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Fleas serve as arthropod vectors for several emerging and re-emerging infectious disease causing agents including, Rickettsia felis. Although the prevalence of R. felis infection in colonies of fleas has been examined, the influence of the R. felis infection on flea microbiota has not been investigated. We identified three colonies of cat fleas, Ctenocephalides felis, with varying prevalence of R. felis infection (Louisiana State University (LSU), 93.8%; Professional Laboratory and Research Services Inc. (PLRS), 16.4%; Elward II (EL), 0%) and subsequently utilized polymerase chain reaction amplification, restriction fragment length polymorphism analysis and sequencing of the 1.4-kb portions of 16S rRNA genes to examine the diversity of bacteria in the flea populations. A total of 17 different bacterial 16S rRNA gene sequences were identified among the C. felis colonies. The prevalence of two Wolbachia species that were identified in each flea colony differed between colonies and R. felis-uninfected and -infected fleas. Species richness was unchanged among the R. felis-uninfected (LSU, PLRS and EL colonies) and -infected (LSU and PLRS colonies) fleas; however, between R. felis-uninfected and -infected fleas within both the LSU and PLRS colonies, R. felisuninfected fleas have greater species richness. Diversity indices did not identify a difference in diversity between any of the flea samples. The interaction of endosymbionts within arthropods can widely impact the dissemination of vertically transmitted pathogenic bacteria; and the reciprocal may be true. These results suggest that carriage of R. felis has an impact on the richness of flea microbiota.

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Section: Discussionmentioning

confidence: 99%

Comparative microbiota of Rickettsia felis-uninfected and -infected colonized cat fleas, Ctenocephalides felis

et al. 2007

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“…PFGE was performed following essentially the same method as described previously (30). Total genomic DNA was isolated in an agarose-embedded form and was subjected to enzymatic digestion with 50 U of XbaI.…”

Section: Methodsmentioning

confidence: 99%

“…However, PFGE has some limitations, like expense, equipment required, and limitations on the number of isolates that can be analyzed simultaneously. The PFGE profiles of some strains cannot be resolved due to strong DNase activity or degradation by free radical or peroxide produced during electrophoresis, resulting in smeared profiles (21,30). In addition, variation in PFGE patterns has been reported for STEC strains that are passaged through the bovine or human gastrointestinal tract (4,18).…”

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confidence: 99%

“…Changing PFGE patterns have also been reported after subculture of STEC strains in vitro (13). To overcome such problems, we recently developed a rapid and simple DNA fingerprinting method for molecular epidemiological analysis of STEC strains (30). The PCR-restriction fragment length polymorphism (PCR-RFLP) assay exploits the nucleotide sequence diversity within the region V defined as the regulatory region of Stx phage.…”

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confidence: 99%

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Comparison of a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Pulsed-Field Gel Electrophoresis To Determine the Effect of Repeated Subculture and Prolonged Storage on RFLP Patterns of Shiga Toxin-Producing Escherichia coli O157:H7

Shima

Sugimoto

et al. 2006

J Clin Microbiol

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In this study, we compared a recently developed PCR-restriction fragment length polymorphism (PCR-RFLP) assay with pulsed-field gel electrophoresis (PFGE) using three different Shiga toxin-producing Escherichia coli (STEC) strains to understand whether repeated subculture in vitro and prolonged storage at room temperature affect the RFLP patterns of STEC. The PFGE profiles of the STEC strains changed by 1 to 8 fragments after repeated subculture and prolonged storage; one strain was no longer clonal after repeated subculture compared to the original isolate according to the Tenover criteria. In contrast, RFLP patterns obtained by PCR-RFLP were identical after repeated subculture and prolonged storage. These data clearly indicate that the PCR-RFLP assay which is based on the diversity of region V, a regulatory region of Stx-phage, was not affected by repeated subculture and prolonged storage and is a more practical and reliable method for molecular typing of STEC strains.

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“…19) Pulsed-field Gel Electrophoresis --Pulsed-field gel electrophoresis (PFGE) analysis was performed with the protocol described elsewhere. 20,21) Briefly, the genomic DNA isolated from each P. aeruginosa strain was fixed into the agarose plugs, and the plugs were equilibrated with the restriction enzyme buffer (pH 7.5) for 1 hr at room temperature. Thereafter, the DNA was digested with 50 U of XbaI for 12 hr at an appropriate temperature.…”

Section: Methodsmentioning

confidence: 99%

A Plasmidic Class 1 Integron from Five Pseudomonas aeruginosa Clinical Strains Harbored aacA4 and Nonsense-mutated cmlA1 Gene Cassettes

Shi

Yamasaki

et al. 2007

JOURNAL OF HEALTH SCIENCE

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Five strains of multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) were isolated from inpatients at a local hospital in China. The most frequent resistance was to cefoperazone, ciprofloxacin, ceftriaxone, cefotaxime, gentamicin, piperacillin, trimethoprim sulfamethoxazole, and tobramycin. These strains were found to contain the class 1 integron, in which the 2360-bp gene cassettes were flanked by 5 -and 3 -conserved segments. Sequence analysis revealed that the gene cassettes contained aacA4 and cmlA1 genes; however, the latter gene had a nonsense mutation resulting in the production of a truncated protein. To the best of our knowledge, this is the first report of a nonsense mutation in the cmlA1 gene. Moreover, the P. aeruginosa strains showed identical profiles in pulsed-field gel electrophoresis, suggesting that they were derived from the same clone. These results emphasize the importance of controlling the spread of multidrug-resistant pathogens in hospitals.

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Development of a PCR-Restriction Fragment Length Polymorphism Assay for the Epidemiological Analysis of Shiga Toxin-Producing Escherichia coli

Cited by 18 publications

References 58 publications

Comparative microbiota of Rickettsia felis-uninfected and -infected colonized cat fleas, Ctenocephalides felis

Comparative microbiota of Rickettsia felis-uninfected and -infected colonized cat fleas, Ctenocephalides felis

Comparison of a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Pulsed-Field Gel Electrophoresis To Determine the Effect of Repeated Subculture and Prolonged Storage on RFLP Patterns of Shiga Toxin-Producing Escherichia coli O157:H7

A Plasmidic Class 1 Integron from Five Pseudomonas aeruginosa Clinical Strains Harbored aacA4 and Nonsense-mutated cmlA1 Gene Cassettes

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