Comparison of PCR-RFLP and PFGE for determining the clonality of enterohemorrhagicEscherichia colistrains

Abstract

We report here on a comparative evaluation of PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) assays, and ascertain the clonal relationship between 13 enterohemorrhagic Escherichia coli O157 : H7 strains isolated from fecal samples collected from three cows over a period of 2 months. PCR-RFLP analysis was carried out with either BglI or EcoRV digested LA-PCR amplicons, generated by targeting region V of the Stx-phage. While PCR-RFLP analysis placed these 13 strains into a single clonal type, pulsotyping analysis, as reported earlier, grouped these strains into four different PFGE subtypes of which three were closely related, while the other appeared to be different. The comparative analysis was extended further using two clonally different wild-type (3-0 and Sakai 215) strains and 17 derivative strains which had passed through an animal's gastrointestinal tract. The PCR-RFLP assay, which was not only able to differentiate the wild-type strains, but also placed the passaged derivative strains into their respective parental group, although PFGE patterns of the same set of strains resulted from different PFGE subtypes. These data indicate that PCR-RFLP is the more reliable and useful assay for a molecular epidemiological survey of enterohemorrhagic E. coli strains.

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

“…We recently dem- onstrated alteration of PFGE profiles of STEC strains caused by in vivo passages through the bovine intestine (4), while PCR-RFLP profiles remained unaltered (31). This study further demonstrates that RFLP patterns of STEC strains by PCR-RFLP assay were not affected due to in vitro clonal turnover caused by subculture and prolonged storage.…”

Development of a Multiplex PCR-Based Rapid Typing Method for EnterohemorrhagicEscherichia coliO157 Strains

“…It is also difficult to obtain consistently reproducible results among different laboratories, which hinders interlaboratory data comparisons. Other strain-typing methods for EHEC O157, including PCR-restriction fragment length polymorphism (15,16), polymorphic amplified typing sequences (5,6), and multiplelocus variable-number tandem repeat analysis (9), have also been developed. Although these methods have their own advantages, they require special techniques and/or equipment and are time-consuming.…”

Rapid Determination ofEscherichia coliO157:H7 Lineage Types and Molecular Subtypes by Using Comparative Genomic Fingerprinting

“…Likewise, ribotyping alone does not offer the discriminatory power needed to effectively fingerprint E. coli O157:H7 strains, although it has been successfully used in conjunction with PFGE to offer additional discrimination (3,38). PCR-RFLP based on virulence regions (such as the regulatory region of Stx phage) has been used to unambiguously determine clonality among E. coli O157:H7 strains but lacks the resolution to differentiate among strains that are temporally or geographically distant (42,43). The analysis of tandem duplication in the genome with MLVA has been useful in characterizing clonal organisms such as E. coli O157:H7 (20) with greater discriminatory power than PFGE (32,36), although its usefulness in higher-level analyses, such as lineage typing, has not been determined.…”