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

Abstract

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.

FIG. 1.

PFGE patterns of XbaI-digested fragments obtained when wild-type and successive-passage-derived STEC strains RIMD0509952 (A), EC1015 (B), and 83-1385 (C). (A) PFGE profiles obtained with STEC O157:H7 strain RIMD0509952 and its passage-derived strains. Lanes: 1, I-a (original pattern); 2, I-b; 3, I-c; 4, I-d; 5, I-e; 6, I-f. (B) PFGE profiles obtained with STEC O157:H7 strain EC1015 and its passage-derived strains. Lanes: 1, II-a (original pattern); 2, II-b; 3, II-c; 4, II-d; 5, II-e; 6, II-f; 7, II-g; 8, II-h; 9, II-i; 10, II-j; 11, II-k; 12, II-l; 13, II-m; 14, II-n; 15, II-o; 16, II-p; 17, II-q. (C) PFGE profiles obtained with STEC O157:H7 strain 83-1386 and its passage-derived strains. Lanes: 1, III-a (original pattern); 2, III-b; 3, III-c; 4, III-d; 5, III-e; 6, III-f; 7, III-g. Lambda ladders were used as molecular size markers. Positions of DNA fragments with known molecular masses are indicated. Parentheses and asterisks represented additional and lost bands, respectively.

FIG. 2.

(A) Agarose gel electrophoresis patterns of PCR amplicons obtained against region V of Stx phage. Lanes: 1, I-A (RIMD0509952, original pattern); 2, II-A (EC1015, original pattern); 3, II-B (EC1015, lost Stx2 phage pattern); 4, III-A (83-1386, original pattern); 5, C600. Ladders (2.5 kb) were used as molecular mass markers on both sides of the gel. (B and C) BglI digest (B) and EcoRV digest (C) of LA-PCR products obtained from region V in STEC O157:H7 strains RIMD0509952, EC1015, and 83-1386. Lanes: 1, I-A (RIMD0509952, original pattern); 2, II-A (EC1015, original pattern); 3, II-B (EC1015, lost Stx2 phage pattern); 4, III-A (83-1386, original pattern). Lambda-HindIII digest and 1-kb ladders were used as molecular mass markers on both sides of each gel. Positions of DNA fragments with known molecular masses are indicated.