Development of a PCR-restriction fragment length polymorphism assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli

Abstract

Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control E. coli strains carrying six different Stx phages such as E. coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks.

FIG. 1.

(A) Restriction map of the Stx2φ-I genome and the location of the six characteristic regions (I to VI). B and X represent BamHI and XhoI, respectively. Arrows indicate the location of primers. (B) Restriction map of region V in six Stx phages. Bg and E represent BglI and EcoRV, respectively.

FIG. 2.

(A) LA-PCR products of region V in six Stx-phages. Lanes: 1 and 8, 5-kb DNA ladder; 2, C600; 3, C600 (Stx1φ); 4, C600 (Stx2φ-I); 5, C600 (Stx2φ-II); 6, C600 (933W phage); 7, RIMD0509952 (Sakai strain; VT1-Sakai phage, VT2-Sakai phage). BglI digest (B) and EcoRV digest (C) of LA-PCR products obtained from region V in six Stx phages. Lanes: 1, λ-HindIII digest; 2 and 8, 1-kb DNA ladder; 3, C600 (Stx1φ); 4, C600 (Stx2φ-I); 5, C600 (Stx2φ-II); 6, C600 (933W phage); 7, RIMD0509952 (Sakai strain; VT1-Sakai phage, VT2-Sakai phage).

FIG. 3.

(A) Field inversion gel electrophoresis of LA-PCR products obtained from 6 control strains and 24 representative strains differentiated by the PCR-RFLP assay. Lanes: 1 and 32, 5-kb DNA ladder; 2, C600; 3, C600 (Stx1φ); 4, C600 (Stx2φ-I); 5, C600 (Stx2φ-II); 6, C600 (933W phage); 7, RIMD0509952 (Sakai strain; VT1-Sakai phage, VT2-Sakai phage); 8, 1-A (Okayama, Japan); 9, 2-B (Osaka, Japan); 10, 3-C (Okayama, Japan); 11, 4-D (Okayama, Japan); 12, 5-E (Okayama, Japan); 13, 6-F(Okayama, Japan); 14, 7-G (Osaka, Japan); 15, 8-H (Osaka, Japan); 16, 9-I (Osaka, Japan); 17, 10-J (Osaka, Japan); 18, 11-K (Nagano, Japan); 19, 12-L (Nagano, Japan); 20, 13-M (Nagano, Japan); 21, 14-N (Nagano, Japan); 22, 15-O (Nagano, Japan); 23, 16-P (United States); 24, 17-Q (United States); 25, 18-R (United States); 26, 19-S (United States); 27, 20-T (United States); 28, 21-U (Canada); 29, 22-V (Canada); 30, 23-W (Canada); 31, 24-X (Canada). Field inversion gel electrophoresis of BglI digest (B) and EcoRV digest (C) of LA-PCR products obtained from 6 control strains and 24 representative strains differentiated by the PCR-RFLP assay. Lanes: 1, λ-HindIII digest; 2, 32, 1-kb DNA ladder; 3, C600 (Stx1φ); 4, C600 (Stx2φ-I); 5, C600 (Stx2φ-II); 6, C600 (933W phage); 7, RIMD0509952 (Sakai strain; VT1-Sakai phage, VT2-Sakai phage); 8, 1-A (Okayama, Japan); 9, 2-B (Osaka, Japan); 10, 3-C (Okayama, Japan); 11, 4-D (Okayama, Japan); 12, 5-E (Okayama, Japan); 13, 6-F (Okayama, Japan); 14, 7-G (Osaka, Japan); 15, 8-H (Osaka, Japan); 16, 9-I (Osaka, Japan); 17, 10-J (Osaka, Japan); 18, 11-K (Nagano, Japan); 19, 12-L (Nagano, Japan); 20, 13-M (Nagano, Japan); 21, 14-N (Nagano, Japan); 22, 15-O (Nagano, Japan); 23, 16-P (United States); 24, 17-Q (United States); 25, 18-R (United States); 26, 19-S (United States); 27, 20-T (United States); 28, 21-U (Canada); 29, 22-V (Canada); 30, 23-W (Canada); 31, 24-X (Canada).

FIG. 4.

Comparison of the typing results between PCR-RFLP and PFGE. (A) PFGE patterns of STEC strains isolated in Okayama and Nagano digested by XbaI or ApaI. Lanes: 1 and 6, lambda ladder; 2, 1-A (Okayama, Japan), XbaI; 3, 3-C (Nagano, Japan), XbaI; 4, 1-A (Okayama, Japan), ApaI; 5, 3-C (Nagano, Japan), ApaI. Field inversion gel electrophoreses of BglI digest (B) and EcoRV digest (C) of LA-PCR products of 1-A (Okayama, Japan) and 3-C (Nagano, Japan). Lanes: 1, λ-HindIII digest; 2 and 5, 1-kb DNA ladder; 3, 1-A (Okayama, Japan); 4, 3-C (Nagano, Japan).