Comparison of molecular methods for typing Vibrio parahaemolyticus

Abstract

An outbreak of Vibrio parahaemolyticus gastroenteritis on Canada's west coast in 1997 emphasized the need to develop molecular methods for differentiation and typing of these organisms. Isolates were analyzed by enterobacterial repetitive intergenic consensus sequence (ERIC) PCR, detection of restriction fragment length polymorphisms (RFLP) in rRNA genes (ribotyping), pulsed-field gel electrophoresis (PFGE), and RFLP analysis of the genetic locus encoding the polar flagellum (Fla locus RFLP analysis). ERIC PCR and ribotyping were the most informative typing methods, especially when used together, while Fla locus RFLP analysis was the least discriminatory. PFGE exhibited good discrimination but suffered from a high incidence of DNA degradation. ERIC PCR and ribotyping will be useful for the evaluation of genetic and epidemiological relationships among V. parahaemolyticus strains.

FIG. 1

Representative ribotype patterns of BglI-digested DNA. Pattern designations are given in parentheses. Lane 1, T97-31 (A); lane 2, T97-32 (B); lane 3, T97-33 (C); lane 4, T97-54 (D); lane 5, T97-57 (degraded DNA from isolate exhibiting pattern E in subsequent analysis); lane 6, T97-45 (F); lane 7, T97-58 (G); lane 8, T97-60 (H); lane 9, T97-52 (I); lane 10, T97-61 (J); lane 11, T97-65 (K); lane 12, T97-66 (L); lane 13, T97-472 (M); lane 14, T97-474 (N); lane M, PvuII-digested supercoiled DNA, with sizes of selected standard bands shown at the right.

FIG. 2

Representative PFGE patterns for V. parahaemolyticus chromosomal DNA digested with ApaI. Pattern designations are given in parentheses. Lane M, λ DNA concatemer size standard, with sizes of selected bands given at the left; lane 1, T97-31 (A1); lane 2, T97-34 (A2); lane 3, T97-450 (B); lane 4, T97-42 (C); lane 5, T97-45 (D); lane 6, T97-52 (E); lane 7, T97-61 (I); lane 8, T97-65 (K).

FIG. 3

ERIC 1R PCR profiles from selected V. parahaemolyticus isolates. Pattern designations are in parentheses. Lane M, 100-bp ladder; lane 1, T97-31 (A1); lane 2, T97-32 (B1); lane 3, T97-33 (C1); lane 4, T97-56 (D); lane 5, T97-65 (G); lane 6, T97-450 (I); lane 7, T97-474 (L); lane 8, negative control.

FIG. 4

RFLP patterns obtained by RsaI digestion of the product of PCR of the V. parahaemolyticus Fla locus. Pattern designations are given in parentheses. Lanes M, 100-bp ladder; lane 1, T97-31 (A); lane 2, T97-32 (B); lane 3, T97-33 (C); lane 4, T97-58 (D); lane 5, T97-56 (E).