Clonal diversity among recently emerged strains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread

Abstract

The genomes of the O3:K6 strains of Vibrio parahaemolyticus which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of V. parahaemolyticus. Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the rrn operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.

FIG. 1

Representative results for the five ribotypes of the O3:K6 V. parahaemolyticus strains encountered in this study. R1, strain VP86; R2, strain VP199; R3, strain VP208; R4, strain VP155; and R5, strain VP96. Numbers on the right are molecular sizes (in kilobases).

FIG. 2

PFGE of NotI-digested genomic DNAs of V. parahaemolyticus O3:K6 strains. The gel was stained with ethidium bromide. Numbers indicate the sizes of the molecular size markers (in kilobases). The RFLP patterns of strains isolated between February and August 1996 (strains VP61, VP100, VP122, and VP136) compared to the RFLP patterns of strains isolated between August 1996 and March 1998 (strains VP144, VP165, VP210, and VP218) are distinct. No RFLP was observed between the strains isolated in the later phase (June 1998) of the outbreak (strains VP238 and VP239).

FIG. 3

PFGE of CeuI-digested genomic DNAs of V. parahaemolyticus O3:K6 isolates and identification of number of rrn operons. The enzyme-digested DNAs were electrophoresed, with pulse times interpolated between 20 and 200 s for 24 h at 10 V/cm at 4°C to separate CeuI fragments (operons C1 to C9) (A), and for better resolution of CeuI operons C4 to C8, the pulse time was interpolated between 5 and 100 s for 24 h at 10 V/cm at 4°C (the region for operon C4 to C8 is expanded in panel B. Since CeuI has sites only in rrn operons in the bacterial genomes examined so far, it appears that there are nine CeuI sites in the genome of V. parahaemolyticus and hence probably nine rrn operons. Lanes: a, VP100; b, VP122; c, VP136; d, VP144; e, VP165; f, VP165; g, VP210; h, VP218; i, VP238; and M, bacteriophage λ multimeric DNA as marker. Numbers indicate the sizes of the molecular size markers (in kilobases). The arrowhead indicates RFLP between initial isolates only in operons C5 and C6 (lanes a to d). Strains isolated from the later phase of the outbreak had identical CeuI pulsotypes (lanes e to i).