Genotyping of pandemic Vibrio parahaemolyticus O3:K6 still open to question.

Vibrio parahaemolyticus is one of the major seafood-borne gastroenteritis-causing bacteria, frequently associated with consumption of raw or inappropriately cooked seafood. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered major virulence factors for the organism (7). V. parahaemolyticus can be classified into 13 O serotypes and 71 K serotypes (3). Although various serovars of the bacterium can cause infections, O3:K6 has been recognized as the predominant serovar responsible for most outbreaks worldwide since 1996 (5). The pandemic strains and other recently emerged serovars such as O4:K68 and O1:K untypeable showed almost identical pulsed-field gel electrophoresis (PFGE) patterns (1), suggesting that these strains are clonally related. Matsumoto et al. (5) reported that the pandemic strains exhibit a unique sequence within the toxRS operon, which encodes transmembrane proteins in the regulation of virulence-associated genes conserved in the genus Vibrio. On the other hand, Nasu et al. (6) isolated filamentous phage possessing a unique open reading frame, ORF8, from one of the pandemic strains, and Iida et al. (4) claimed that ORF8 was a useful genetic marker for identifying strains. It was thus necessary to evaluate the use of the toxRS sequence or ORF8 as a reliable genetic marker for the identification. A total of 24 strains of V. parahaemolyticus that had been isolated from various sources with known serological identities were used in the present study and are listed in Table 1. These include 21 strains of O3:K6, consisting of 12 strains isolated before 1996 and 9 strains isolated since1996, and 3 strains of O4:K68 isolated since 1999. PFGE typing was performed on genomic DNAs of the O3:K6 and O4:K68 strains digested with the restriction enzyme SfiI, following a method described previously (1). Of 21 strains of O3:K6 subjected to the typing, 9 strains isolated since 1996 were classified as type A, 2 strains isolated in 1981 were classified as type B, and 5 strains isolated between 1981 and 1996 were classified as type C (Table 1). The 5 strains isolated between 1982 and 1988 showed PFGE fragment patterns different from any of the above PFGE types (Table 1). The presence of TDH gene (tdh) and TRH gene (trh) was determined by PCR with a set of primers, 5 -GGTACTAAATG GCTGACATC-3 and 5 -CCACTACCACTCTCATA-TGC-3 , and another set of primers, 5 -GGCTCAAAATGGTTAAGC G-3 and 5 -CATTTCCG-CTCTCATATGC-3, respectively, fol-