Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus O3:K6 in seafood and the coastal environment in Japan

Abstract

Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.

FIG. 1.

Schematic representation of qualitative and quantitative analyses of total and tdh-positive V. parahaemolyticus in seafood. APW, alkaline peptone water; SPB, salt polymyxin broth; CV, CHROMagar Vibrio; RPLA, reversed passive latex agglutination test; TSI, triple sugar iron.

FIG. 2.

Relationship between total number of V. parahaemolyticus organisms and number of tdh-positive V. parahaemolyticus organisms in seafood. Each circle indicates the number in each sample. ND, tdh gene not detected by qualitative analysis (<1 CFU/25 g), NC, tdh gene not detected by quantitative analysis (<3 MPN/10 g) but detected by qualitative analysis (>1 CFU/25 g).

FIG. 3.

Representative AP-PCR patterns exhibited by selected strains. The results obtained with primer 2 and primer 4 are shown as indicated. Lanes: 1, VP1281; 2, VP1282; 3, VPFO1-5; 4, APCCVP9810; 5, 1001A46; 6, VPFOO-10; 7, VP-81; M1, φX174 DNA digested with HaeIII; M2, 1-kb DNA ladder. Arbitrarily designated AP-PCR patterns are indicated at the bottom. The test strains and their AP-PCR patterns are listed in Table 3.

FIG. 4.

PFGE profiles of selected strains of V. parahaemolyticus and their relations. (A) PFGE profiles of NotI-digested genomic DNAs. The profiles were considered different if they differed by one or more DNA fragment. The profiles were classified into 15 profiles, arbitrarily designated A through P (indicated at the bottom). The asterisk denotes the profile obtained from reference strains. Lanes: M, molecular size markers (DNA size standard maker, lambda ladder; Bio-Rad); 1, FIHES98V1-32-4; 2, JKY-VP6; 3, VP-2; 4, BE98-2062; 5, VP1280; 6, KX-V225; 7, VP80; 8, VPF00-18; 9, VP1152; 10, APCC VP 9810; 11, 1001A44; 12, 1001A46; 13, APCC VP 00030; 14, APCC VP 00031; 15, 4917; 16, APCC VP 00157; 17, APCC VP 00190; 18, 5095. Test strains and their PFGE profiles are listed in Table 3. (B) Dendrogram constructed from the 15 PFGE profiles shown in panel A. The scale for the similarity coefficient is indicated as S_AB.

FIG. 4.

PFGE profiles of selected strains of V. parahaemolyticus and their relations. (A) PFGE profiles of NotI-digested genomic DNAs. The profiles were considered different if they differed by one or more DNA fragment. The profiles were classified into 15 profiles, arbitrarily designated A through P (indicated at the bottom). The asterisk denotes the profile obtained from reference strains. Lanes: M, molecular size markers (DNA size standard maker, lambda ladder; Bio-Rad); 1, FIHES98V1-32-4; 2, JKY-VP6; 3, VP-2; 4, BE98-2062; 5, VP1280; 6, KX-V225; 7, VP80; 8, VPF00-18; 9, VP1152; 10, APCC VP 9810; 11, 1001A44; 12, 1001A46; 13, APCC VP 00030; 14, APCC VP 00031; 15, 4917; 16, APCC VP 00157; 17, APCC VP 00190; 18, 5095. Test strains and their PFGE profiles are listed in Table 3. (B) Dendrogram constructed from the 15 PFGE profiles shown in panel A. The scale for the similarity coefficient is indicated as S_AB.