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Journal of Clinical Microbiology, June 2000, p. 2156-2161, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Filamentous Phage Associated with Recent Pandemic Vibrio parahaemolyticus O3:K6 Strains

Hatsumi Nasu,1 Tetsuya Iida,1,* Tomomi Sugahara,1 Yoshiharu Yamaichi,1,dagger Kwon-Sam Park,1 Katsushi Yokoyama,2 Kozo Makino,2 Hideo Shinagawa,2 and Takeshi Honda1

Department of Bacterial Infections1 and Department of Molecular Microbiology,2 Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan

Received 15 November 1999/Returned for modification 22 January 2000/Accepted 11 March 2000

A specific serotype, O3:K6, of Vibrio parahaemolyticus has recently been causing epidemics of gastroenteritis in Southeast Asia, Japan, and North America. To examine whether the new O3:K6 strains possess characteristics that may exacerbate outbreaks, we compared V. parahaemolyticus O3:K6 strains with non-O3:K6 strains using strains isolated from individuals with traveler's diarrhea at Kansai Airport Quarantine Station, Osaka, Japan. All 24 O3:K6 strains possessed a common plasmid, pO3K6 (DNA size, 8,782 bp, with 10 open reading frames [ORFs]). The gene organization of pO3K6 was similar to that of Vf33, a filamentous phage previously described in V. parahaemolyticus. We isolated a phage (phage f237) from the culture supernatant of V. parahaemolyticus O3:K6 strain KXV237, which formed a turbid plaque on an indicator strain. The genome of f237 was single-stranded DNA, and the double-stranded DNA obtained by treatment of the genome with DNA polymerase was identical to that of pO3K6 when analyzed by agarose gel electrophoresis after HindIII digestion. Furthermore, the N-terminal amino acid sequence of the f237 major coat protein was found in ORF4 of pO3K6. Our results showed that pO3K6 is a replicative form of f237. Among the ORFs found in the f237 genome, the sequence of ORF8 had no significant homology to those of any proteins in databases. ORF8 was located on a region corresponding to the distinctive region of Vf33, and its G+C content was apparently lower than that of the remaining DNA sequence of f237. By colony hybridization, ORF8 was detected only in O3:K6 strains isolated since 1996 and was not found in O3:K6 strains isolated before 1996 and clinical V. parahaemolyticus strains other than those of serotype O3:K6. Thus, this study shows that f237 is exclusively associated with recent V. parahaemolyticus O3:K6 strains. The ORF8 gene can be a useful genetic marker for the identification of the recently widespread O3:K6 strains of V. parahaemolyticus.

* Corresponding author. Mailing address: Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8276. Fax: 81-6-6879-8277. E-mail: iida{at}biken.osaka-u.ac.jp.

dagger Present address: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 4-24-1 Kuhonji, Kumamoto 862-0976, Japan.

Journal of Clinical Microbiology, June 2000, p. 2156-2161, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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