Filamentous phage associated with recent pandemic strains of Vibrio parahaemolyticus.

A group of pandemic strains of Vibrio parahaemolyticus has recently appeared in Asia and North America. We demonstrate that a filamentous phage is specifically associated with the pandemic V. parahaemolyticus strains. An open reading frame unique to the phage is a useful genetic marker to identify these strains.


The Study
We studied 96 strains of V. parahaemolyticus isolated from diarrheal patients during January to May 1999 at the Kansai International Airport quarantine station, Osaka, Japan. Detection of f237 was by colony hybridization using a digoxigenin-labeled DNA probe targeted for ORF8 (8) and prepared as described (8). In brief, after amplification of a partial DNA sequence of ORF8 (746 bp in size) by polymerase chain reaction (PCR) with the genomic DNA of V. parahaemolyticus KXV237 strain (RIMD2210633) (8) as a template, the amplified DNA was labeled with digoxigenin using a PCR DIG labeling kit (Boehringer GmbH, Mannheim, Germany). PCR primers for ORF8 were 8S (5'-GTTCGCATA-CAGTTGAGG-3') and 8A (5'-AAGTACAGCAGGAGTGAG-3').
The conditions for PCR were as follows: After heating at 94°C for 3 minutes, a cycle of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute was repeated 30 times, followed by end extension step at 72°C for 5 minutes. PCR was done on a Perkin-Elmer (Foster City, CA) thermal cycler type 9700. Pulsed-field gel electrophoresis (PFGE) and Southern hybridization were performed as described (10). For PFGE, the V. parahaemolyticus genomic DNA in agarose blocks was digested with restriction enzymes NotI or SfiI. A contourclamped homogeneous electric field method was used for PFGE on a CHEF Mapper System (Bio-Rad Laboratories, Richmond, CA). The conditions for PFGE were 1% agarose gel in 0.5x Tris-borate-EDTA buffer at 6 V cm -1 , with pulse times increasing linearly from 1 to 60 seconds within 24 hours.
Of the 96 strains examined, 53 tested positive for ORF8, suggesting the presence of phage f237 (Table). In addition to O3:K6, the ORF8-positive strains were found in strains with serotypes O4:K68 and O1:KUT. None of the strains with other serovars showed evidence of ORF8. When the genomic DNA of the strains was digested with NotI or SfiI endonuclease, PFGE showed very similar restriction patterns for the bacterial genomes of the ORF8-positive strains, irrespective A group of pandemic strains of Vibrio parahaemolyticus has recently appeared in Asia and North America. We demonstrate that a filamentous phage is specifically associated with the pandemic V. parahaemolyticus strains. An open reading frame unique to the phage is a useful genetic marker to identify these strains. of their serovars (data not shown). Southern hybridization with a probe for ORF8 after PFGE demonstrated that, in all the ORF8-positive strains, the largest NotI fragment (size approximately 1,080 kb) reacted with the probe, suggesting that f237 integrated into the bacterial chromosome.

Conclusions
Our study shows that the filamentous phage f237 is associated not only with O3:K6 serovar but also with other recently emerging serovars of V. parahaemolyticus. In the 55 strains showing PFGE genotypes closely related to those of the recent O3:K6 strains, >96% was positive for the ORF8 (Table). Such high prevalence of the phage f237 in the V. parahaemolyticus strains showing pandemic spread suggests that the phage might confer a so-far-unknown phenotype to the bacterium. The phenotype might, in turn, protect the organism against selective pressure in a certain environment before it infects humans. Whether phage f237 has played a part in the recently increasing pandemic potency of strains of V. parahaemolyticus is a subject for further study.
The virulence of V. parahaemolyticus extends beyond acute gastroenteritis: it can also cause wound infections and septicemia (5). Physicians everywhere need to be alert to the possibility of infection from these recently emergent strains. To distinguish the new and increasingly common V. parahaemolyticus strains from classic ones, ORF8 is a useful genetic marker.