Environmental Microbiology (2003) 5(8), 706–710 © 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology1462-2920Blackwell Publishing Ltd, 20035 8706710Short CommunicationPathogenic V. parahaemolyticus in the Seto Inland SeaM. J. Alam, S.-I. Miyoshi and S. Shinoda Received 3 February, 2003; accepted 10 March, 2003. *For correspondence. E-mail alammj30@hotmail.com; Tel. and Fax (+81) 86 251 7967. Brief report Studies on pathogenic Vibrio parahaemolyticus during a warm weather season in the Seto Inland Sea, Japan M. Jahangir Alam,* Shin-Ichi Miyoshi and Sumio Shinoda Department of Environmental Hygiene, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan. Summary Vibrio parahaemolyticus is a potentially pathogenic bacterium, occurring naturally in estuarine and marine environments throughout the world. The inci- dence of this organism in an aquatic environment depends upon many ecofactors. Sea water and organic material were collected during the warm weather season from a coast of the Seto Inland Sea, Japan, and analysed to determine V. parahaemolyti- cus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by polymerase chain reaction (PCR), using iso- lated DNA from enrichment culture of the samples. About 99% of samples were positive for V. para- haemolyticus with densities of 3 to > 1400 cells per 100 ml of water or 10 g of organic samples by the most-probable-number (MPN)-PCR technique, but only 76.6% were positive by the conventional MPN culture technique, with densities ranging from 3 to > 1400 cells per 100 ml of water or 10 g of organics. Furthermore, the tdh and trh genes were positive in 41.5% and 8.5% of samples, respectively, by the MPN- PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN culture proce- dure. The difference in detection between the MPN culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detec- tion sensitivities and other factors. The aquatic environment has been implicated as a prin- cipal reservoir of many pathogenic vibrios. Vibrio para- haemolyticus is a well-known human pathogen, causing gastroenteritis through consumption of raw or under- cooked seafood (Honda and Iida, 1993). This bacterium has been found to account for about 20–30% of all food poisoning cases in Japan. It is also reported to be an important agent of travellers’ diarrhoea. Thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH), encoded by the tdh and trh genes, respectively, are the best recognized pathogenic factors of V. para- haemolyticus (Honda and Iida, 1993). Such pathogenic factors have rarely been detected in environmental V. parahaemolyticus isolates. The reason for this discrep- ancy in virulence between clinical and environmental iso- lates is not well understood. Vibrio parahaemolyticus and many other vibrios become abundant during warmer months in the sea waters of Japan (Miyamoto et al., 1962; Chowdhury et al., 1990) and other parts of the world but, in the cold weather months, they are rarely isolated (Miyamoto et al., 1962; Kelly and Stroh, 1988; Chowdhury et al., 1990). The ecology and cultura- bility of V. parahaemolyticus present in food and environ- mental samples have not been fully understood. Environmental conditions impose a variety of stresses on bacteria, which in turn have developed different strategies to survive, including adaptation and transmission. There- fore, the incidence of a species in a water environment is dependent upon many ecofactors. In environmental and food samples, vibrios may be injured by various physico- chemical factors and, therefore, may not grow in selective and/or non-selective culture media. Furthermore, in unfavourable environmental conditions, formation of via- ble-but-non-culturable (VNC) cells has been reported for more than 30 bacterial species (Xu et al., 1982; Colwell, 1993; Oliver, 1995; Oliver et al., 1995), but the role that these cells might play in the environment and in patho- genesis has so far not been resolved (Colwell, 1993; Bogosian et al., 1998; Barer and Harwood, 1999). Cells in the VNC state are unable to form colonies on plates but are still alive and capable of metabolic activity (Colwell and Huq, 1994). Moreover, VNC cells may remain poten- tially pathogenic (Colwell and Huq, 1994), and can resume active growth when environmental conditions are restored (Colwell, 1993; Oliver, 1995; Whitesides and Oliver, 1997). Detection of such cells requires alternative methods.