Pandemic spread of an O3 : K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses

Vibrio parahaemolyticus O3:K6 strains responsible for the increase in the n umber of cases of diarrhea in Calcutta, India, beginning in February 1996 a nd those isolated from Southeast Asian travelers beginning in 1995 were sho wn to belong to a unique clone characterized by possession of the tdh gene but not the trh gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopa dhyay, S. Garg S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:3150-3155, 1997), Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study. Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the tdh and trh typing, and AP -PCR profiles). The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone. The clonality of the new O3:K6 strains was also confirmed by analysis of the toxRS sequence, which h as been shown to be useful for phylogenetic analysis of the members of the genus Vibrio. The toxRS sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 a t least at 7 base positions within a 1,346-bp region. A new PCR method targ eted to 2 of the base positions unique to the new O3:K6 clone was developed . This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier. One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also exami ned by the new PCR method. The tdh-positive and trh-lacking strains that be longed to the O4:K68 and O1:K untypeable serovars and were isolated in thre e countries and from international travelers beginning in 1997 gave positiv e results. The AP-PCR profiles of these strains were nearly identical to th ose of the new O3:K6 clone, and their toxRS sequences were 100% identical t o that of the new O3:K6 clone. The results suggest that these strains may h ave diverged from the new O3:K6 clone by alteration of the O:K antigens. In conclusion, this study presents strong evidence for the first pandemicity in the history of V. parahaemolyticus and reports a novel toxRS-targeted PC R method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.