Susceptibility to environmental stresses. Bacteria that arehighly resistant to environmental stress may survive better in
the environmental substrate and have a greater chance tospread. Thirty of the recently isolated O3:K6 strains from Taiwan,Korea, and Japan were examined for their susceptibilities to different environmental stresses and compared to those of other reference strains (Table 1). The average D values at 50°C, at pH 4, and at 0.2% NaCl were about 3 to 4 min, 6 to 11 min, and 80 to 120 min for different categories of strains, respectively. About 5 to 12% of the strains of O3:K6 and non-O3:K6 serovars isolated before 1996 survived at 4°C for 6 days (Table 4). No particularly stress-resistant strain was discerned. V. parahaemolyticus is a vulnerable species and is usually rapidly inactivated at 48 and 55°C, at salinity below 0.5%, or at pH 4.0 (2). When the strains were grouped according to their location of isolation and compared, some groups differed significantly. For example, the recently isolated O3:K6 strains from Japan had a significantly lower low-temperature survival rate while having a higher resistance to mild acid and lowsalinity
treatments. When all the recently isolated O3:K6strains as a group were compared with other strains, the group
of recently isolated O3:K6 strains basically did not show any specific trait that would enhance its survival in the environment. ***There should be undiscovered characteristics in these O3:K6 strains that enable their pandemic spread.
As compared by the arbitrarily primed PCR method, the O3:K6 strains isolated between 1982 and 1993 differed from
those isolated after 1996; those recently isolated O3:K6 strains belonged to a unique clone (19). Clonality of the new O3:K6 strains was also confirmed by the analysis of the toxRS sequence, which differs from those of the old O3:K6 strains at at least seven base positions within a 1,346-bp region. A new PCR method targeted for the toxRS sequence was developed to detect this new group of O3:K6 strains (16). While analyzing these O3:K6 strains recently isolated in India, Bag et al. observed the presence of a major ribotype, R4. By another PFGE method using NotI digestion, they showed the presence of one clone (1). The PFGE procedure used in this study was more discriminative, and the recently isolated O3:K6 strains could show eight different patterns. In conclusion, our PFGE analysis has demonstrated that the
new O3:K6 strains isolated after 1996 in Taiwan, Korea, Japan, and India form a genetically closely related group that is distinct from the O3:K6 and non-O3:K6 strains isolated before 1996. The results provide a line of evidence that the new clone of O3:K6 has caused a pandemic spread. However, the new O3:K6 strains and the earlier isolates did not differ in biological characteristics such as TDH production and susceptibilities to antibiotics and environmental stresses. A future study is needed to find out what other characteristic(s) is associated with the pandemicity of the clone.