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

Abstract

Vibrio parahaemolyticus O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown 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. Mukhopadhyay, 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 has 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 at least at 7 base positions within a 1,346-bp region. A new PCR method targeted 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 examined by the new PCR method. The tdh-positive and trh-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results. The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their toxRS sequences were 100% identical to that of the new O3:K6 clone. The results suggest that these strains may have 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 PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.

FIG. 1

Target positions of the PCR primers used to amplify the toxRS sequences and the essential base difference in the toxRS sequence between the old O3:K6 strain group and the new O3:K6 clone. The bases described are those in the coding strand, except that the bases in the GS-VP.1 and GS-VP.2 primers are those incorporated in the actual oligonucleotides. Numerals indicate the base positions that correspond to those in the reported toxRS sequence of strain AQ3815 (Lin et al. [7] and GenBank accession no. L11929).

FIG. 2

Results of AP-PCR assay for O3:K6 strains isolated in Bangladesh. The results obtained with primer 1 are shown in A1 and B1, and those obtained with primer 2 are presented in A2 and B2. (A1 and A2) Lanes: 1 and 13, molecular size markers (phage λ DNA digested with HindIII); 2 and 14, molecular size markers (phage φX174 DNA digested with HaeIII); 3 and 12, strain VP47 (a control strain isolated in Calcutta in 1996 [16]); 4 through 9, Bangladeshi strains isolated in 1980; 10 and 11, Bangladeshi strains isolated in 1981. (B1 and B2) Lanes: 1 and 15, molecular size markers (phage λ DNA digested with HindIII); 2 and 14, molecular size markers (phage φX174 DNA digested with HaeIII); 3 and 13, VP47; 4 through 10, Bangladeshi strains isolated in 1996; 11 and 12, Bangladeshi strains isolated in 1997.

FIG. 3

Results of AP-PCR assay for representative O3:K6, O4:K68, and O1:KUT strains isolated in various geographical locations and from international travelers. Results obtained with different primers are shown in different panels as indicated. For all panels, lane designations correspond to the experimental strain numbers listed in Table 2. M1, molecular size markers (mixture of phage λ DNA digested with HindIII and phage φX174 DNA digested with HaeIII).

FIG. 4

GS-PCR results for representative strains of serovars O3:K6, O4:K68, and O1:KUT, isolated in various geographical locations and from international travelers. M2, molecular size markers (phage φX174 DNA digested with HaeIII). Lane numbers correspond to the experimental strain numbers listed in Table 2.