Virulence genes of clinical Vibrio cholerae O1 isolates in Thailand and their ribotypes

Abstract

Objective: To determine virulence associated-genes and ribotypes of Vibrio cholerae epidemic strains isolated from cholera patients in Thailand. Method: A total of 240 V. cholerae El Tor, O1 strains, isolated from patients with cholera in Thailand during two different periods, i.e. 1999-2000 (200 strains; 193 Ogawa and 7 Inaba) and 2001-2002 (40 strains; all Inaba), were analyzed for the presence of virulence genes, namely ctxA, ctxB, zot, ace, toxR, tcpA, hlyA, nanH and ninT by PCR. For ribotyping, genomic DNA segments of the 240 strains and 10 reference V. cholerae strains isolated before 1999 from Thailand and elsewhere were digested with BglI endonuclease, subjected to a 0.8% agarose gel electrophoresis, blotted onto a nylon membrane and probed with enzyme-labeled Escherichia coli rRNA. The DNA bands were visualized by autoradiography. Results: Genes encoding the A and B subunits of CT, Zot, Ace, ToxR, TcpA, HlyA, NanH and NinT could be amplified from all of the 10 V. cholerae O1 reference strains and from 239 of the 240 studied isolates. One Inaba isolate of 2001-2002 gave only amplicons of toxR and hlyA. For ribotyping, the 10 reference strains revealed six different patterns designated A to F. None of the 240 strains isolated in Thailand during the two periods had the A-C, E and F ribotypes. The isolates of 1999-2000 revealed ribotype D and three other ribotypes, designated G, H and I. The majority of the isolates of 2001-2002 showed ribotype G. The remaining showed other new ribotypes, J and K. Conclusions: The clinical V. cholerae isolates of two epidemics from Thailand showed a sustained appearance of one epidemic V. cholerae clone, and a constant, but gradual and minor change in the genetic constituent of the other V. cholerae strains as indicated by the change of the ribotypes of the strains in the two study periods. Moreover, we found that a V. cholerae strain which cannot produce CT, Zot, Ace, TcpA, NanH and NinT can still cause symptomatic cholera.