Classical ctxB gene in Vibrio cholerae O1 and O56 serogroups from Kerala, South India

Vibrio cholerae is the causative agent of the life-threatening diarrhoeal disease cholera. Cholera toxin (CT), the major enterotoxin produced by V. cholerae, is responsible for profuse diarrhoea. The major biotypes of V. cholerae O1 (El Tor and classical) are differentiated on the basis of some phenotypic and genotypic traits (Safa et al., 2010). The genotypic traits are based on variations in tcpA, rstR (Safa et al., 2010) and hlyA gene sequence (Rivera et al., 2001). The tcpA, hlyA and rstR genes encode a major pilin subunit, a cytotoxic haemolysin and a repressor (which regulates the replication of phage DNA), respectively. Strains other than O1 and O139 (collectively called non-O1/nonO139) are ubiquitously distributed in aquatic environments and are not associated with epidemic cholera (Islam et al., 1994). However, sporadic cases of diarrhoea and extraintestinal infections caused by non-O1/non-O139 strains have been reported widely (Sharma et al., 1998; Cheng et al., 2004; Restrepo et al., 2006). Recently, many new genotypes of El Tor strains have emerged carrying variants of CT (Bhuiyan et al., 2009; Joshi & Albert, 2009; Taneja et al., 2009). These variants have spread to countries of Asia and Africa including India and Bangladesh (Safa et al., 2010). In the present study, the biotypeassociated phenotypic and genotypic characteristics of recently isolated O1 and O56 strains from southern Kerala, India, were analysed. The ctxB genes from all the strains were amplified by using Phusion High-Fidelity DNA Polymerase (NEB) and ctxB-F and ctxB-R primers (Olsvik et al., 1993). The purified PCR products were ‘A’ tailed and subsequently ligated to the pGEM-T Easy vector and sequenced on both strands using T7 and SP6 primers with the BigDye Terminator kit (Applied Biosystems). Four strains were analysed in the present investigation. Strains A199 and A217 were isolated from Vembanad Lake, Allapuzha district, whereas O1 strain A880 was isolated from a drinking water source in Allapuzha district, Kerala. MCV09, a clinical O1 strain, was isolated from a hospitalized patient admitted to Medical College Hospital, Trivandrum, Kerala. All the test strains were positive in the Voges–Proskauer test and for b-haemolysis of sheep erythrocytes and exhibited resistance to polymyxin B (Table 1). The O1 strains showed El Tor-specific tcpA and hlyA genes. However, O56 serogroup strains exhibited the El Tor-specific hlyA gene and a variant allele of the tcpA gene (GenBank accession no. EU362122). The strains of serogroup O56 showed both the classical and El Tor type of rstR gene whereas O1 strains showed only the El Tor type of rstR. This indicates the presence of two different copies of CTX prophages in O56 strains. Unexpectedly, ctxB gene sequencing in all test strains revealed 100 % identity to ctxB of classical strain 569B (GenBank accession no. U25679). Hence according to the new genotyping scheme developed by Safa et al. (2010), the O1 and O56 serogroup strains investigated could be classified into ctxB genotype 1 since they possess the ctxB1 allele.