A Method for the Simultaneous Detection of Vibrio cholerae Strains and Drug-Resistant Genes in their Genome by Real-Time PCR

Resumo

A method was developed for the simultaneous detection of V. cholerae strains and the presence of drug-resistant genes in their genomes by real-time PCR. Resistance to four antibiotics used for cholera treatment (tetracycline, trimethoprim, chloramphenicol and ciprofloxacin) was analyzed. The sensitivity of the panel was 1 × 10³ CFU/mL for pure cultures, simulated clinical material, and environmental samples. PCR efficiency was confirmed by the analysis of 60 natural V. cholerae strains isolated from patients and the environment in different years. It was established that all of the studied V. cholerae strains of the O1 serogroup El Tor biovar isolated before 1993 did not contain the tested drug-resistant genes. At the same time, 18 toxigenic clinical strains (90%) imported within 1993–2010 were characterized by multiple drug resistance; their genomes contained trimethoprim resistance genes (dfrA1) and chloramphenicol resistance genes (floR), and 11 strains additionally contained a tetracycline-resistant gene tetR. In addition, nontoxigenic clinical and aqueous V. cholerae strains carrying a ciprofloxacin-resistant gene, the qnrVC (qnrVC1) gene, have been detected in the last few years (Kalmykia, 2011–2013).