Phenotypic and molecular characterization of vibrio cholerae causing 2012 diarrheal outbreak in kathmandu

Abstract

Cholera is characterized by perfuse watery diarrhea which removes essential body fluid, nutrients leading to a condition of dehydration and malnutrition and often proved to be fatal if not treated. It is one of the common among diarrheal diseases and is emerging as a huge health burden in developing countries like Nepal. Etiological agent of Cholera namely Vibrio cholerae, in current scenario is transforming itself in to various forms and variants gaining novel pathogenic attribute and multidrug resistant property creating hurdles in clinical management. This research primarily aims to analyze genetic diversity, characterize V. cholerae strains prevalent in 2012 Kathmandu diarrheal outbreak according to various phenotypic, serological, genetic markers and pulsotyping. In this research probable Cholera samples were collected from diarrheal patients and V. cholerae strains were identified and screened using recent, precise microbiological, serological and molecular assays. Thus screened strains were further subjected to phenotypic tests, antibiotic susceptibility test, PCR based genotyping assays and Pulsed Field Gel Electrophoresis. Additionally each patient was directly interviewed for clinical history as well demographic information. Among selected highly probable Cholera samples (N=72) V. cholerae O1 (32%), V. cholerae non O1 (7%) and non specified pathogens (61%) were found. Similarly antibiotic susceptibility profile of selected strains identified that all isolated V. cholerae O1 strains were 100 % resistant to Trimethoprim/Sulfamethoxazole (SXT) ,Nalidixic Acid (NA); Streptomycin and 100 % susceptible to Erythromycin; Gentamicin; Tetracycline, Ampicillin ,Azithromycin and Ciprofloxacin. Combined result from phenotypic and PCR based genotypic assays showed that V. cholerae O1 strain possessed Cholera toxin (ctxAB),rfbO1 O1 specific antigen, toxin coregulated pilli allele specific to ET Tor biotype tcpA , Regulatoryelements of Ctxф namely rstRETand rtxC, Toxin linked cryptic plasmid (tlc). Another genotyping test based on Double Mismatch Amplification Mutation Assay (DMAMA-PCR) assay also identified a variation pattern in ctxB gene ,which categorized these strains in to ctxB genotype 7 and revealed that these strain carried point mutation at 20 aminoacid position of ctxB sequence which caused change in code for amino acid (Histidine→Asparagine,(58th nucleotide change C →A)). Further, PFGE pulsotype analysis of selected strains and control strain N19691 predicted 100% correlation among them and 90% correlation with N19691, hence suggesting all strains to be of seventh pendamic EI Tor ancestry and are of same clonal origin. Keywords: Cholera, EI Tor, antibiotic susceptibility, ctxB genotype, PFGE, clonal