Environmental water is an important source for Vibrio cholerae, which is autochthonous to the aquatic environment, monitoring this bacterium in water is important for control of cholera. Vibrio cholerae represents an enormous public health problem around the world, especially in developing countries. One hundred samples were collected and selected. The samples were filtered and transferred to slants containing 2ml of alkaline peptone water, then subcultured on selective medium Thiosulphate Citrate Bile Salt Sucrose agar. All presumptive isolates were confirmed by using a series of biochemical tests including Oxidase test, Simmon Citrate test, DNase test, Indole test, Klingler Iron Agar (KIA) test, MacConkey agar test and motility. Confirmed Vibrio cholera strains were then screening for slide agglutination test by using commercially antisera polyvalent and monovalent O1 and O139 for determining strain serotype. The DNA extracted from pure culture and Polymerase Chain Reaction assay was used for molecular detection of Vibrio cholerae, a specific primer which designed according to ctxA gene sequences. This primer was detection and amplifying 241 base pairs of the ctxA gene. The resistance to antibiotics by Vibrio cholerae was determining by using thirteen standardized disc diffusion including Amikacin, Ceftriaxone, Ceftazidime, Gentamycin, Tetracycline, Streptomycin, Tobramycin, Cephotaxime, Nalidixic Acid, Norfloxacin, Cephalothin, Rifampicin, Cefixime. From one hundred water samples were detected, fifty-six samples were motile and positive for biochemical tests. Fifteen isolates confirmed as Vibrio cholera by Polymerase Chain Reaction (PCR) assay with primers designed for ctxA and 241bp band was observed. These fifteen isolates showed agglutination with polyvalent and monovalent O1 antisera, and two strains represented Ogawa from other strains that showed Inaba. The fifteen isolates exhibited an identical response to each antibiotic examined. They showed sensitive to all antibiotics except Amikacin, Streptomycin, Cefixime, Norfloxacin, Cephalothin. the aim of this study was determined the accurate method for detection of Vibrio cholerae in environmental water. In the current study, we found that the molecular method using Polymerase Chain Reaction performance using the ctxA gene-specific primers for detection of Vibrio cholerae was faster and accurate and specific.
Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples
