vanA harboring enterococcal and non-enterococcal surface water isolates monitored by an oligonucleotide DNA probe /

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2016

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Nakipoğlu, Mustafa

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Untreated wastewaters and treated effluents even after final disinfection inhabitate antibiotic resistant bacteria and resistance genes before they are released into surface waters. A correlation between resistant bacteria with antibiotic resistance genes in surface waters has been found. Of particular interest are vancomycin-resistant enterococci (VRE) harboring vanA gene that confers their high-level resistance to glycopeptide antibiotics including teicoplanin. Therefore, in this study, river water samples were analyzed to investigate vancomycin- and teicoplanin-resistant bacterial isolates harboring vanA gene. Out of 290, 18 surface water isolates displayed resistance to both antibiotics. These glycopeptide resistant enterococcal and non-enterococcal isolates, identified by 16S rRNA sequencing, were found to harbor vanA gene with sequence similarities of 58 to 100%. The presence of D-alanine-D-lactate ligase encoded by vanA gene was also shown for all vancomycin- and teicoplanin-resistant isolates through western blotting. The fate of vanA gene in surface waters provides information on the exposure and potential threats of those bacteria for the environment and human health. For this purpose, a 25-mer-oligonucleotide DNA probe based on the 909 bp BamHI-ClaI fragment from Enterococcus faecium plasmids pVEF1 and pVEF2 was also prepared by using Vector NTI Express software. Under the hybridization stringency conditions of 46 °C, 55 % formamide and 0.020 M NaCl, designed vanA probe appeared to be highly specific to vanA-positive Enterococcus faecalis tested. In situ fluorescent hybridizations under the same stringency conditions were also used to monitor the river water samples by using fluorescent microscopy. The results indicated that newly designed vanA-targeted oligonucleotide DNA probe was highly specific and quantitative tool for monitoring vancomycin- and teicoplanin-resistant bacteria in surface waters. Due to reuse of treated wastewater, antibiotic resistant bacteria and resistance genes are being introduced into surface waters and possess human health risks. Therefore, surface waters are not only hot spots for vanA harboring enterococcal isolates but also non-enterococcal ones due to gene dissemination and require special scientific consideration.

Subject Keywords

Vancomycin., Glycopeptides., Oligonucleotides., Sewage

URI

http://etd.lib.metu.edu.tr/upload/12619768/index.pdf
https://hdl.handle.net/11511/25487

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

M. Nakipoğlu, “vanA harboring enterococcal and non-enterococcal surface water isolates monitored by an oligonucleotide DNA probe /,” M.S. - Master of Science, Middle East Technical University, 2016.