Isolation and characterization of Oxytetracyclin resistance E. coli in Al Muthanna Veterinary hospital using Multiplex PCR

Different antibiotics have been used in the cure and control of many forms of infections in a variety of animal species. The non-prudent use of antibiotics in veterinary clinic can lead to develop of resistant forms of micro-organisms to antibiotics. This phenomenon is a natural and unavoidable and associated with routinely use of antibiotic in many species, including man. With amplified antibiotics usage over current decades, there has been an emerging alarm about the speeded development of antibiotics resistance in the environment. This study intends to investigate the antimicrobial resistance profile in different micro-organism for some antimicrobials agents that are frequently used to treat the animals in veterinary hospitals/ Al Muthanna province. Moreover, to determine the tetracycline and oxytetracycline resistance genes in Escherichia coli that revealed resistance for antibiotic disc diffusion test. A total of 53 clinical cases ( sheep, goat, cow and cat) were presented to Al Muthanna veterinary hospital with different clinical signs. A sterile fecal and nasal swabs, milk samples and 4 pus samples were collected from these cases. All samples were cultured in preliminary culture media consequently, Gram stain and biochemical tests were done for identification. Antibiotic susceptibility analysis were done for all bacterial isolates by standardized disc diffusion method. The isolates were tested with a panel of 6 antibiotic discs (tetracycline (T30), and oxytetracycline ( TE10), streptomycin ( S25), penicillin (P10), trimethoprim- sulphamethoxazoles (SXT25) and Chloramphenicol ( C10) discs. Totally 53samples including 9, 37,6 and 1 were collected from cow , sheep, goat and cat respectively. The total number of isolated bacteria were 53 isolates. Ten out of 53 bacterial isolates from different clinical cases ( sheep, goat, cow) were identified using biochemical tests as E. coli. These E. coli were tested by disk diffusion method to determine resistance patterns to 6 antimicrobial agents. Later on, Tetracycline and oxytetracycline resistance E.coli in disk diffusion were tested by PCR to identified the presence of tet (A) resistance gene. Ten E.coli isolates was resistance for tetracycline and oxytetracycline , these isolates were tested for identify the prevalence of tetracycline resistance genes tet (A). Nine out ten 9/10 (90 %) of E. Coli isolates were carried tetA gene. The tet (A) gene of strains were amplified by PCR with two sets of primers targeting tetracycline efflux gene (tetA). In conclusion, this study approved the presence of antimicrobial resistant (AMR) bacteria that isolated from different clinical cases refereed to Al Muthanna veterinary hospital. Moreover, this study investigated the spread of resistant E. coli in Iraqi animals, with special emphasis on tetracycline and oxytetracycline resistant E. coli. This study also iv approved the presence of tetA resistance gene that found in 90% of the tested resistance E.coli. The Authors recommend to do another future study that include high number of bacterial isolates and determine another antimicrobial genes responsible for transfer the resistance between other kind of bacteria. Besides, the authors recommend to use the antimicrobial wisely and prohibited providing this products without receipt.