Nisha. A. R.VANISHREE2020-07-232020-07-232018http://krishikosh.egranth.ac.in/handle/1/5810149684‘Antimicrobial resistance’ is a global epidemic issue nowadays. In the recent past, role of efflux pumps in development of resistance is intervened so as to overcome bacterial resistance to wide class of chemotherapeutic agents. In the current study an attempt has been made to investigate the ability of ‘piperine’, a phytochemical to overcome the efflux pump (AcrAB-TolC) mediated resistance in E. coli isolates from bovine mastitis. Antibiotic disc diffusion assay was carried out to determine the minimum inhibitory concentration (MIC) for enrofloxacin, ciprofloxacin (fluoroquinolones) tetracycline and oxytetracycline (tetracyclines) in the presence of piperine (20 ppm, 40 ppm, 60 ppm, 80 ppm, 100 ppm). Further, piperine alone at various concentrations was also tested for its antibacterial activity if any. Gene expression of acr A, acr B and tol C genes were studied by reverse transcription and real time PCR. Biofilm formation was examined in the resistant colonies in the presence of each of the above antibiotics and piperine by congo red assay. In silico analysis to determine piperine with acr B of efflux pump was carried out by using autodock software. The study revealed that piperine exposure in combination with enrofloxacin, ciprofloxacin, tetracycline or oxytetracycline resulted in dose-dependent significant (p < 0.05) increase in ‘zone of inhibition’ in the disc diffusion assay. Further, piperine reduced the MIC of enrofloxacin, ciprofloxacin, tetracycline or oxytetracycline against E.coli isolates from mastitis and showed synergistic action at a concentration of 100 ppm. A 16 fold reduction in MIC of enrofloxacin and ciprofloxacin, 5.2 fold reductions in the MIC of tetracycline and 4.6 fold reduction in the MIC of oxytetracycline were observed upon coexposure at concentration of 100 ppm. Gene expression studies indicated down regulation of expression of acr A, acr B and tol C efflux pump in E. coli isolates resistant to enrofloxacin, ciprofloxacin and oxytetracycline, while tetracycline inhibited expression of only acr B. Piperine downregulated acrB gene in both fluoroquinolones and tetracyclines resistance E.coli isolates. Relatively piperine showed more efflux pump inhibitory activity in ciprofloxacin resistant E. coli isolates. However fluoroquinolones and tetracycline tested failed to prevent biofilm formation in E. coli isolates. In silico studies revealed that piperine bind with acr B protein of efflux pump of E coli. To conclude, piperine co-exposure with enrofloxacin, ciprofloxacin, tetracycline or oxytetracycline potentiate the antibacterial activity against E. coli isolates from mastitis through inhibition of AcrAB-TolC pump. Thus, piperine can be used as an adjuvant with fluoroquinolones or tetracyclines against resistant E.coli isolates causing mastitis.ennullThesis