EFFECT OF PIPERINE AS AN EFFLUX PUMP INHIBITOR FOR FLUROQUINOLONES AND TETRACYCLINES IN ESCHERICHIA COLI ISOLATES FROM MASTITIS
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Date
2018
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD
Abstract
‘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.
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