PCR detection of three antibiotic resistance genes and preliminary safety assessment of local probiotic bacteria for bovine mastitis control

Abstract

Antibiotic resistance among bacteria has been identified as a global threat for public and animal health. Mastitis is a widespread disease among dairy cows and treatment mainly includes antibiotics as bacteria being the most prevalent etiological agents. The emergence of resistant bacteria is a challenge for effective control of bovine mastitis due to frequent therapeutic failures. Therefore, exploring alternatives to reduce antibiotic usage is crucial to mitigate the risk of resistance development. Probiotics may inhibit the growth of pathogenic bacteria, potentially reducing the need for antibiotic use against mastitis. Probiotic characterization is required to ensure their safety before application. The present study aimed to screen probiotic bacteria for selected antibiotic resistance genes and evaluate their safety for external usage in cattle. Isolates of bacteria taken from fermented coconut water, curd, and rice water, bacterial colonies were grown on MRS (De-Man Rogosa Sharpe Agar) agar and 15 colonies consisting of 6, 5 and 4 strains from each of these products respectively, were further proceeded to extract DNA using a commercial kit (Promega, USA). PCR was then performed to detect sul1, bla SHV and tet(A) genes representing resistance to antibiotics sulfonamide, β – lactams and tetracycline respectively, using established protocols. PCR Products were visualized on 1.5% agarose gels after electrophoresis and the images were analyzed by Gel documentation system. For safety evaluation on dairy cattle, three probiotic strains which demonstrated in vitro antagonistic activity to bacterial mastitis pathogens were selected. MRS broths containing log 7 CFU/mL were kept in contact with the skin of six healthy animals and observed for 72 hours for any hypersensitivity reactions or behavioral changes. Two control animals were tested applying normal saline instead of probiotics. The results revealed that all 15 cultures did not amplify PCR products of 822 bp, 768 bp and 577 bp corresponding to sul1, bla SHV and tet(A) genes. Positive controls produced the respective gene amplifications. In safety evaluation, no adverse reactions were observed in tested cattle within 72 hours. Tested probiotic strain from coconut water and two strains from curd did not result in hypersensitivity reactions such as swelling, reddening or thickening. The coat condition remained unchanged. Cattle continued their normal behavior. Posture and movements of cattle were not affected. Feeding and drinking behavior and social interactions were not changed. There were no observed differences between the performance of the control cattle and tested cattle. In conclusion, the absence of examined antibiotic resistance genes and the clinical safety of the probiotic cultures indicated their suitability as beneficial product formulation that can be applied externally to antagonize causative agents of mastitis in dairy farming. Potential transfer of the particular antibiotic resistant genes from these probiotic cultures to other bacteria could be excluded. Further research is in progress for additional characterization of the probiotic strains to develop ecofriendly products.