Resistance-guided isolation and identification of antibiotic-resistant bacteria from bull semen: exploring alternatives to antibiotics in semen extenders.

Bacterial contamination in bull semen adversely affects semen quality and fertility, with rising antibiotic resistance highlighting the need for effective alternatives. This study focused on isolating antibiotic-resistant bacteria from Sahiwal bull semen using antibiotics like penicillin and streptomycin, followed by identification through 16S rRNA gene sequencing. The predominant resistant bacteria included Bacillus cereus (50%), Proteus mirabilis (17%), Enterococcus spp. (17%), Pseudomonas aeruginosa (5%), Klebsiella variicola (5%), and others like Niallia circulans. From these, Bacillus cereus (Gram-positive) and Proteus mirabilis (Gram-negative) were selected for further analysis. The antimicrobial efficacy of zinc oxide nanoparticles (ZnO NPs) and a cyclic hexapeptide (c-WFW AMP) was evaluated as potential antibiotic alternatives (ATAs). ZnO NPs inhibited both bacteria at 60 µg/ml, while c-WFW AMP was effective at 4 µg/ml for Bacillus spp. and 6 µg/ml for Proteus spp. The minimum bactericidal concentrations were 80 µg/ml for ZnO NPs and 6 µg/ml for c-WFW AMP against both strains, indicating potent bactericidal action. These findings support the use of ZnO NPs and c-WFW AMP as promising ATAs in semen extenders to control bacterial contamination and reduce reliance on conventional antibiotics in bull semen preservation.