UTTAR PRADESH JOURNAL OF ZOOLOGY

Background: Bovine mastitis remains one of the most significant diseases affecting dairy animals, causing major economic losses through reduced milk production, treatment costs, and culling, with subclinical mastitis often remaining undetected. Among the emerging mastitis pathogens, multidrug-resistant Escherichia coli mastitis has gained importance due to its increasing antimicrobial resistance and the presence of multiple resistance genes that complicate treatment and pose public health concerns.

Aim: The present study investigated bovine mastitis and associated antimicrobial resistance patterns in Escherichia coli organisms from samples of 400 dairy animals.

Study Design: Milk samples were screened using the California Mastitis Test (CMT) and cultural examination with special reference to E. coli organisms and analysis of phenotypic and genotypic characterization for antimicrobial drug resistance.

Place and Duration of Study: Animals were screened from Pune and Satara districts of Maharashtra and CMT positive samples were processed in the Veterinary Microbiology Laboratory at KNP College of Veterinary Science, Shirwal.

Methodology: CMT positive milk samples were processed for bacterial isolation and antibiotic resistance by disc diffusion technique. Further the E. coli confirmed by molecular detection with PCR were processed for antibiotic resistance studies with MIC and genotypic identification of resistance genes.

Results: Total 210 (52.50%) cows were confirmed positive comprising 33 (15.71%) clinical and 177 (84.29%) subclinical mastitis cases. Bacteriological analysis revealed 86 Staphylococcus spp. as the predominant pathogen (40.95%), followed by 33 (15.71%) other Gram-positive bacteria, while E. coli accounted for 22.85% (48/210) and 43 (20.48%) other enterobacterials. Antimicrobial susceptibility testing with disc diffusion technique of bacterial isolates demonstrated highest sensitivity to Amikacin (88.09%) followed by Ciprofloxacin (82.38%) and Enrofloxacin (62.38%), whereas highest resistance was observed against Ampicillin/Sulbactam (71.42%) followed by Gentamicin (69.52%). Amongst the phenotypically identified 48 E. coli isolates using molecular confirmation with 16S rRNA PCR yielded a specific 401 bp amplicon. These isolates showed highest susceptibility for Amikacin (89.58%) followed by Cefotaxime/Clavulanic acid (83.33%) and Ceftriaxone/Tazobactum (79.16%), while highest resistance was observed against Gentamicin (68.75%) and Ampicllin/Sulbactum (68.75 %). Minimum inhibitory concentrations (MICs) of antibiotics against E. coli isolates showed a great variation with Resazurine micro-broth dilution method in which highest number of isolates showed MIC at >0.781 or lower dilutions for Quinolones, Amikacin, Amoxicillin/Clavulanic acid and Ceftriaxone/Tazobactum, whereas, a some of the isolates did not show MIC at >25 dilution for most of the antibiotics used. A high level of antimicrobial resistance was observed in E. coli isolates, with 53.33% being multidrug-resistant; the isolates harboured resistance genes blaTEM (70.83%) followed by tetA (64.58%), aac(3)-iv (62.5 %), qnrA (52.08 %) and blaSHV (8.33 %) indicating resistance to β-lactams, tetracyclines, quinolones, and aminoglycosides. Multiple resistance gene combinations were observed in E. coli isolates in bovine mastitis, with a high prevalence of two different resistance genes in 04.16-45.83% isolates, three resistance genes in as high as 31.25 % isolates and four resistance genes in 16.66% isolates.

Conclusions: The study highlights the high prevalence of subclinical bovine mastitis in Maharashtra, driven by multidrug-resistant Escherichia coli carrying multiple resistance genes, underscoring the urgent need for rational antibiotic stewardship and continuous AMR surveillance in dairy herds.