Molecular and Phenotypic Characterization of Antimicrobial Resistance in Gram Negative Bacteria Recovered from Subclinical Mastitis

This study was undertaken to estimate the prevalence and antimicrobial susceptibility of gram negative bacteria (GNB) recovered from dairy cows with subclinical mastitis (SCM) with detection of β-lactamase encoding genes, sulphonamide encoding gene and intergron classes by polymerase chain reaction (PCR). In total, 180 cow’s milk samples were collected from three different dairy farms. Milk samples were subjected to physical examination and subsequently to California mastitis test (CMT). Subclinically infected milk samples were subjected to bacterial isolation and GNB isolates were subsequently tested for their antimicrobial susceptibility to 11 antimicrobial agents by disc diffusion method. All GNB isolates were investigated for the presence of integron classes, β-lactamase encoding genes (blaTEM, blaSHV, blaCTX) and sulphonamide encoding gene (sul1) gene by using PCR.The overall prevalence of subclinical mastitis (SCM) in lactating dairy cows was 55.56% (100/180). Bacteriological analysis revealed the presence of gram negative bacteria in 35% (35/100) of the tested samples. E. coli was found to be the most prevalent organism (10) followed by Klebsiella pneumonia (5), Proteus mirabilis (4) Enterobacter aerogenes (3), Serratia liquefaciens (3), Providencia rettgeri (2), Proteus vulgaris (2),Citrobacter freundii (2), Citrobacter diversus (1), Enterobacter agglomerans (1), Enterobacter cloacae (1) and Yersinia enterocolitican (1). Antimicrobial susceptibility results revealed that the highest resistance was observed for amoxicillin, clindamycin, trimethoprim/sulfamethoxazole, vancomycin and rifapime. All bacterial isolates revealed a multidrug resistance (MDR). By PCR 100%, 80% and 60% of gram negative isolates harbored blaTEM, blaSHV and blaCTX , respectively. Meanwhile, sul1 was detected in 80% (28/35) of the tested isolates. Class 1 intergron was detected in 91.4% (32/35) and Class 2 intgrons could not be identified in all the tested isolates. This study indicates the need for effective control measures to challenge the increase in occurrence of subclinical mastitis. The overall proportion of antimicrobial resistance was high. As a result, this study suggests that the risk of dissemination of resistance gram negative bacteria through the food chain.