Antibiotic Resistance Phenotypes And Genotypes Of Staphylococcus Aureus Isolated From Milk Submitted To The Central Veterinary Laboratories

Abstract

Bovine milk provides an ideal environment for the spread and exchange of genetic material between bacterial species therefore constituting a significant reservoir for antibiotic resistant Staphylococcus aureus (S. aureus). In Kenya, studies on antibiotic resistance in S. aureus have primarily been limited to phenotypic methods yet there is lack of adequate information on their genotypes. This study determined the genetic and phenotypic antibiotic resistance patterns of S. aureus to antibiotics commonly used in the treatment of bovine mastitis. A total of 280 milk samples were obtained from Central Veterinary Laboratories (C.V.L) at Kabete after a questionnaire survey, which identified the commonly used antibiotics. Identification and confirmation of S. aureus was by morphological and biochemical tests. Phenotypic antibiotic susceptibility tests (ASTs) were done by Kirby Bauer’s disc diffusion method. S. aureus antibiotic resistance markers for tetracycline (tetM and tetK) and methicillin (mecA) were used to design primers. Polymerase chain reaction (PCR) was used to screen for the three resistance genes among phenotypically resistant isolates. The amplicons were then sequenced and analyzed. The questionnaire survey revealed that most respondents had administered tetracycline (38%) and penicillin (24%) antibiotics to the sick cows. A total of 108 (33%) samples, were positive for S. aureus. Phenotypic (AST) revealed that, of the 108 S. aureus isolates investigated, 11 (10.1%) and 6 (5.6%) were resistant to tetracycline and methicillin respectively. The other 6 (5.6%) isolates were resistant to both antibiotics. Overall, 15/17 (88%) of the phenotypically tetracycline resistant isolates revealed genetic resistance; 10/17 (59%) of the isolates had tetM gene, while tetK was detected in 5/17 (29.4%). Blast analysis showed that tetK and tetM genes had been identified in isolates from humans and animals. The resistance genes were harboured in mobile genetic elements such as plasmids and transposons respectively, suggesting a potential transfer of antibiotic resistance determinants. Phylogenetic analysis revealed that the sequences in this study clustered together, suggesting genetic relatedness. This study reaffirms the presence of phenotypic and genetic resistant S. aureus as a potential health hazard in Kenya. It demonstrates that tetM and tetK primers targeting resistance genes may be used for the detection of tetracycline resistance. However, the genetic basis of resistance towards methicillin could not be established, necessitating further studies to confirm the role of other genetic antibiotic resistance determinants. The combined use of genetic and phenotypic antibiotic resistance detection strategies will enable more precise and faster responses to future infections of S. aureus, and mitigate its impact.