Identification of methicillin-resistant Staphylococcus aureus in horses using conventional and molecular techniques

Staphylococcus aureus is a coagulase-positive, Gram-positive, coccal bacterium. It is one of the leading causes of both skin and invasive infections. It plays an important role in diagnostics and treatment due to its ability to develop resistance to antimicrobial drugs. Methicillin-resistant Staphylococcus aureus or MRSA is an important nosocomial pathogen in both humans and animals due to its resistance to all β-lactam antimicrobial agents. Colonization of MRSA in horses poses a great concern. This is considered an important risk factor for development of staphylococcal related diseases in horses admitted to veterinary hospitals. Colonized horses can also be a source of zoonotic MRSA infections. Methicillinresistant Staphylococcus aureus detection based on a PCR reaction is commonly used and various types of PCR-based assays were developed to assist in early detection of MRSA. The main aim of the study was to compare the currently used conventional microbiological techniques with a published multiplex PCR assay targeting the mecA, spa and pvl genes for the rapid and accurate identification of MRSA in horses admitted to the Onderstepoort Veterinary Teaching Hospital, University of Pretoria. A total of 50 isolates, which consists of isolates from horses and their immediate environment, were included in the study of which 94% (n=47) were shown to be infected with methicillin resistant Staphylococcus aureus using conventional microbiological techniques. The remaining three gave inconsistent results. Their isolates were obtained, DNA was extracted and subjected to the multiplex PCR assay. The PCR results indicated that both the mecA and spa genes were present in 72% © University of Pretoria 2 (n=36) of these isolates, indicative of MRSA strains. In 20% (n=10) of the isolates, only the spa gene could be detected; suggesting that these cannot be classified as being methicillin resistant. The pvl gene could not be detected in any of the isolates tested. A total of four isolates (8%) yielded results that were inconsistent with being MRSA using molecular identification. Overall there was a good correlation between genotypic analysis by PCR and phenotypic determination using S. aureus species identification and susceptibility testing methods. The multiplex PCR assay had a detection limit of 2.18 x 10 colony-forming units (cfu)/ml. This detection limit is higher compared to other published molecular identification techniques used for Staphylococcus aureus but sensitive enough for the accurate detection of MRSA in overnight cultured isolates. Results suggest that the current PCR assay could be used as a supplementary diagnostic method in the routine diagnosis for rapid, sensitive, and specific detection of S. aureus and its associated antibiotic resistance genes in equine samples. © University of Pretoria