2.4.1. Phenotypic

All isolates confirmed to be *S. aureus* by PCR were subjected to antibiotic susceptibility testing to oxacillin (5 μg) [37] by disc diffusion test as well as growth on Brilliance MRSA II agar [38], to determine phenotypic resistance to methicillin. Inoculated plates were incubated at 37 ◦C for 24 h [38]. All isolates that tested positive on Brilliance MRSA II agar (blue to violet colonies) or resistant by oxacillin disc were considered to be presumptive MRSA.

#### 2.4.2. Molecular Confirmation of MRSA

Presumptive isolates from Brilliance MRSA II agar, as well as isolates that were phenotypically resistant to oxacillin, were further confirmed by PCR detection of the *mec*A gene (responsible for methicillin resistance) using specific primers (Table 1) as earlier described [20,39]. The *fem*A gene, a factor essential for methicillin resistance, was also evaluated [40] by PCR using specific primers (Table 1). A 25 uL reaction was set up consisting of 12.5 μL master mix, 0.5 μL forward primer, 0.5 μL reverse primer, 6.5 μL nuclease-free water and 5 μL of DNA. PCR was conducted using a T1000 Touch Thermal Cycler (Bio-Rad, Johannesburg, SA, USA). The cycling conditions used for confirmation of the *mec*A and *fem*A gene are shown in Table 1**.** The amplicons were separated using 1.5% agarose stained with ethidium bromide and visualized under a transilluminator (UVITEC Alliance 4.7, Bio-Active., Ltd., Bangkok, Thailand).



 genes.
