Isolation and Molecular Confirmation of *S. aureus*

Sand samples were vigorously hand shaken in Phosphate Buffered Saline (PBS), where a ratio of 2 g of sand to 80 mL of PBS was used [20,33]. Both sand and water samples were enriched in tryptone soy broth and incubated at 37 ◦C for 24 h, followed by sub-culturing on mannitol salt agar (MSA), and further incubated at 37 ◦C for 24 h. Presumptive *S. aureus*, identified by the fermentation of mannitol (yellow colonies) were purified on nutrient agar. Presumptive isolates were stored in 25% glycerol at −80 ◦C.

Polymerase chain reaction (PCR) was used for confirmation of *S. aureus* as previously described [20]. DNA was extracted using the boiling method where 2 mL of overnight pure Nutrient broth cultures were transferred to sterile eppendorf tubes and centrifuged at 13,000 rpm for 3 min. The supernatant was discarded and cells re-suspended in 200 μL sterile distilled water. The cell solution was then heated at 100 ◦C in an Accu dri-block (Lasec, SA) for 10 min, followed by centrifugation at 13,000 rpm for 2 min to pellet the cells [34]. The supernatants were transferred to clean, sterile tubes and used directly as templates for PCR assay or stored at −20 ◦C for subsequent use.

A method previously described by Maes [35] was used for identification of *S. aureus*, based on the detection of a specie-specific nuc-gene. *S. aureus* ATCC 25923 was used as a positive control. Each 25 μL PCR reaction mix constituted 12.5 μL of 2X PCR master mix, 0.5 μL each of both reverse and forward primers (Table 1), 6.5 μL nuclease-free water and 5 μL of template DNA. PCR was conducted in a T1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA). Cycling conditions are shown in Table 1. The PCR products were separated by agarose gel electrophoresis in 1% agarose, stained with ethidium bromide. A 100 bp DNA ladder was included in each run.
