Mannitol salt agar

Nutrient broth (LENNOX), Nutrient agar medium (SIGMA-ALDRICH), Luria Bertani (LB) broth (LENNOX), McConkey agar (SIGMA-ALDRICH), mannitol salt agar (OXOID), skim milk agar (NEOGEN), 3% KOH, starch (SIGMA-ALDRICH), Gram staining kit (MERCK), bacteriological peptone (OXOID), hydrogen peroxide, Kings B medium (SIGMA), Wattman No. 1 disc, oxidase reagent, phenol, 0.5% picric acid (SIGMA-ALDRICH), Kovacs reagent, 2% Sodium carbonate (MERCK), Nessler’s reagent (SIGMA-ALDRICH), dilute iodine, Lead (III) nitrate (Sigma- Aldrich), cadmium nitrate tetrahydrate (Sigma- Aldrich), chromium (III) nitrate (Sigma- Aldrich). Analytical balance (SARTORIUS GMBM GOTTINGEN, Germany), digital weighing machine (Jeweler Precision Balance Model: DH-V600A)steam sterilizer (autoclave), 37ºC incubator (MMM group Medcenter Enrich tungsten GmbH), 37ºC shaker (Irmeco GmbH, Germany), Laminar flow (ESCO Prod Model; EQU/03-EHC; Serial # 2000–0052), sterile dissecting pins, Sterile distilled water, dissecting box, gloves, dissecting board, sterile bottles, 70% ethanol, 500 ml beakers, micropipette, 250 ml conical flasks, test tubes, bacteriological wire loop, Petri plates, glycerol, glass rod, glass slides, coverslips, spirit lamp, microscope, and toothpicks.

Meat samples were homogenized in a stomacher using a sterile plastic bag. This involved 25 g of each meat sample and 225 mL of buffered peptone water with 6.5% NaCl. Petrifilm™ Staph Express count plate (3M™, St. Paul, MN, USA), a modified chromogenic Baird-Parker medium, was used as a selective and differential medium for S. aureus detection. The Petrifilm™ Staph Express disk contained toluidine blue O and DNA. DNase-positive organisms degrade DNA that reacts with toluidine blue O to form pink zones. This allowed for a distinction between S. aureus colonies and other staphylococci. Single S. aureus colonies were transferred to mannitol salt agar (Neogen; Lansing, MI, USA). All S. aureus isolates were identified and confirmed using the classical method, which included Gram staining, catalase testing, and coagulase testing (PROLEX™, Neston, UK). All S. aureus isolates were further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) (Bruker, Bremen, Germany) using the 70% formic acid protein extraction method.

The frozen isolates were recovered by culturing on a selective culture media, mannitol salt agar (Neogen company, UK), and produce bacterial colonies. The isolates were confirmed as S. aureus through tests for mannitol fermentation, gram stains and biochemical tests, and catalase and tube coagulase activity [10 ]. All isolates that were used were further tested for antibiotic susceptibility against oxacillin, and were confirmed to be MRSA. An agar dilution assay was performed on Mueller-Hinton agar (MHA) (Neogen, UK) containing oxacillin (6 μg/mL) and 4% NaCl. A bacterial suspension equivalent to a 0.5 McFarland standard was cultured on MHA plates and incubated under aerobic conditions overnight at 35 ˚C. The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [11 ].