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Hospital acquired-Staphylococcus aureus (HA-Staphylococcus aureus), particularly methicillin-resistant Staphylococcus aureus (MRSA), are an important source of nosocomial infections with high morbidity and mortality rates. Few reports showed that infections due to HA-Staphylococcus aureus in Saudi Arabia is increasing, particularly infections attributed to HA-MRSA. The study aimed to explore the prevalence and clinical characteristics of HA-Staphylococcus aureus for the first time in Medina, Saudi Arabia. A total of 1262 clinical samples of hospitalized patients were examined for the presence of Staphylococcus aureus through selective culturing on mannitol salt agar. Vitek Compact System and conventional methods were followed to confirm the isolates. Vitek Compact System tested the antimicrobial susceptibility of isolates whereas the standard PCR was employed to detect the genes encoding antimicrobial resistance (mecA and vanA) and virulence factors (tst, et, and LukS-PV). The overall HA-Staphylococcus aureus prevalence was low (6.58%, n = 1262) of which 84.34% (n = 83) were MRSA. Approximately, 57 samples of the 70 MRSA (81.5%) exhibited a multidrug-resistance (MDR) pattern. All the 83 HA-Staphylococcus aureus isolates were negative for the genes encoding toxic shock syndrome toxin, exfoliative toxin, and Panton-Valentine leukocidin. The study was conducted during the Covid-19 pandemic under partial lockdown, restricted hospitalization, and increased disinfection and infection control measures. Therefore, the low prevalence of HA-Staphylococcus aureus should be carefully interpreted and further multicenter investigations could reveal its true incidence in the city. The high prevalence of MDR HA-MRSA is alarming as it highlights inappropriate antibiotic prescriptions to counter staphylococcal infections. HA-Staphylococcus aureus investigated in this study might lack certain virulence factors. However, their MDR traits and invasive nature could worsen the situation if not properly handled. [ABSTRACT FROM AUTHOR]