Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 5 2026 10 05 799 806 EN Zeinab Abiri Department of Health & Food Quality Control, Faculty of Veterinary Medicine, Ardakan University, Ardakan, Iran; Biology and Animal Reproduction Science Research Institute, Ardakan University, Ardakan, Iran Samira Ghorbani Department of Health & Food Quality Control, Faculty of Veterinary Medicine, Ardakan University, Ardakan, Iran; Biology and Animal Reproduction Science Research Institute, Ardakan University, Ardakan, Iran Mohammad Sadegh Safaee Firouzabadi Biology and Animal Reproduction Science Research Institute, Ardakan University, Ardakan, Iran; Department of Clinical Sciences, Faculty of Veterinary Medicine, Ardakan University, Ardakan, Iran 2025 08 19 2026 09 20 Background and Objectives: In mammals, milk provides the nutrients required for growth and development, including proteins, fats, carbohydrates, vitamins, and essential minerals. Donkey milk has recently attracted increasing interest as an alternative to cow’s milk, particularly for infants with cow’s milk protein allergy, due to its favorable nutritional composition. However, limited information is available on the microbiological quality of donkey milk based on molecular confirmation of Staphylococcus aureus and the detection of antimicrobial resistance genes, particularly in Iran. The present study assessed the microbiological quality of raw donkey milk through conventional bacteriological culture, PCR confirmation of Staphylococcus aureus, antimicrobial susceptibility testing, and molecular detection of the mecA and blaZ resistance genes. Materials and Methods: Between January and April 2023, a total of 50 raw donkey milk samples were collected and subjected to conventional bacteriological examination. Presumptive Staphylococcus aureus isolates were identified by culture-based methods and confirmed by PCR targeting the 23S rRNA gene. PCR assays were also performed to detect the coa, blaZ, and mecA genes. Antimicrobial susceptibility testing was conducted using the CLSI disk diffusion method with ten antimicrobial agents. Results: Of 12 bacterial isolates, 8 (16%) were confirmed as S. aureus. The blaZ gene, was present in all isolates (100%), while mecA, was absent. Antibiotic susceptibility testing showed variable resistance among isolates, with notable resistance against penicillin, with all isolates showing resistance (100%). The second highest resistance rate was observed for trimethoprim- sulfamethoxazole, where 25% of the isolates showed resistance. Conclusion: These findings indicate the presence of β-lactam resistance determinants despite the absence of molecular evidence of methicillin resistance. The study provides baseline data on the occurrence and antimicrobial resistance characteristics of S. aureus in raw donkey milk in Iran and highlights the value of combining phenotypic methods with PCR for microbiological surveillance of dairy products. https://ijm.tums.ac.ir/index.php/ijm/article/view/5712 https://ijm.tums.ac.ir/index.php/ijm/article/download/5712/1940