AYANSA KEBENESSA MEDEKSA2024-04-052024-04-052023-11Staphylococcus aureus is one of the most frequent organisms known to cause food poisoning and other diseases including humans and animals. Raw milk is known to be a major means of transmission of this pathogenic bacterium to humans andanimals. This study aims to isolate, characterize, and molecular detection ofantibiotic resistance genes for Staphylococcus aureus from cow milk samplescollected fromwolkite, Ethiopia. A total of 385 samples of cow milk were collected randomly. Each of the samplescollected was serially diluted, cultured on blood agar, and incubated at 37 oC for 24hr and preliminary screening for possible Staphylococcus species isolate was conducted. The secondary screening for possible Staphylococcus aureus such as coagulase test, fermentation of mannitol salt agar, anti-biotic sensitivity tests, and molecular characterization for Nicene and mecA genes were conducted. A total of 30 possible Staphylococcus species were isolated and 21 isolates were screened as S. aureus based on the advancedbiological identification software. The rest of the isolates were found to be positive for catalase, triple sugar iron tests, cocci in shape, positive to gram staining, non motile, and negative to vogues pressure tests. Some of them were coagulase positive and 60% were positive to mannitol salt agar. All tested isolates were resistant to penicillin, 53.9% were multidrug-resistant and most of them were sensitive to clindamycin. Under the PCR detection of antibiotic resistance genes, 70% of the possible isolates were found to have the mecA gene while 80% were positive for the nuc gene. These isolates were predicted to be S. aureus subsp. aureus, S. cohnii, S. intermedius, and S. vitulinusin which nearly all of them have belonged to S. aureus subsp. aureus following the methods of advanced biological identification software identification tools. In conclusion, the recent isolates obtained from raw milk harbored specific genes responsible for disease-causing that were reported and available in the genomic DNA of S. aureus throughout the history of evolutionenAntibiotics,PCR,Raw milkStaphylococcusZoonotic diseaseThesis