Our study aims to investigate some tetracycline resistance genes in isolates of Staphylococcus aureus that collected from ewes milk that collected randomly. Sixty Milk samples are taken from ewes randomly and submitted to culturing on blood agar then submitted to PCR methods for final confirming detection of Staphylococcus aureus by using specific primers designed for (Glpf) gene depending on NCBI and Primer3 then making sensitivity test for tetracycline then make PCR for detection of some tetracycline resistance genes. Our results showed positive samples for Staphylococcus aureus was 14/60 (23.3%) by using PCR. Besides, all Staphylococcus aureus isolates were submitted to the tetracycline sensitivity test (disk diffusion method (Kirby‐Bauer) by tetracycline. Moreover, showing the percentage of tetracycline resistance isolates was 8/14(57.1%), and tetracycline sensitive isolates were 6/14 (42.8 %). Also, our results showed the percentage of tetracycline resistance genes tet(L) and tet(k) were 9/14 (64.2%) and 7/14 (50%) respectively, and percentage of isolates that carry gene tet(L) and tet(K) together was 2/14 (14.2)% by using polymerase chain reaction technique. Our study concluded ewes milk that contaminated by Staphylococcus aureus at a high rate, and the non-response to treatment by tetracycline is occurred due to present tetracycline resistance genes such as tet(L) and tet(k), were causes treatment failure by tetracycline.

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