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Abstract Milk may serve as a vehicle for great varieties of food borne pathogens, especially staphylococci as one of the most important pathogen in milk. The pathological role of CoNS in bovine mastitis has increased inevitably and there is an urgent need to highlight the threat to both human and animal health, originating from CoNS. There are many genes engaged in CoNS pathogenicity and virulence during dairy cattle mastitis. The importance of CoNS in bovine mastitis depends on factors that facilitate evasion of phagocytosis. In this study was designed to throw spot lights upon CoNS isolated from clinical and sub clinical mastitic milk of cattle, studing its cultural, biochemical and In-vitro anti-microbial sensitivity for it with special reference to some virulence genes and antibiotic resistant genes. So the present study was performed on a total of Two hundred and five milk samples collected from lactating cows raised in small-scale farming systems located in the Monofeya province (Egypt). Clinical examination of cows and macroscopic evaluation of milk reveled forty clinical mastitis cases (19.51%) in which the udder was hot, red, painful, swollen and sometimes atrophied. Macroscopic changes in milk were recorded in all cows. Some animals suffered from systemic reaction (slightly increased temperature). California Mastitis Test (CMT) were applied on rest of milk samples (165) where 110 cows (53.66%) were diagnosed subclinically mastitic and 55 (26.83%) were negative. A total of 128 Staphylococcus spp. isolates were obtained from milk samples from cows diagnosed with clinical and subclinical mastitis. from these, 30 (23.44%) coagulase-positive staphylococci (CPS) and 98 (76.56%) coagulase-negative staphylococci (CoNS). The following CoNS species S.epidermidis, S.haemolyticus and S. saprophyticus were isolated from the milk of clinically and subclinically mastitic cows. S. saprophyticus (10.20%) constituted the highest percentage of CoNS species isolated from the milk of cows with clinical mastitis followed by S.haemolyticus (8.16%) and S.epidermidis (6.12%) whereas, S. epidermidis (34.69%) constituted the highest percentage of CoNS species isolated from the milk of cows with subclinical mastitis followed by S. saprophyticus (28.57%) and S. haemolyticus (12.24%). Concerning evaluation of CoNS virulence characteristics, our study revealed 20 CoNS isolates have hemolytic activity (alpha heamolysis), 6 isolates showed biofilm production on The Congo Red agar. Four CoNS isolates carried both characters (Heamolysis and biofilm production). The highest resistance rate of CoNS observed to penicillin G (84%) and Oxacillin (50%). The highest rate of sensitivity observed in this report were to Gentamicin (96%, for each), Ciprofloxacin and Vancomycin (84%, for each), followed by Novobiocin (80%). Of 15 phenotypically methicillin resistance strains selected at random, methicillin resistance was confirmed by amplification of the mecA gene for 11(73.33%) isolates. Concerning toxin detection, only 5 isolates (33.3 %) carried seb gene. Toxic shock syndrome toxin1 (tst) gene could not be detected in all tested isolates. With respect to biofilm gene icaD, six isolates were tested positive by PCR. MRCoNS need special attention because they can be implicated in cow mastitis. Furthermore, MRCoNS may constitute a reservoir of novel and diverse antimicrobial resistance genes and antimicrobial genetic structures that can be transferred to other pathogenic ba |