## 2 Materials and Methods
### Microbiological Analyses  
Microbiological analyses were performed on the obtained milk samples as follows:  
* Detection of _Staphylococcus_ sp. [31; 32; 33]: 1 mL of milk samples was enriched in 9 mL of Mueller-Hinton broth supplemented with 6.5% NaCl (Oxoid Ltd., Basingstoke, UK). Incubation at 37 \({}^{\circ}\)C for 20 h followed and 1 mL was enriched in 9 mL of Tryptone Soya broth with 10% NaCl and 1% sodium pyruvate (HiMedia Labs, Einhausen, Germany). Incubation followed at 35 \({}^{\circ}\)C for 20 h and then the enrichment broth was inoculated onto Baird-Parker agar (Oxoid Ltd., Basingstoke, UK) containing 30% egg yolk with 1% tellurite (Oxoid Ltd., Basingstoke, UK) and mannitol salt phenol red agar (Merck, Darmstadt, Germany) plates. Another quantity of milk sample was inoculated into 5.0% Sheep Blood Agar [SBA, (Becton Dickinson, Sparks, NV, USA)] plates. Anaerobic incubation followed for the SBA plates at 37 \({}^{\circ}\)C and were evaluated after 48 and 72 h. Taxonomic classification of the potentially _Staphylococcus_ sp. isolates followed by using routine microbiological procedures such as: colony morphology, Gram staining, catalase and oxidase reactions and coagulase test [free coagulase (Coagulase plasma--EDTA, bioMerieux, Lyon, France) and bound coagulase production (Staphylase test, bioMerieux, Lyon, France)]. After the basic microbiology, the _Staphylococcus_ sp. isolates were processed by using VITEK-2 (bioMerieux, Lyon, France), according to the manufacturer's instructions. Finally, the strains were then frozen at -80 \({}^{\circ}\)C in BHI broth (Oxoid Ltd., Basingstoke, UK) with 20% glycerol. * Enumeration of _E. coli_ was carried out according to ISO 16649-2:2001 [34]. A 1-mL aliquot of each sample and series of 10- fold dilutions were set using peptone tryptone water (Condalab, Madrid, Spain) and were transferred into petri dishes, to which Tryptone Bile X-glucuronide medium (TBX; Oxoid, Hampshire, UK) was immediately added. Incubation at 44 \({}^{\circ}\)C for 24 h followed, and then the presumptive colonies were enumerated. The quantification limit was 1 CFU/mL. The primary identification was carried out using API 20 E (bioMerieux) and the identification was completed using the Vitek\({}^{\copyright}\)2 system (bioMerieux, Marcy I'Etoile, France). Antimicrobial Susceptibility Testing  
The reference strains _S. aureus_ ATCC 29213 and _E.coli_ ATCC 25922 were used for quality control and were the first passage from ATCC's freeze-dried vial. In the present study all stains which were identified as _S. aureus_ or _E. coli_ isolates were screened for susceptibility using the disk diffusion test which was performed and evaluated according to the guides of Clinical and Laboratory Clinical Institute (CLSI) [35]. The zone of inhibition was recorded in millimeters and results were interpreted [35]. * For _S. aureus_ were used; ampicillin (20 \(\upmu\)g), erythromycincin (15 \(\upmu\)g), tetracycline (30 \(\upmu\)g), clindamycin (2 \(\upmu\)g), ciprofloxacin (10 \(\upmu\)g), ceftiofur (30 \(\upmu\)g, 3 rd generation cephalospororin), rifampicin (5 \(\upmu\)g), teicoplanin (30 \(\upmu\)g), pefloxacin (10 \(\upmu\)g), amoxicillin-clavulanate (30 \(\upmu\)g), streptomycin (30 \(\upmu\)g), gentamicin(10 \(\upmu\)g), cefuroxime (20 \(\upmu\)g), norfloxacin (NX-10 \(\upmu\)g), ceftaxone (25 \(\upmu\)g), halidixic acid (NA-30 \(\upmu\)g), vancomycin (VA-30 \(\upmu\)g), sulphamethoxazole-trimethoprim (30 \(\upmu\)g), chloramphenicol (30 \(\upmu\)g) and Cef (cefoxitin, 30 \(\upmu\)g). All _S. aureus_ isolates were evaluated for growth using the Oxacillin salt agar screen susceptibility test (OSAS). The phenotypic identification of methicillin-resistant _S. aureus_ (MRSA) was performed as follows: Coagulase-positive _S. aureus_ (CoPS) isolates were first identified phenotypically by placing them on Mueller-Hinton agar plate containing 4% NaCl and 6 ug/mL of oxacillin.