Coagulase gene polymorphism of Staphylococcus aureus isolated from clinical and sub-clinical bovine mastitis in Isfahan and Chaharmahal va Bakhtiari provinces of Iran

A total of 86 S. aureus isolates from Chaharmahal va Bakhtiari (n = 30) and Isfahan (n = 56) were isolated from 360 clinical and sub-clinical bovine mastitic milk samples (140 samples from Chaharmahal va Bakhtiari and 220 samples from Isfahan). Milk samples were inoculated onto blood agar base (Merck) supplemented with 5% defibrinated sheep blood. Isolates were identified by conventional methods, including Gram staining, colony morphology, hemolysis, tests for catalase, clumping factor, DNase, acetoin, and anaerobic fermentation of mannitol.

Bacterial DNA extraction was carried out according to Rodrigues da Silva and da Silva (2005). Bacterial cell lysate was prepared from 0.5 ml of overnight trypticase soy broth cultures. After centrifugation at 12,000×g for 10 min, bacterial pellets were washed with 500 μl of Tris–hydrochloride–ethylene diamine tetra acetic acid (TE) buffer (10 mM Tris–HCl, pH 7.5, and 1 mM EDTA) and centrifuged again. The pellets were re-suspended in 200 μl of TE buffer (pH 7.5) with 15 U of lysostaphin (2 mg, Sigma) per milliliter and incubated at 37°C for 1 h. Next, 15 μl of proteinase K (20 mg/ml; Fermentas) was added, and the suspension was incubated at 56°C for 1 h. The suspension was then heated at 95°C for 15 min to inactivate the proteinase K. An equal volume of phenol–chloroform was added and the mixture centrifuged at 12,000×g for 10 min. The upper phase was carefully transferred into another Eppendorf tube and mixed with two volumes of 95% ethanol and stored overnight at –20°C. The mixture was then centrifuged at 12,000×g for 5 min. The DNA pellet was washed with ice-cold 70% ethanol, re-centrifuged, and dried by tube inversion. The DNA was suspended in 100 μl of sterile TE, quantified in a spectrophotometer (at 260 nm), and kept frozen at –20°C.

PCR was performed in a 50-μl reaction mixture containing 2 μl of template DNA (approximately 500 ng/μl), 5 μl of 10 × PCR buffer (750 mM Tris–HCl (pH 8.8), 200 mM (NH₄)₂SO₄, and 0.1% Tween 20), 200 μM of each of the four deoxynucleotide triphosphates, 1 U of Taq DNA polymerase (Roch Applied Science), and 50 pmol of each primer (COAG2: CGA GAC CAA GAT TCA ACA AG; COAG3-AAA GAA AAC CAC TCA CAT CA). The PCR reaction was performed in a thermocycler (Eppendorf, Mastercycler® 5330, Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) using the following cyclic conditions: initial denaturation at 95°C for 2 min, 30 cycles of 30 s each with denaturation at 95°C, 2 min annealing at 58°C, 4 min extension at 72°C, and a final 7 min extension at 72°C (Goh et al. 1992).

The PCR products were digested with AluI (Fermentas) for restriction analysis. For this aim, 12.5 μl of PCR products was mixed with 10 U of enzyme and 10 × 1.5 μl restriction buffer and then incubated at 37°C overnight (Aslantas et al. 2007).

The PCR products and the digested fragments were separated in 1% and 3% agarose gel (Fermentas), respectively, with 10 mg/ml aqueous solution of ethidium bromide (Fermentas) and then were photographed under ultraviolet illumination. The 100-bp marker (Fermentas) was used as a size standard for the calculation of the sizes of the coa and AluI-generated coa fragments.