Bacterial strains
A selection of 56 MSSA isolates strains from cases of bovine mastitis clinical or subclinical cases of dairy bovine (n=118) in Oran were performed by bacteriological methods and by different molecular techniques (Maldi Tof and MLST, spa typing). The milk samples were collected all over Oran region during a year. The strains were selected as pure cultures obtained after mastitic milk cultivation on agar plates. Screening for pathogenic S. aureus was done by performing a determination of toxic profile by PCR in real time [4]. andvarious biochemical assays, including the coagulase test, ²-hemolysis, DNase test and growth on S. aureus agar medium. Methicillin resistance was tested on columbia agar with 5.0% salt (Oxoid) with BBL Sensi-Disc 1 ¼g Oxacillin discs (BD)⁴.

Mixed glycerol stocks of S. aureus cultures were prepared by suspending several loopfuls of bacteria taken by sweeping across the plate in 1.5 ml of saline with 200 ¼l of 45% glycerol for storage at “80°C. Taking a sweep across the plate rather than picking a single colony for glycerol stocks allowed us to maintain the genetic diversity of S. aureus strains in the sample for later analyses⁸.

Identification Maldi Tof (Matrix Assisted Laser Desorption Ionisation Time Of Flight):

The mass spectrometry allowed us to determine and confirm the identification of the species of Staphylococcus aureus strains⁸.

DNA extraction
To extract bacterial DNA requires the preparation of a DNA suspension from a bacterial strain⁸. Prepare a suspension of 4 to 5 colonies in 1 ml of sterile distilled water. Centrifuge 1 minute at 10000-12000 rpm and then remove the supernatant, Add 200 .µl of InstaGene Matrix to the pellet and incubated for 30 min at 56°C. High Speed Vortex for 10 seconds then places the tube at 100°C for 8 minutes. Collect the supernatant and store at -20°C until PCR [8].

DNA isolation was done following the mini preparation method. Isolated DNA samples were checked for purity and quantified by spectrophotometric analysis. The samples were then resolved on agarose gel (0.8%) with 1 ml of template DNA mixed with 3 ml of loading dye (xylene cyanol + bromophenol blue) and electrophoresed at 100 volts for 1 h. DNA samples showing intact bands were used for PCR amplifications.

Fig. 1. Results of different types divided according to the spa isolation season during the study

PCR amplifications of four virulence genes was done as described by Peacock et al¹⁴, Specific primers (Table 1) were used to amplify the four virulence genes in all S. aureus isolates. To amplify the, 25 ml of reaction mixture was made containing 20 ng of template DNA, 100 ng of primers genes, 160 mM of dNTP mix, 1.25 U Taq polymerase, 1X Taq buffer, and 0.5 mM MgCl2. All 67 isolates were amplified individually for all four genes using the specific primers, with 32 cycles of denaturation at 95°C for 1 min, annealing at 50°C for icaA, 45°C for sdrE, and 55°C for hlg and cna for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 2 min, on a DNA engine (PTC-100, MJ Research, USA). PCR products were resolved on 1.0% agarose gel at 60 volts for 2.5 h. Gels were stained with Ethidium Bromide solution (0.5 mg/ml) and documentation was done using the Gel Doc system (Bio-Rad).
Table (1):
Characteristics of 56 Staphylococcus aureus isolates recovered from bovine mastitis cases.

PVL: Planton-Valentino Leukocidin, MSSA: Methicillin sensible Staphylococcus aureus
+: Characteristics positives -: Characteristics negatives

Genotyping data production and analysis
Spa-typing
A staged spa-typing protocol was developed to enable identification of multiple-strain colonization on a large-scale. The polymorphic X region of the protein A gene (spa) was amplified with primers 1095 F: 52 -AGACGATCCTTCGGTGAGC-32  and 1517R: 52 -GCTTTTGCAATGTCATTTACTG-32  PCR reactions consisted of 0.25 mM dNTPs (Qiagen), 0.5 U of GoTaq Flexi DNA Polymerase (Promega), Colorless GoTaq Flexi Buffer, 2.5 mM of Magnesium Chloride and 0.25 ¼M of primers in a volume of 10 ¼l. PCR conditions were 94°C for 2 min, 35 cycles each of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s, and a final extension at 72°C for 5 min. PCR products were purified using Agent court AM Pure XP beads (Beckman Coulter)[8].

Fig. 2. Revelation of spa typing on electrophoresis gel

Samples were sequenced with the same primers as used in PCR. Sequencing reactions used BigDye v3.1 sequencing mix (Applied Biosystems) and were cycled using 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 2 min. Products were purified with Agent court Clean SEQ beads (Beckman Coulter) and separated on an ABI 3730 DNA Analyzer (Applied Biosystems)[8].

Chromatograms were analyzed using Ridom Staph Type v2.0.3 software (Ridom GmbH). The relationships between spa-types were investigated using the BURP clustering algorithm incorporated into Ridom Staph Type.

Identification of rearrangements in spa-gene
A small proportion of isolates did not yield clean sequence traces with the original primers indicating the presence of rearrangements in the spa-gene. To identify possible rearrangements, primers spa-3 F: 52 -ATAGCGTGATTTTGCGGTT-32  and spa-3R: 52 -CTAAATATAAATAATGTTGTCACTTGGA-32  were used to amplify the whole spa-gene. As some isolates failed to amplify even with this extended set of primers, an alternate forward primer, spa T3-F: 52 -CAACGCAATGGTTTCATCCA-32  binding upstream from 1095 F was used together with standard reverse primer 1517R. Primer spaT3-F binds to a part of sequence encoding an IgG-binding domain of the spa-gene that is repeated five times in the gene. Due to presence of multiple binding sites for the spaT3-F primer within spa-gene, only the reverse primer (1517R) was used for sequencing¹⁰.

MLST typing
Highlighting and sequencing of a fragment of seven genes present in Staphylococcus aureus providing classification after sending the database ‘’Mlst.net’’. The seven genes are: arc (carbamate kinase), aroE (shikimate dehydrogenase ), glpF (glycerol kinase ), gmk (guanylate kinase ), pta (phosphate acetyl transferase), tpi (triosephosphate isomerase), yqiL (acetyl coenzyme A acetyl transferase). The spa tandem repeats amplification ands and condition used for MLST were performed as previously described [10]. for a subset of strains selected as representative isolates of the different spa lineages. Sequence types (STs) were assigned through the MLST database (www.mlst.net). Isolates were classified in clones by combining ST and spa lineage.

Fig. 3. Revelation of MLST typing on electrophoresis gel

Fig. 4. Different clonal complexes of methicillin-sensitive Staphylococcus aureus those are isolated in dairy farms