2.1. Clinical isolates
The study was carried out at hospitals (Khartoum state, in Soba teaching hospital and Military hospital). The process was accomplished in Medical Laboratory College at Khartoum University, Department of Microbiology and Molecular Biology. The study population includes patients who attended to hospitals for wound infections or post-operative surgical site infections. Questionnaires were used for patients to get their socio-demographic data. Forty-six wound swabs were collected from patients; records/information was anonymized and de-identified prior to analysis.
2.2 Bacterial Identification
After the samples were received to the laboratory, they inoculated on Mannitol salt agar (Oxoid CM0085B), each isolated strains of Gram positive cocci were sub cultured on nutrient agar (Oxoid CM0 003B) and incubated at 37ºC over night for biochemical reactions. S.aureus were identified by fermentation of mannitol, colonial morphology, Gram stain, Catalase test and coagulase test using conventional methods.
2.3. Molecular Characterization
2.3.1. PCR Amplification of 16S rRNA Gene
The genomic DNA of S.aureus isolates were extracted from nutrient agar plates by guanidine chloride method as described previously by Alsadig et al., . Then, PCR was carried out using universal oligonucleotide primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) And 1492R (5′ TACGGTTACCTTGTTACGACTT-3′) . The reaction mixture was included 1 μl of bacterial DNA, 22μl of δdH2O, and l μl each primer in a final reaction volume of 25 μl. This mixture was added to the PCR master mix (GoTaq, Promega, USA) following the manufacturing guide. Then, run with a thermal cycler (SensoQuest, Germany) as follows: 30 cycles were performed in a thermocycler, each cycle has three steps of denaturation (95°C for 1 min), annealing (54°C for 1 min), extension (72° C for 3 min) and final extension time of 72°C for 5 min . Amplified products were analyzed by conventional electrophoresis, Bands were determined using an Imagemaster VDS image analysis system (SCIE- PIAS VISION U.K). The Sizes of the amplified products using universal primer were 1500 bp which suggesting that bands is 16srRNA gene.
2.3.2. Sequencing of 16S rRNA gene
DNA purification and Standard Sanger sequencing was conducted for ten isolates which were packaged according to the National Health Research Ethics Committee authorization and following the instructions of the sequencing company (Macrogen Inc. Seoul, Korea). The sequences was submitted in NCBI: https: // www.ncbi.nlm.nih.gov, with the accession numbers: from MT154222 to MT154231.
2.3.3. Bioinformatic Analysis
Nucleotide sequence isolates were visualized using Finch TV program ( version 1.4.0). In order to search for nucleotide sequences similarity, Genbank databases were used by online program nucleotide BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Closely related sequences were explored from NCBI and subjected to multiple sequence alignment by BioEdit program (version 7.2).[18, 19]. Gblocks was used to estimate the quality of each sequence, edit and eliminate poor quality sequences. The Neighbor-Joining phylogenetic tree was carried out by MEGA software using default parameters (http://www.megasoftware.net/index.html)[22, 23].