A total of 200 nasal swabs were collected between April 2017 to December 2018 from two different communities. First hospital samples (n=83) included physicians (n=7), nurses (n=12), radiologists (n=8), laboratory medical specialists (n=8), ECHO (n=1), medical residents (n=1), hospital admins (n=6), clinical nutrition (n=1), internship students (n=35) and medical research assistants (n=4) at King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. Second community samples (n=117), we concentrated on academic faculties (n=10), admins (n=36), and students (n=71) at King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Riyadh, Saudi Arabia. The exclusion for hospital were patients, and personnel who worked part-time at KAMC; for the community were students who were in direct contact with patients, and those with incomplete records were excluded from this study.
Specimen collection and transport
A dry polyester swab was inserted into the naris, parallel to the roof of the mouth, and left in situ for some seconds. Specimens from each nostril were obtained with an identical swab. The collected nasal swabs were transported to the lab research as soon as possible. If not were kept at 2-30oC until transported to the lab. If the process of collected nasal swabs could not be performed within 36 hr, they were stored in the refrigerator at 2-8oC for a maximum of five days. The collection of nasal swabs was daily during the working day for up to 3 months.
Isolation and identification of coagulase-positive staphylococcus
Initial screening and identification of CoPS isolates were performed according to the standard laboratory protocols. Briefly, the collected nasal swabs were streaked directly on mannitol salt agar (MSA), and S. aureus isolates were tested for methicillin resistance using a standard oxacillin salt agar screening plate procedure and cefoxitin susceptibility as indicated by the Clinical and Laboratory Standards Institute (CLSI) . For confirmation, chromogenic agar was used for rapid MRSA screening since this agar had high sensitivity for MRCoPS (>98%) . Methicillin-resistant and methicillin-sensitive staphylococcus strains were included as controls. Confirmation of methicillin-resistance was done by inoculating a 10 L aliquot of a bacterial suspension with 3 X 106 CFU/mL (McFarland turbidity no. 1) on Mueller-Hinton agar (MHA) supplemented with calcium (50 mg/L), magnesium (25 mg/L), sodium chloride (4%) and oxacillin (4 g/mL). The growth of at least one well-defined colony on these plates incubated at 30oC for 48 hr was suggestive of MRSA. The methicillin-resistance strain was confirmed by microdilution in Muller-Hinton broth supplemented with cations and sodium chloride, using a final inoculation of 7.5 x 104 CFU. MRSA was identified with a minimal inhibitory concentration (MIC) of oxacillin >8 g/mL, and those with a MIC <1g/mL were considered as methicillin-sensitive staphylococcus (MSSA). A MIC of oxacillin between >1g/mL and <8 g/mL was defined as a methicillin-intermediate staphylococcus (MISA) .
Antimicrobial susceptibility testing
Antimicrobial susceptibility of isolated coagulase positive staphylococcus towards 7 antimicrobials was determined by the agar dilution method in Mueller-Hinton agar according to the Clinical and Laboratory Standard Institute (CLSI) guidelines  and the results were evaluated after incubation at 35oC for 24 hr. The discs were penicillin (P, 1 μg), cefoxitin (FOX, 30 μg), vancomycin (VA, 30 μg), clindamycin (CC, 2 μg), sulfamethoxazole/trimethoprim (SXT, 25 μg), rifampin (RA, 25 μg), and quinupristin (SYN, 15 μg). A fresh subculture of isolates was prepared on MSA and incubated at 37°C for 18–24 hr. With the help of a wire loop, 4–5 well-isolated colonies of comparable look were picked and transferred into the tube of sterile traditional saline. The clumping of cells was avoided by emulsifying the inoculum inside the tube. The inoculums were adjusted to 0.5 McFarland (McFarland 0.5 equals approximately 108 CFU/mL). After adjusting the inoculum within 15 min, a sterile cotton swab was swayback into the inoculum. The swab was patterned over the complete surface of the MHA plate, rotating the plate just about 60° three times to make sure merging growth. Inoculation was completed by running the swab around the surface of the agar. Excess moisture on the agar surface could be absorbed before applying the antimicrobial discs. MSSA ATTC 25923, MSSA ATCC 229213, MRSA ATCC BAA1026, and Staphylococcus epidermidis ATCC 1228 were used as control strains.
Genetic characterization of methicillin-resistant coagulase-positive staphylococcus
Ten microliters of overnight Lysogeny broth culture in a tube were centrifuged at 8000 rpm for 10 min and the supernatant was discarded. The pellet was suspended in phosphate buffer saline using vortex then centrifuged again and the supernatant was discarded. Then the pellet was suspended in 2 mL of TE buffer and mixed by the vortex. A 500 L was transferred to 1.5 ml Eppendorf and 50 L of SDS (10%) and 5 L of proteinase K were added and left in the incubator for 1.5 hr at 37oC. An equal volume of (5 L) phenol, chloroform, isoamyl mixture was added, vortexed for 1 min, centrifuged for 4 min, and the aqueous supernatant containing DNA was transferred to another tube. A fifty-five L of 3 M sodium acetate and double volume of cold alcohol (100%) were added and mixed gently 2-3 times and centrifuged for 10 min. the supernatant was removed and the pellet was washed by mixing gently with 1 mL of 70 % alcohol and centrifuged for 1 min. The supernatant was removed, and the pellet was left to dry then suspended in 10 L of TE buffer and incubated for 30 min at 55oC and stored at -20oC .
Detection of the coa and spa genes
The identified CoPS strains subject to a polymerase chain reaction (PCR) for coa gene detection, using the following primers: forward (5'-CGA GAC CAA GAT TCA ACA AG-3') and reverse (5'-AAA GAA AAC CAC TCA CAT CA-3'), which design to amplify the 3’ ends hypervariable region containing 81-base pair (bp) tandem repeats of coa gene. The amplification reaction consisted of associate degree initial denaturation step at 94°C for 5min, followed by thirty cycles of denaturation at 95°C for 30 sec, tempering at 55°C for 45 sec, extension at 72°C for 2 min, followed by a final extension at 72°C for 7 min . The tested CoPS strains focus on PCR for spa gene detection using the following primers: forward (5'-ATC TGG TGG CGT AAC ACC TG-3'), and reverse (5'-CGC TGC ACC TAA CGC TAA TG-3') which scheme to amplify the polymorphic region that contains a variable number of 24 bp tandem repeats of the spa gene coding for protein A. Amplification reaction consisted of associate degree initial denaturation step at 94°C for 4 min, followed by thirty-five cycles of denaturation at 94°C for one min, annealing at 56°C for one min, extension at 72°C for 3 min, followed by a final extension at 72°C for 5 min . The PCR product and restriction digest fragments were detected by electrophoresis in 2% agarose gel. The pictures were pictured and analyzed in Gel Doc (BIORAD).
Detection of the mecA, SCCmecII, SCCmecVIa, and SCCmecVIb genes
All MRCoP isolates were tested for confirmation of the presence of the 310 bp PCR product of the mecA gene, using the following primers: forward (5'-TGG CTA TCG TGT CAC AATCG-3') and reverse (5'-CTG GAA CTT GTT GAG CAG AG-3'). The amplification reaction was carried out in 25 L volumes, under the following conditions: initial denaturation at 92°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 seconds, and extension at 72°C for 30 sec, followed by a final extension at 72°C for 3 min . Identification of the SCCmec complex was performed by a PCR screen for SCCmecII (287 bp) and SCCmeIVa (776 bp) and SCCmecIVb (1000 bp) using the DNA extract prepared above as a template. DNA samples were tested for the presence of the SCCmec complex, using the following primers: for SCCme II gene, forward 5'-CAA AAG GAC TGG ACT GGA GTC CAA A-3' and reverse 5'-CAA GTG AAT TGA AAC CGC CT-3'; for SCCmecIVa gene, forward 5'-TTT GAA TGC CCT CCA TGA ATA AAA T-3' and reverse 5'-AGA AAA GAT AGA AGT TCG AAA GA-3'; for SCCmecIVb gene, forward 5'-AGT ACA TTT TAT CTT TGC GTA-3'and reverse 5'-AGT CAT CTT CAA TAT CGA GAA AGT A-3'. Samples were heated for 1 min at 95oC, 30 cycles of [30 sec at 94oC, 60 sec at 50oC and 120 sec at 72oC], and 2 min at 72oC . The PCR product and restriction digest fragments were detected by electrophoresis in 2% agarose gel. The pictures were pictured and analyzed in Gel Doc (BIORAD).
Detection of the aae, aap, emb, IcaD, and PVL genes
DNA samples were tested for the presence of the 110 to 220 bp PCR product of aae gene, using the following primers: forward 5'-AAC AAA TTG ATA AAG CAA CG-3' and reverse 5'-GTT GTC TTT CCT TTA GTG TC-3'. Samples were heated at 95oC for 10 min before cycling for 45 cycles of 95oC for 10 sec, 5oC for 20 sec, and 72oC for 25 sec . The aap gene was detected using PCR with a specific primer: forward 5'-TCA CTAAAC AAC CTG TTG ACG AA-3' and reverse 5'-AAT TGA TTT TTA TTA TCT GTT GAA TGC-3'. Samples were heated at 94oC for 4 min, followed by 25 cycles of 94oC for 30 sec, 55oC for 30 sec, 72oC for 1 min, and 72oC for 5 min . All isolated MRSA were tested for the presence of the 455 bp PCR product of the embp gene, using the following primers: forward 5'-AGC GGT ACA AAT GTC AAT ATC-3'and reverse 5'-AGA AGT GCT CTA GCA TCA TCC-3'. Amplification reaction consisted of an initial denaturation step (96°C for 2 min), 40 amplification cycles of 94°C for 1 min, 55°C for 30 sec and 72°C for 1 min, with a final 10-min extension step at 72°C . DNA samples were tested for the presence of the 188 bp PCR product of IcaD gene, using the following primers: forward 5'-ATG GTC AAG CCC AGA CAG AG-3'and reverse 5'-CGT GTT TTC AAC ATT TAA TGC AA-3'. Samples were heated at 94°C for 5 min, followed by 50 cycles at 94°C for 30 sec, 55.5°C for 30 sec, 72°C for 30 sec, and 72°C for 1 min after the conclusion of the 50 cycles . The primer sequence for the PVL gene was described previously by McClure et al. . The primer was used for the co-amplification of PVL genes: forward 5'-ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A- 3' and reverse 5'-GCA TCA ACT GTA TTG GAT AGC AAA AGC- 3' procedure as indicated by the CLSI [18, 30]. Amplification reaction consisted of an initial denaturation step at 94°C for 3 min followed by 30 cycles of the following steps: denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and a final extension at 72°C for 5 min . The PCR products and restriction digest fragments were identified by electrophoresis in 2% agarose gel. The images were visualized and analyzed in Gel Doc (BIORAD).
Statistical analysis was performed with SPSS (v.22.0) statistics software. The variation between Healthy and healthcare communities by several variables. Observed differences were assessed for statistical significance by chi-square. A significant difference was statistically accounted for at a p-value of <0.05.
The Institutional Review Board (IRB) at King Abdullah International Medical Research Centre (KAIMRC) granted permission to conduct the project after reviewing the ethical aspects of the proposal (File#3190/2015).