Study population
The study population included medical students at the Faculty of Medicine in the Galilee of Bar-Ilan University, in Safed, Israel. The Faculty has two academic classes: 4-year and 3-year tracks. The 3-year track students finished 3 years of pre-clinical medical education in medical faculties across Europe (such as Hungary, Italy, and Lithuania). Almost all of these students reported no patient encounters during their studies, as most of their education was lecture-based. The 4-year track students have graduated from various pre-med Israeli academic institutions. As no dormitories are available, most students live separately. The study was approved by the local Ethics Committees of Baruch Padeh Medical Center, Poriya, and Bar-Ilan University.
Sampling
Samples were collected three times for each participant during 2012–2014. Sampling was made by a sterile swab (COPAN, Italy) from the nostrils, independently by the participant. First sampling was performed towards the end of pre-clinical studies, with second and third samplings collected at 13 and 19 months post-initial sampling while students were rotating in clinical clerkships. All samples were kept at room temperature for a maximum of 24 h before being transferred to the microbiology laboratory.
Staphylococcus aureus identification
Microbiological examinations were performed at the Baruch Padeh Medical Center Microbiology Laboratory in Poriya, Israel. Primary culture was performed by striking technique in order to perform culture and colony isolation on a selective chromogenic growth medium (Chromagar MRSA/MSSA, hylabs, Israel). Confirmatory tests for SA included gram staining, catalase test, and rapid agglutination test for simultaneous detection of the fibrinogen affinity antigen (clumping factor), protein A, and the capsular polysaccharides (Pastorex™ Staph Plus, BIO RAD, France).
Susceptibility tests
Antimicrobial susceptibility tests were performed by disc diffusion method on Muller-Hinton agar (BD Diagnostics, Sparks, MD) in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines for rifampicin, vancomycin, erythromycin, gentamicin, cefotaxime, clindamycin, penicillin, oxacillin, mupirocin, fusidic acid, tetracycline, trimethoprim-sulfamethoxsazole (TMP-SMX), and cefoxitin [33]. MDR for MRSA was defined as resistance to at least three antimicrobial agents in addition to β-lactams.
Molecular characteristics
Typing of MRSA strains was performed at the National Staphylococcus aureus Reference Center, Central Laboratories, Israel Ministry of Health, Jerusalem, Israel.
PCR detection of mecA and PVL genes
PCR reactions in a total volume of 23 mL included 10.5 mL DDW, 12.5 mL PCR mix (ReddyMix, Tamar Laboratory Supplies, Mevaseret Zion), MecA1 and MecA2 primers or Luk-PV-1 and Luk-PV-2 primers (1 mM each). Thermocycling conditions were set at 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s, followed by a final step of 72 °C for 2 min [34, 35].
Detection of SCCmec types
SCCmec types were detected using the method of Zhang et al. [35]. PCR reactions were performed in a total volume of 20 mL, including 9.5 mL DDW, 9.5 mL PCR mix (ReddyMix, Tamar Laboratory Supplies, Mevaseret Zion), and 1 mL primer mix with a final concentration as recommended [35]. Thermocycling conditions were set at 95 °C for 5 min, followed by 11 cycles of 94 °C for 45 s, 65 °C for 45 s, 72 °C for 1.5 min, followed by 26 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 1.5 min, and a final step of 72 °C for 10 min [35].
spa typing
spa typing was performed in a 20 mL reaction including 9.5 mL DDW, 9.5 mL PCR mix (ReddyMix, Tamar Laboratory Supplies, Mevaseret Zion), and primers 1514R, 1113 F (4pmol each). Thermocycling conditions were set at 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 60 °C for 45 s, 72 °C for 90 s, followed by a final extension step of 72 °C for 10 min. Cycle sequencing was carried out using the BigDye Terminator v1.1 chemistry (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s protocol. Cycle sequencing products were purified by gel using Performa DTR according to the manufacturer’s recommendation. Electrophoresis was carried out on a 3730 × l Genetic Analyzer (Applied Biosystems) and the analysis was done using sequencing Analysis v.5.3.1 software (Applied Biosystems). spa typing analysis was performed using BioNumerics 7.5 software [36].
PFGE
Agarose-embedded (Lonza, USA) SA DNA was digested with SmaI (Fermentas, Lithuania) followed by gel electrophoresis in the CHEF MAPPER (Bio-Rad) system. Electrophoresis conditions were 14 °C, 0.5 × Tris-borate-EDTA buffer (Sigma, Switzerland), initial pulse 5 s, final pulse 50 s, 6 V, 18 h. Salmonella Braenderup H9812 restricted with XbaI (Fermentas, Lithuania) was used as a marker. PFGE restriction patterns were analyzed by the BioNumerics software (Applied Maths). The pulsotypes were compared using the band-based DICE similarity coefficient with 1% optimization and tolerance. The un-weighted pair group method with arithmetic mean (UPGMA) algorithm was used for cluster analysis [37].
Statistical analysis
Statistical analysis was performed using SPSS for windows version 21 (IBM Analytics). Proportion tests, Chi-square tests, and Wilcoxon signed ranks test were used for the univariate analysis using α = 0.05 and 95% confidence interval (CI). Statistical significance was based on p ≤ 0.05.