Study design
This one-year prospective study was conducted from January to December 2015 at the National Cheng Kung University Hospital (NCKUH), Tainan, Taiwan, local fire department and long-term facilities. Healthcare providers who met the study criteria were offered the opportunity to participate in the study. The target population included out-of-hospital and in-hospital healthcare providers. Out-of-hospital providers were EMTs, student EMTs and staffs of LTCF, and in-hospital care providers included nurses and physicians working in the ED and ICU.
Selection of participants
Study samples were collected from student EMTs, EMTs, physicians and nurses in different departments of the healthcare facilities. The inclusion criteria for participants included HCWs such as EMTs, physicians, and nurses who have had more than 6 months of working experience at the acute and chronic healthcare facilities. Student EMTs, regarded as representatives of the general population with limited exposure to patients, were enrolled from the annual routine new EMT training program; paramedics and private ambulance EMTs with short term urgent care, transport to hospitals, or transfer of patients between healthcare facilities were selected from two local private ambulance groups. Physicians and nurses who worked in the adult medical ICU (MICU) and the medical ED (MED) were asked to participate. The exclusion criteria included HCWs who had active infections such as fever and known respiratory tract infections, urinary tract infections or other occult illnesses, and HCWs who have taken an antibiotic during the prior 21-days. The protocol and consent forms were approved by the Ethical Review Committee of NCKUH (B-ER-104-029). Written informed consents were obtained from participants prior to taking nasal swabs. Participants were asked to fill out an anonymous questionnaire regarding their place of work (current and previous), wearing adequate personal protective equipment and washing hands before and after patient care.
Microbiologic methods
One sample was taken from each participant. A sterile cotton swab was used to circle the anterior 1 cm of the nasal vestibule both nares. The swabs were immediately placed into transport medium (Venturi Transystem, Copan Innovation Ltd.) and brought to the microbiology laboratory. Swab samples were inoculated by the streak plate method on Trypticase soy agar with 5% sheep blood plates and incubated overnight at 37°C. S. aureus was identified by colony morphology, gram stain and the coagulase test. The presumptive S. aureus isolates were confirmed by coagulase (coa) gene-based PCR [19]. MRSA were identified by the cefoxitin disk-diffusion method according to the recommendations of Clinical and Laboratory Standard Institutes[20].
PFGE
MRSA isolates were identified further by PFGE analysis with chromosomal DNA using the enzyme SmaI. The relatedness of strains was determined by comparison of restriction fragment-length polymorphism in accordance with the guidelines published by Tenover et al.[21] PFGE patterns resulting in 2-3 band differences were considered to be closely related, those with 4-6 band differences were considered to be possibly related, and those with ≥7 band differences were considered to be unrelated.
Susceptibility testing
The antimicrobial susceptibility of MRSA isolates to 10 antibiotics, including oxacillin, trimethoprim/ sulfamethoxazole, penicillin, teicoplanin, linezolid, clindamycin, doxycycline, fusidic acid, vancomycin, and erythromycin, was determined in accordance with the guideline of Clinical Laboratory Standards Institute[20].
Biofilm formation assay
Four microliters of a bacterial overnight culture were inoculated into 1 ml of tryptic soy broth containing 0.25% glucose. An aliquot (200 µl) of the sample was poured into each of a 96-well polystyrene microplate (167008, Thermo Fisher Scientific, Waltham, MA, USA), and incubated for 3 days at 37°C. The fluid was removed and the plate was stained with 0.1% safranin solution. The OD490 was measured using a microplate reader (µQuant, BioTek Instruments, Winooski, VT, USA).
Molecular characterization
Genomic DNA was obtained from the MRSA isolates by a Qiamp DNA mini kit protocol (Qiagen, Hilden, Germany) for molecular characterization. The presence of Panton-Valentine leucocidin (pvl) gene and genes for fibronectin binding protein A and B (fnbA, fnbB) were determined by PCR as previous described[22, 23]. The S. aureus multilocus sequence typing (MLST) scheme uses internal fragments of the following seven house-keeping genes: arc, aro, glp, gmk, pta, tpi and yqi. PCR amplification was carried out on chromosomal DNA using an extension time of 30 seconds, and an annealing temperature of 55°C, with Taq polymerase. The PCR products were then sequenced and the data were uploaded to the MLST website (http://www.mlst.net) for further analysis[24]. Typing of the staphylococcal chromosomal cassette mec (SCCmec) was done by PCR with primers and by the methods published previously[25]. PCR for mecA, mupA, and qacA/B were performed by the methods described previously[24, 26].
Methods of measurements
Primary outcome
Positive nasal swabs for MSSA and MRSA were reported. Further nasal carriage of the MSSA and MRSA prevalence were calculated by descriptive statistics and cross tabulations to determine the frequency distribution of the MRSA nasal carriage among the different groups of healthcare professionals. Pearson’s chi-square test, Fisher’s exact test Cochran–Mantel–Haenszel test, logistic regression, and generalized linear models were used to compare MRSA colonization between groups. Odd ratios (ORs) were calculated with 95% confidence intervals (CIs). Student t test or Mann–Whitney U test were used to compared continuous variables. SAS software version 9.4 (SAS, Inc., Cary, North Carolina, USA) was applied for data entry, processing and statistical analysis.
Secondary outcome analysis
Positive MRSA samples were enrolled for further pathogenicity by molecular analysis. The drug susceptibility, basic molecular typing, virulence factors such as pvl gene and genes for fibronectin binding protein and the biofilm formation assay were conducted.