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The Detection of Mupirocin Resistance and Nasal Carriage of Methicillin Resistant Staphylococcus aureus among Healthcare Workers at University Hospitals of Tehran, Iran.

Background: Nasal mupirocin is found to eradicate effectively methicillin-resistant Staphylococcus aureus (MRSA) from colonized patients, but there are concerns about resistant strains. The aim of this study was to detect the mupirocin resistance and nasal carriage of methicillin resistant S. aureus among healthcare workers (HCWS) at the university hospitals of Tehran. Methods: Totally 270 nasal swabs were collected and S. aureus were identified by confirmatory tests in 2013. Determination of oxacillin and mupirocin resistance was performed by disk diffusion method and the mupirocin MIC assessed using E-test. The ileS-2 (mupA) and mecA genes were detected in DNA extracts by multiplex PCR. Results: The prevalence of S. aureus nasal carriage among HCWs was 14.44%. E-test and disk diffusion methods showed 5 and 4 mupirocin resistant isolates, respectively. Statistically significant difference was observed between sex (P=0.035), hospitals (P=0.0001) and occupation (P=0.009) with nasal carriage of S. aureus. A significant difference was found between sex (P=0.041) and occupation (0.034) with regard to MRSA carriage. All MRSA isolates were susceptible to linezolid, fusidic acid and vancomycin. Conclusion: Since the HCWs play an important role as a reservoir for resistant isolates in the hospital setting, regularly screening should be performed for identification of nasal carriers.


Introduction
The global emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become a thoughtful problem for public health. MRSA has been identified as one of the major cause of nosocomial infections, which is resistant to various classes of antibiotics (1). Staphylococci could colonize the skin and nasal mucosa (2). Eradication or inhibition of staphylococcal colonization is still considered as an important strategy to prevent infection and transmission of these strains. Rationale behind such a strategy is that the most staphylococcal infections are caused by endogenous strains; so, carriage of S. aureus is a major risk factor for subsequent infections (3). The eradication of this organism in nasal carriers with mupirocin has been shown to reduce the rate of nosocomial infections in hospitalized patients (4). Mupirocin is available as an antimicrobial agent. Nasal formulation of mupirocin was recommended by the Food and Drug Administration of United States (US FDA) as part of an infection control for using in the eradication of nasal carriage of S. aureus in adult patients and healthcare workers (HCW) to reduce the risk of infection among high-risk patients for acquisition of MRSA. Using mupirocin is limited for infection control and other prophylactic functions in order to tackle the concerns of the emergence of resistance (5). There are three groups of susceptibility to mupirocin that include susceptible to mupirocin (MIC≤4μg/ml), low level resistant to mupirocin (MIC to64μg/ml) and high level resistant to mupirocin (MIC≥512 μg/ml) (6). Currently there is no desirable method for interpreting the susceptibility tests of mupirocin. Existing methods based on MIC determination by E-test method and detection of resistance genes by PCR are used (5). Since HCWs are in direct contact by the patients, they have a significant role in dissemination of resistant isolates in the hospital setting. Therefore, we decided to do present study in order to evaluate the prevalence of MRSAs that are resistant to mupirocin, isolated from HCWs nasal carriers and accomplish molecular techniques and the MIC together for epidemiological purposes. Antibiotic susceptibility pattern of MSSA and MRSA isolates was determined as well. If nasal carriers identified, the therapeutic measures can be proceed to prevent the dispersion of resistant isolates in hospital setting.

Study design
In this cross sectional survey 270 HCWs nasal swabs were collected from five hospitals affiliated to Tehran University of Medical Sciences (TUMS) in 2013. All participants gave written informed consent.

Bacterial isolation
After sampling, using sterile swabs, samples immediately transferred to transport medium moved to the Department of Pathobiology, then sub cultured on blood agar and incubated for 24 hours at 37 °C. Identification of S. aureus was performed by confirmatory tests [Gram's stain, catalase, coagu-lase and DNase tests and mannitol fermentation on mannitol salt agar (MSA)].

Determination of minimal inhibitory concentration (MIC)
The mupirocin MIC assessed using E-test® mupirocin strips (AB Biodisk, Solna, Sweden) according to the manufacture's guidelines. Strains were considered susceptible if MIC was ≤4 mg/l and levels of mupirocin resistance were defined as low-level with MIC 8-256 mg/l and high-level with MIC ≥512 mg/l.

DNA extraction
DNA was extracted using a DNeasy kit (Qiagen, Valencia, CA) as recommended by the manufacturer.

Multiplex PCR
The ileS-2 (mupA gene ( and mecA genes were detected in DNA extracts by multiplex PCR assay, as described previously (8) with some modifications ( Table 1). The genes were amplified on an Eppendorf (Hamburg, Germany) thermocycler with the final volume of 50 μlit containing 24 μlit of Qiagen HotStarTaq master mix (Invitrogen, Carlsbad, CA, contain PCR buffer with 3 mM MgCl2, 400 µM of each dNTP and 2.5 units HotStarTaq DNA Polymerase), 2 μlit of each primer (20 pMol, MecA and Mup), 14 μlit of RNase-free water and 8 μlit of DNA template. Then products were elec-trophoresed on agarose gel and the presence or absence of resulting bands was evaluated.

Statistical analysis
Data were analyzed using SPSS statistical software, version 16.0. The categorical data were compared using the Chi-square test or Fisher's exact test and quantitative variables were compared by one-way ANOVA. Statistical significance was set at 0.05 levels.  Table 3.     Table 7.

Discussion
S. aureus nasal colonization appears to play a significant role in the epidemiology and pathogenesis of infection (9). Mupirocin is a topical antibiotic agent that interferes with bacterial protein synthesis, which can be used for eradication of staphylococcal nasal colonization and control of MRSA transmission in Health Care Facilities (10). Today, the emergence of MuRSA strains has been reported from many parts of the world. The prevalence of these strains in Korea, India, South Africa and Nigeria has been reported 5%, 14.6%, 7% and 0.5 % respectively (11)(12)(13)(14). Currently prevalence of mupirocin resistance in MRSA is increasing in areas where are widely used these antibiotics (15). There are scattered reports and limited number of study conducted to detect staphylococcal nasal colonization in Iran. In a study of hospitalized patients in Kermanshah, on nasal swabs, all isolates were susceptible to mupirocin that reflected the limited use of this antibiotic, especially in that region (16).
In present study the prevalence of S. aureus nasal colonization was 14.44% among which 17(43.58%) were MRSA and others were MSSA and 5 (1.85%) isolates found to be mupirocin-resistant. Of MuRSA isolates 3 were high level resistant. Prevalence of nasal carriage of S. aureus varied between hospitals and statistical information showed a significant relationship between the hospitals and presence of S. aureus nasal carrier as well. This seems to be due to differences in infection prevention and control measures. There were statistically significant relationship between nursing job and nasal carriage of S. aureus and MRSA but not with MuRSA carriage. This could be a warning because of the high possibility of nurse-to-patient transmission of these bacteria and dissemination of them in hospital setting. Therefore, practice of sanitary principles along with the routine screening of nasal carriers for the treatment purposes can be very useful in preventing the creation of epidemics in hospital setting.
None of the nasal carriers had a history of hospitalization or antibiotic use within the previous three months in our study. A significant relation was found between the sexes and nasal carriage of S. aureus (P=0.035) and MRSA (P=0.041) but not with MuRSA (P=0.182). This result is likely due to sample size or gender distribution of HCWs in the hospitals under studied. It is notable that we did not found any significant difference between age and years of working in hospital with regard to the nasal carriage of MSSA, MRSA and MuRSA.
In present study mupA gene PCR results were positive for two isolates and both of them were showed high-level resistance (MIC>1024) to mupirocin (isolates SH4, SH10). In addition, both of these two isolates were MRSA. Studies show that almost all of mupirocin resistant MRSA isolates with high resistance to mupirocin have positive PCR result for mupA gene (17,18). Among these studies, there are some exceptions; this means that there are strains with high-level resistance to mupirocin, which are negative for the mupA gene. In these strains mupA gene, may located on chromosome rather than being on plasmids (19). This may justify and explain our result about strain S9. The results of our study indicated that one of the MRSA isolates (SH4) showed high level resistance to mupirocin by E-test method while was sensitive to mupirocin in disk diffusion method (this test was repeated 3 times), and it is noteworthy that this strain had positive PCR for mupA gene. Treatment with mupirocin in the presence of high-level resistance strains is not effective. In addition, there is evidence suggesting that presence of low-level resistant strains may cause failure in treatment (20)(21)(22). This emphasizes the importance of identification of both high and low level resistant strains. So, due to some discrepancy in these two tests (disk diffusion test and E-test) it seems that the screening results obtained from disk diffusion method and MIC determination must be confirmed by other methods such as detection of resistance genes by PCR analysis in order to avoid false-negative results. Hence, an unnecessary use of mupirocin and spread of resistant strains in hospital settings can be avoided by appropriate treatment decisions. In this study, antibiotic susceptibility pattern of MRSA and MSSA strains were examined, that indicated antibiotic resistance among MRSA was much higher than MSSA. The highest resistance of MRSA isolates was related to erythromycin and all MSSA and MRSA strains were susceptible to linezolid, fusidic acid and vancomycin.

Conclusion
Since the HCWs play an important role as a reservoir for resistant isolates in the hospital setting, regularly screening should be performed for identification of nasal carriers. Consequently, because of antibiotic resistance among staphylococcal strains, thus antibiogram is a recommended method prior to treatment in order to select the best remedy.

Ethical considerations
Ethical issues (Including plagiarism, Informed Consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.