Prevalence and genotypic relatedness of methicillin resistant Staphylococcus aureus in a tertiary care hospitalBA Fomda1, MA Thokar1, G Bashir1, A Khan1, A Kour1, D Zahoor1, P Ray2
1 Department of Microbiology, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India
2 Department of Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, Punjab and Haryana, India
Correspondence Address: Source of Support: Funded by ICMR,, Conflict of Interest: None DOI: 10.4103/0022-3859.143964
Source of Support: Funded by ICMR,, Conflict of Interest: None
Background: Methicillin-resistant Staphylococcus aureus (MRSA) is the most common multidrug-resistant pathogen causing nosocomial infections across the world. MRSA is not only associated with significant mortality and morbidity but also places a large economic strain on our health care system. MRSA isolates are also typically resistant to multiple, non-β-lactam antibiotics. We conducted a prospective study in a tertiary care hospital, to determine the prevalence of MRSA and to establish the clonal distribution of MRSA isolates recovered from various clinical specimens. Materials and Methods: Clinical samples were cultured and S. aureus was identified as per standard microbiological procedures. Susceptibility testing was done by agar disk diffusion and minimum inhibitory concentration (MIC) method as recommended by CLSI. Methicillin resistance was detected by phenotypic methods namely, oxacillin disc diffusion (ODD), minimum inhibitory concentration (MIC) of oxacillin, cefoxitin disk diffusion (CDD), and MIC of cefoxitin. Amplification of mecA gene by PCR was used as gold standard for detection of methicillin resistance. Pulsed field gel electrophoresis (PFGE) typing was performed for MRSA isolates. Results: Out of 390 S. aureus isolates, 154 (39.48%) isolates were MRSA and 236 (60.51%) isolates were MSSA. Penicillin was the least effective antibacterial drug against the hospital associated S. aureus isolates with 85.64% resistance rate. All the isolates were susceptible to vancomycin. The MRSA showed a high level of resistance to all antimicrobials in general in comparison to the MSSA and the difference was statistically significant (P < 0.05). Multiplex PCR performed for all strains showed amplification of both the mecA and nucA genes in MRSA strains whereas MSSA strains showed amplification of only nucA gene. PFGE of these isolates showed 10 different patterns. Conclusion: Prevalence of MRSA in our hospital was 39.48%. Most of these isolates were resistant to erythromycin, clindamycin, cotrimoxazole, and ciprofloxacin, whereas high sensitivity was seen to vancomycin followed by gentamicin. CDD and MIC for cefoxitin showed 100% sensitivity, specificity, PPV and NPV as compared to PCR for mecA gene. In maximum number of isolates PFGE type A pattern was seen suggesting clonal relatedness.
Keywords: CDD, MRSA, MIC, multiplex PCR, ODD,pulse field gel electrophoresis typing
Staphylococcus aureus is a versatile human pathogen that causes diseases ranging from relatively mild infections of the skin and soft tissue to life-threatening sepsis, pneumonia, and toxic shock syndrome (TSS).  The emergence of strains resistant to methicillin and other antimicrobial agents has become a cause of major concern because methicillin resistant S. aureus (MRSA) infections are not only a significant cause of morbidity and mortality, but they also place a large economic strain on our health care system. . This study was undertaken to determine the prevalence of MRSA and to establish the clonal distribution of methicillin resistant S. aureus isolates recovered from various clinical specimens in our hospital setting.
The study was carried out in Department of Microbiology; Sher-i-Kashmir Institute of Medical Sciences, Kashmir, India over a period of 3 years. Ethical clearance was obtained from institute's ethical clearance committee.
Isolation and identification of S. aureus
Samples like blood, pus, urine, wound swabs, and other body fluids were inoculated onto 5% sheep blood agar, MacConkey agar and Hi-chrome UTI agar and incubated at 37°C for 24 h. Identification of S. aureus was done by standard microbiological tests.
Antimicrobial susceptibility pattern
The antibiotic susceptibility testing was performed by using the Kirby-Bauer disc diffusion method according to CLSI guidelines.  The antibiotic discs used in our study [Table 1], were procured from Hi-media Mumbai, India. Inducible clindamycin resistance was determined by D-test. ATCC 25923 was used as control.
Determination of minimum inhibitory concentration
Minimum inhibitory concentration (MIC) of antibiotics such as oxacillin and cefoxitin (Sigma St. Louis, Mo, USA) against S. aureus was determined by using micro broth dilution method according to CLSI guidelines.  ATCC 29213 was used as control.
PCR for mecA and nucA gene was carried out for 390 S. aureus strains by the methodology described by Mason et al. Isolates that were mecA and nucA positive were considered as MRSA. ATCC 43300 were used as positive control.
Genotyping by pulsed field gel electrophoresis
Pulsed field gel electrophoresis (PFGE) typing of SmaI (Fermentas, UK)-digested DNA was carried out by a protocol described previously by Bannerman et al. The criteria proposed by Tenover et al. were employed to analyze the DNA fingerprints generated by PFGE. 
Frequencies were obtained by Chi square and Fisher's exact test (two tailed) with the SPSS statistical program (version 15).
Of the 1200 specimens screened, S. aureus was isolated from 390 (32.5%). Most of the isolates were recovered from pus (44.01%) followed by blood (30.33%), body fluids (17%), and (8.66%) urine samples. Maximum number 334 (85.64%) of isolates were resistant to penicillin while 100% were susceptible to vancomycin.
Out of 390 isolates, 154 (39.48%) were MRSA and 236 (60.51%) were MSSA by PCR [Figure 1], CDD and MIC of cefoxitin. All the isolates resistant to cefoxitin had an MIC of >32 μg/ml. Using ODD method 143(36.66%) isolates were identified as MRSA and 247(63.33%) as MSSA. MIC of oxacillin showed 148(37.94%) isolates as MRSA, all having MIC over >4 μg/ml. The MRSA isolates showed a high level of resistance to all antimicrobials in general in comparison to the MSSA and the difference was statistically significant [Table 1].
Phenotypic methods compared with PCR as gold standard showed 100% sensitivity and specificity for CDD and MIC for cefoxitin, while as 92.85% and 96.1% for ODD and MIC for oxacillin respectively. All the four methods showed 100% specificity and PPV [Table 2].
All isolates were subjected to PFGE. Ten common patterns were identified among the MRSA isolates. Strains showing same PFGE pattern were grouped as a pulsotype and assigned alphabetically (A to J). Maximum strains (n = 40) belonged to band type A [Figure 2].
The accurate and early determination of methicillin resistance is of key importance in the prognosis of infections caused by S. aureus. Detection of mecA gene or its product, penicillin binding proteins (PBP2a), is considered the gold standard for MRSA confirmation. 
Sensitivity and specificity for CDD and MIC for cefoxitin was 100% in our study and MIC for cefoxitin of >32 μg/ml was seen in all mecA positive isolates, showing that there is a direct correlation of presence of mecA and cefoxitin MIC >32 μg/ml as reported by Salimnia et al. Results of CDD and MIC for cefoxitin were in concordance with PCR for mecA gene in our study, as reported earlier. ,
A high negative and positive predictive value of 100% for CDD in our study correlates well with the findings of Velasco et al. CDD and MIC for cefoxitin are both equally good for detection of MRSA; however, CDD because of its simplicity is more suitable and can be an alternative to PCR for detection of MRSA in resource constraint settings.
A high specificity and low sensitivity of ODD as compared to CDD as seen in our study has also been reported by Anand et al. while a higher (100%) sensitivity but lower specificity (89%) has been reported by Adaletti et al.
The prevalence of MRSA was 39.48% in our study. A high prevalence of MRSA (51.8%) has been reported in India by Mallick et al. while an MRSA prevalence of 23% has been reported from Australia, 67% from Japan, 40% from South Pacific, and 32% from USA. 
Probable reasons for good susceptibility of MRSA isolates to vancomycin (100%) and gentamicin (81.81%) could be due to the fact that these drugs are infrequently used as first line therapy in our hospital. The high resistance rates of our MRSA isolates to other classes of antibiotics reflect the excessive use of these antibiotics. Thus, they can no longer be relied upon for empirical treatment of suspected staphylococcal infections in our setting.
PFGE, because of its great discriminatory power and high degree of specimen typeability is accepted as the gold standard for the molecular typing of S. aureus.  Although, PFGE is time-consuming and labor intensive, in this study it was used as it helps in tracking genetic changes which occur in a relatively short time, while multilocus sequence typing (MLST) another typing method is more suitable for studying long-term genetic variations in S. aureus. 
PFGE analysis of the MRSA strains in our study demonstrated that most of the strains were highly related to one another, suggesting clonal expansion in this population; however, Gyathri et al. have suggested diverse PFGE pattern in their MRSA isolates that were related to each other by MLST and spa typing. 
From time to time measures were taken to control MRSA infection by MRSA screening, use of contact precautions and promotion of hand hygiene in our hospital. Given the questionable benefit of MRSA screening and isolation strategies for preventing healthcare-associated infections as well as the fact that such isolation is not cost effective and can result in social isolation of patients, many in the infection control community have begun to explore other strategies like decolonization of MRSA positive patients or universal decolonization of all patients with mupirocin and chlorhexidine. 
Hence, we conclude that the prevalence of MRSA in our hospital is 39.48% with maximum isolates being clonally related belonging to PFGE type A pulsotype. The resistance of MRSA isolates to other classes of antibiotics except vancomycin and gentamicin is quite high. CDD can be recommended for testing methicillin resistance in S. aureus isolates routinely in microbiology laboratories.
We are thankful to Indian Council of Medical Research (ICMR) for financial assistance in this study. File no-5/3/3/5/2006-ECD1.
[Figure 1], [Figure 2]
[Table 1], [Table 2]