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A Prospective Study on the Prevalence and Antibiotic Sensitivity Pattern of Methicillin Resistant Staphylococcus aureus isolated from Various Clinical Specimen at a Tertiary Care Post Graduate Teaching Institute

Staphylococcus aureus, the most clinically significant species of Staphylococci has been recognized as an important cause of human disease for more than 100 years (Ankur et al., 2013). It is one of the pathogens of greatest concern because of its intrinsic virulence factors, its ability to cause diverse array of life threatening infections, it’s competency to adapt to different environmental conditions and its nasal carriage, which accounts for possible spread and re infection (Anupurba et al., 2003). Infections by Staphylococcus aureus are continuously challenging the clinicians despite the availability of antibiotics from nearly 70 yrs. This was due to the emergence of various types of antibiotic In recent times, the treatment of Staphylococcus aureus infection has become problematic because of emergence of resistance to antibiotics which is a cause of concern for all the clinicians and microbiologists worldwide. Infections caused by MRSA have been associated with high morbidity and mortality rates. Hence this study was conducted to know the prevalence of Methicillin resistant Staphylococcus aureus [MRSA] isolates in various clinical specimens and to determine the sensitivity of these isolates to different antimicrobial agents. Among the 161 clinical isolates of S. aureus, highest isolation was from pus samples (64%), followed by blood (13%) and respiratory secretions (13%). 82 (51%) were identified as MRSA by Cefoxitin disc diffusion method and maximum MRSA were again isolated from pus (61%). Comparatively MRSA prevalence is more in males (73%) and most common affected age group was 21-30 years. All MRSA strains were resistant to penicillin (100%), followed by ciprofloxacin (93%) and erythromycin (61%). We found statistically significant differences in the drug susceptibility pattern of MRSA & MSSA for Penicillin, Clindamycin, Erythromycin, Ciprofloxacin, Co-trimoxazole and Levofloxacin. MDR-MRSA strains in our study is 45%. Hence we suggest, more and more studies in future are needed to fight against rising menace of antibiotic resistance among Staphylococcus aureus International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 1859-1869 Journal homepage: http://www.ijcmas.com


Introduction
Staphylococcus aureus, the most clinically significant species of Staphylococci has been recognized as an important cause of human disease for more than 100 years (Ankur et al., 2013). It is one of the pathogens of greatest concern because of its intrinsic virulence factors, its ability to cause diverse array of life threatening infections, it's competency to adapt to different environmental conditions and its nasal carriage, which accounts for possible spread and re infection (Anupurba et al., 2003). Infections by Staphylococcus aureus are continuously challenging the clinicians despite the availability of antibiotics from nearly 70 yrs. This was due to the emergence of various types of antibiotic In recent times, the treatment of Staphylococcus aureus infection has become problematic because of emergence of resistance to antibiotics which is a cause of concern for all the clinicians and microbiologists worldwide. Infections caused by MRSA have been associated with high morbidity and mortality rates. Hence this study was conducted to know the prevalence of Methicillin resistant Staphylococcus aureus [MRSA] isolates in various clinical specimens and to determine the sensitivity of these isolates to different antimicrobial agents. Among the 161 clinical isolates of S. aureus, highest isolation was from pus samples (64%), followed by blood (13%) and respiratory secretions (13%). 82 (51%) were identified as MRSA by Cefoxitin disc diffusion method and maximum MRSA were again isolated from pus (61%). Comparatively MRSA prevalence is more in males (73%) and most common affected age group was 21-30 years. All MRSA strains were resistant to penicillin (100%), followed by ciprofloxacin (93%) and erythromycin (61%). We found statistically significant differences in the drug susceptibility pattern of MRSA & MSSA for Penicillin, Clindamycin, Erythromycin, Ciprofloxacin, Co-trimoxazole and Levofloxacin. MDR-MRSA strains in our study is 45%. Hence we suggest, more and more studies in future are needed to fight against rising menace of antibiotic resistance among Staphylococcus aureus resistance mechanisms especially to methicillin and vancomycin, which was the theme of several epidemiological studies (Arora et al., 2010;Assadullah et al., 2003).
With the introduction of Penicillin in 1940, it was thought that we can escape from the deadly threats of Staphylococcal infections, but no sooner in 1944 first strain of Staphylococcus aureus resistant to penicillin G appeared in London, mediated by the presence of penicillinase enzyme which hydrolyses the β-lactam ring of penicillin (Astagneau et al., 1996). By 1950s, penicillinase producing strains of Staphylococcus aureus were so common that penicillin became useless against Staphylococcal infections. To resolve this issue, in 1960 semi-synthetic penicillin (penicillinase stable penicillin) -like Methicillin came in picture, but availability of these agents did not stem the tide of resistance as within a year the first MRSA was reported from U.K. This was mediated by the presence of PBP-2a which is expressed by an exogenous gene, mecA (Bandaru, 2010).
In the past few decades MRSA has emerged as an important nosocomial pathogen worldwide. In India, prevalence rate varies from 30-85% in different parts and has now become endemic (Barber, 1961;Boucher, 2010). A multicentric study done in India involving 17 tertiary care Hospitals reported MRSA prevalence of 41% in 2008-2009(Clinical and Laboratory Standards Institute, 2012, MRSA is of serious therapeutic concern not only due to its resistance to Methicillin, but also because of resistance to many other antimicrobials that are used on regular basis in Hospitals. Therefore, the most reliable and sustained therapeutic agent against methicillin-resistant Staphylococcus aureus (MRSA) strains is Vancomycin. Increasing prevalence of MRSA has lead to the extensive use of vancomycin. This inturn has lead to the decreased susceptibility to vancomycin. Such resistance is a serious clinical and public health consequences because currently few licenced alternatives are available to treat vancomycin resistant Staphylococcus aureus infections.
The association of Multidrug resistance with MRSA adds to the problem and the presence of MDR strains in the hospital cannot be neglected. Several studies are prevailing for Methicillin resistance from India but few have worked on Multi-drug resistance. MRSA strain showing resistance towards ≥ 3 antibiotics is defined as MDR strain and its prevalence ranges from 23% -60% in India (Diep et al.,). The emergence of MDR MRSA infections is worrisome in the present therapeutic scenario. Keeping the above points in view, the present study was planned to find out the prevalence of MRSA among isolates of Staphylococcus aureus in various clinical specimens along with their antibiotic sensitivity pattern so as to guide the clinicians of our hospital to select appropriate antimicrobial agents and also to make them aware, that if inappropriate use these antibiotics is continued it may lead to impending public health disaster.

Source of material
The present study was conducted in the Department of Microbiology, GMCH, Udaipur, during the year 2013-2014. A total of 161 non-duplicate Staphylococcus aureus isolates from various clinical specimens [pus, wound or vaginal swabs, blood, body fluids (csf, pleural fluid, ascitic fluid) urine, sputum, ET secretion etc] were included in the study. Isolates from both in-patients and out-patients were considered. Institutional Ethical clearance was obtained. Data regarding age, sex, patient location, history of any clinical illness etc. was obtained from the requisition form submitted to Microbiology Department, GMCH.

Inclusion criteria
All Staphylococcus aureus strains isolated from various clinical specimens, were included in the study.

Exclusion criteria
Clinical specimen's yielding growth of Gram positive cocci other than Staphylococcus aureus and all gram negative bacteria were excluded.

Isolation and identification of Staphylococcus aureus
All the isolates were identified by standard procedures (gram staining, catalase test, mannitol fermentation, Hugh-Leifson OF media, slide coagulase and tube coagulase test). Tube coagulase was taken as the main criteria of identification and was performed by diluting plasma in freshly prepared normal saline (1:6). Three to four colonies were emulsified in 1 ml of diluted plasma and the tubes were incubated at 37ºC. Readings were taken at 1, 2, 3 and 4 h and further incubated at room temperature if no clot formation was observed (Yogesh et al., 2013).

Detection of methicillin resistance
Cefoxitin disc (30 µgm) was used to detect methicillin resistant isolates as Cefoxitin, which is a potent inducer of the mecA regulatory system, is being widely used as a surrogate marker for detection of mecA genemediated methicillin resistance. MRSA strains exhibiting inducible resistance to methicillin grow much more readily in the presence of cefoxitin than oxacillin, due to an enhanced induction of PBP 2a by cefoxitin. Isolates with zone diameter of ≤21 mm were considered resistant to methicillin and zone of ≥22 mm were sensitive (Harcharan et al., 2014).

Staphylococcus aureus is a major human pathogen and is one of the commonest causative agent of Community and Hospital acquired infections. The treatment of
Staphylococcus aureus infection has become problematic because of emergence of resistance to Penicillin, Methicillin, Vancomycin and many other antibiotics, by acquiring several resistance mechanisms. Increased antimicrobial resistance for such an organism is, therefore a cause of concern.
In the past few decades MRSA has emerged as an important nosocomial pathogen worldwide. In India, prevalence rate varies from 30-85% in different parts and has now become endemic (Lowy, 1998). A multicentric study done in India involving 17 tertiary care Hospitals reported MRSA prevalence of 41% in 2008-2009(Mallick et al., 2010. MRSA is of serious therapeutic concern not only due to its resistance to Methicillin, but also because of resistance to many other antimicrobials that are used on regular basis in Hospitals. Therefore, the most reliable and sustained therapeutic agent against methicillin- In the present study a total of 161 nonduplicate Staphylococcus aureus strains were isolated from various clinical specimens. Among all these samples highest isolation was from pus 103 (64%). Harcharan singh et al., (2014) in Udaipur (65%), Manu Chaudhary et al., (2013) in H.P (63%) and Ankur Goyal et al.,in Agra (66.03%), also reported the highest isolation of Staphylococcus aureus from pus.
All the Staphylococcus aureus strains were screened for Methicillin resistance using Cefoxitin disc diffusion method. Since it is a potent inducer of mecA gene, hence considered as a surrogate marker for detection of methicillin resistance by CLSI 2012, in our study 82 (51%) isolates turned out to be MRSA and 79(49%) as MSSA, from a total of 161 Staphylococcus aureus strains. The prevalence rate of MRSA in our institute is 51%, which is similar to the studies conducted by Vidhani and Mehndiratta et al., in 2001 showing a prevalence rate of 51.6% and almost comparable to the study conducted by Majumdar et al., in 2001 andAssadullah et al., in 2003 showing 52.9% prevalence rate. The higher rate in their studies may been attributed to the fact that the studies were conducted at a tertiary care multispecialty center with more and more patients coming from pheriphery and small nursing homes, where injudious use of antibiotics and inadequate infection control policies are prevalent. Verma et al., in 2000 reported a higher prevalence rate of 80.89% in Indore and Mehta et al., in 2007 reported a lower prevalence rate of 24% in Chandhigarh. This variation in prevalence may be because of several factors like different Geographical and environmental conditions, population group under study, healthcare facilities available in the particular hospital, implementation and monitoring of infection control committee, rationale antibiotic usage which varies from hospital to hospital. We have 51% prevalence rate of MRSA in our hospital setup. It therefore calls in for better vigilance and implementation of more effective MRSA surveillance programme complemented with improved infection control practices.
In the present study, maximum MRSA were isolated from pus 50 (61%), followed by blood 13(15%), respiratory secretions 8 (10%), Swabs and body fluids 4(5%) each and least from Urine 3(4%). This pattern correlates with studies conducted by Vidya Pai et al., in 2010 andNitish Kumar Sharma et al., 2013. This is due to the reason that Staphylococcus aureus accounts for most of the skin and soft tissue infections, septicemia and also respiratory tract infection.
Comparatively MRSA prevalence was more in males (73%) than in females (23%) in our study. Similar findings was also reported by  and Abhishek Mewar et al., The increased rate of MRSA infections among males could be due to their more outdoor activities, inturn exposing them to contaminated environment and also compared to females, accidental injuries are more common among men.
Most of the MRSA strains were isolated from 21-30 yrs of age group (ie 32%) and in 51-60yrs (ie 26%), indicating MRSA infection is more common in working and old age group. The reason for this may be that younger age group are more involved in outdoor activities inturn exposing them to contaminated environment and in older age group it may be due to waning immunity, hormonal abnormalities and co-morbid conditions. Similar pattern of affected age group was also reported by Ankur Goyal et al., in 2013. Isolation was more from the IPD patients (88.8%) than from the OPD patients (11.2%). Among IPD patients, highest prevalence was seen in orthopedics, oncology and surgical wards. Similarly Mallick and Basak reported 84.8% MRSA from IPD patients, in which maximum strains were isolated from surgical and orthopedics ward. This might be because the patients in these wards are usually with open wounds and are debilitated. They undergo multiple interventions in the hospital which further increases the risk of MRSA infection due to multiple people involvement as well as prolonged stay in the wards. Along with these factors, the patient is usually on multiple antibiotics.
Resistance of MRSA to a wide range of antibiotics is well documented 35 . In the present study antibiotic resistance is significantly more in MRSA strains of Staphylococcus aureus as compared to MSSA strains. Statistically significant difference was observed for Penicillin, Fluoroquinolones, Erthromycin, Clindamycin and Cotrimoxazole. Such similar statistically significant difference between the sensitivity pattern of MRSA and MSSA was also reported by Vidhani et al., (2013), Shilpa Arora et al., (2013) Majumder et al., (2013 Our study had also showed high degree of resistance to Erythromycin and Co-trimoxazole. Such high resistance pattern observed for these antibiotics in our study may be due to the differential clonal expansion and indiscriminate empirical use of these drugs. The present study also showed a low level of resistance to Clindamycin, similar to the study conducted by Ankur Goyal et al., (2013). This may be due to the antibiotic recycling and the dependence of the clinicians more on beta lactam drugs. In the light of the present study, we would recommend use of Clindamycin for the management of soft tissue infections, with MRSA strains that are sensitive to Clindamycin since it has better soft tissue penetration and no renal dosing adjustments and thus by reserving Vancomycin for life threatening infections.
The association of Multidrug resistance with MRSA adds to the problem and the presence of MDR strains in the Hospital can't be neglected. Several studies are prevailing for Methicillin resistance from India but few have worked on Multi-drug resistance. MRSA strain showing resistance towards ≥ 3 antibiotics is defined as MDR strain. MDR MRSA strains in our study is 45%, a study conducted by Majumder et al., (2013) reported 23.2% MDR-MRSA, 32% by Anupurva et al., and as high as 63.6% by Rajaduraipandi et al., (2000). The emergence of MDR MRSA infections is worrisome in the present therapeutic scenario, hence a continuous antibiotic surveillance is required for all the Staphylococcus aureus isolates. This inturn will help in formulation of effective antibiotic policies in the health care set-up or else the threat will increase.
In the present study 3% resistance was observed for Vancomycin by disc diffusion test, Harcharan Singh et al., (2013) conducted a study in Udaipur and observed a higher resistance of 13% to Vancomycin by using Disc diffusion method only. Similarly Yogesh Kumar Gupta et al., and Ankur Goyal et al., had reported no vancomycin resistance by Disc diffusion in Rajasthan and Agra respectively.
To conclude, the result of our present study indicated high antibiotic resistance to commonly used antibiotics by MRSA isolates. The increase in vancomycin resistance among MRSA and MDR-MRSA and excessive use of antimicrobial agents has worsened the sensitivity. Hence prudent and responsible usage of newer antibiotics is advocated to preserve their continued effectiveness in the management of difficult to treat infections caused by MRSA and VRSA. We should undertake more and more such studies in future to fight against rising menace of antibiotic resistance. Also more research should be done to find better treatment policies, effective and cheaper alternative antibiotics in developing countries like ours. The findings of the studies should be shared with hospital infection control committee to help in the formulation of infection control polices and also antibiotic policies. So that the primary care givers can use antibiotics rationally.