1-43 1 Prevalence and antimicrobial susceptibility pattern of methicillin-resistant Staphylococcus aureus (MRSA) in CMS-teaching hospital: a preliminary report R. K. Sanjana 1, Rajesh Shah2, Navin Chaudhary2, Y.I. Singh 3 1Assistant Professor, 2Lecturer, 3Prof & Head, Dept. of Microbiology, College of Medical Sciences-Teaching Hospital, Bharatpur, Chitwan District, Nepal. Abstracts Aims: Nosocomial infection is a major problem in the world today. Methicillin- resistant Staphylococcus aureus (MRSA) strains, usually resistant to several antibiotics and also intrinsic resistance to ß- lactam antibiotics, shows a particular ability to spread in hospitals and now present in most of the countries. The present study was carried out to investigate the prevalence of MRSA and their rate of resistance to different antistaphylococcal antibiotics. Materials and methods: Between April 2007 and December 2009, the clinical specimens submitted at the microbiology laboratory were processed and all Staphylococcus aureus (S. aureus) isolates were included in this study. All isolates were identified morphologically and biochemically by standard laboratory procedures and antibiotic susceptibility pattern including oxacillin was determined by modified Kirby Bauer disc diffusion method. Results: Out of a total of 348 Staphylococcus aureus strains isolated from various clinical samples, 138 (39.6%) were found to be Methicillin- resistant. Among MRSA isolates, 86(62.3%) were from different inpatient departments, whereas, 52(37.7%) of the isolates were from outpatients. All MRSA were resistant to penicillin. More than 70% of the MRSA strains were resistant to cephalexin, ciprofloxacin and cloxacillin, while less than 10% of them were resistant to azithromycin, amikacin and tetracycline. Many MRSA strains were multidrug resistant. However, no strains were resistant to vancomycin. Conclusion: This preliminary report showed a high prevalence of MRSA in our hospital. To reduce the prevalence of MRSA, regular surveillance of hospital acquired infection and isolation is the need of the hour. Key words: Nosocomial infection, methicillin-resistant Staphylococcus aureus (MRSA), multidrug resistant. Correspondence: Dr. R. K. Sanjana E-mail: Rajkumari_sanjana@yahoo.co.in , 1-6 Original ArticleJournal of College of Medical Sciences-Nepal, 2010, Vol. 6, No. 1 2 Introduction Staphylococcus aureus is a leading cause of hospital acquired infection (HAI) and over the past 50 years it has acquired resistance to previously effective antimicrobials including the penicillinase resistant ones like methicillin.1 Today, methicillin resistant Staphylococcus aureus(MRSA) has emerged as one of the most important nosocomial pathogens.2 The percentage of hospitals isolating MRSA in the developed countries has increased from 2% in the 70’s to 30% in the 90’s.3 Moreover, half of S. aureus in many centres are methicillin resistant(multidrug resistant) posing major therapeutic challenge.4 MRSA causes more than 50% of HAI and are more virulent than the methicillin sensitive strains.5,6 Prompt diagnosis of MRSA infection is, therefore, important for patients, health care givers and for epidemiological purposes. Hospital acquired infection (HAI) gives an enormous burden to the health care system significantly affecting the patient’s morbidity and mortality. It results in prolongation of hospital stay and hence higher bed occupancy rate with an attendant increase in the cost of hospitalisation.7, 8 Surveillance of MRSA related infections especially in the hospital set up is required and has been doing in the developed countries. Not only that, the magnitude of the problem is yet to be quantified. This study is an attempt to assess the prevalence of methicillin resistant S. aureus (MRSA) infection and its antibiotic susceptibility pattern in this hospital. Materials and methods This study was based on retrospective data of samples sent from different wards and OPDs of College of Medical Sciences-teaching hospital, Bharatpur. Total strains of 348 S. aureus were isolated from pus, urine, sputum, wound swab, aural swab, blood, throat swab and urethral swab during April 2007 and Dec 2009. S. aureus was identified by conventional method. 9 The antimicrobial susceptibility test was carried out using Kirby-Bauer’s disc diffusion method modified and updated by Clinical and Laboratory Standards Institute guidelines (CLSI). 10 Each of the strain was screened for oxacillin resistance using American Type Culture Collection (ATCC) 43300 as the control. A standard inoculum was prepared by direct colony suspension in and comparing it with 0.5 Mc. Farland turbidity. Using a sterile cotton swab and after removing the excess of the inoculum by pressing against the side of the tube, the suspension was inoculated on a Mueller Hinton Agar medium by lawn culture method all over the surface of the medium. Oxacillin disc (1µg), (HI Media Laboratories, Pvt. Ltd. Mumbai) was applied along with other antimicrobials for testing sensitivity and the plates were examined after an over night incubation at 37 º C. Zone of inhibition diameter (in mm) were measured and results were interpreted as sensitive, resistant as per recommendation of Clinical and Laboratory Standards Institute guidelines (CLSI). Other antimicrobials tested were chloramphenicol (30µg), tetracycline (30 µg), gentamicin (10µg ), erythromycin (15µg), co- trimoxazole (25µg), cephalexin (30µg ), ciprofloxacin (5µg), amikacin (30µg ), cefotaxime (30µg) and vancomycin (10µg ). Results Isolation of Staphylococcus was maximum in pus samples. Out of the 348 strains of S. aureus examined 138 (39.6%) were found to be Methicillin- resistant and of which 86 (62.3%) were from inpatient departments. Amongst them only 9 (10.4%) of the Journal of College of Medical Sciences-Nepal, 2010, Vol. 6, No. 1 3 isolates were from intensive care units (ICU). A total of 52 (37.7%) MRSA strains were from outpatients. Maximum isolation of MRSA was from pus (53.3%), followed by wound swabs (44.4%), sputum (37.5%), aural swabs (33.3%) etc. (Table- 1) shows detection of MRSA in different samples. All the strains of MRSA were found to be resistant to Penicillin. (Table -2) depicts the antibiotic susceptibility data for all the S. aureus isolates. Among MRSA, resistance to cephalexin was 81.8%, ciprofloxacin -71.0%, cloxacillin -70.6%, erythromycin -58.0%, gentamicin -38.0%, cefotaxim -31.6%, cotrimoxazole -20.4%, while amikacin, azithromycin and tetracycline were resistant to less than 10% of the MRSA strains. Many MRSA strains were multidrug resistant. No strain was resistant to vancomycin. However, 41.2% of Methicillin sensitive S. aureus (MSSA) were resistant to penicillin, 25.7% resistance to cephalexin, 25.4% resistance to ciprofloxacin, 16.6% resistance to cloxacillin, 14.5% resistance to erythromycin, 32.3 % resistance to gentamicin as compared with MRSA. MSSA isolates also revealed higher susceptibility to cefotaxime, cotrimoxazole with a resistance rate of 9.3% and 9.8% of the strains respectively. None of the MSSA was resistant to azithromycin. Table- 1: Isolation of MRSA from Specimens of outdoor and indoor patients in CMS-teaching hospital, Bharatpur, Nepal R.K. Sanjana et al. Prevalence and antimicrobial susceptibility..............................: a preliminary report OPD Ward & ICU Total S.No Specimens S. aureus MRSA (%) S. aureus MRSA (%) S. aureus MRSA (%) 1 Pus 60 32 53.33 80 53 66.25 140 85 60.71 2 Urine 20 04 20.00 30 08 26.66 50 12 24.00 3 Wound swab 09 04 44.44 10 06 60.00 19 10 52.63 4 Sputum 08 03 37.50 15 06 20.00 23 09 39.13 5 Aural swab 09 03 33.33 20 06 30.00 29 09 31.03 6 Blood 18 02 11.11 34 03 08.82 52 05 09.61 7 Throat swab 08 02 25.00 12 03 25.00 20 05 25.00 8 CSF 00 00 00 01 00 00 01 00 00 9 Urethral swab 04 01 25.00 03 01 33.33 07 02 28.57 10 Bone cartilage 00 00 00 02 00 00 02 00 00 11 Semen 03 01 33.33 02 00 00 05 01 20.00 Total 139 52 37.68 209 86 41.14 348 138 39.65 4 Discussion MRSA is a global phenomenon with a prevalence rate ranging from 2% in Netherland and Switzerland, to 70% in Japan and Hong Kong.11, 12 In this study, the prevalence of MRSA was found to be 39.6%. Prevalence of MRSA was higher among inpatients (41.1%) than outpatients (37.4%). This difference could be due to prolonged hospital stay, instrumentation and other invasive procedures. A comparable prevalence rate of 34.7%, 31.0% and 38.5% were also reported from Assam, Tamil Nadu and Delhi13, 14, 15 whereas, in some studies the rate is comparatively low. In a study in Eastern part of Nepal in Dharan, the rate of MRSA was (26.4%),16 which was low as compared to this study. In another study in Nagpur the rate of MRSA (19.5%) 17 was also low compared to our study. However, in another study it was very high (80.8%).18 Analysis from previous studies revealed a relationship between methicillin resistance and Table- 2: Resistance to individual antimicrobials in MRSA and MSSA isolated in CMS-teaching hospital, Bharatpur,Nepal Journal of College of Medical Sciences-Nepal, 2010, Vol. 6, No. 1 MRSA MSSA Total S. No Antimicrobials Tested Resistance (%) Tested Resistance (%) Tested Resistance (%) 1 Penicillin G 126 126 100 189 78 41.25 315 204 64.76 2 Cephalexin 22 18 81.81 69 25 36.23 91 43 47.25 3 Ciprofloxacin 83 59 71.08 138 35 25.36 221 94 42.53 4 Cloxacillin 136 96 70.58 187 80 42.78 323 127 39.31 5 Erythromycin 62 36 58.06 124 18 14.51 186 54 29.03 6 Gentamicin 21 08 38.09 65 21 32.30 86 29 33.72 7 Cefotaxim 57 18 31.57 118 11 09.32 175 29 16.57 8 Co-trimoxazole 44 09 20.45 132 13 09.84 176 22 12.50 9 Ofloxacin 68 12 17.64 154 18 11.68 222 30 13.51 10 Amoxyclav 28 04 14.28 84 07 08.33 112 11 09.82 11 Azithromycin 52 05 09.61 158 00 00 210 08 03.80 12 Amikacin 65 06 09.23 112 07 06.25 177 13 07.34 13 Tetrcyclin 50 04 08.00 59 06 10.16 109 10 09.17 14 Vancomycin 128 00 00 186 00 00 324 00 00 5 resistance to other antibiotics.19, 20 This study showed that all MRSA isolates were significantly less sensitive to antibiotics as compared with MSSA isolates. Many of the isolates were resistant to commonly used antistaphylococcal agents except vancomycin. Anupurba et al. also observed that 32% of MRSA isolates are resistant to all commonly used antibiotics for S. aureus except vancomycin.21 Because of the resistance of MRSA to all commonly used antibiotics, it is necessary to test newer group of antibiotics such as vancomycin and teicoplanin routinely. Resistance to (cephalexin) was much higher (81.8%) in this study. This is comparable to the study done by Namrata et al. in the eastern part of Nepal who reported the resistant rate to be above (65%).16 Resistance to quinolones (ciprofloxacin) was also high (71%) in this study . In the study reported by Lahari Sakia et al., the resistant rate was also high (87.5%) in Assam.13 However, in the same institute, a previous study, in 2001, reported the resistant rate of ciprofloxacin to be only (22.8%).16 The rapid emergence of ciprofloxacin is probably due to the indiscriminate and empirical use of these drugs. MSSA isolates shows higher susceptibility to penicillin and cloxacillin (100% vs. 41.3%) and (70.6% vs.43.0%) respectively than MRSA strains. The epidemiology of MRSA is gradually changing since its emergence was reported. Initially there were occasional reports but now it has become one of the established hospital acquired pathogen. Moreover, the association of multidrug resistance with MRSA had added to the problem. ß – lactam antibiotics like penicillin and cephalexin resistance were 100% and 81% respectively. Resistance to amino glycosides was more in gentamicin (38%) than amikacin (9.2%) in this study, however, it cannot be recommended for empirical treatment of MRSA associated infections. Vancomycin seems to be the only antimicrobial agent which showed 100% sensitivity and may be used as the drug of choice for treating multidrug resistant MRSA infections. However, regular monitoring of vancomycin sensitivity and routine testing of other newer glycopeptides like teicoplanin should be carried out. Further, the regular surveillance of hospital associated infections including monitoring antibiotic sensitivity pattern of MRSA and formulation of definite antibiotic policy may be helpful for reducing the incidence of MRSA infection. Conclusion This preliminary report showed a high prevalence of MRSA in our hospital. There is a need for surveillance of MRSA and its antimicrobial profile. 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