DR.HIRAK JYOTI RAJ,KUMUDINI, F-8, Aurobindonagar, Judges' Court Midnapore, Dist.-West Midnapore,West Bengal; PIN

SPECIES IDENTIFICATION OF STAPHYLOCOCCI FROM PUS SAMPLES USING COMMERCIAL IDENTIFICATION SYSTEM AND THEIR ANTIMICROBIAL SUSCEPTIBILITY PATTERN WITH SPECIAL REFERENCETO METHICILLIN. Tanusri Biswas, Munmun Das(Sarkar), Hirak Jyoti Raj, RastonMondal, C.Srilatha. Tanusri Biswas MD, Assistant Professor, Hirak Jyoti Raj MD (ExMD-PGT),Department of Microbiology: Raston Mondal MBBS, Department of Community Medicine,,Burdwan Medical College, Burdwan, West Bengal, India. Munmun Das (Sarkar) MD, Assistant Secretary( Medical Education), Department of Health & Family Welfare, West Bengal,(ExMD-PG):CSrilatha MD,Professor, Department of Microbiology. Mamata Medical College, Khammam, Andhra Pradesh, India Received: March 2012 Accepted June 2013 Correspondence: DR.HIRAK JYOTI RAJ,KUMUDINI, F-8, Aurobindonagar, Judges' Court Midnapore, Dist.-West Midnapore,West Bengal; PIN-721101 Keywords: Abstract The genus 'Staphylococcus' is widely distributed in nature. Some of its species are important pathogens in skin and soft tissue infections causing substantial rates of morbidity and mortality. A hospital based cross sectional study was undertaken to find out different species of staphylococcus in pus samples along with their recent antimicrobial susceptibility pattern including methicillin resistance. Total of 134(67%) staphylococci were isolated from 200 clinical specimens of pus samples by using conventional techniques for identification of the genus Staphylococcus. Among them 116(86.57%) were coagulase positive Staphylococcus aureus. The coagulase negative strains were identified by using "Hi-Staph Identification Kits" through twelve different biochemical tests, based on the principle on ph change and substrate utilisation. Total 18(13.43%) coagulase negative staphylococci were isolated.16 (11.94%) isolates out of 134 were identified as Staphylococcus epidermidis and 2(1.49%) were Staphylococcus haemolyticus. Total 76(56.72%) isolates were methicillin resistant, methicillin resistant Staphylococcus aureus were 62(46.27%), methicillin resistant Staphylococcus epidermidis 12(8.96%) and methicillin resistant Staphylococcus haemolyticus were 2(1.49%). Antimicrobial susceptibility testing revealed that methicillin resistant isolates were significantly more resistant to other commonly used antimicrobials like amoxicillin, ciprofloxacin, gentamicin, co-trimoxazole, tetracycline, than the methicillin sensitive ones. All the isolates were distinctly sensitive to linezolid, rifampicin as well as vancomycin. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Hi-Staph Identification Kits, methicillin resistance. JK-Practitioner 2013;18(3-4):21-30 INTRODUCTION: Staphylococci are ubiquitous bacteria in nature. They are parasites of the body surface of animals and their ability to cause disease appears to be an incidental character. Nevertheless staphylococcal diseases are very common because the conditions that permit the organism to gain access to the tissues are numerous and frequent in occurrence. 1 21 JK- Practitioner Vo 1.21, No (3-4) July-Dec 2013 12

Apart from Rosenbachs Staph pyogenes aureus, now validly named Staphylococcus aureus, the genus also contains twenty three other described species, most of which are composed of coagulase negative staphycoccus (CoNS). The latter may be pathogenic for man, especially members of the species Staphylococcus epidermidis and Staphylococcus saprophyticus. The increasing interest shown in the coagulase negative staphylococci helps to redress a tendency to measure staphylococci solely in terms of their relationship with the coagulase positive Staphylococcus aureus. 2 Serious infections produced by Staphylococcus aureus include bacteraemia, osteomyelitis, acute endocarditis, myocarditis, mastitis, staphylococcal scalded skin syndrome (SSSS), abscess of muscles, infections of urogenital tract. 1 In yesteryears Staphylococcus aureus were very much sensitive to different commonly used antimicrobial agents. But recently due to natural and acquired genetic processes they show multidrug resistance which becomes a major challenge in control of severe life threatening infections among the hospital patients and also in the community. The most notable of this phenomenon is the emergence of Methicillin resistance Staphylococcus aureus (MRSA). 3 Methicillin was the first compound developed to combat resistance due to penicillinase ( lactamase) production of staphylococci. MRSA first appeared in 1961 in Europe. 4 MRSA is often subcategori ed as community acquired MRSA (CA-MRSA) and hospital acquired MRSA (HA-MRSA) depending upon the circumstances of acquiring diseases. It is often referred to in the press as a Superbug." 3 MRSA is a resistant variant of the common bacteria Staphylococcus aureus with the ability to survive treatment with - lactam antibiotics such as penicillin, methicillin and cephalosporins 6 Indiscriminate use of multiple antibiotics, prolonged hospital stay, intravenous drug use and carriage of MRSA in nose are some of the important risk factors for MRSA acquisition. 7 MRSA is neither more infectious nor more virulent than Methicillin sensitive Staphylococcus aureus (MSSA) 8 Currently 30-50% of Staphylococcus aureus and more than 50% of coagulase negative staphylococci are becoming resistant to methicillin. Resistance occurs as a result of acquisition of meca gene that codes for a novel penicillin binding protein PBP2. Expression of PBP2 renders the bacteria resistance to all -lactam antibiotics (including cephalosporins and carbapenems). 9 Recent break points (NCCLS, 1999) have shown an acceptable correlation with the presence or absence of the meca gene in Staphylococcus epidermidis, Staphylococcus hominis and Staphylococcus haemolyticus isolates. 10 Currently the treatment option for MRSA infections is limited to very few and expensive drugs like teicoplanin and vancomycin. 7 The knowledge regarding prevalence of methicillin resistant staphylococci and their current antimicrobial JK- Practitioner Vo 1.22, No (3-4) July-Dec 2013 12

profile become necessary in the selection of appropriate empirical treatment of infection caused by them. 3 So the present study was undertaken to identify different species of staphylococci as aetiological agents in skin and soft tissue infections (SSTI-s) and other pyogenic infections, to elicit their recent antimicrobial susceptibility pattern with special reference to methicillin resistance with a view to create awareness among clinicians about the prevalence of such infection and to guide them for proper medical management. Methods Study Design: It was a cross sectional hospital based study. Period of study: July, 2009 to June, 2010. Place of study: Mamata Medical College and Hospital, Khammam, Andhra Pradesh. Sample size: Total 200 pus samples were collected from in-patient and out-patient department. Sample collection: Pus samples were collected aseptically in sterile vials or by sterile swab sticks (in duplicate) from patients with their prior consent and institutional ethical committee s permission. While collecting pus from the abscesses, wounds or other sites, special care was taken to avoid contamination with commensal organisms from the skin. For deep seated chronic infections (i.e. osteomyelitis, empyema, lung abscess and muscle abscess), specimens were collected from the operation theatre at the time of operation and drainage. Specimens were labelled and immediately transported to the laboratory for microbial processing. Isolation and Identification: Only those samples showing violet coloured Gram positive cocci in grapes like clusters in preliminary Gram s staining were processed further by culturing and subculturing on Nutrient, Blood and MacConkey agar and confirmed for Staphylococcus on the basis of morphology, colony characteristics, catalase, slide and tube coagulase test. 11 ' 12 ' 13 ' 14 ' 13 Biochemical profiles for species identification of CoNS were performed by using KB004 Hi-Staph Identification Kit -a standardi ed colorimetric identification system utilising 12 conventional biochemical tests based on principle of ph change and substrate utili ation, indicated by colour change in the media 16 ' Procedures: A homogenous sterile saline suspension was made with 1-3 well isolated colonies; the density was matched with 0.5 Mc-Farland standards. Each well in the strip was inoculated with 501 of this inoculum by surface inoculation and incubated at 37 C for 18-24hrs with addition of reagents in well no.l 2 after incubation. 16 (Picture 1) Interpretation of tests is as follows: Tests Staph Staph epidermidis haemolyticus Voges Proskauer s + + Alkaline phosphatase ONPG Urease + _ Arginine + + Mannitol Sucrose + + Lactose + Arabinose Raffinose Trehalose _ + Maltose + + 23 JK- Practitioner Vo 1.23, No (3-4) July-Dec 2013 12

The antimicrobial susceptibility test was performed for all the staphylococci isolated in Mueller Hinton agar (MHA) according to the standardi ed Kirby- Bauer disc diffusion method by using different antimicrobial discs as amikacin (Ak, 30 g), amoxicillin (Ac, 30 g), cefepime (Cpm, 30 g), cephalexin (Cx, 30 g), ciprofloxacin (Cf, 5 g), cotrimoxa ole (Co, 1.25/23.75 g), erythromycin (E, 15 g), gentamicin (G, 10 g), line olid (L, 30 g), penicillin G (P, lounits), rifampicin (R, 5 g), tetracycline (T, 30 g) and vancomycin (Va, 30 g) 17 ' 18 Staphylococcus aureus ATCC 29213 strain was used as control strain for the standardi ation of antimicrobial susceptibility testing and other tests. 3 After incubation at 35 C for 24 hours, one diameters were documented in millimetres by measuring the diameter of clear one around the discs and were interpreted as resistant, intermediate resistant or susceptible. 18 Methicillin sensitivity was tested on MHA supplemented with 5% NaCl and Mannitol salt agar (MSA), after uniform streaking with the swab, with 1 g oxacillin discs and incubated at 30-32 C for 24 hours. For MHA disc diffusion method, 10mm one si e was accepted as resistant for Staphylococcus aureus and for CoNS one si e was accepted as resistant at 17mm. For MSA disc diffusion method, both for Staphylococcus aureus and CoNS, one si es were accepted as resistant at 16mm 19 Statistical Analysis: Analysed with SPSS10.0 (Statistical Package for Social sciences)and percentage, chi-square test and Fisher s exact (two tailed) tests were used. A p-value of 0.05 was taken as significant.results From a total of 200 pus samples, staphylococci were isolated from 134 (67%) samples. Out of these 134 isolatesll6 (86.57%) were found to be coagulase Figurel. Species distribution ofmethicillin resistant & methicillin sensitive staphylococcal isolates JK- Practitioner Vo 1.24, No (3-4) July-Dec 2013 12

positive Staphylococcus aureus andl8 (13.43%) were CoNS. Only 2 species of CoNS were isolated-16 (11.94%) Staphylococcus epidermidis and 2 (1.49%) Staphylococcus haemolyticus (Table 1) Staphylococcal Coagulase No. of Percentage Isolates Test Species Staph aureus Positive 116 86.57 Staph epidermidis Negative 16 11.94 Staph haemolyticus Negative 2 1.49 Total 134 100 The reports on methicillin resistance of 116 Staphylococcus aureus confirmed the presence of 62 (53.45%) as MRSA, rest 54 (46.55%) as MSSA; methicillin resistance among 18 coagulase negative isolates were 14 (77.78%) and 4 (22.22%) were methicillin sensitive. Although presence of methicillin resistance in coagulase positive isolates (53.45%) in comparison to coagulase negative isolates (77.78%) was not statistically significant (p0.05) in this study but among total 134 samples containing staphylococci, MRSAs were prevalent in 62 (46.27%) samples whereas methicillin resistant Staphylococcus epidermidis (MRSE) were prevalent in 12(8.96%) and methicillin resistant Staphylococcus haemolyticus in 2 (1.49%) samples. Isolation of MRSA (62) from different wards: Surgery 37(59.67%), Orthopaedic: 12(19.35%), Gynaecology Obstetrics:3(4.84%), Dermatology: 7(11.29%) and other wards:3(4.84%). Isolation of MRSE (12) from Surgery wards: 5(41.67%), Orthopaedic: 2(16.67%), Gynae Obst.:l(8.33%) and 4(33.33%) from Dermatology. Two (100%) methicillin resistant Staphylococcus haemolyticus were isolated from Gynae Obst wards. (Table 2) Table 2. Wards -wise frequency distribution of coagulase positive and Meth. Species Surg. Ortho. Gynae. Derm. Other T ' Resist. Wards 'Wards & Wards Wards o Staph. (%) (%) Obst. (%) (%) T Wards A (%) L Coag. Staph. 37 12 3 7 3 62 ( ve) aureus (59.67) (19.35) (4.84) (11.29) (4.84) Coag. Staph. 5 2 1 4 0 12 (-ve) epider- (41.67) (16.67) (8.33) (33.33) (0) -midis Staph. 0 0 2 0 0 2 haem - (0) (0) (100) (0) (0) lyticus TOTAL 42 14 6 11 3 76 Methicillin resistance was more (65%) among hospital acquired isolates HA- MRSA-responsible for causing infections 48hrs after hospital admission and with any type of hospital intervention 7 in comparison to (53.19%) community acquired isolates CA-MRSA-causing infections within 48hrs of hospital admission and without any hospital intervention 7. Methicillin sensitivity, on the contrary, was more (46.80%) among community acquired isolates in respect to (35%) hospital acquired ones, though p value (0.05) was not statistically significant. Antimicrobial susceptibility pattern also showed in general resistance of staphylococcal isolates to the most commonly used antimicrobials (Table3). All were sensitive to line olid, rifampicin and vancomycin. Remarkably high degree of resistance was detected against cefepime (35.48%), cephalexin (29.03%), cotrimoxa ole (61.29%), tetracycline (48.39%), gentamicin (70.97%), ciprofloxacin (87.10%), penicillin G (100%) and erythromycin (80.65%) (p 0.05, in cases of all above drugs) for coagulase positive methicillin resistant staphylococci (MRSA) and against tetracycline (85.71%) and erythromycin (85.71%) for coagulase negative methicillin resistant staphylococci (MR-CoNS) (Table 4,) 25 JK- Practitioner Vo 1.25, No (3-4) July-Dec 2013 12

Discussion The present study highlighted the problem of MRSA in a tertiary care hospital which provides the health care need of the whole district and adjoining areas. Here percentage (86.57%) of Staphylococcus aureus was more than that (13.43%) of CoNS (Staphylococcus epidermidis-11.94%, Staphylococcus haemolyticus-\.49%) (Table 1) because Staphylococcus aureus is the main aetiological agent and most frequently isolated micro-organism in various SSTIs. Although Staphylococcus epidermidis accounts for majority of CoNS infections, other species have also been identified in association with human infections. Study by Majumder et al showed Staphylococcus aureus -71.25% and CoNS-28.75%,which was more than that in present study as they included isolates from both clinical specimens as well as carriers. 2 " Among CoNS, 88.89% (16/18) were Staphylococcus epidermidis and 11.11% (2/18) Staphylococcus haemolyticus. Study by Caierao Juliana et al reported 41.10"/(/Staphylococcus epidermidis and 23.40% Staphylococcus haemolyticus. 10 Study by Shobha et al revealed Staphylococcus epidermidis 49.23% and Staphylococcus haemolyticus 1.53%. 17 Mohan et al reported Staphylococcus epidermidis 82.29% and Staphylococcus haemolyticus 1.59' 21 Here increased prevalence of Staphylococcus epidermidis compared to previous studies indicated their increasing role as an aetiological agent of nosocomial infections. Prevalence of Table 3. Antimicrobial susceptibility pattern of staphylococci iso lated Coagulase positive Coagulase negative Staphylococci (n 116) Staphylococci (n 18) Antimicro bials Sensitive Sensi- tant tive Sensitant Intm.* Resis- Sensi- Intm. Resis- -tive tive (%) (%) (%) (%) (%) (%) Amikacin 106 6 4 12 2 4 (91.30) (5.17) (3.45) (66.67 (11.11) (22.22) Amoxicillin 0 0 116 0 0 18 (0) (0) (100) (0) (0) (100) Cephalexin 78 16 22 14 0 4 (67.24) (13.79) (18.97) (77.78) (0) (22.22) Cefepime 84 8 24 12 2 4 (72.41) (6.90) (20.69) (6(5.67) (11.11) (22.22) Ciprofloxacin 32 14 70 4 6 8 (27.59) (12.27) (60.34) (22.22) (33.33) (44.44) Cotrimoxa ole 38 24 54 4 8 6 (32.76) (20.69) (46.55) (22.22) (44.44) (33.33) Erythromycin 30 44 42 4 4 10 (25.86) (37.93) (36.21) (22.22) (22.22) (55.56) Gentamicin 46 22 48 2 4 12 (39.66) (18.97) (41.38) (11.11) (22.22) (66.67) Line olid 116 0 0 18 0 0 (100) (0) (0) (100) (0) (0) Penicillin G 18 0 98 0 0 18 (15.52) (0) (84.48) (0) (0) (100) Rifampicin 116 0 0 18 0 0 (100) (0) (0) (100) (0) (0) Tetracycline 38 32 46 2 4 12 (32.76) (27.59) (39.66) (11.11) (22.22) (66.67) Vancomycin 114 0 2 18 0 0 (98.28) (0) (1.72) (100) (0) (0) *Intm. Intermediate JK- Practitioner Vo 1.26, No (3-4) July-Dec 2013 12

Staphylococcus haemolyticus is also observed in pus samples as it may cause wound infection, bacteraemia and endocarditis in spite of urinary tract infection. In this study prevalence of MRSA (46.27%) (Figurel) was nearer to the results i.e. 51.60% reported from L N Hospital. 7 In India, the prevalence is changing likewise 32.80% in 1994, 24% in 1996, 32% in 1997. 7 Chaudhury et al in 2007, reported it as 52.80%. 22 So the present study is also keeping pace with the trend. Methicillin resistance, in present study, was found in 77.78% (14/18) of CoNS isolates, among them Staphylococcus epidermidis accounted for 66.67% (12/18) and Staphylococcus haemolyticus 11.11% (2/18). Another two studies also revealed 87.70% and 54.40% of CoNS respectively as methicillin resistant. 10 ' 19 These suggested an increasing prevalence of MR-CoNS in hospital isolates. In case of both MRSA and MR- CoNS, isolation was more from Surgery wards (59.67% 41.67%) and Orthopaedic wards (19.35%, 16.67%) followed by Gynaecology and Obstetric wards (G O) (4.84%,8.33%) and Dermatology (11.29%, 33.33%) (Table2). Two Staphylococcus haemolyticus were isolated from G O wards only. Study by Srinivasan et al showed prevalence of MRSA as 80%from Surgical and 28% from Orthopaedic ward. <4> This was probably due to prolonged stay in hospital with greater chance of acquiring infections. Actually more thanl5 days stay in hospital is associated with increased risk of getting infections. Vidhani et al reported the prevalence of HA-MRSA (87.30%) and CA-MRSA (12.70%) which were comparable with present study findings of 65.00% HA-MRSA and 53.19% CA-MRSA; although it was not statistically significant (p0.05) here. Multiple antimicrobial resistances of the isolates to commonly used drugs (Table 3) like amoxicillin 100% (116/116), penicillin G 84.48% (98/116), ciprofloxacin 60.34% (70/116), gentamicin 41.38% (48/116), erythromycin 36.21% (42/116) and co-trimoxa ole 46.55% (54/116) in case of Staphylococcus aureus and for CoNS, like amoxicillin 100% (18/18), penicillin G 100% (18/18), gentamicin 66.67% (12/18), tetracycline 66.67% (12/18), erythromycin 55.56% (10/18), ciprofloxacin 44.44% (8/18) and co-trimoxa ole 33.33% (6/18), were due to rapid increase in penicillinase producing hospital strains as a consequence of cross infections leading to multidrug resistance. This included both penicillinase production as well as non-en ymatic resistance (methicillin resistance). The genetic determinants for such resistance comprise a series of plasmid borne and chromosomal genes. Selection and cross infections were the main factors responsible for building up and maintaining population of resistant staphylococci. 1 Moreover expression of PBP-2 genes renders the bacteria resistant to penicillins, cephalosporins and carbapenems. 9 In present study, the co- existing resistance of MRSA-s to different antimicrobials like tetracycline (48.39%), co-trimoxa ole (61.29%),gentamicin 27 JK- Practitioner Vo 1.27, No (3-4) July-Dec 2013 12

(70.97%),erythromycin (80.65%) ciprofloxacin (87.10%), penicillin G (100%),and amoxicillin (100%) was statistically significant (p0.05) than that of MSSA isolates (Table 4). High resistance to these antimicrobials has also been reported in other studies. 7 ' 23 Remarkable high resistance (87.10%) of MRSA to ciprofloxacin (87.10%) also reported from other studies. 3 ' 7 An increase in gentamicin resistance from 0% before 1996 to 80% after 1996 has been reported in a study by Rajaduraipandi et al in which it was (63.20%). 3 Increased tetracycline resistance (54.20%) was also observed in another study. 2 " In case of MR-CoNS also higher degree of resistance detected against other anti-microbials like amoxicillin( 100%), penicillin G (100%) followed by erythromycin (85.71% ), tetracycline (85.71%), gentamicin (57.14%) and ciprofloxacin ( 57.14% ) (Table 4) was corelated with study findings of Shobha et al and Mohan et al. 17 ' 21 Erythromycin resistance (85.71%) was higher here as compared to their findings. These multidrug resistant isolates with their propensity to spread and coloni e debilitated patients pose a major difficulty in treating systemic infections. However almost all methicillin resistant isolates (both MRSA and MR- CoNS) were recorded as 100% sensitive to line olid, rifampicin followed by vancomycin (96.77%,100%), amikacin (8 7. 10%, 5 7. 14%), cefepime ( 5 8. 0 6 %, 5 7. 1 4 %), cephalexin (54.84%,71.43%). This type of sensitivity pattern was observed in many studies. 3 ' 4 ' 17 So these drugs can now be used for treating Table 4. Different antimicrobial resistance pattern of coagulase positive and coagulase negative, methicillin resistant Antimicrobials No. of different Antibiotic p value No. ofdifferent Antibiotic p resistant coagulase(+ ve) resistant Coagulase(-ve) value staphylococci staphylococci MR (n-62) MS (n-54) MR (n-14) MS (n-4) (%) (%) (%) (%) linezolid 0 0 0 0 (0) (0) (0) (0) Rifampicin 0 0 0 0 (0) (0) (0) (0) Vancomycin 2 0 0.538 0 0 (3.23) (0) (0) (0) Amikacin 4 0 0.165 6 0 0.245 (6.46) (0) (42.86) (0) Cefepime 22 4 <0.001 4 0 0.524 (35.48) (7.41) (28.57) (0) Cephalexin 18 2 <0.001 4 0 0.524 (29.03) (3.70) (28.57) (0) Co-trimoxazole 38 14 <0.001 6 0 0.245 (61.29) (25.93) (42.86) (0) Tetracycline 30 11 0.003 12 0 0.005 (48.39) (20.37) (85.71) (0) Gentamicin 44 6 <0.001 8 2 1.000 (70.97) (11.11) (57.14) (50.00) Ciprofloxacin 54 20 <0.001 8 0 0.092 (87.10) (37.04) (57.14) (0) Amoxicillin 62 54 14 4 (100) (100) (100) (100) Penicillin G 62 37 <0.001 14 4 (100) (68.52) (100) (100) Erythromycin 50 5 <0.001 12 0 0.005 (80.65) (9.26) (85.71) (0) Chi square test, Yates correction, Fishers exact test (two-tailed) MR Methicillin Resistant, MS Methicillin Sensitive. JK- Practitioner Vo 1.28, No (3-4) July-Dec 2013 12

multidrug resistant MRSA and MR-CoNS infections. Line olid appears to be one of the few available antimicrobial agents with proven activity against multidrug resistant Staphylococcus aureus including strains with reduced susceptibility to glycopeptides (i.e. vancomycin). However, the drug being bacteriostatic, has to be given for longer duration, which may counterbalance its cost efficacy. 4 Considering the results of present study, it can be concluded that prevalence of methicillin as well as other multiple drug resistant strains of staphylococci are now increasing day by day in a hospital environment and that is going to make treatment of patients with skin and soft tissue and other life threatening infections, gradually unresponsive. Line olid, vancomycin, rifampicin and fourth generation cephalosporins should be kept as reserve drugs to be used cautiously and judiciously, only when badly needed. Intense attention should be given on prevention and control of nosocomial infections by formulating a proper hospital infection control policy. References 1. Easman CSF Staphylococcal diseases. In: Smith GR, Easman CSF, editors Topley Wilsons Principles of Bacteriology, Virology and Immunity. Vol 3. 8 th ed. Philadelphia, Hamilton: B. C. Decker Inc, 1990: 215-38. 2. Easman CSF, Goodfellow M Staphylococcus and Micrococcus. In: Parker MT, Duerden BI, editors Topley and Wilson s Principles of Bacteriology, Virology and Immunity. Vol 2. 8 th ed. Philadelphia, Hamilton. B.C. Decker Inc, 1990:161-86. 3. Rajaduraipandi K, Mani KR, Panneerselvam K, Mani M, Bhaskar M, Manikandan P Prevalence and antimicrobial susceptibility pattern of methicillin resistant Staphylococcus aureus'. A multicentric study. Indian J Med Microbiol 2006; 24(1): 34-8. 4. Srinivasan S, Sheela D, Shashikala, Mathew R, Ba roy J, Kanungo R Risk factors and associated problems in the management of infections with methicillin resistant Staphylococcus aureus. Indian J Med Microbiol 2006; 24 (3): 182-5. 5. Wikipedia Contributors, Methicillinresistant Staphylococcus aureus Internet. Wikipedia, The Free Encyclopaedia cited 26 June 2009. Available from: http://en. Wikipedia. Org./w/index. Php Title Methicillin-resistant-staphylococcus aureus oldid 298683773. 6. Goyal R, Das S, Mathur M Coloni ation of methicillin resistant Staphylococcus aureus among health care workers in a tertiary care hospital of Delhi. Indian J Med Sci. 2002; 56 (7): 321-4. 7. Vidhani S, Mehndiratta PL, Mathur MD Study of methicillin resistant S. aureus (MRSA) isolates from high risk patients. Indian J Med Microbiol 2001; 19(2): 13-6. 8. Ashok R, Anuradha K, Babu SS, Bheerappa N, Sastry RA, Lakshmi V Post operative infection of an abdominal mesh due to methicillin resistant Staphylococcus aureusa case report. Indian J Med Microbiol 2004; 22(4): 260-2. 9. Murray PR, Rosenthal KS, Pfaller MA Staphylococcus and related organisms. In:. Murray PR, Rosenthal KS, Pfaller MA, editors Medical Microbiology. 5 th ed. Philadelphia: Elsevier Mosby; 2005:221-36. 10. Caierao J, MusskopfM, Superti S, Roesch E, 29 JK- Practitioner Vo 1.29, No (3-4) July-Dec 2013 12

Dias CG, da evedo PA Evaluation of phenotypic methods for methicillin resistance characteri ation in coagulase negative Staphylococci (CNS). J Med Microbiol 2004; 53:1195-9. 11..Duguid JP Staining methods. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors Mackie McCartney Practical Medical Microbiology. 14 th ed. New Delhi, India: Elsevier, Reed Elsevier India Private Limited, 1999:793-812. 12. Collee JG, Marr W Culture of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors Mackie McCartney Practical Medical Microbiology. 14 th ed. New Delhi, India: Elsevier, Reed Elsevier India Private Limited, 1999:113-30. 13. Collee JG, Marr WSpecimen collection, culture containers and media. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editorsmackie McCartney Practical Medical Microbiology. 14 th ed. New Delhi, India: Elsevier, Reed Elsevier India Private Limited, 1999:95-112. 14.Barid DStaphylococcus: Cluster-forming Gram-positive cocci. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors Mackie McCartney Practical Medical Microbiology. 14 th ed. New Delhi, India: Elsevier, Reed Elsevier India Private Limited, 1999: 245-62. 15. Collee JG, Miles RS, Watt B Tests for the identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors Mackie McCartney Practical Medical Microbiology. 14 Sl ed. New Delhi, India: Elsevier, Reed Elsevier India Private Limited, 1999:131-50 16. KB004 HiStaph Identification Kit. Rapid Biochemical Identification Test Kits. Hi Media Laboratories Pvt. Limited. A-406 Bhaveshwar Pla a, LBS JK- Practitioner Vo 1.30, No (3-4) July-Dec 2013 Marg, Mumbai-400086, India. Literature Code: Pr in 004/ KB004/0904. 17.Shobha KL, Rao PS, Thomas J Staphylococcus isolates among Survey of hospital personnel, environment and their antibiogram with special emphasis on methicillin resistance. Indian J Med Microbiol 2005; 23(3): 186-8. 18. one Si e Interpretative Chart. Antimicrobial Susceptibility Test Discs. Hi Media Laboratories Pvt. Limited. A-406, Bhaveshwar Pla a, LBS Marg, Mumbai-400086, India. 19. Ercis S, Sancak B, Hascelik G A comparison of PCR detection of MEC A with oxacillin disc susceptibility testing in different media and sceptor automated system for both Staphylococcus aureus and coagulasenegative staphylococci isolates. Indian J Med Microbiol 2008; 26(1): 21-4. 20.Majumder D, Sharma BJN, Phukan AC, Mahanta J Antimicrobial susceptibility pattern among methicillin resistant Staphylococcus isolates in Assam. Indian J Med Microbiol 2001; 19(3): 138-40.21. 21. Mohan U, Jindal N, Aggarwal P Species distribution and antibiotic sensitivity pattern of coagulase negative Staphylococci isolated from various clinical specimens. Indian J Med Microbiol 2002; 20(1): 45-6. 22.Chaudhury A, Kumar AG In vitro activity of antimicrobial agents against oxacillin resistant staphylococci with special reference to Staphylococcus haemolyticus. Indian J Med Microbiol 2007; 25(1): 50-2. 23.Tahnkiwale Supriya S, Roy S, Jalgaonkar SV Methicillin resistance among isolates of Staphylococcus aureus: Antibiotic sensitivity pattern and phage typing. Indian J Med Sci. 2002; 56 (7): 330-4. original article 12