Abstract

Background :There is a marked variability in the prevalence of Staphylococcal infections in different specialties. This study was done to find the prevalence of Methicillin Resistant Staphylococcus aureus (MRSA) in different specialties of our hospital.

Methods : Various clinical specimens (Pus, Blood, Urine, CSF, Sputum, Ascitic and Pleural fluids) were processed and the pathogens were identified as per the standard bacteriological techniques

Results : A total of 4847 (8.89%) Staphylococcus aureus isolates were isolated from 54,486 clinical specimens. Amongst these, MRSA were 40.21%. Majority of the MRSA were from wound swab (68.19%). Amongst indoor patients, 91.49% MRSA were from wards and 8.51% were from Intensive Care Units. The antibiotic susceptibility pattern of MRSA showed 100% sensitivity to Vancomycin and Linezolid followed by Netilmicin (39.4%). All MRSA isolates were resistant to Penicillin.

Conclusion : The determination of prevalence and antibiotic sensitivity pattern of MRSA will help the treating clinicians for first line treatment in tertiary care hospitals.

Introduction

taphylococcus aureus is one of the most
versatile nosocomial (i.e. acquired in hospital) pathogen.1,2 Methicillin-resistant S. aureus (MRSA) has emerged as a major pathogen world-wide.3-8 Several surveys have confirmed that the incidence of MRSA varies by region; during the last 20 years, the proportion of isolates resistant to methicillin has ranged from less than 1% in Scandinavia to more than 30% in Spain, France, Italy and India.9-11 in recent years, MRSA has become a particularly significant problem in Indian hospitals. In one study conducted in a tertiary care hospital in India, MRSA carriage rate ranged between 28.4% in outpatients and 33.5% in in-patients.12 MRSA is often seen in tertiary care hospitals with special care units such as burns, surgical, pulmonary and before coming to the hospital etc. are the possible predisposing factors of MRSA emergence. The development of resistance to multiple antibiotics and control of disease transmission by MRSA isolate in hospitals / communities have been recognized as the major challenges as the bacterial population that expresses the resistance phenotype varies according to the environmental conditions.2 Therefore, the knowledge of prevalence of MRSA and their current antimicrobial profile become necessary in the selection of appropriate empirical treatment of these infections. We determined the prevalence of MRSA from different clinical and MRSA carrier screening samples and their in vitro susceptibility pattern to various antimicrobial agents to record the current status of MRSA response to commonly used anti-Staphylococcus antibiotics in our hospital.

Material and Methods

The study was carried out in a tertiary care hospital over a period of four years (2002-2005) with the aims:

1. To isolate and identify Staphylococcus aureus from different clinical specimens across various specialities and screen these isolates for Methicillin resistance.
2. To study the distribution of MRSA in different Intensive Care Units.
3. To study the antimicrobial susceptibility pattern of these isolates.
4. To determine the Minimal Inhibitory Concentration (MIC) of MRSA for Oxacillin by agar dilution method
5. To correlate the different phage types with different clinical specialities.

A total of 54,486 clinical specimens such as urine, pus, sputum /throat swab, blood, pleural and synovial fluid, catheter tip, bedsore, tissue exudates, conjunctival, ear, nasal, tracheal swabs and oral and MRSA carrier screening samples were collected.

All the samples were aseptically handled and processed. The morphotypes were done for all the samples based on the Gram staining method to determine the likely organism present. Subsequently, the clinical specimens and carrier screening samples were inoculated on to Blood agar plates, MacConkey agar and incubated at 37°C for 24 hours. All strains were further tested for the production of free coagulase enzyme using tube coagulase test based on standard methods. Staphylococcus aureus ATCC 25923 of known coagulase production was included as control strain.

All the confirmed S. aureus strains were subsequently tested for methicillin resistance based on Kirby-Bauer disk diffusion method using oxacillin discs (1 mg) obtained from Hi-Media Laboratories Pvt. Ltd. The isolates were considered methicillin resistant, if the zone of inhibition was 13 mm or less. Further, the antibiotic susceptibility pattern of methicillin resistant S. aureus strains was determined on the day of their isolation by the modified Kirby-Bauer disc diffusion method on Muller Hinton agar using the criteria of standard zone sizes of inhibition to define sensitivity or resistance to different antimicrobials. The antibiotics used were amikacin (30 mg); amoxy-clav (20/10 mg); cefazolin (30 mg); cephotaxime (30 mg); ciprofloxacin (10 mg); penicillin-G (10 unit); cloxacillin (30 mg); netilmicin (10 mg); vancomycin (30 mg); linezolid (30 mg). Finally, the data were recorded and analysed at the completion of the study.

Results

A total of 4847 Staphylococcus aureus was isolated from 54,486 clinical specimens (Table 1). In our study, we have reported a prevalence of 8.89% of Staphylococcus aureus from 54,486 clinical specimens. In a study conducted in 1991, Pal and Ayyagiri14 reported a low prevalence of Staphylococcus aureus. They isolated 1770 isolates of Staphylococcus aureus from 91,367 different clinical specimens i.e. 1.93%. Durmaz et al15 reported a prevalence of 2.86% Staphylococcus aureus from a total of 13,348 different clinical specimens in a study conducted in Turkey during 1997. The MRSA Surveillance Study group in 1997 studied a total of 13,610 various clinical specimens across three centres in India and reported prevalence of 5.42% of Staphylococcus aureus.16 Mehta et al17 in 1998, reported a high prevalence of Staphylococcus aureus (20.65%) from 26,261 clinical specimens in a five year study.

Our study showed an MRSA prevalence of 40.21%. Pal and Ayyagiri14 in 1991 reported 22.6% methicillin resistance; while Durmaz et al15 reported 31.3% methicillin resistance from Turkey. The MRSA Surveillance Study group reported 31.8% methicillin resistance across three centres across India.16 Mehta et al reported 29% MRSA prevalence.¹⁷

In a study of Staphylococcus aureus strains submitted to the Indian National Reference Laboratory, Maulana Azad Medical College, New Delhi it was found that prevalence ofMRSA has increased from 9.83% in 1988 to 45.44% in 1992.18

In our study only 3.33% (65 isolates) of the MRSA isolates were from the outdoor patients and the prevalence of MRSA in Intensive care units was 8.51% (166). The maximum prevalence of MRSA 38.53% was in the burns unit (Table 2).

The antibiotic sensitivity pattern of MRSA showed 100% sensitivity to Vancomycin and Linezolid followed by Netilmicin (39.4%), Amoxycillin-Clavulinic acid (34%) and Amikacin (30.9%). All the MRSA isolates were resistant to Penicillin (Table 3).

In our study, maximum isolates had MIC values between 64 –128 mg /ml (Table 4). No isolate was found to have MIC values more than 128 mg/ml for Oxacillin. Barrett et al19 in 1968 reported MIC values ranging from 50-100 mg/ml. Shanson et al20 (1976), have reported MIC of 25 mg/ml; Peacock et al21 (1980), have reported values > 64 mg/ml; Bhatia et al22 (1982), have reported values > 10 mg/ml; Craven et al23 (1986) reported values of 6.25 mg/ml while Santos et al24 (1999) have reported values as high as 512 mg/ml.

The phage typing was done at National Staphylococcal Phage Typing Centre, Maulana Azad Medical College, New Delhi. Of the 575 isolates, 343 (59.65%) were non typable by routine sets of phage with RTD Table 5). Of the 232 isolates that were typable, 179 (73.66%) belonged purely to phage group III. Of these, the common phages were 47/54/75/85. Colley et al3 isolated 79 MRSA isolates, which belong to phage group III. Craven et al23 performed phage typing on 52 isolates of MRSA. The isolates belonged to group III phages, type 47, 54, 75, 83A. They stated that Staphylococcus aureus strains lysed by group III phages have often been resistant to multiple antibiotics and have frequently have been associated with many outbreaks of infection.

References

1. Lowy FD. Staphylococcus aureus infections. New Engl J Med 1998; 339 : 520-32.
2. Archer GL. Staphylococcus aureus: a well-armed pathogen. Clin Inf Diseases 1998; 26 : 1179-81.
3. Colley EW, McNicol MW, Bracken PM. Methicillin resistant Staphylococcus aureus in a general hospital. Lancet 1965; 1 : 595-7.
4. Crossley K, Landesman B, Zaske D. An outbreak of infections caused by strains of Staphylococcus aureus resistant to methicillin and aminoglycosides. J Inf Diseases 1979; 139 : 280-7.
5. Fluckiger U. Epidemiology of methicillin-resistant Staphylococcus aureus. Chemotherapy 1979; 45 : 121-34.
6. Cars O. Colonisation and infection with resistant gram-positive cocci. Epidemiology and risk factors. Drugs 1997; 54 (Suppl 6) : 4-10.
7. Michel M, Gutman L. Methicillin-resistant Staphylococcus aureus and Vancomycin resistant enterococci: therapeutic realities and possibilities. Lancet 1997; 349 : 1901-6.
8. Ayliffe GA. The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clin Inf Diseases 1997; 24 (Suppl 1) : S74-79.
9. Herwalt AH. Control of methicillin-resistant Staphylococcus aureus in the hospital settings. Am J Med 1999; 106 : 11S-18S.
10. Voss A, Milatovic D, Wallrauch_Schwarz C, et al. Methicillin-resistant Staphylococcus aureus in Europe. European J Clin Microbiol Infect Diseases. 1994; 13 : 50-5.
11. Mehta A, Rodrigues C, Kumar R, et al. A pilot programme of MRSA surveillance in India. J Post Med 1996; 42 : 1-3.
12. Mathur SK, Singhal S, Prasad KN, et al. Prevalence of methicillin-resistant Staphylococcus aureus in a tertiary care hospital in India. Indian J Med Microbiol 1994; 12 : 96-101.
13. Villanova PA. Performance standards for antimicrobial disk susceptibility tests. In National Committee of Clinical Laboratory Standards. Approved standards. 7th Edition National Committee for Clinical Laboratory Standards; 2000 : M2-M7.
14. Pal N, Ayyagiri A. Drug resistance pattern of Methicillin-resistant Staphylococcus aureus. Ind Pead 1991; 28 : 725-9.
15. Durmaz B, Durmaz R, Sahin K. Methicillin resistance among Turkish isolates of Staphylococcus aureus strains from nosocomial and community infections and their resistance pattern various antimicrobial agents. J Hosp Inf 1997 ; 37 : 325-9.
16. MRSA Surveillance Study Group. A pilot programme of methicillin resistant Staphylococcus aureus surveillance in India. JAPI 1997; 45 (6) : 443-5.
17. Mehta A, Rodrigues C, et al. Control of methicillin resistant Staphylococcus aureus in a tertiary care centre – a five year study. Indian J Med Microbiol 1998; 16 (1) : 31-4.
18. Mehendiratta PC, Vidhare S, Mehta MD. A study of Staphylococcus aureus isolates submitted to a reference laboratory. Indian J Med Res 2001; 114 : 90-4.
19. Barrett FF, McGehee RF, Finland M. Methicillin resistant Staphylococcus aureus at Boston City Hospital. New Engl J Med 1969; 279 (9) : 441-8.
20. Shanson DC, Kensit JG, Duke R. Outbreak of hospital infections with a strain of Staphylococcus aureus resistant to gentamicin and methicillin. Lancet 1976; 1347-8.]
21. Peacock JE, Marsik FJ, Wenzel R. Methicillin resistant Staphylococcus aureus: Introduction and spread within a hospital. Ann Intern Med 1980; 93 : 526-32.
22. Bhatia R, Baveja UK, et al. Methicillin resistant Staphylococci. Indian J Med Res 1982; 76 : 365-7.
23. Craven DE, Reed C, et al. A large outbreak of infections caused by a strain of Staphylococcus aureus resistant to Oxacillin and aminoglycosides. Am J Med 1981; 71 : 53-8.
24. Santos KRN, Teixeria LM, Leal GS, et al. DNA typing of methicillin resistant Staphylococcus aureus isolates and factors associated with nosocomial acquisition in two Brazilian University Hospitals. J Med Microbiol 1999; 48 : 17