Antibiotic Susceptibility Pattern of Nosocomial Isolates of Staphylococcus aureus in a Tertiary Care Hospital, Nepal

Results: Among 149 Staphylococcus aureus isolates, highest resistance was observed against Penicillin (91.94%) followed by Fluoroquinolone (61.74%), Erythromycin (52.94%), Gentamicin (46.98%), Cotrimoxazole (42.95%), Tetracycline (40.94%) and others, whereas susceptibility was observed maximum against Chloramphenicol (94.85%) followed by Rifampicin (92.61%), Tetracycline (59.06%), Cotrimoxazole (57.04%), and others. None of the isolates were resistant to Vancomycin and Teicoplanin. Of these isolates 44.96 % of the isolates were Methicillin resistant S. aureus (MRSA). Resistance to Penicillin, Fluoroquinolone, Erythromycin, Gentamicin, Co-trimoxazole and Tetracycline were associated signifi cantly with MRSA isolates (X2= 8.779, p<0.05, X2= 74.233, p<0.05, X2= 84.2842, p<0.05, X2= 108.2032, p<0.05, X2= 88.1512, p<0.05 and X2= 79.1876, p<0.05 respectively). Although most of the Methicillin sensitive S. aureus (MSSA) isolates were susceptible to both Rifampicin and Chloramphenicol, only Rifampicin susceptibility was signifi cantly associated with them (X2= 10.1299, p<0.05). Among three Biochemical tests for the detection of β lactamase detection namely chromogenic, iodometric and acidimetric test, chromogenic test method had highest sensitivity and specifi city.


INTRODUCTION
Staphylococcus aureus acquired resistance to Methicillin soon after its introduction in therapy. These strains called Methicillin resistant S. aureus (MRSA) are a major cause of morbidity and mortality around the world and have been a most common cause of nosocomial infection since late 1970s. 1 After acquisition of Methicillin resistance in S. aureus, there has been a steady rise in the prevalence of MRSA in many countries.
The prevalence rate of MRSA had approached to 50% in the US hospitals. 2 In the UK, 44% of S. aureus isolated from health care system were MRSA and in Japan 60-70% of S. aureus were MRSA in inpatients. 3,4 It has been stated that hospital associated MRSA are often multiple resistant to other commonly used antibiotics while, community acquired MRSA are often resistant only to β lactam antibiotics and Erythromycin. 5 The aim of this study was to fi nd out the prevalence of nosocomial MRSA in a tertiary care center Kathmandu.

METHODS
A prospective descriptive study was conducted in Tribhuvan University teaching hospital, Kathmandu, Nepal during November 2007 to June 2008. Consecutive clinical samples submitted in microbiology department for culture and sensitivity from patients with nosocomial infection were processed and inoculated onto chocolate agar (CA), blood agar (BA) and MacConkey agar (MA) as required by following standard methodology. 6 The S. aureus isolates (n=149) were identifi ed on the basis of Gram's reaction, biochemical tests and agglutination test. Gram positive cocci in clusters and in short chains, catalase positive, oxidase negative, fermentative, Voges Proskauer positive, mannitol fermenter, clumping factor positive, DNase positive, coagulase positive and Staphytect plus latex agglutination (Oxoid, UK) positive were identifi ed as S. aureus.
Isolates resistant to both Oxacillin and Cefoxitin were identifi ed as MRSA and those susceptible were identifi ed as Methicillin sensitive S. aureus (MSSA).
β lactamase test was performed by three biochemical methods namely chromogenic method, acidimetric method and iodometric method on the colony growing around the penicillin disc. 6,8 For chromogenic test method, Nitrocefi n disc (BBL, USA) was used and reagents for acidimetric and iodometric method were prepared in house.
S. aureus ATCC 43300 and S. aureus ATCC 25923 were used as MRSA and MSSA reference strains. Chi square test was used for the statistical analysis of the data.
The study was approved by the institutional review board, TUTH.

RESULTS
Among 149 nosocomial S. aureus isolates (including 13 isolates from urine and urinary catheter), highest resistance was observed against Penicillin (91.94%) followed by Fluoroquinolone, Erythromycin, Gentamicin, Cotrimoxazole and Tetracycline (Table 1). On the other hand, susceptibility was observed maximum against Chloramphenicol (94.85%) followed by Rifampicin, Tetracycline and Cotrimoxazole. None of the isolates were resistant to glycopeptides Vancomycin and Teicoplanin.
Of 149 isolates 44.96% (n=67) isolates were resistant to both Oxacillin and Cefoxitin and hence were identifi ed as MRSA. Obviously 82 isolates were susceptible to these antibiotics and were identifi ed as MSSA.
A uniform pattern of antibiotic susceptibility/ and resistance was observed among the 38 MRSA isolates. They exhibited a uniform pattern of resistance to Cotrimoxazole, Fluoroquinolone, Gentamicin, Tetracycline and Erythromycin; and susceptibility to Rifampicin and Chloramphenicol. Similarly out of 82 MSSA isolates, 41 isolates exhibited a uniform pattern of susceptibility to Cotrimoxazole, Fluoroquinolone, Gentamicin, Tetracycline, Erythromycin, Rifampicin and Chloramphenicol.

DISCUSSION
Small percent (8%) of the nosocomial S. aureus isolates were susceptible to Penicillin which was in concordance with the reported 10 % in USA. 5 Greater susceptibility to Chloramphenicol was observed without any predilection either to MRSA or to MSSA in present study. Chloramphenicol used to be a very effective antibiotic and its overwhelming use in clinical practice in past has led to emergence of resistance due to which its use has been discontinued. 9 Later as suggested by present fi nding, the organisms have lost resistance to this antibiotic thus rendering them susceptible to Chloramphenicol in these days.
Most MSSA isolates were susceptible to almost all of the FDA recommended antibiotics. On the contrary, most MRSA isolates were resistant to most of the FDA recommended antibiotics similar to what has been stated in USA. 5 In present study probably the fi rst of its kind, the prevalence of nosocomial MRSA isolates obtained   In β lactamase test few false positive and false negative results were obtained with all the three test methods. Chromogenic test had high sensitivity and specifi city as compared to other test methods. But expensiveness and local unavailability of Nitrocefi n disc may impede its regular use, whereas the reagents for acidimetric and iodometric test can be prepared in house using benzyl Penicillin G. One penicillin resistant MRSA isolate that was negative with all three tests had decreased the sensitivity and specifi city of these three tests. Therefore, as recommended by American Society of Microbiology 2004 for suitability of all three test methods for testing β lactamase production in S. aureus and on the basis of present fi nding the acidimetric and iodometric test can be followed. 6

CONCLUSIONS
Nosocomial MRSA strains being signifi cantly associated with resistance to many FDA recommended antibiotics and being a major cause of nosocomial infection, such strains should be stopped from spreading to other patients. Therefore, patients infected with such strains should be identifi ed, kept in isolation and treated with other drug of choice for MRSA. Further, healthcare workers should be trained to control hospital infection and infection control program should be conducted effectively in all health care centers.