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 ~  Abstract
 ~  Material and methods
 ~  Results
 ~  Material and methods
 ~  Results
 ~  Discussion
 ~  References

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Year : 2001  |  Volume : 19  |  Issue : 2  |  Page : 13-16

Study of methicillin resistant S. aureus (MRSA) isolates from high risk patients

Department of Microbiology, Maulana Azad Medical College, New Delhi, India

Correspondence Address:
Department of Microbiology, Maulana Azad Medical College, New Delhi, India

 ~ Abstract 

MRSA is an important hospital pathogen, the incidence of which is increasing every year especially in high risk groups. The present study was performed in high risk patients admitted in burns and orthopaedic units of LN hospital to study the infection rate of MRSA from these units. The proportion of MRSA amongst S. aureus isolates was found to be 51.6% and these isolates were multidrug resistant. Phage typing of these isolates gave a typeability of 41.8% using the MRSA set of phages. Biotyping of these isolates could divide them into four groups. The study shows a high incidence of MRSA from burns and orthopaedic units with a high level of antibiotic resistance amongst these isolates.

How to cite this article:
Vidhani S, Mehndiratta P L, Mathur M D. Study of methicillin resistant S. aureus (MRSA) isolates from high risk patients. Indian J Med Microbiol 2001;19:13-6

How to cite this URL:
Vidhani S, Mehndiratta P L, Mathur M D. Study of methicillin resistant S. aureus (MRSA) isolates from high risk patients. Indian J Med Microbiol [serial online] 2001 [cited 2020 May 30];19:13-6. Available from:

Staphylococcus aureus has been reported as a major cause of community and hospital acquired infections.[1] The organism has a differential ability to spread and cause outbreaks in hospitals.[2] Infections causes by S.aureus used to respond to b lactam and related group of antibiotics. However, due to development of methicillin resistance amongst S.aureus isolates (MRSA); treatment of these infections has become problematic. Indiscriminate use of multiple antibiotics, prolonged hospital stay, intravenous drug abuse, carriage of MRSA in nose are few important risk factors for MRSA acquisition.[3] Burns and Orthopaedics are two such high risk units where patients are on multiple antibiotics and have a long stay in hospital. Currently, the treatment options for MRSA infections are limited to very few and expensive drugs like Teicoplanin and Vancomycin.
Thus, control of MRSA is essential to curtail the introduction and spread of infection. This can be achieved by observing universal precautions and conducting regular epidemiological studies like the present one to know the changing trends. It has been seen that MRSA are generally not typeable by routine set of phages and henceforth, CPHL, London have developed a new set of MRSA phages which were propagated and standardized in the present study.

 ~ Material and methods Top

A total of four hundred and fifty patients from Orthopaedics and Burns units of LN Hospital, New Delhi were studied over a period of ten months from March 1998 to Dec. 1998. Pus samples for bacteriological examination were taken from orthopaedic wound and burnt sites at the time of admission followed by 2nd day and weekly thereafter.
A preliminary Gram staining was performed to determine the likely organism present. The samples were inoculated on blood agar, MacConkey's agar and glucose broth which were incubated for 24 hours at 37oC aerobically. Subculture from liquid media on to solid media were done after 24 hours of incubation. S.aureus from pus samples was identified by standard techniques[4] based on colony morphology, gram stain, catalase, slide and tube coagulase and Hugh Leifson's oxidative fermentation test.
Antibiotic sensitivity testing was performed on allS. aureus isolates using modified stokes same plate comparative disc diffusion test.[5] The antibiotics used were Penicillin (10IU); Amoxycillin (10 mg); Augmentin (25 mg); Cephalexin (30 mg); Cefotaxime (30 mg); Cefuroxime (30 mg); Gentamicin (10 mg); Netilmicin (10 mg); Amikacin (30 mg); Erythromycin (5 mg); Roxithromycin (15 mg); Methicillin (5 mg); Rifampicin (2 mg); Ofloxacin (10 mg) and Vancomycin (30 mg). Separate plates were used for methicillin testing on Mueller Hinton agar and these plates were incubated at 30oC x 24 hours. All methicillin resistant strains were also confirmed by oxacillin screening[4] using 6 mg/ml of oxacillin in Mueller Hinton agar with 4% NaCl. Plates were incubated at 30oC and strains showing growth on this medium were taken as methicillin resistant Staphylococcus aureus (MRSA). All strains of MRSA were subjected to minimum inhibitory concentration estimation against oxacillin using agar dilution method as per standard recommendations.[6]
Phage typing was performed at National Phage Typing Centre, MAMC. Typing was done by standard method[7] at 1 and 100 RTD (routine test dilution) using a basic set of 23 phages obtained from CPHL, Colindale, UK. All MRSA strains were further phage typed using a set of 9 MRSA phages. The 9 phages used were M3, M5, M12, MR8, MR25, C30, C33, C38. All MRSA were characterized by biotyping into 4 groups[8] in the following way:
Statistical analysis was performed using chi-square test and Z test. A p value of <0.05 was taken as statistically significant.

 ~ Results Top

A total of 1,426 wound swabs were taken from 450 patients from Orthopaedics and Burn units.
Amongst these high risk patients, 407 patients (90.4%) showed evidence of bacterial growth from their wound swabs. S. aureus was isolated from 188 patients (41.8%) out of which the proportion of MRSA was found to be 51.6% i.e. 97 out of 188 S. aureus isolates were resistant to methicillin. There was a marked difference in antibiotic sensitivity pattern of these MRSA vs the MSSA isolates [Figure - 1]. None of the MRSA isolate was found to be sensitive to Penicillin and Amoxycillin while 6(5.5%) and 12(11%) respectively of MSSA were sensitive to these antibiotics. 85(77.9%) of MSSA were sensitive to Cefotaxime while only 17(21.5%) of MRSA were sensitive to this antibiotic. Sensitivity to macrolide group of antibiotics like Erythromycin and Roxithromycin was seen in 77(70.6%) of MSSA in comparison to 14(17.7%) of MRSA. Amongst the Aminoglycosides; maximum sensititivity was seen with amikacin and 74(67.9%) of MSSA were sensitive to this antibiotic while only 21(26.6%) MRSA were sensitive to the same. Fifty three (67%) of MRSA and 76(69.7%) of MSSA were found to be sensitive to fluoroquinolone group i.e. ofloxacin. All S.aureus isolates (MSSA and MRSA) were found to be uniformly sensitive to vancomycin which is the drug of choice for treating infections caused by MRSA.
All MRSA isolates were further confirmed by testing the minimal inhibitory concentration of oxacillin for them. All isolates had an MIC value above 4 mg/ml. MIC50 was 64 mg/ml and MIC90 was 256 mg/ml. [Table - 1].
Majority of MRSA isolates were found to be hospital acquired (87.3%) i.e. the isolate was obtained after 48 hours of admission or was directly related to the hospital intervention while only 12.7% were acquired in community i.e the isolate was obtained within 48 hours of admission and was unrelated to the hospital intervention [Table - 2].

All S.aureus isolates were phage typed using the routine set of phages and all MRSA isolates were further typed using MRSA phages. Amongst the S.aureus isolates only 35.6% were typeable using the conventional phages and amongst these maximum belonged to phage group III (49.3%) followed by phage group of mixed phages (32.8%), group I (16.4%) group II (1.5%).
Majority of MRSA were nontypeable by conventional set of phages and hence these isolates were phage typed using a new set of MRSA phages which gave a typeability of 41.8% at 100 RTD.
Typeability by biotyping was found to be 91.2%. Maximum number of isolates belonged to group B (49.4%) followed by Gp D (34.2%) and Gp A (7.6%). None of the isolates belonged to Gp C and 8.8% of isolates could not be categorised into any of the above mentioned groups and hence were called non typeable group.

 ~ Discussion Top

The epidemiology of MRSA has continued to evolve since its first appearance more than three decades ago. Initially, there were sporadic reports of methicillin resistance amongst nosocomial S. aureus isolates but later MRSA became a well established hospital acquired pathogen with few reports of community acquired isolates. Recent studies report an increased prevalence of community acquired MRSA with different risk factors compared to the earlier investigations from Detroit which first reported community acquired MRSA. [9]
Our study indicates that the epidemiology of MRSA in our country is also changing over the past few decades. 51.6% of S.aureus isolates were found to be resistant to methicillin in the present study while in the previous studies the incidence was found to be 32.8% in 1994; [10] 24% in 1996,[11] 32% in 1997.[12] This implies that the incidence of infection by MRSA isolates keeps changing every year and it is on a rise compared to last few years. The incidence of community acquired MRSA was found to be 12.7% while 87.3% of MRSA isolated were hospital acquired.
The antibiotic sensitivity results showed that all MRSA isolates were significantly more resistant to antibiotics as compared to MSSA isolates (p<0.05). The resistance of MRSA to b lactams like penicillin and amoxycillin was 100% while 87.4% isolates were resistant to augmentin. Cefotaxime resistance was seen in 78.5% of isolates. This was much higher than the resistance obtained in another study in 1995[13] (67%) from the same institution. This has serious implications as far as the treatment of MRSA infections is concerned. It also implies the need to test newer group of antibiotics routinely like vancomycin and teicoplanin.
High MIC values of oxacillin for MRSA isolates was obtained in the present study. MIC50 was found to be 64 g/mL and MIC[90] was 256 g/mL which was higher than results obtained in previous studies[13],[15] and indicates increasing emergence of highly resistant strains over the years.
Bacteriophage typing is an established method of epidemiological typing for S.aureus but due to emergence of methicillin resistance typeability has decreased substantially. Only 35.6% of S.aureus isolates were typeable by the routine set of phages. Amongst these, maximum belonged to group III (49.3%). A total of 64.4% isolates could not be typed by this set of routine phages and this was comparable to nontypeability of 60% in a study in 1997.[15] Typeability of MRSA by the routine set of phages was found to be very low when compared to the typeability in previous years.[15] This signifies an increasing problem of non typeability amongst MRSA and need for newer set of phages for MRSA typing. CPHL Colindale, London has developed a newer set of MRSA phages. These phages were propagated and standardized for the first time in the present study. Typeability of MRSA isolates using these phages was found to be 41.8% which is a significant figure compared to the typeability pattern of MRSA using the routine set. Due to high level of non typeability another scheme of typing using biochemical reactions was performed for all MRSA isolates. According to this method, MRSA isolates could be divided into 4 groups but no correlation between these 4 biotypes and phage types could be established. In the present study only 49.4% of MRSA belonged to biotype B. MRSA biotyping in a study conducted in 1993 categorized 60% of MRSA into B biotype[16] while another study conducted in 1995 reported all MRSA (100%) as biotype B[13] . This implicates that biotyping alone cannot be used for typing purposes. Thus MRSA poses an important problem for epidemiological typing by phenotypic methods due to changing pattern every year. This problem can be solved to some extent by using a combination of these phenotypic methods like the present study to trace the source of infection. It also implies the need to develop a local set of MRSA phages pertaining to a particular area so as to increase the typeability by phage typing. 

 ~ References Top

1.Sheagren JN. Staphylococcus aureus. The persistent pathogen. New Engl J Med 1984; 310: 1368-73, 1437.  Back to cited text no. 1    
2.Williams DN, Peterson PK. Endocarditis caused by coagulase negative staphylococci. Infection 1979;7:5-9  Back to cited text no. 2    
3.Kluytmans J, Belkum AV, Verbrugh H. Nasal Carriage of Staphylococcus aureus; Epidemiology, underlying mechanisms and associated risk. Clin Microbiol Rev 1997; Volume 505-520.  Back to cited text no. 3    
4.Baird D. Staphylococcus. Cluster forming gram positive cocci. Mackie and McCartney Practical Medical Microbiology (4ed) 1996; 2 :245-58.  Back to cited text no. 4    
5.Stokes EJ, Ridgeway GL, Wren MWD. Antimicrobial drugs in clinical microbiology 7th ed. Arnold, London, 1993 : 234-280.  Back to cited text no. 5    
6.Reeves DS, Philip J, William JD, Wise R. Laboratory methods in antimicrobial chemotherapy. Churchill Livingstone, Edinburgh 1978; 202-26.  Back to cited text no. 6    
7.Blair JE, William REO. Phage typing of Staphylococci. Bull WHO 1961; 24 : 771-754.  Back to cited text no. 7    
8.Coia JE, Thomson CF, Baird D. Characterization of MRSA by biotyping, immunoblotting and restriction enzyme fragmentation patterns, J Med Microbiol 1990; 31 : 125-32.  Back to cited text no. 8    
9.Layton MC, Hierholzer WJ, Patterson JE. The evolving epidemiology of methicillin resistant Staphylococcus aureus at a university hospital. Infection Control and Hospital Epidemiology, January 1995; Volume 12-17.  Back to cited text no. 9    
10.Mathur SK, Singhal S, Prasad KN, Kishore J, Ayyagiri A. Prevalence of MRSA in a tertiary care hospital. Ind J Med Microbiol 1994; 12(2) : 96-101.   Back to cited text no. 10    
11.Pulimood TB, Lalitha MK, Jesudason MV. The spectrum of antimicrobial resistance amongst MRSA in a tertiary care centre in India. Ind J Med Res 1996; 103 : 212-215.  Back to cited text no. 11    
12.MRSA surveillance study group. A pilot programme of MRSA surveillance in India. J Assoc Physicians India 1997; 45 : 443-445.  Back to cited text no. 12    
13.Gupta N. Nosocomial infections by coagulase positive Staphylococci in a surgical ward, MD thesis, Delhi University, 1995.  Back to cited text no. 13    
14.Kloos WE. Staphylococcus. Topley and Wilson's Microbiology and Microbial Infections 1998; 9 ed. Vol.2; 577-618.  Back to cited text no. 14    
15.Goel N. MRSA. Evaluation of phage typing methods, MD Thesis, Delhi University 1998.  Back to cited text no. 15    
16.Krishna Prakash S, Mehndiratta P, Mathur MD. Typing and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus. abstract 2nd Indian Conference of Hospital Infection. Hospital Infection Society of India, 1993; 56.  Back to cited text no. 16    
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