Prevalence and Antibiogram of Methicillin Sensitive and Methicillin Resistant Staphylococcus aureus Isolated from Pus Samples in a Tertiary Care Teaching Hospital

Staphylococcus aureus is an important pathogen causing bacterial infection of skin lesions, such as burns, traumatic lesions, diabetic ulcers and post operative infections. The most common organism isolated from wound infections in our hospital is Staphylococcus and there have been several reports of MRSA isolates from pus samples during previous years. The aim of the present work is to study the rate of Staphylococcal wound infection and also to know the Antibiogram of Methicillin Sensitive and Methicillin Resistant Staphylococcus aureus. Staphylococcus aureus isolated from pus samples were identified based on colony morphology, Gram stain, Catalase and Coagulase test. Antibiotic susceptibility testing by disc diffusion was performed by Kirby Bauer method as per CLSI guidelines. Out of 595 samples collected, 534 [89.7 %] yielded growth of one or more bacterial species. Among 534 culture positive samples, 152 yielded pure growth of only staphylococcal species. 40.3% of the wound infections were caused by Staphylococcus species and among them 81.7% were caused by Staphylococcus aureus. The overall rate of MRSA infection in the present study was 29.6%. Significant difference was observed in the susceptibility pattern of Methicillin Sensitive and Methicillin Resistant Staphylococcus aureus. MRSA continues to be a major threat in the health care setting. The increased level resistance pattern of Methicillin Resistant Staphylococcus aureus observed in our study may be due to the fact that ours is a tertiary care hospital with extensive usage of broad spectrum antibiotics leading to survival advantage and persistence of drug resistant pathogens.


INTRODUCTION
Staphylococci are an important pathogen causing human disease. It has an exceptional ability to colonise, spread and cause outbreaks in hospitals. They can be cultured even from dried clinical samples after several months, and are very hardy bacteria. 1 Therefore, it is not surprising that even in spite of availability of effective antimicrobial agents, improved health care conditions and stringent hospital infection control measures, Staphylococcus aureus has established itself as a major human pathogen.
Staphylococcus aureus is a major pathogen causing secondary bacterial infection complicating pre-existing skin lesions, such as burns, traumatic lesions, diabetic ulcers, osteomyelitis and surgical site infections. Staphylococcus isolated in hospitals has shown an alarming tendency to develop resistance to antimicrobial drugs 2 and has been recognized as one of the important challenges in control of hospital infections.
Resistance to penicillin appeared in 1942. The emergence of infections due to Methicillin Resistant Staphylococcus aureus [MRSA] during late 1970s and 1980s world over 3 and their introduction and spread in hospitals increased the rate of hospital acquired infections in the hospitals. The emerging threat of vancomycin resistance among coagulase negative Staphylococcus and Enterococci poses a serious public health concern as vancomycin has been the drug of choice for treatment of resistant gram positive organisms.
Wound infections are the most common type of pyogenic infections that contributes for prolonged hospital stays, significantly increasing the cost of medical care and also have an important role in the development of multi drug resistance. Staphylococcus is the most common organism causing wound infections and isolated from pus samples in our hospital and, there have been several reports of MRSA isolates from pus samples during previous years. Thus a study was taken up to detect the number of wound infections due to Staphylococci and also to study their susceptibility pattern which helps in better management of the infection. The main aim of the present study was to find out the rate of Staphylococcal wound infection and also to know the Antibiotic susceptibility pattern of Methicillin Sensitive and Methicillin Resistant Staphylococcus aureus isolated from pus samples.

Methodology
All patients admitted and/or attending surgery and orthopedics OPD with suspected microbial wound infection and the discharge, which yielded growth of staphylococci, were included in the present study.

Sample collection
Pus from the wound was collected using two sterile swabs after cleaning the wound with sterile normal saline and surrounding skin with alcohol and transported immediately to Microbiology laboratory. Care was taken not to touch the wound edges inadvertently to avoid unnecessary skin contamination. A second sample was collected in all culture positives for staphylococci, for reproducibility of result, to confirm the presence of true infection and to rule out colonization and contamination.

Direct smear study
Direct smears were made from the first swab and stained with Grams stain. 4 The smear was screened for the presence of inflammatory cells and the type of microbial flora.

Culture
The second swab was inoculated onto Mac Conkey agar and Blood agar. Inoculated plates were incubated aerobically at 37°C for complete 24 6 All the discs were procured commercially [Hi-media Laboratories limited].
The methicillin resistant staphylococcal strains were tested for vancomycin susceptibility by "Macrodilution broth susceptibility method". The MIC [Minimum Inhibitory Concentration] of the test strain was interpreted as the lowest concentration of vancomycin at which there was no visible staphylococcal growth judged by lack of turbidity and interpreted according to CLSI guidelines. 7 ATCC S.aureus 25923 was used as control strain for the susceptibility testing.
All the statistical analysis was performed using the software SPSS for Windows (Statistical Presentation System Software, SPSS Inc, New York) version 22.

RESULTS
In the present study, 595 pus samples were collected from clinically suspected wound infection cases. Clinical diagnoses of the cases selected in the present study were diabetic foot, cellulitis, traumatic wound, osteomyelitis, nonhealing ulcer, post-operative wound infection, gangrene and burns.
Out of the 595 subjects, 425 [71.4%] were male patients and 170 [28.6%] were female patients. The male to female ratio in our present study was 2.5:1. The age and sex distribution of the samples is depicted in Table 1.
152 cases of diabetic foot and 122 cases of cellulitis were the predominant clinical cases in the present study. These were found more  Table 2. In the present study overall rate of wound infection based on direct microscopic examination

DISCUSSION
The discovery and development of miracle antibiotics is undoubtedly one of the utmost advances of modern medicine. But, unfortunately, the emergence of multidrug resistant bacteria, is threatening the effectiveness of many of these antimicrobial agents.
When Penicillin was first introduced in early 1940s, almost all staphylococci were susceptible. By 1942, there were few isolated reports of penicillin resistance among S.aureus isolates, by 1950s almost 50% of all strains were resistant to penicillin. In 1960, penicillinase resistant drugs such as methicillin, oxacillin, nafcillin etc., were introduced. By late 1970s Methicillin resistant strains of staphylococci emerged and they have become increasingly more established as nosocomial pathogens.
The Male to Female ratio in our study was 2.5:1. The increased rate of wound infections among males could be due to their outdoor occupation, more prone for injuries and infections occur due to exposure to contaminated environment. A similar observation has been made by Siddiqi et al 8 who have reported a male to female ratio of 2.6:1.
Out of 595 cases, 149 [25.1%] were found in the sixth decade. Majority of these were cases of diabetic foot wounds and cellulitis. This is because of diminishing immunity as age advances and also the possibility of underlying hormonal abnormalities.
The direct gram stain smear study of the samples helps to confirm the presence of true infection, to identify the type and number of bacteria causing infection. It also gives a clue   16 [38.56%]. The higher rate in their studies has been attributed to fact that the study group was from a tertiary care multispecialty center with no admission policy and indiscriminate use of antibiotics.
The multidrug resistance of MRSA to a wide range of antibiotics is well known. The antibiotic susceptibility results showed that all MRSA isolates were significantly highly resistant to antibiotics than MSSA isolates [P<0.00]. The resistance pattern of MRSA to b lactams like penicillin and ampicillin was 100% in our study. Similar findings were seen in the studies by Gupta et al, 17 Uma choudhary et al, 11 Anupurba et al 13  Linezolid, the oxazolidinone has shown 100% efficacy against Staphylococcus aureus in the present study. Similar consistent activity of linezolid against MRSA has also been shown by Stevens et al. 24 However, linezolid resistance in S.aureus has been reported by Tsiodras et al. 25 As linezolid's antibacterial activity is comparable with that of vancomycin in the present study, linezolid can be used as another option to vancomycin in treating MRSA infections.

CONCLUSION
The observations of the present study conclude that staphylococcal infections are the most common cause of wound infections among hospitalized patients and continue to be a growing thorn in the Health care system. Despite the latest reports that Gram Negative bacteria have overtaken staphylococci as the leading cause of nosocomial infections, MRSA continues to be the major threat in the health care setting. The results of this study would not only contribute a great deal in the prevention of such infections but also help us in formulating an antibiotic policy for the treatment of Staphylococcal lesions in our hospital.