Antimicrobial Resistance Pattern of Staphylococcus aureus Isolated from Skin and Soft Tissue Infection in Patients at a Tertiary Care Hospital, Southern India

Introduction

Staphylococcus aureus is a highly virulent human pathogen known to cause various hospital-acquired and community-acquired infections and is a pathogen of concern due to its capacity to cause a wide range of mild to lethal infections, its ability to adapt to diverse environmental conditions and its distinctive ability to develop resistance to all antibiotics.^1,2 Infection caused by S. aureus ranges from mild skin infection to soft tissue infection, bacteremia, necrotizing disease, osteomyelitis, meningitis, staphylococcal toxic shock syndrome and others.²

Penicillin, which was the first antibiotic to have been used against S. aureus in the 1940s, rapidly lost its sensitivity due to the production of beta-lactamase enzyme by resistant isolates. To overcome penicillin resistance in Staphylococci, the penicillinase-resistant penicillin (methicillin) was introduced in 1959. Nevertheless, with in a year of its introduction, the first isolate of methicillin resistant Staphylococcus aureus (MRSA) was discovered.³ Since then, MRSA has assumed epidemic proportions, spreading throughout hospitals in Europe in the 1960s and later in the USA, in the 1970s. By the 1990s, MRSA was reported from virtually all countries. Of all the different antibiotics to which resistance was described, methicillin resistance turned out to be the most clinically significant, as all available penicillins and other beta-lactams, monobactams and carbapenems were totally ineffective against such isolates.³

For a long time, the only effective antibiotic against such isolates was vancomycin. Escalating prevalence of MRSA infections led to the increased usage of vancomycin and ultimately to the development of reduced susceptibility and rarely, full blown resistance to this antibiotic.⁴ Currently, resistance to higher antibiotics such as vancomycin, teicoplanin and linezolid has started emerging at a slower pace across the world.^5,6

The present study aimed to determine the distribution of S. aureus infection in the skin and soft tissue infections and its drug resistance pattern. The infection rate among male and female patients were analyzed and antimicrobial sensitivity was assessed towards commonly used antibiotics. The antibiotic susceptibility pattern was compared between MSSA (Methicillin Sensitive S. aureus) and MRSA (Methicillin Resistant S. aureus) infections.

Materials and Methods

A prospective observational study with one year of duration (Jan-Dec 2020) was conducted in the department of Microbiology, in a tertiary care hospital, located in Mangalore, Karnataka state, India. The demographic details were collected from all the patients. One hundred non-duplicate pus /wound swabs from 50 males and 50 females attending the hospital from both inpatient and outpatient departments were collected from skin and soft tissue infections. The participants were categorized into four groups based on their age (years) - 15-20, 20-40, 40-60 and 60-65.

Pus samples from pyogenic lesions were submitted to microbiology laboratory for analysis and all the isolates of Staphylococcus aureus isolated from pus samples were included for the study. There were no exclusion criteria. The study was approved by the Institutional ethical committee.

Isolation and Identification of Staphylococcus aureus

The pus/wound swab samples were processed using standard microbiological methods. The samples were inoculated on to Blood Agar and Mac Conkey agar and incubated at 37^oC for 24-48 hours. For the detection of S. aureus and differentiation from related species, Gram stain was performed to look for Gram positive cocci arranged in clusters and colony morphology was observed on both Blood and Mac Conkey agar. The confirmation of the S. aureus colonies was done using routine standard biochemical tests such as, Catalase test, Coagulase test and Mannitol fermentation. The antimicrobial susceptibility testing was performed by Kirby-Bauer disk diffusion method.

A 0.5 McFarland standard bacterial growth was inoculated on Mueller Hinton agar (MHA) with 4% NaCl.⁷ The antibiotic discs were placed over this. After 24 hours of incubation at 37ºC, zone diameter was measured to determine the sensitivity as per 2019 Clinical Laboratory Standard Institute (CLSI) guidelines.⁷ The following antibiotic disks were tested to determine their sensitivity - Cefoxitin (30 µg), Vancomycin (30 µg) Teicoplanin (30 µg), Rifampicin (30 µg), Erythromycin (15 µg), Amikacin (30 µg), and Linezolid (30 µg). Methicillin resistance was determined by using cefoxitin disk. The frequency of S. aureus infections and their sensitivity was calculated and expressed as percentage.

Results and Discussion

The results of the study showed that out of 100 nonduplicate pus/wound swab cultures, S. aureus was the most common organism isolated, with a recovery rate of 77%, followed by Pseudomonas spp, E. coli, Proteus spp, Candida spp, Streptococci spp, Salmonella typhi, and Streptococcus pneumonia (Table 1). Among the S. aureus isolates, 49.3% were found to be MRSA, and moderate resistance was observed for rifampicin, erythromycin, and amikacin. However, all S. aureus strains (both MRSA and MSSA) were 100% sensitive to higher antibiotics such as vancomycin, teicoplanin, and linezolid (Table 2).

The study found that male patients had a higher percentage of S. aureus infection compared to female patients, with 90% of male patients being positive for S. aureus infection compared to 64% of female patients (Table 3). Age-wise analysis revealed that among males, the 40-60 age group had the highest infection rate, followed by the 15-20 age group, while among females, the 20-40 age group had the highest infection rate, followed by the 15- 20 age group. In the combined age group analysis, the 40-60 age group had a slightly higher rate of infection, followed by the 20-40 age group (Table 4).

Overall, the study provides valuable insights into the prevalence of different organisms causing pus/wound infections and the sensitivity patterns of S. aureus to different antibiotics. The findings can help healthcare professionals make informed decisions regarding the appropriate use of antibiotics for the treatment of these infections.

This study was conducted in a tertiary care center located in Mangalore city, catering to patients from both Mangalore city and surrounding small towns. The results indicated a high S. aureus infection rate of 77% in pus/ wound swab samples. This finding is consistent with previous studies from Odissa, Sharma et al., (2013), and Pai et al., (2010), which reported S. aureus isolation rates of 55%, 65%, and 76%, respectively.^8-10 Of note, our study found a high proportion of MRSA strains among S. aureus isolates, with a rate of 49% indicating a high level of resistance. Similar studies conducted in Karnataka, Maharashtra, North East India, and Delhi have reported MRSA isolation rates ranging from 46% to 54% in various clinical samples.^10-13 The high prevalence of MRSA strains in our study highlights the need for effective infection control measures to prevent the spread of resistant strains in the community. Further research is needed to determine the factors contributing to the high prevalence of MRSA strains in our study population.

Our study revealed a higher rate of resistance to different antibiotic groups (rifampicin, erythromycin, and amikacin) among MRSA isolates compared to MSSA isolates. This finding is consistent with previous studies by Mallick et al., (2011), Gupta et al., (2014), and Bhowmik et al., (2019), which reported similar observations of higher resistance rates among MRSA isolates compared to MSSA isolates.^8,13-14 These results highlight the importance of judicious use of antibiotics to prevent the emergence of antibiotic-resistant strains and the need for continued surveillance of MRSA and MSSA isolates to inform treatment decisions. Further research is needed to determine the underlying mechanisms of antibiotic resistance in MRSA isolates.

Our study found that all the MSSA and MRSA isolates were susceptible to vancomycin, teicoplanin and linezolid which are standard treatments for MRSA infections. This is consistent with the findings of most published studies on S. aureus infections in India, which also reported high sensitivity rates for these antibiotics.^9-10,15 However, resistance to these antibiotics is beginning to emerge in some areas. For example, Tiwari et al., (2008) reported the emergence of Vancomycin-resistant Staphylococcus aureus (VRSA) in Varanasi, Uttar Pradesh, with rates of 1% in 2006 and 0.3% in 2008.^16-17 Additionally, two other studies by Mallick et al., and Mandal et al., reported vancomycin-resistant S. aureus rates of 15% in Rajasthan and West Bengal, respectively.^13,18 This suggests that vancomycin resistance is becoming an increasingly important issue in India and worldwide, and its prevalence may continue to rise if its usage is not managed properly. In addition to complete resistance, intermediate resistance to vancomycin (VISA) and hetero-resistance to vancomycin intermediate S. aureus (hVISA) have also been reported globally.^5,19

In our study, the antimicrobial sensitivity report showed that glycopeptides and linezolid antibiotics were 100% sensitive. However, other antibiotics such as rifampicin (83%), erythromycin (80.5%), and amikacin (79.2%) also demonstrated good sensitivity rates. These findings suggest that first and second-line antibiotics can be used as treatment options based on the aspartate transferase (AST) report. Our results are consistent with previous studies conducted in Mangalore.^9,10 Continuous surveillance of MSSA and MRSA antimicrobial resistance is necessary to monitor the trend of drug resistance. To address this issue, an antibiotic stewardship program should be implemented by the infection control team in the hospital. Additionally, increasing awareness among healthcare professionals and the public regarding the rational use of antibiotics may help reduce the emergence and spread of drug-resistant pathogens.

Our demographic analysis revealed a higher S. aureus infection rate among males (90%) compared to females (64%). In males, the 40-60 age group had the highest infection rate at 42%, while in females, the 20-40 age group had the highest infection rate at 43%. However, we were unable to determine the reason for this discrepancy, and further studies with more detailed clinical information are necessary to explore this observation. A more comprehensive investigation may help us better understand the underlying factors associated with S. aureus infection rates among males and females in different age groups, which could inform targeted interventions to reduce infection rates.

Conclusion

In conclusion, the study found a high prevalence of S. aureus infection in pus/wound swab samples, with a 49% rate of MRSA strains indicating high resistance. The study also revealed that all MRSA and MSSA isolates were susceptible to vancomycin, teicoplanin, and linezolid, which are the standard treatments for MRSA infections. Our study demonstrated a higher rate of resistance to different antibiotic groups among MRSA isolates compared to MSSA isolates, highlighting the importance of judicious use of antibiotics. The demographic analysis revealed a higher S. aureus infection rate among males compared to females, and the 40-60 age group had the highest infection rate. Effective infection control measures are necessary to prevent the spread of antibiotic-resistant strains.

Conflict of Interest

None