European Journal of Cardiovascular Medicine

Wound infections represent a significant clinical burden, contributing to delayed healing, prolonged hospitalization, and elevated healthcare costs. Among the bacterial pathogens, Staphylococcus aureus remains a predominant etiological agent in both hospital acquired and community acquired wound infections due to its array of virulence factors, adaptability, and antibiotic resistance mechanisms [1,2].

A critical factor in the persistence of S. aureus in chronic and non-healing wounds is its ability to form biofilms structured microbial communities embedded within a self produced extracellular matrix. Biofilm formation confers protection against host immune defenses and markedly reduces the efficacy of antimicrobial agents, often necessitating prolonged or combination therapy [3,4]. Biofilm producing Staphylococcus aureus isolates are commonly linked with increased rates of treatment failure, especially in the presence of methicillin resistance.

Emerging studies have demonstrated a strong association between biofilm formation and antimicrobial resistance phenotypes, particularly methicillin resistant Staphylococcus aureus (MRSA), which complicates therapeutic outcomes and limits the choice of effective oral antibiotics [2,3,5]. This phenomenon is further exacerbated in clinical settings such as urinary tract infections and nasal carriage, where biofilm producing MRSA strains demonstrate widespread dissemination and colonization potential [5,6].

Despite the growing concern, routine clinical microbiology laboratories seldom assess biofilm forming ability or link it to resistance profiles. In this context, the present study was undertaken to determine the distribution of biofilm forming Staphylococcus aureus isolates in wound infections and evaluate their antibiotic susceptibility patterns, with an emphasis on the relationship between biofilm formation and methicillin resistance.

This study aims to provide insight into the local microbiological trends and guide appropriate empirical therapy.

Study Design and Setting

This cross sectional, microbiological surveillance study was conducted in the Department of Microbiology at Nimra Institute of Medical Sciences, Jupudi, Vijayawada, a tertiary care teaching hospital in Andhra Pradesh, India. The study was carried out over a period of seven months, from November 2023 to May 2024.

Sample Collection and Processing

A total of 100 wound swab samples were aseptically collected from patients presenting with clinically diagnosed wound infections, including surgical site infections, traumatic wounds, and chronic ulcers. Samples were immediately transported to the microbiology laboratory and processed within two hours of collection.

Isolation and Identification of Staphylococcus aureus

The samples were inoculated onto blood agar and MacConkey agar and incubated aerobically at 37°C for 24–48 hours. Colonies suggestive of Staphylococcus aureus were further identified by Gram staining, catalase test, slide and tube coagulase tests, and mannitol fermentation on mannitol salt agar.

Detection of Biofilm Formation

Biofilm formation was assessed by the tissue culture plate (TCP) method, the gold standard for quantitative biofilm detection. Isolates were grown in brain heart infusion broth supplemented with 1% glucose and incubated in 96 well polystyrene microtiter plates at 37°C for 24 hours. Wells were stained with 0.1% crystal violet and absorbance was measured at 570 nm. Based on optical density values, isolates were categorized as strong, moderate, weak, or non-biofilm producers.

Interpretation of biofilm production based on optical density values of tissue culture plate method

Antibiotic Susceptibility Testing

Antibiotic susceptibility of the Staphylococcus aureus isolates was determined by the Kirby Bauer disc diffusion method on Mueller Hinton agar, as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Antibiotics tested included cefoxitin (30 µg), vancomycin (30 µg), linezolid (30 µg), clindamycin (2 µg), erythromycin (15 µg), ciprofloxacin (5 µg), gentamicin (10 µg), and trimethoprim-sulfamethoxazole (1.25/23.75 µg). MRSA detection was inferred using cefoxitin disc screening.

Statistical Analysis

Data were entered into Microsoft Excel and analyzed using SPSS version 25. Descriptive statistics were used to calculate frequencies and percentages. Chi-square test was applied to assess the association between biofilm formation and methicillin resistance. A p-value < 0.05 was considered statistically significant.

Of 100 wound swab samples collected from patients with clinically diagnosed wound infections, Staphylococcus aureus was isolated in 38 cases, accounting for a prevalence rate of 38.0%. Among these, biofilm formation was detected in 26 isolates (68.4%) using the tissue culture plate method, while 12 isolates (31.6%) did not exhibit biofilm forming capacity (Table 1).

Table 1: Distribution of Staphylococcus aureus and Biofilm Formation Among Wound Isolates (N = 100)

Further stratification based on biofilm intensity revealed that 12 isolates (31.6%) were strong biofilm producers, 9 (23.7%) were moderate producers, and 5 (13.1%) were weak producers. The remaining 12 isolates (31.6%) were categorized as non-biofilm producers (Table 2). This distribution suggests a significant burden of biofilm-associated S. aureus in wound infections, particularly in the context of moderate-to-strong biofilm-forming phenotypes.

Table 2: Intensity of Biofilm Formation Among S. aureus Isolates (n = 38)

The antibiotic susceptibility pattern of the 38 S. aureus isolates demonstrated complete susceptibility to vancomycin (100%) and high sensitivity to linezolid (97.4%) and clindamycin (76.3%).

Figure 1. Intensity of Biofilm Formation Among S. aureus Isolates

Conversely, elevated resistance rates were observed against cefoxitin (52.6%), indicating a high prevalence of methicillin-resistant S. aureus (MRSA). Resistance to commonly used oral agents such as erythromycin (44.7%), ciprofloxacin (50%), and trimethoprim-sulfamethoxazole (39.5%) was also notable (Table 3).

Table 3: Antibiotic Susceptibility Pattern of S. aureus Isolates (n = 38)

An analysis of the relationship between biofilm production and methicillin resistance revealed a strong association.

Figure 2. Antibiotic Susceptibility Pattern of S. aureus Isolates

Among the 26 biofilm-producing isolates, 17 (65.4%) were MRSA and 9 (34.6%) were methicillin-sensitive S. aureus (MSSA). In contrast, among the 12 non-biofilm-producing isolates, only 3 (25.0%) were MRSA and 9 (75.0%) were MSSA. This indicates a significantly higher prevalence of methicillin resistance among biofilm-forming isolates (Table 4), highlighting the clinical importance of biofilm detection in predicting antibiotic resistance patterns.

Table 4: Association Between Biofilm Production and Methicillin Resistance

This study offers valuable insights into the microbiological profile of wound infections, emphasizing the prevalence of Staphylococcus aureus, its biofilm-forming ability, and associated antimicrobial resistance. Among 100 wound samples analyzed, S. aureus was isolated in 38% of cases—consistent with previous findings from regional and global surveillance studies reporting similar frequencies in hospitalized patients with infected wounds [7,8].

The ability of S. aureus to form biofilms was observed in 68.4% of isolates, with strong and moderate producers accounting for a substantial proportion. This supports the growing body of evidence highlighting biofilm formation as a key virulence factor that enhances persistence in wound sites, impairs antibiotic efficacy, and contributes to chronicity [9,10]. Biofilm matrices restrict antimicrobial penetration and promote survival of persister cells, rendering standard therapy ineffective in many clinical scenarios [11].

The antibiotic susceptibility pattern in our study revealed universal sensitivity to vancomycin and high susceptibility to linezolid (97.4%), in agreement with previous studies highlighting these agents as reliable choices against multidrug-resistant strains [12]. However, the 52.6% resistance to cefoxitin underscores a high burden of MRSA in wound infections, paralleling similar reports from India, China, and Southeast Asia [13,14]. Notably, resistance to ciprofloxacin (50%) and erythromycin (44.7%) limits the utility of these agents in community and outpatient settings.

A key observation was the significant association between biofilm formation and methicillin resistance. In our study, 65.4% of biofilm-producing isolates were MRSA compared to only 25% among non-biofilm formers. This association is supported by recent literature demonstrating that biofilm-forming S. aureus strains tend to harbor multiple resistance genes, including those associated with integrons, agr dysregulation, and adherence-related elements [8,9,14].

Limitations of the study include its single-center design and limited sample size, which may restrict generalizability. Nonetheless, the findings underscore the necessity of integrating biofilm detection and resistance profiling into routine microbiological surveillance to guide effective antimicrobial stewardship.

This study highlights the high prevalence of biofilm-forming Staphylococcus aureus in wound infections, with a significant proportion demonstrating methicillin resistance. The strong association between biofilm production and MRSA underscores the clinical challenge in managing such infections, particularly due to limited efficacy of conventional antibiotics. While vancomycin and linezolid remain highly effective, rising resistance to commonly used agents like cefoxitin and ciprofloxacin necessitates judicious antibiotic use. Routine biofilm screening and susceptibility profiling should be integrated into microbiological diagnostics to guide targeted therapy. Strengthening infection control and antimicrobial stewardship programs is essential to curb the spread of biofilm-associated, drug-resistant S. aureus in healthcare settings.

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