Chronic non-healing wounds represent a silent epidemic, imposing a substantial and growing burden on global healthcare systems, patients, and society. A chronic wound is typically defined as one that fails to proceed through the orderly and timely reparative process of healing within an expected timeframe, generally considered to be 6 to 12 weeks [1]. These wounds, which include diabetic foot ulcers, venous leg ulcers, pressure ulcers, and arterial insufficiency ulcers, are characterized by a prolonged inflammatory phase, persistent infection, and a failure of re-epithelialization [2]. This pathological state leads to significant morbidity, including chronic pain, immobility, limb amputation, and a profound reduction in the quality of life for affected individuals [3].

The pathophysiology of chronic wound healing failure is complex and multifactorial. Local factors such as tissue hypoxia, repetitive ischemia-reperfusion injury, and a high bacterial burden contribute to the arrest of the normal healing cascade [4]. Systemic factors, including advanced age, malnutrition, immunosuppression, and co-morbidities such as diabetes mellitus, peripheral vascular disease, and chronic venous insufficiency, play a crucial permissive role [2]. Among these, diabetes mellitus is particularly significant, as it leads to peripheral neuropathy, micro- and macro-vascular disease, and impaired immune function, all of which synergistically create a wound environment highly susceptible to infection and resistant to healing [5]. The wound bed in chronic wounds is often not sterile; instead, it is colonized by a complex, polymicrobial community of bacteria and fungi [6].

The role of microorganisms in wound chronicity has evolved from a simple concept of infection to a more nuanced understanding of bacterial biofilms. Biofilms are structured communities of bacteria encased in a self-produced extracellular polymeric matrix that adheres to the wound bed [7]. Within a biofilm, bacteria exhibit altered phenotypes, including a significantly increased tolerance to antibiotics and host immune defenses, making them exceptionally difficult to eradicate [7, 8]. It is now widely recognized that biofilms are present in a majority of chronic wounds and represent a primary barrier to healing [9]. The microbial burden and the specific species present can shift over time, influenced by prior antibiotic exposure, local wound conditions, and the patient's underlying health status [10].

In the Indian subcontinent, the challenge of chronic wounds is particularly acute. The high prevalence of diabetes, combined with tropical climates, under-resourced rural healthcare, and a high incidence of road traffic accidents, contributes to a large patient population presenting with advanced, complex wounds [11]. Data on the clinical and microbiological profiles of these wounds is essential for guiding empirical antimicrobial therapy, implementing effective infection control practices, and developing targeted wound care strategies. However, regional data from non-metropolitan tertiary care centres in South India is often limited. A combined perspective from both dermatology and surgery is vital, as these wounds often present with both cutaneous manifestations and deeper tissue involvement requiring surgical intervention. This study aims to bridge this gap by providing a comprehensive clinical and microbiological analysis of chronic non-healing wounds at a tertiary care hospital in Tamil Nadu, India.

OBJECTIVE

The primary objective of this study is to characterize the clinical and microbiological profile of chronic non-healing wounds in patients presenting to the Dermatology and Surgery departments of a tertiary care centre in Tamil Nadu, India. This includes a detailed analysis of patient demographics, common comorbidities, wound types, and wound characteristics such as duration, size, and clinical signs of infection. Understanding these factors is crucial for identifying the at-risk population and tailoring preventive and therapeutic strategies.

The secondary objective is to isolate, identify, and determine the antimicrobial susceptibility patterns of the aerobic bacterial pathogens colonizing or infecting these chronic wounds. This data will delineate the local microbiological landscape and the prevailing antibiotic resistance trends. The findings will serve to inform clinicians about the most likely pathogens and the most effective empirical antibiotic choices, thereby promoting rational antibiotic stewardship, reducing treatment failures, and potentially curbing the rise of antimicrobial resistance in this vulnerable patient population.

Study Design and Setting: This was a prospective, observational, cross-sectional study conducted over a period of 16 months from September 2021 to December 2022. The study was carried out in the outpatient departments (OPDs) and inpatient wards of Dermatology and General Surgery at a tertiary care teaching hospital located in Tamil Nadu, India. The institution serves a large, diverse population from both urban and rural areas, providing a representative sample of the region's wound care challenges. Patient Recruitment: A total of 50 consecutive patients who fulfilled the study criteria and provided written informed consent were enrolled. A detailed history was obtained from each patient, encompassing demographic data, presenting complaints, duration of the wound, history of trauma or prior surgery, and any underlying co-morbidities such as diabetes mellitus, hypertension, or peripheral vascular disease. A thorough clinical examination was performed, focusing on the wound’s anatomical location, size, depth, edge characteristics, presence of slough, granulation tissue, and clinical signs of infection (e.g., erythema, purulent discharge, malodor, local warmth). Inclusion Criteria: • Patients of either sex, aged 18 years and above. • Patients presenting with a non-healing wound of more than 6 weeks duration. • Wounds of any aetiology, including diabetic foot ulcers, venous stasis ulcers, pressure ulcers, and post-traumatic/post-surgical wounds. • Patients willing to provide written informed consent for participation in the study. Exclusion Criteria: • Patients with wounds of less than 6 weeks duration (acute wounds). • Patients who had received systemic or topical antibiotics within the 72 hours prior to sample collection. • Patients with a known diagnosis of autoimmune ulcers (e.g., pyoderma gangrenosum, vasculitic ulcers). • Patients with malignant ulcers or wounds with active bleeding. • Patients who were immunocompromised due to conditions like HIV/AIDS or those on long-term immunosuppressive therapy. Sample and Data Collection Procedure: Following patient enrollment and clinical assessment, wound samples were collected using a standardized aseptic technique. The wound surface was first cleansed with sterile normal saline to remove surface contaminants and exudate. Two types of samples were then obtained. For a swab sample, a sterile cotton swab was rotated over a 1 cm² area of the viable wound bed using the Levine technique, applying sufficient pressure to express tissue fluid [12]. For a tissue biopsy, a small, deep portion of tissue was obtained from the wound bed using a sterile scalpel or punch biopsy after local anesthesia, when feasible. Tissue samples were considered the gold standard for microbiological analysis [13]. All samples were immediately placed in sterile containers and transported to the microbiology laboratory within one hour of collection to ensure the viability of aerobic organisms. Statistical Data Analysis: In the laboratory, the collected samples were processed using standard microbiological methods. Specimens were inoculated onto 5% sheep blood agar and MacConkey agar plates and incubated aerobically at 37°C for 24 to 48 hours. Significant bacterial growth was identified based on colony morphology, Gram staining, and a panel of biochemical tests. Antimicrobial susceptibility testing was performed on the isolated pathogens using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar, following the Clinical and Laboratory Standards Institute (CLSI) guidelines [14]. The data was entered into a Microsoft Excel spreadsheet. Descriptive statistics were used for analysis. Categorical variables were expressed as frequencies and percentages, while continuous variables were summarized using mean and standard deviation. The analysis was performed using a standard statistical software package (e.g., SPSS version 21.0 or similar).

Clinical Profile (Table 1): A total of 50 patients with chronic non-healing wounds were included in the study. The study population showed a male predominance, with 32 (64%) males and 18 (36%) females. The age of the patients ranged from 24 to 78 years, with the majority (n=30, 60%) belonging to the 41-60 years age group. The most common underlying comorbidity was diabetes mellitus, present in 28 (56%) patients, followed by hypertension in 15 (30%) and peripheral vascular disease in 8 (16%) patients. The most frequently encountered wound types were diabetic foot ulcers (n=17, 34%), followed by post-traumatic wounds (n=13, 26%), venous leg ulcers (n=8, 16%), and post-surgical wound dehiscence (n=7, 14%).

Wound Characteristics and Microbiological Isolates (Table 2 & 3): The mean duration of the wounds was 4.8 months (standard deviation ± 2.1). The lower limb was the most affected anatomical site, accounting for 40 (80%) of the wounds. Clinically, 41 (82%) of the wounds showed overt signs of infection, including purulent discharge and malodor. A total of 73 bacterial isolates were obtained from the 50 wound samples, resulting in a culture positivity rate of 100%. Polymicrobial growth (≥2 organisms) was observed in 23 (46%) cases, while 27 (54%) samples yielded a monomicrobial infection. Among the isolates, Staphylococcus aureus was the most common, accounting for 22 (30.1%) of all isolates. Pseudomonas aeruginosa was the second most common (n=14, 19.2%), followed by Escherichia coli (n=11, 15.1%), and Klebsiella pneumoniae (n=8, 11%). Other Gram-negative bacteria and Enterococcus species accounted for the remainder.

Antimicrobial Susceptibility Patterns (Table 4): The antibiotic susceptibility profiles revealed concerning resistance patterns. Among the 22 S. aureus isolates, 40% (n=9) were identified as Methicillin-resistant Staphylococcus aureus (MRSA). All Gram-positive isolates were uniformly sensitive to vancomycin and linezolid. Among the Gram-negative isolates, a high degree of resistance was observed against commonly used antibiotics. For Pseudomonas aeruginosa, resistance to ciprofloxacin was seen in 57% of isolates, and to ceftazidime in 43%. However, sensitivity to piperacillin-tazobactam and meropenem was high (86% and 93%, respectively). Similarly, for Escherichia coli and Klebsiella pneumoniae combined, resistance to ciprofloxacin was 68%, and to third-generation cephalosporins was 58%. Carbapenem resistance was noted in 2 (10.5%) of these enterobacterial isolates.

Table 1: Demographic and Clinical Characteristics of Study Patients (N=50)

Table 2: Wound Characteristics of the Study Population (N=50)

Table 3: Microbiological Isolates from Chronic Wounds (N=73 Isolates from 50 Samples)

Table 4: Antimicrobial Resistance Patterns of Key Isolates

The management of chronic non-healing wounds remains a formidable challenge in clinical practice, particularly in a resource-constrained country like India, where the burden is immense. This study, conducted at a tertiary care centre in Tamil Nadu, provides crucial insights into the clinical demographics and evolving microbiological profile of these complex wounds. The findings highlight the intersection of a high prevalence of systemic disease with significant local microbial pathology, underscoring the need for a comprehensive, multidisciplinary approach to care [15]. Our study found a clear male predominance (64%) and a peak incidence in the 5th to 6th decades of life, which is consistent with previous Indian and international literature [16, 17]. This demographic trend is likely multifactorial, reflecting higher rates of outdoor labor, trauma, and delayed healthcare-seeking behavior among men. The most significant comorbidity was diabetes mellitus, present in over half of our patients (56%). This finding reinforces the well-established link between diabetes and impaired wound healing, driven by a combination of peripheral neuropathy, vascular insufficiency, and dysfunctional immune responses [5]. The high prevalence of diabetic foot ulcers (34%) as the most common wound type further emphasizes the critical need for integrated diabetic foot care programs within the healthcare system. These programs should focus on patient education, regular foot screening, and early intervention to prevent the progression of minor injuries into chronic, limb-threatening ulcers [18]. The microbiological analysis revealed a high rate of wound contamination and infection, with 100% culture positivity. This is in line with studies showing that most chronic wounds are heavily colonized by bacteria, often in biofilm form [6, 7]. The predominance of Staphylococcus aureus (30.1%) as the most common isolate is a globally consistent finding in chronic wound research [19]. However, the high prevalence of Gram-negative organisms, particularly Pseudomonas aeruginosa (19.2%) and members of the Enterobacteriaceae family (26.1%), is noteworthy. This polymicrobial profile, observed in 46% of our cases, is characteristic of wounds of longer duration and often indicates a shift in the wound microbiome towards more virulent and antibiotic-resistant species [10, 20]. The high proportion of Gram-negative bacteria, especially P. aeruginosa, has significant therapeutic implications, as it is inherently resistant to many commonly used oral antibiotics and is a prolific biofilm producer. Perhaps the most alarming finding of this study is the high level of antimicrobial resistance. A MRSA prevalence of 40% among S. aureus isolates is a significant concern, as it limits empirical therapeutic options and necessitates the use of costlier, last-resort antibiotics like vancomycin or linezolid. This rate is comparable to, or higher than, data from other recent Indian studies on wound infections, indicating a rapidly escalating problem of Gram-positive resistance [11, 16, 21]. Similarly, the resistance rates observed in Gram-negative organisms were substantial. More than half of the P. aeruginosa isolates were resistant to ciprofloxacin (57%), and a large proportion of the enteric Gram-negative bacteria were resistant to fluoroquinolones (68%) and third-generation cephalosporins (58%). This is highly problematic, as these are among the most widely prescribed antibiotics for skin and soft tissue infections in the community [22]. While piperacillin-tazobactam and carbapenems showed better in-vitro activity, their use is associated with higher costs, the need for intravenous access, and the risk of promoting even more resistant pathogens, including Carbapenem-Resistant Enterobacterales (CRE) [23]. This data strongly supports the argument for abandoning blind, empirical therapy in favor of culture-guided, targeted treatment for all chronic wounds. The combined Dermatological and Surgical perspective of this study highlights the need for a dual-pronged approach. Dermatologists are often the first point of contact for managing the wound bed, addressing issues like maceration, contact dermatitis, and performing debridement. Surgeons are essential for addressing deeper structural issues, such as revascularization in ischemic limbs, aggressive surgical debridement of necrotic tissue and biofilm, and managing osseous involvement. The high burden of infection seen in this study reinforces the surgical principle that effective source control through debridement is paramount. Antibiotics alone cannot penetrate a biofilm or avascular tissue; therefore, surgical removal of the nidus of infection is often a prerequisite for successful antimicrobial therapy and wound closure [24]. A collaborative, protocol-driven approach that integrates best practices from both specialties offers the best chance for limb salvage and wound resolution. Limitations of the Study This study, while providing valuable local data, has certain limitations that must be acknowledged. The most significant is the relatively small sample size of 50 patients, which may limit the generalizability of the findings to the broader population. The study was conducted at a single tertiary care centre, which likely sees a higher proportion of complex and referred cases, potentially skewing the microbiological profile towards more resistant organisms than might be seen in a primary or secondary care setting. The observational design allows us to identify associations but cannot establish causality. Furthermore, the sampling period of 16 months may not account for seasonal variations in microbiology. Methodologically, while we collected both swabs and tissue biopsies, the standard culture techniques used were primarily for aerobic bacteria; we did not employ molecular methods to identify the full microbiome or specifically culture for anaerobic bacteria and fungi, which are known components of wound biofilms [6, 13]. Finally, the antibiotic susceptibility patterns reflect in-vitro data, which may not always perfectly correlate with clinical outcomes in the complex environment of a chronic wound. Acknowledgement The authors express their sincere gratitude to the Institutional Ethics Committee for their approval and guidance. We are deeply thankful to the Department of Microbiology, particularly the laboratory technical staff, for their diligent work in processing the samples and performing the culture and sensitivity analyses. Their expertise was invaluable to the successful completion of this research. Our appreciation extends to the nursing staff of the Dermatology and Surgery wards and outpatient departments for their assistance in patient recruitment and sample collection. We are most grateful to the patients who consented to participate in this study, without whom this research would not have been possible.

In conclusion, this study from a tertiary care centre in Tamil Nadu demonstrates that chronic non-healing wounds impose a significant clinical burden, predominantly affecting middle-aged diabetic males. The clinical and microbiological picture is dominated by diabetic foot ulcers and traumatic wounds that are heavily colonized with a complex polymicrobial flora. Staphylococcus aureus remains the most common isolate, but a high prevalence of Gram-negative organisms like Pseudomonas aeruginosa and Escherichia coli was noted, reflecting the chronicity and severity of these wounds.

The most critical finding is the alarming level of antimicrobial resistance, particularly the 40% MRSA rate and high resistance to fluoroquinolones and cephalosporins among Gram-negative pathogens. This underscores the urgent need for a paradigm shift in clinical practice. Empirical antibiotic therapy should be strongly discouraged. Instead, management must be rooted in obtaining deep tissue cultures followed by targeted, narrow-spectrum antimicrobial therapy based on susceptibility results. This approach, integrated within a multidisciplinary framework involving both Dermatology and Surgery, is essential. By combining aggressive surgical debridement, optimal wound bed preparation, and rational antibiotic stewardship, we can improve patient outcomes, reduce the risk of amputation, and combat the growing threat of antimicrobial resistance. Future research should focus on molecular diagnostics to better characterize the wound microbiome and its biofilms, enabling the development of novel, non-antibiotic-based therapeutic strategies.