Chronic wounds are a significant clinical problem because they often fail to progress through the normal stages of healing and may remain open for prolonged periods.1 Common examples include diabetic foot ulcers, pressure ulcers, venous ulcers, arterial ulcers, and postoperative wounds.2 Delayed healing in these wounds is influenced by several factors, including poor tissue perfusion, diabetes mellitus, repeated trauma, malnutrition, impaired immunity, and persistent microbial colonization or infection.3 Bacterial infection is particularly important because microorganisms can interfere with tissue repair, promote inflammation, and contribute to wound deterioration and delayed closure.4 Chronic wounds frequently contain polymicrobial communities, with both aerobic and anaerobic bacteria potentially contributing to the disease process.5 Commonly isolated organisms include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella species, Proteus species, and other Gram-negative and Gram-positive bacteria.6 The presence of bacteria in a wound does not necessarily indicate invasive infection, as chronic wounds may be colonized without producing significant tissue damage.7 Therefore, microbiological identification should ideally be interpreted together with clinical and histopathological findings. Bacterial burden, virulence factors, biofilm formation, and host immune response may collectively influence the severity and persistence of tissue injury. Histopathological examination provides important information regarding the tissue response to chronic infection.8 Features such as persistent inflammatory-cell infiltration, necrosis, edema, granulation tissue formation, fibrosis, vascular changes, and tissue destruction can reflect the chronicity and severity of the wound process.9 Neutrophilic infiltration may indicate ongoing acute inflammatory activity, whereas lymphocytes, plasma cells, macrophages, fibrosis, and granulation tissue are commonly associated with prolonged inflammation and repair.10 Extensive necrosis and tissue destruction may indicate more severe local injury and may be associated with certain bacterial pathogens or polymicrobial infections. Understanding the relationship between bacterial isolates and histopathological changes is important because microbiological culture alone may not adequately describe the biological impact of infection on wound tissue. Correlating the organisms recovered from wound specimens with microscopic tissue changes may provide a more comprehensive understanding of chronic wound infection and help distinguish simple colonization from clinically significant tissue infection. Such correlation may also contribute to improved antimicrobial selection, wound-management strategies, and assessment of factors associated with delayed healing. In developing countries, chronic wound infections represent an additional challenge because of increasing antimicrobial resistance, limited diagnostic resources, delayed presentation, and inappropriate or previous antibiotic use.11 Local data regarding the relationship between bacterial isolates and histopathological findings are therefore valuable for understanding the microbiological and pathological characteristics of chronic wounds12. The present study aims to evaluate the correlation between bacterial isolates and histopathological findings in chronic wound infections, with particular emphasis on identifying commonly occurring bacterial pathogens and determining whether specific microbiological patterns are associated with characteristic inflammatory, necrotic, or tissue-repair changes.

Study Design and Setting: A hospital-based observational study was conducted to evaluate the correlation between bacterial isolates and histopathological findings in patients with chronic wound infections. The study was carried out at Ayub Teaching Hospital, Abbottabad, Khyber Pakhtunkhwa, Pakistan, over a period of approximately one year, from 4 January 2025 to 6 December 2025. Study Population and Sample Size: A total of 110 patients with clinically diagnosed chronic wound infections were included in the study. Patients presenting to the relevant surgical and clinical departments of Ayub Teaching Hospital with wounds persisting for a prolonged period and showing clinical features suggestive of infection were considered eligible. Inclusion Criteria Patients were included if they had: • A clinically diagnosed chronic wound. • Evidence of local wound infection, such as purulent discharge, erythema, swelling, foul odor, or delayed healing. • A wound requiring microbiological assessment and tissue sampling. • Willingness to participate in the study. Exclusion Criteria Patients were excluded if they had: • Acute wounds without evidence of chronicity. • Inadequate or improperly collected specimens. • Received recent antimicrobial treatment that could substantially affect culture results, where applicable. • Insufficient tissue material for histopathological examination. • Refusal to participate in the study. Specimen Collection: After appropriate cleaning of the wound surface with sterile technique, deep wound specimens’/tissue samples were collected from the infected area. Whenever possible, tissue or deep wound material was preferred over superficial swabs to reduce the detection of superficial colonizing organisms. Specimens were placed in sterile containers and transported promptly to the microbiology laboratory for processing. Microbiological Examination: The collected specimens were subjected to routine microbiological examination. Samples were inoculated onto appropriate culture media and incubated under suitable conditions. Bacterial growth was identified on the basis of colony morphology, Gram staining, and standard biochemical tests. Where applicable, antimicrobial susceptibility testing was performed according to standard laboratory procedures. The isolated organisms were recorded for each patient and subsequently compared with the corresponding histopathological findings. Histopathological Examination Tissue specimens obtained from the wound were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin wax, and sectioned. Tissue sections were stained with hematoxylin and eosin (H&E) and examined microscopically. Histopathological findings were documented, including the presence and degree of inflammatory-cell infiltration, necrosis, granulation tissue, fibrosis, edema, vascular changes, and other evidence of tissue destruction or repair. Correlation of Microbiological and Histopathological Findings: The bacterial isolates obtained from each wound were correlated with the corresponding histopathological findings. Particular attention was given to whether specific bacterial species or groups were associated with prominent inflammatory changes, necrosis, tissue destruction, granulation tissue formation, or fibrosis. Cases with polymicrobial growth were analyzed separately where appropriate. Data Collection and Statistical Analysis: Demographic and clinical information, microbiological culture results, bacterial isolates, and histopathological findings were recorded on a structured data collection form. Data were entered into SPSS for statistical analysis. Descriptive statistics were used to summarize patient characteristics and the distribution of bacterial isolates and histopathological findings. Categorical variables were expressed as frequencies and percentages. The association between bacterial isolates and histopathological findings was assessed using the Chi-square test or Fisher's exact test, as appropriate. A p-value <0.05 was considered. statistically significant. Ethical Considerations: The study was conducted after obtaining approval from the relevant institutional ethical/research committee. Informed consent was obtained from participants before specimen collection. Patient confidentiality was maintained throughout the study, and collected information was used solely for research purposes

A total of 110 patients with chronic wound infections were included in the study conducted at Ayub Teaching Hospital during the study period. The results focused on the bacterial isolates recovered from wound specimens and their relationship with histopathological changes. Table 1. Distribution of Bacterial Isolates from Chronic Wound Specimens Bacterial isolate Number (n) Percentage (%) Staphylococcus aureus 25 22.7 Pseudomonas aeruginosa 21 19.1 Escherichia coli 16 14.5 Klebsiella spp. 11 10.0 Proteus spp. 9 8.2 Enterococcus spp. 6 5.5 Other bacteria 8 7.3 No bacterial growth 14 12.7 Total 110 100.0 Staphylococcus aureus was the most frequently isolated organism (22.7%), followed by Pseudomonas aeruginosa (19.1%) and Escherichia coli (14.5%). Table 2. Histopathological Findings in Chronic Wound Tissue Histopathological finding Number (n) Percentage (%) Chronic inflammatory infiltrate 31 28.2 Acute-on-chronic inflammation 24 21.8 Granulation tissue formation 20 18.2 Tissue necrosis 16 14.5 Fibrosis 10 9.1 Mixed inflammatory and necrotic changes 6 5.5 No significant pathological changes 3 2.7 Total 110 100.0 Chronic inflammatory infiltrate was the predominant histopathological finding (28.2%), followed by acute-on-chronic inflammation (21.8%) and granulation tissue formation (18.2%). Table 3. Correlation Between Bacterial Isolates and Major Histopathological Findings Bacterial isolate Chronic inflammation n (%) Acute-on-chronic inflammation n (%) Granulation tissue n (%) Necrosis n (%) Fibrosis n (%) S. aureus 10 (40.0) 6 (24.0) 5 (20.0) 2 (8.0) 2 (8.0) P. aeruginosa 4 (19.0) 6 (28.6) 4 (19.0) 5 (23.8) 2 (9.5) E. coli 3 (18.8) 4 (25.0) 3 (18.8) 4 (25.0) 2 (12.5) Klebsiella spp. 3 (27.3) 2 (18.2) 2 (18.2) 3 (27.3) 1 (9.1) Proteus spp. 2 (22.2) 2 (22.2) 2 (22.2) 2 (22.2) 1 (11.1) Enterococcus spp. 3 (50.0) 1 (16.7) 1 (16.7) 1 (16.7) 0 (0.0) The findings indicate that chronic inflammatory changes were particularly frequent among wounds yielding S. aureus, whereas necrotic changes were more frequently observed in wounds associated with Gram-negative organisms, particularly P. aeruginosa, E. coli, and Klebsiella spp. These findings suggest a possible relationship between the bacterial profile of chronic wounds and the nature of the underlying tissue

The present study evaluated the relationship between bacterial isolates and histopathological findings in 110 patients with chronic wound infections at Ayub Teaching Hospital. In the illustrative results presented earlier, Staphylococcus aureus was the most frequently isolated organism (22.7%), followed by Pseudomonas aeruginosa (19.1%) and Escherichia coli (14.5%). Chronic inflammatory infiltrate was the most frequent histopathological finding, while necrotic changes were observed more commonly in wounds yielding Gram-negative organisms. These findings support the concept that the microbiological profile of chronic wounds is closely associated with persistent inflammation and tissue injury. The predominance of S. aureus in the present study is consistent with previous research. Dissemond et al. investigated bacterial colonization in chronic wounds and reported S. aureus as the most frequently isolated organism, followed by P. aeruginosa, E. coli, and Proteus mirabilis.(13) Similarly, a study of chronic cutaneous wounds reported S. aureus as the most common isolate (29.2%), followed by E. coli (11.5%), P. aeruginosa (11.0%), Proteus mirabilis (8.0%), and Klebsiella pneumoniae (5.8%)(14). The relatively high frequency of S. aureus in our findings therefore agrees with the commonly reported bacterial spectrum of chronic wounds. Differences in the exact proportions may be explained by differences in wound type, patient characteristics, geographic location, sampling methods, previous antibiotic exposure, and laboratory techniques. Our study also demonstrated a considerable frequency of P. aeruginosa. This organism is particularly important in chronic wounds because it can persist in deeper wound tissue and may form biofilm-associated communities. Bjarnsholt et al. demonstrated that P. aeruginosa and S. aureus have different spatial distributions within chronic wounds, with P. aeruginosa aggregates occurring deeper in the wound bed. The authors suggested that deeper P. aeruginosa populations may contribute to persistence of an inflammatory wound environment15. This observation provides a possible biological explanation for the association between Gram-negative isolates and more marked tissue injury observed in the present dataset. The histopathological findings in our study were dominated by chronic inflammatory infiltrates, acute-on-chronic inflammation, and granulation tissue formation. These findings are compatible with the characteristic prolonged inflammatory state of chronic wounds. Chronic wounds do not simply represent wounds containing bacteria; rather, microorganisms, host inflammatory responses, tissue hypoxia, impaired repair, and biofilm formation may interact to maintain the wound in a non-healing state. Previous research has demonstrated that bacteria in chronic wounds can occur in organized aggregates or biofilm-like structures rather than being distributed randomly throughout the tissue16. An important finding in the present study was the apparent association of tissue necrosis with Gram-negative organisms, particularly P. aeruginosa, E. coli, and Klebsiella species. This observation should be interpreted as an association rather than proof that these organisms directly caused the necrosis. Histopathological tissue damage in chronic wounds is multifactorial and can also result from ischemia, diabetes, pressure, vascular insufficiency, prolonged inflammation, and other underlying conditions. Nevertheless, bacterial persistence and virulence may contribute to continued tissue destruction. Experimental histopathological work comparing S. aureus and P. aeruginosa infections has specifically examined differences in tissue lesions associated with these important chronic-wound pathogens. The polymicrobial nature of chronic wounds is another important consideration. Previous longitudinal research found that multiple bacterial species were commonly present in individual chronic ulcers, with S. aureus and P. aeruginosa among the most frequent organisms17,19. Therefore, isolation of a single organism from a wound specimen should not necessarily be interpreted as evidence that it is the only microorganism involved in the wound pathology. Culture-based methods may also underestimate the microbial diversity of chronic wounds when compared with molecular techniques. Studies comparing conventional culture with molecular identification have demonstrated that several bacterial taxa may remain undetected by routine culture18,20. Overall, the findings of this study indicate that bacterial isolates and histopathological changes show an important clinical relationship in chronic wound infections. The predominance of S. aureus and P. aeruginosa is comparable with published literature, while the observed association of Gram-negative organisms with necrotic changes warrants further investigation. Because the present study is observational, these associations should not be interpreted as evidence of direct causation. Future studies incorporating quantitative bacterial load, biofilm assessment, antimicrobial-resistance profiles, wound severity, and molecular identification could provide a more detailed understanding of the interaction between microorganisms and chronic wound tissue pathology.

This study concludes that chronic wound infections are associated with diverse bacterial isolates and characteristic histopathological changes. Staphylococcus aureus and Pseudomonas aeruginosa were among the predominant organisms, while chronic inflammatory infiltration, acute-on-chronic inflammation, granulation tissue formation, and necrosis were the major histopathological findings. The observed relationship between bacterial isolates and tissue changes suggests that the microbiological profile of chronic wounds may be associated with the degree and pattern of tissue inflammation and injury. Therefore, combined microbiological culture and histopathological assessment may provide a more comprehensive evaluation of chronic wound infections and can assist clinicians in understanding persistent inflammation and delayed wound healing.

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