Bacterial biofilm in chronic lower limb ulcers: Associations with comorbidities, wound characteristics, and therapeutic approaches

Chronic ulcers are wounds characterized by impaired tissue repair, often resulting from multifactorial causes such as underlying diseases or trauma. These lesions frequently harbor microorganisms capable of forming biofilms and exhibiting antimicrobial resistance, thereby worsening the clinical condition and limiting the effectiveness of therapeutic interventions. To characterize bacterial biofilm formation in chronic lower-limb ulcers of patients treated at the outpatient wound clinic of the Instituto Lauro de Souza Lima, Bauru, São Paulo, Brazil, using microbiological, molecular, and histopathological techniques, and to investigate its association with clinical and sociodemographic parameters. Phenotypic and molecular biofilm production was evaluated in microbial isolates obtained from chronic lower-limb ulcers collected by swab and biopsy. Tissue samples were subjected to histopathological analysis using Hematoxylin and Eosin (H&E) and Periodic Acid–Schiff (PAS) staining to assess tissue morphology and detect microbial biofilms. Clinical signs and sociodemographic variables were recorded at two distinct time points to monitor patient outcomes. Samples from 33 patients (mean age 63.8 years, predominantly male, with low educational level) yielded 107 bacterial isolates. Maceration (94%) and ochre dermatitis (82%) were the most prevalent inflammatory signs. Biofilm production by Staphylococcus spp. was significantly associated with the presence of slough (adjusted p = 0.0012). Phenotypic biofilm detection was observed in 59.8% of isolates using Congo red agar and in 67.3% using the polystyrene microplate assay, with higher production among Gram-positive bacteria. Molecular analysis confirmed these findings, revealing a high prevalence of the icaD gene in Staphylococcus spp. (93%) and the pelF gene in Pseudomonas aeruginosa (87%). Polymicrobial samples predominantly exhibited strong biofilm adherence. Comparison between the two clinical assessments demonstrated statistically significant differences in pain levels, wound temperature, and ulcer area, indicating partial clinical progression despite the persistence of certain inflammatory features. Chronic ulcers have a multifactorial etiology, with microbial biofilms playing a central role in delayed healing. These findings underscore the need for integrated therapeutic approaches targeting both microbial and host-related factors.