A multidisciplinary approach to the treatment of long-term-to-heal wounds: targeting the disruption of biofilms

Background. The persistent problem of long-term non-healing wounds, particularly in patients with diabetic foot syndrome, is increasingly associated with the formation of polymicrobial biofilms, which significantly reduce the effectiveness of standard antimicrobial therapy. The present study purposed to investigate the molecular mechanisms of antibiotic resistance among key pathogens involved in wound infection and to assess the efficacy of photodynamic therapy (PDT) compared to conventional antimicrobial agents against biofilms in vitro. Materials and methods. A total of 66 clinical isolates were obtained from 41 patients with diabetic foot syndrome undergoing treatment at the V.T. Zaitsev Institute of General and Urgent Surgery of the NAMS of Ukraine. The cohort (mean age 61.0 ± 6.3 years) consisted of 58.5 % men and 41.5 % women. The microbial spectrum included Staphylococcus aureus (30 isolates) and various Gram-negative pathogens (36 isolates). Genetic markers of resistance were identified by multiplex polymerase chain reaction, and susceptibility phenotypes were determined using standard disk diffusion and minimum inhibitory concentration (MIC) assays. Biofilms were modelled on 96-well plates and 35-mm dishes. The efficacy of PDT (6% 5-aminolevulinic acid + red light 635 nm) was assessed in comparison with standard antibio­tics. Results. Results demonstrated a high prevalence of resistance genes: CTX-M-2 (26.6 % in S.aureus, 16.7 % in Gram-negatives), VIM (5.6 %), NDM (2.8 %), VanA and VanB (6.7 % in S.aureus), Erm (16.6 % in S.aureus), Tet (23.3 % in S.aureus), QnrB (13.3 % in S.aureus). Phenotypic resistance was highest for metronidazole (52.3 %) and cephalosporins (22.7 %), with strong correlations between genetic markers and phenotypic results. MIC testing revealed that conventional antibiotics, while effective against planktonic cells, failed to eradicate mature biofilms. PDT exhibited significant antibiofilm activity against both planktonic and biofilm forms of S.aureus, E.coli, K.pneumoniae, P.aeruginosa, A.baumannii, and C.albicans. Combined biofilms (C.albicans + S.aureus; S.aureus + K.pneumoniae) showed limited response to combined antibiotic/antifungal therapy, whereas PDT induced notable structural disruption. Microscopy confirmed biofilm damage and planktonic dispersion post-PDT, likely mediated by singlet oxygen generation. These findings underscore the need for integrating PDT into the multidisciplinary management of chronic wound infections, especially where antibiotic resistance and biofilms are prominent. Conclusions. PDT represents a promising adjunctive modality for enhancing wound healing outcomes by effectively targeting biofilms. Future clinical studies are needed to validate these in vitro results and to optimize treatment protocols.