A potential therapeutic strategy of an innovative probiotic formulation toward topical treatment of diabetic ulcer: an in vivo study

The study addresses the challenge of chronic diabetic ulcers (DUs), which have high morbidity and risk of amputation and often respond poorly to conventional therapies (debridement, off-loading, antibiotics), in part due to antibiotic-resistant infections. The authors hypothesize that topical probiotic Lactobacillus species formulated in a stable oleogel can enhance wound healing by promoting key processes (e.g., VEGF-mediated angiogenesis, collagen deposition, fibroblast activity, re-epithelialization) and by exerting antipathogenic/antibiofilm effects. The purpose is to evaluate, in vivo, the wound-healing efficacy of four Lactobacillus species applied topically to diabetic rat wounds compared with tetracycline and controls, using morphological, biochemical, and histopathological endpoints, and to assess antibiofilm activity against common pathogens.

The paper frames probiotics, particularly lactic acid bacteria (LAB), as beneficial in diverse medical applications via mechanisms such as immune modulation, strengthening barrier function, modulation of microbiota, and antipathogenic activity through organic acid production. Prior studies indicate Lactobacillus strains can inhibit pathogens (e.g., Staphylococcus aureus, Pseudomonas aeruginosa) and can stimulate VEGF expression, fibroblast migration, and collagen synthesis, thereby accelerating healing. Evidence from animal and human studies suggests probiotics (or postbiotics/lysates) can improve re-epithelialization, reduce biofilms, and enhance collagen deposition and angiogenesis in wounds, including diabetic models. However, the precise molecular mechanisms remain incompletely defined. This study builds on these findings by testing viable Lactobacillus species in a topical oleogel formulation for DU healing.

Study design: In vivo controlled experimental study in diabetic male Sprague-Dawley rats with full-thickness dorsal skin wounds; parallel in vitro antibiofilm assay. Probiotic formulation: Stable oleogel-based formulation (per prior study), containing glycerol and PEG (400/4000) with precipitated viable Lactobacillus cells as API at 1×10^8 CFU/ml. Species tested individually: L. rhamnosus (IBRC-M 11409), L. acidophilus (IBRC-M 10815), L. casei (IBRC-M 10711), L. fermentum (IBRC-M 10816). Comparator: Tetracycline 3% ointment (TC). Animals: 48 male Sprague-Dawley rats (200–300 g, 8–10 weeks), housed under controlled conditions. Ethics approvals obtained (ir.sums.aec.1400.027). Diabetes induction: Single intraperitoneal streptozotocin (STZ) 60 mg/kg in citrate buffer (pH 4.5) after overnight fast; after 1 week, rats with fasting blood glucose >300 mg/dl included. Anesthesia and wound model: Xylazine 5 mg/kg + ketamine 80 mg/kg IP; hair removed and skin prepped; circular full-thickness wound (2 cm diameter) created dorsally under aseptic conditions. Group allocation (n=6 per group): - Control: nondiabetic, no treatment. - DM: diabetic, no treatment. - DM + B.g: diabetic, base gel without bacteria, once daily for 14 days. - DM + TC: diabetic, topical tetracycline, once daily for 14 days. - DM + LBF: diabetic, gel with L. fermentum, once daily for 14 days. - DM + LBR: diabetic, gel with L. rhamnosus, once daily for 14 days. - DM + LBC: diabetic, gel with L. casei, once daily for 14 days. - DM + LBA: diabetic, gel with L. acidophilus, once daily for 14 days. Outcomes and assessments: - Morphological: Wound photographs on days 0, 3, 7, 14; wound area quantified with Image Pro Plus V6; wound closure percentage = (Initial area − Open area)/Initial area ×100. - Biochemical: Hydroxyproline content (index of collagen) measured from homogenized tissue samples collected on days 7 and 14; acid hydrolysis (6N HCl, 120°C, 8 h), chloramine-T, Ehrlich’s reagent; absorbance at 550 nm. - Histopathological and stereological analyses: Formalin-fixed, paraffin-embedded sections; Masson’s trichrome staining; volume densities (epidermis/re-epithelialization, collagen fibrils, hair follicles, neovascularization) via Delesse point-counting; fibroblast numerical density via optical dissector method (Nv = ΣQ / (ΣP × a/f) × h / BA). Tissue sampling by Vertical Uniform Random (VUR) method on days 7 and 14. - Antibiofilm assay (in vitro): L. rhamnosus biofilms grown in 12-well plates (MRS, 30°C, 48 h); challenged with pathogens at 10^8 CFU/ml (E. coli ATCC 35150, Listeria monocytogenes ATCC 7644, Salmonella typhimurium ATCC 14028) for 24, 48, 72, 96 h; planktonic cultures removed, biofilm-associated viable counts determined on selective media (SM, MOX, XLD) after 48 h at 37°C; controls were pathogen-only wells; assays in triplicate. Antibiofilm activity calculated from log CFU reductions relative to control. Statistical analysis: One-way ANOVA with Tukey’s post hoc test (IBM SPSS); experiments in triplicate where applicable; significance at P ≤ 0.05. Euthanasia: Thiopental 100 mg/kg after tissue sampling.

- Antibiofilm activity (L. rhamnosus): Significant inhibition of biofilm formation for E. coli, L. monocytogenes, and S. typhimurium at 24–96 h versus controls (P ≤ 0.05). Maximal inhibition at 24 h: 100% for E. coli and L. monocytogenes; 94.8% for S. typhimurium. Inhibition remained >~70% through 96 h. - Wound closure: All groups showed progressive closure; highest closure in probiotic groups, particularly L. acidophilus (DM+LBA) and L. rhamnosus (DM+LBR) at day 14 (~89–96%); base gel had the least effect (DM+B.g ~62%). L. fermentum (DM+LBF) ~85.31% (not significantly different from TC 89.66%); L. casei (DM+LBC) ~79.96% at day 14. - Hydroxyproline (collagen index): Highest in DM+LBA (116.67 μg/ml) and lowest in DM (92.84 μg/ml) at day 14. DM+LBA and DM+LBR had greater hydroxyproline than DM and DM+TC over time; significant increases noted vs DM on day 7 (DM+LBA, DM+LBR) and day 14 (DM+LBA). - Re-epithelialization (volume density): Increased over time in all groups; highest in DM+LBA (0.23) and lowest in DM+B.g (0.07) at day 14. All lactobacilli groups except L. casei exceeded DM and DM+TC at both time points, significantly vs DM. L. casei was similar to DM. - Hair follicles (volume density): Highest in DM+LBA (0.28) and lowest in DM (0.09) at day 14; probiotics generally exceeded DM and DM+TC, with significant effect for DM+LBA at day 14. - Fibroblast numerical density: Highest in DM+LBA (481.31×10^3/mm^3) and lowest in DM (211.51×10^3/mm^3) at day 14; DM+LBC showed higher than DM (296.59×10^3/mm^3) without statistical significance. - Collagen deposition (volume density): Increased across all groups; highest in DM+LBA (0.23) at day 14; lowest in DM (0.08) and DM+B.g (0.07). DM+LBA and DM+LBR were significantly greater than DM and DM+TC at day 14; DM+LBF and DM+LBC were greater than DM but less than TC (NS). - Neovascularization (volume density): Highest in DM+LBA (0.19) and lowest in DM (0.04) at day 14. All lactobacilli groups exceeded DM at day 14, except DM+LBC, which was comparable to DM+TC. - Microscopy: L. acidophilus and L. rhamnosus groups showed thicker epidermis, more mature collagen bundles, increased fibroblasts, hair follicles, and neovascularization at days 7 and 14 compared with control, DM, base gel, and tetracycline; L. casei showed minimal improvements. Overall, L. acidophilus and L. rhamnosus outperformed tetracycline in multiple healing parameters.

The study demonstrates that topical delivery of viable Lactobacillus species in an oleogel can enhance multiple phases of the wound-healing cascade in diabetic rats: faster wound closure, greater collagen synthesis (hydroxyproline), improved re-epithelialization, increased fibroblast density, and augmented angiogenesis/neovascularization. These effects align with hypothesized probiotic mechanisms, including modulation of growth factors (e.g., VEGF), fibroblast activity, and antipathogenic actions. The strong antibiofilm activity of L. rhamnosus against E. coli, L. monocytogenes, and S. typhimurium suggests that reducing pathogen biofilms may contribute to improved healing outcomes, a key advantage in DU where resistant infections impede recovery. Among strains, L. acidophilus and L. rhamnosus provided the most consistent and significant benefits, often surpassing tetracycline, indicating strain-specific efficacy. L. casei showed limited benefit, underscoring that probiotic species selection is critical to therapeutic performance. Collectively, the data support the central hypothesis that probiotic-containing topical formulations can serve as effective alternatives or adjuncts to antibiotics in DU management by simultaneously addressing microbial burden and promoting tissue regeneration.

A novel oleogel formulation containing viable Lactobacillus, particularly L. acidophilus and L. rhamnosus, significantly accelerated diabetic wound healing in rats, improving wound closure and key biochemical and histopathological markers (re-epithelialization, fibroblast density, collagen deposition, neovascularization). L. rhamnosus also showed robust antibiofilm activity against multiple pathogens. These findings position topical probiotics as promising alternatives or adjuncts to conventional antibiotic treatments for diabetic ulcers. Future studies should include detailed mechanistic investigations, assessment of in vivo microbial load changes, optimization of strain selection and dosing, extended treatment durations, and rigorous clinical trials to evaluate safety and efficacy in humans.

- Preclinical model: Results are from a rat model over a 2-week period and may not fully translate to human diabetic ulcers. - Sample size and duration: n=6 per group with assessments up to 14 days; longer-term outcomes (e.g., tensile strength, scar quality, recurrence) were not evaluated. - In vivo microbiology: While strong in vitro antibiofilm activity was shown, the study did not quantify pathogen load or biofilm status directly in wound tissues post-treatment. - Mechanistic insight: The specific molecular pathways (e.g., cytokines, chemokines, VEGF signaling) were not measured; mechanisms remain inferential. - Strain specificity and formulation variables: Only four Lactobacillus species were tested individually at a single dose (1×10^8 CFU/ml); dose–response, combinations, and stability/performance under clinical conditions require further study.