Comparative Analysis of the Change in the Microbiome of Diabetic Foot Ulcers and Host/Pathogens Interactions in Response to Novel Therapies

Abstract

Diabetic foot ulcers (DFUs) are a tertiary complication of diabetes mellitus, developing from the accumulation of other secondary symptoms such as peripheral neuropathy, peripheral vascular disease and immune system dysfunction. Roughly 25% of diabetic sufferers will develop a DFU in their lifetime. Of these 40-80% develop chronic infections potentially resulting in further complications such as gangrene and osteomyelitis, sepsis and death. DFU infections are often polymicrobial and develop biofilms which complicates treatment strategies from the typical antibiotic therapies of other wound infections. The gold standard treatment for DFUs is surgical debridement, offloading and irrigation with an antimicrobial irrigant, but even with this, standard healing rates for DFUs is >12 weeks increasing the risk for additional complications. Electrolyzed water is a potential new therapy which can aid in reducing the infection rates and healing times for DFU sufferers. We hypothesize that the electrolyzed water will be equally or more effective at treating DFUs than the current Prontosan therapy, both with the removal of infection and improving wound healing rates. The DFUs of patients undergoing irrigant therapy with either the standard (Prontosan) and novel (Salvesan/Electrolyzed water – E.Water) irrigants were swabbed throughout the course of treatment and the microbiomes of these wounds were analyzed using 16S rRNA sequencing and QIMME 2.0. Skin microbiomes of non-diabetic participants were also taken for comparison purposes. Principle component analysis of the microbiome data following 4 weeks of irrigant treatment, the microbiomes of patients treated with the E.water (N=5), were more similar to the non-diabetic phenotype than those treated with Prontosan (N=3). The antimicrobial effect of these two irrigants using clinically isolated and reference strain versions of S. aureus and Proteus mirabilis/ vulgaris were done using minimum inhibitory concentration tests, biofilm prevention/ removal assays, Live/Dead confocal microscopy and growth curve analysis. These demonstrated that E.water had similar effects in antimicrobial activity to Prontosan when tested directly against bacterial biofilms but had no antimicrobial activity when in suspension in a culture medium. Prontosan demonstrated effective antimicrobial activity across all experimental procedures. However, significantly higher concentrations were required against P. mirabilis/vulgaris and other Gram negatives organisms. HaCaT cell viability assays, recovery assays and wounded cell culture assays showed that E. water had significantly less cytotoxic effects than Prontosan with significantly less immediate and long-term impacts on HaCaT (epidermal skin) cells. Lastly, viability assays and IL,6/8 ELISAs showed wide variations in impact of the secreted products of different clinical and reference strain isolates of both S. aureus and P. mirabilis against HaCaT cells indicating negative or null impacts. Whilst there is some evidence to support the use of E.water as a DFU therapy, there are still more research required to fully assess its utility. It has been demonstrated to cause compositional changes in the DFU microbiome which more closely resemble those of non-diabetic phenotypes. Its effectiveness as an effective antimicrobial requires further investigation as it was demonstrated that certain in-vitro experimental procedures significantly reduced its effectiveness which is not representative of the in-vivo environment. Its impact on human epidermal cell lines (HaCaT) was shown to be significantly less cytotoxic than the current irrigant used in DFU treatment. Whist this research fails to confirm the hypothesis, it does provide a framework for additional research to further investigate E. water as a potential new therapy for DFUs.

Date of Award	2024
Original language	English
Awarding Institution	Glasgow Caledonian University
Supervisor	Janice Spencer (Supervisor), Patricia Martin (Supervisor) & Susan Lang (Supervisor)