title: Investigating the clinical importance of fungi in diabetic wounds
Creators: name: Bin Ahmed Bakri, Ahmed Rafezzan
Publisher: name: University of Glasgow
Year: 2025

description: DFU is often manifested as a chronic open wound predisposed to invasion by many pathogenic and opportunistic microorganisms including bacteria and fungi. Impaired wound healing characterised by clinically infected ulcers can be a consequence of ineffective eradication of polymicrobial biofilms by antibiotic treatments, leading to limb amputation. A universal microbiome picture is necessary, especially for empirical therapy. Still, the dynamic of individual microbiomes can only be precisely depicted by periodic sampling of the wounds revealing the ongoing response to the antibiotic treatments.

Initially, standard microbiology culture data was analysed as part of the standard wound care for the patients. Of 306 samples with growth, 85.6% of the wounds were polymicrobial, and Candida was always found in mixed growth. Mixed skin, enterics and S. aureus were the topmost isolated organisms in descending order. Despite stratification of the wounds into different grades and stages according to the University of Texas wound classification systems, wounds were not found to be different. The data obtained from the standard culture showed that DFUs are predominated by bacteria from the skin and gut.

Secondly, Oxford Nanopore sequencing technology was utilised to scrutinize the 16S wound microbiomes of 349 extracted wound DNA. Corynebacterium, Staphylococcus and Streptococcus were the major taxa identified from the wounds. Moreover, Finegoldia magna was the most predominant obligate anaerobes identified, and commonly found with all Candida species. The wound diversity stratified according to the grades and stages showed no clustering. A subset of patients with multiple visits was then analysed for changes in microbiome as the wound progressed. Individual patient microbiomes were indeed unique.

An enhanced mycology culture was performed using Sabouraud dextrose agar (SAB) with chloramphenicol and CHROMagarTM for Malassezia to confirm the presence of fungi in the wounds. Thirty fungi isolated were Candida species mainly C. parapsilosis, C. albicans and C. glabrata. All fungi could form biofilms with C. albicans forming the most robust biofilms.

Lastly, real-world biofilm models grown from individual wounds were assessed for biofilm inhibition using Flucloxacillin, Fluconazole and a combination of both using concentrations typically found in the tissue. This model is based on an undefined microbial species found in the wounds.The bacteria and fungal load were quantified using 16S and ITS, respectively and changes in the 16S microbiome were assessed by nanopore sequencing. Antimicrobial treatments have minimal impact on the interkingdom biofilms that were formed consisting of bacteria and fungi.

In conclusion, the dynamics of diabetic wound microbiomes may represent interkingdom interactions between bacteria and fungi that exist as biofilms. This implicates the potential treatment of DFU, as systemic antibiotics alone may not be adequate to eliminate infection when fungi are present. Therefore, treatment targeting both infective aetiologies should be pursued to enhance the elimination of polymicrobial biofilms in chronic diabetic wounds.