Fungal_DFU_ITS
The microbiome can profoundly influence the balance between human health and disease. Chronic non-healing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial communities, often described as biofilms in the wound bed, are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic wound microbiome have almost exclusively focused on bacterial communities, omitting what we hypothesize are important fungal contributions to impaired healing and development of complication. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with wound necrosis and poor outcomes, and may act synergistically to form mixed fungal-bacterial biofilms. We longitudinally profiled 100 chronic, non-healing diabetic foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi. In contrast, cultures performed in parallel captured fungi in only 5% of colonized wounds. The ‘mycobiome’ exhibited high levels of temporal instability and heterogeneity between subjects. Fungal diversity increased with antibiotic administration, wound deterioration or onset of infection-related complications, such as osteomyelitis. Proportions of the phlyum Ascomycota were significantly greater (p=0.015) at the study onset in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while a second major group of taxa identified as allergenic filamentous fungi, were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that Candida albicans and Trichosporon asahii form mixed biofilms with co-isolated Citrobacter fruendii and Staphylococcus simulans, respectively. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clincal outcomes.
DNA was isolated from swab specimens as previously described. The ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS2R (GCTGCGTTCTTCATCGATGC) primers were used for PCR amplification, each having a linker sequence, a sample specific GoLay12 index, and an Illumina adapter to amplify the ITS1 region of the fungal rRNA region. These indexed primers were used in combinations that made it possible to multiplex up to 576 (24 x 24) samples at a time. Each sample was amplified in duplicate, combined, and cleaned using the Agencourt AMPure XP bead-based PCR purification system (Beckman-Coulter). Purified amplicon pools were quantified using the Quant-IT dsDNA High-Sensitivity Assay Kit (Invitrogen) and a microplate reader (Thermo Scientific). A composite sample for sequencing was made by combining equimolar ratios of amplicons from the samples, followed by gel purification with a Qiagen MinElute Gel Extraction Kit to remove potential contaminants and PCR artifacts (the acceptable size window for amplicons was 200-1000 bp in length). The pooled DNA was quantified using a Qubit Fluorometer (Life Technologies), and PhiX174 genomic DNA was spiked in to the sample at ~40% prior to sequencing.