Microbiome Based Biomarkers of Wound Healing

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Abstract Diabetic foot ulcers (DFU) impact over 2 million Americans annually, result in over 130,000 amputations each year, and are associated with high mortality rates. To date, there has been no single infectious agent of DFU identified as a good marker of healing outcome. This is likely because DFUs are host to a diverse community of microbes (i.e., the wound microbiome). Wound microbiomes analyzed at the community-level are promising predictors of wound healing outcomes. We have shown that wounds persisting beyond 12 weeks exhibit high and persistent proportions of mixed-population, anaerobic bacteria within their microbiomes. Additionally, we found a significant increase of anaerobic transcriptional activity in persistent and amputated wounds, even from species identified as being in low abundance by traditional 16S rRNA based gene sequencing. This suggests microbial transcription, and specifically from anaerobic bacteria, are promising biomarkers of wound healing in diabetic patients. The proposed project will identify microbial biomarkers that can be used as prognostic and monitoring tools for DFU wound healing. We hypothesize using RNA instead of DNA will provide a better snapshot of the wound environment and more sensitive biomarkers: specifically, the proportion and transcriptional activity of anaerobes within the wound microbiome can be used as predictors of wound healing outcomes. Using RNAseq to measure the metatranscriptome of the DFU microbiome, we will leverage advances in machine learning approaches to demonstrate the capacity of the anaerobic component of the wound microbiome to serve as a biomarker for wound healing. We have vigorously evaluated the optimal sample collection techniques and methods of detection, determining a simple swab of the ulcer bed is sufficient to characterize the metatranscriptome and will facilitate clinical implementation. We will develop a multiplexed RT-qPCR assay for the panel of candidate biomarker genes we identify and validate the assay sensitivity, specificity, accuracy and precision. We will work closely with the Diabetic Foot Consortium Steering Committee and Data Coordinating Center to test and validate our biomarkers in a multi-site trial with the DFC. Our goal is to develop a multiplexed biomarker assay integrating the complex interactions occurring within the DFU microbiome and tissue microenvironment. It will use a simple swab of the ulcer bed, rely on objective measurements, and is designed to predict healing trajectories at an early time in the clinical course.