SRP326293 PRJNA741238 Bacterial Bioburden Characterization of Infected Diabetic Foot Ulcers in Hospitalized Patients in Association with Clinical Outcomes: Traditional Cultures vs. Molecular Sequencing Methods. Background: Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus (DM). These potentially limb-threatening ulcers are challenging to treat due to the impairment of wound healing in diabetic patients and the complex microbial environment characterizing these ulcers. Aim: To analyze the bacterial bioburden of IDFU in association with clinical outcomes. Methods: Wound biopsies from IDFU were obtained from hospitalized patients and were analyzed using traditional microbiology cultures, 16S rRNA sequencing and shotgun metagenomic sequencing. Patients' characteristics, culture-based results and sequencing data were analyzed in association with clinical outcomes. Results: 31 patients were enrolled. Significantly more anaerobic and Gram-negative bacteria were detected with sequencing methods compared to conventional cultures (59% and 76% were anaerobes according to 16S rRNA and metagenomic respectively vs. 26% in cultures, p=0.001, and 79%, 59% and 54% were Gram negative bacteria respectively, p<0.001). Culture-based results showed that Staphylococcus aureus was more prevalent among patients who were conservatively treated (p=0.048). In metagenomic analysis the Bacteroides genus was more prevalent among patients who underwent toe amputation (p<0.001). Analysis of metagenomic-based functional data showed that antibiotic resistance genes and genes related to biofilm production and to bacterial virulent factors were more prevalent in IDFU that resulted in toe amputation (p<0.001). Conclusion: Sequencing tools uncover the complex biodiversity of IDFU and emphasize the high prevalence of anaerobes and Gram-negative bacteria in these ulcers. Furthermore, sequencing results highlighted the possible association between certain genera, species, and bacterial functional genes to clinical outcomes.