1018. Bacterial Bioburden Characterization of Infected Diabetic Foot Ulcers in Hospitalized Patients in Association with Clinical Outcomes: Traditional Cultures vs. Molecular Sequencing Methods

Abstract Background Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. 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. Our aim was to analyze the microbiome 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. Study Design 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 16SrRNA 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). Occurrences and mean relative abundances of the most prevalent bacteria of IDFU Comparison between [A] traditional cultures, 16S rRNA sequencing and metagenomic sequencing results (genera level - 12 samples) [B] traditional cultures and metagenomic sequencing results (species level – 30 samples) [C] traditional cultures and 16S rRNA sequencing results (genera level - 30 samples) CUL – cultures; 16S - 16S rRNA sequencing; MTG – metagenomic sequencing Bacteroides genus association with toe amputation Bacteroides genera was more common among samples of patients who underwent toe amputation compared with samples of patients who were conservatively treated (p < 0.001). Species level analysis showed that Bacteroides fragilis and Bacteroides xylanisolvens predominated IDFU of patients who underwent toe amputation (p=0.04, p=0.002 respectively). No – conservative treatment; Yes – toe amputation. Functional genes differentiating patients who underwent toe amputation from conservatively treated Yellow stars – indicate genes that were associated with bacterial virulent factors, biofilm formation and resistant mechanisms – all were more prevalent in patients who underwent toe amputation (with p values<> Conclusion Molecular 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 Disclosures Yossi Paitan, PhD, Ilex Medical Ltd (Employee, Other Financial or Material Support, As of 01.01.2021 I am the Laboratories Manager of Ilex Labs)

Background. Liver transplant (LT) recipients have abnormal microbiota before and after transplantation. (1,2) Associations between fecal microbiota, microbial metabolites, and clinical outcomes in liver transplantation are not well established. We correlated fecal microbiota composition and metabolite concentrations with early LT outcomes, including infection.
Methods. In a prospective observational study, we collected peri-transplant fecal samples and determined microbiota composition by 16S ribosomal RNA gene sequencing in LT recipients. Fecal short chain fatty acid (SCFA) and bile acid concentrations were measured by targeted GC-and LC-MS analyses, respectively. Inverse Simpson index was used to determine microbiota alpha-diversity in subjects and healthy controls. Clinical outcomes including length of stay, ICU admission, liver function, antibiotic use, immunosuppressive requirement and post-operative infection were correlated with microbiota composition.
Results. 69 patients were enrolled, 70 liver transplants were performed and 307 peri-transplant fecal samples were collected and analyzed. Compared to healthy controls, the fecal microbiota of LT recipients had reduced alpha-diversity (p< 0.001).
(3) Intestinal domination ( >30% frequency) by Enterococcus or Proteobacteria species was common and occurred in 36% of LT recipients. 76 post-operative infections occurred in 40 LT recipients, with Enterococci causing 52% and Proteobacteria 41% of bacterial infections. In subjects with fecal samples collected within 5 days of infection, 9/17 infections were caused by the organism dominating the microbiota. [Fig2] Microbiota Composition and Metabolite Production 16s gene sequencing color coded by taxonomy. Each bar represents one stool sample nearest to LT compared to healthy controls. Alpha diversity measured by inverse simpson index. Absolute values of microbial metabolites and ratio of primary to secondary bile acids.

Comparison of Microbiota Composition and Post Operative Infection
All bacterial infections captured with a microbiota sample within 5 days of infection.
Conclusion. Microbiota diversity and microbially derived metabolites are markedly reduced in >50% of LT recipients. Intestinal domination and post-operative infections caused by antibiotic-resistant Enterococcus and Proteobacteria correlate with loss of Bacteroidetes, Ruminococcaceae, and Lachnospiraceae species, suggesting a potential role for microbiota reconstitution therapy in LT patients.
Disclosures. Eric G. Pamer, MD;FIDSA, Nothing to disclose Background. Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. 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. Our aim was to analyze the microbiome of IDFU in association with clinical outcomes.

Bacterial Bioburden Characterization of Infected Diabetic Foot Ulcers in
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.

Study Design
Results. 31 patients were enrolled. Significantly more anaerobic and Gramnegative bacteria were detected with sequencing methods compared to conventional cultures (59% and 76% were anaerobes according to 16SrRNA 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).
Occurrences and mean relative abundances of the most prevalent bacteria of IDFU Bacteroides genera was more common among samples of patients who underwent toe amputation compared with samples of patients who were conservatively treated (p < 0.001). Species level analysis showed that Bacteroides fragilis and Bacteroides xylanisolvens predominated IDFU of patients who underwent toe amputation (p=0.04, p=0.002 respectively). No -conservative treatment; Yes -toe amputation.
Functional genes differentiating patients who underwent toe amputation from conservatively treated Yellow stars -indicate genes that were associated with bacterial virulent factors, biofilm formation and resistant mechanisms -all were more prevalent in patients who underwent toe amputation (with p values<> Conclusion. Molecular 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 Disclosures. Yossi Paitan, PhD, Ilex Medical Ltd (Employee, Other Financial or Material Support, As of 01.01.2021 I am the Laboratories Manager of Ilex Labs)