Microbiome Characterization of Infected Diabetic Foot Ulcers in Association With Clinical Outcomes: Traditional Cultures Versus Molecular Sequencing Methods

Hadar Mudrik-Zohar, Shaqed Carasso, Tal Gefen, Anat Zalmanovich, Michal Katzir, Yael Cohen, Yossi Paitan, Naama Geva-Zatorsky, Michal Chowers, Hadar Mudrik-Zohar, Shaqed Carasso, Tal Gefen, Anat Zalmanovich, Michal Katzir, Yael Cohen, Yossi Paitan, Naama Geva-Zatorsky, Michal Chowers

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 impaired wound healing characterizing diabetic patients and the complex microbial environment of these ulcers.

Aim: 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 metagenomic sequencing. Patients' characteristics, culture-based results and sequencing data were analyzed in association with clinical outcomes.

Results: A total of 31 patients were enrolled. Gram-negative bacteria dominated the IDFU samples (79%, 59% and 54% of metagenomics, 16S rRNA and cultures results, respectively, p<0.001). 16S rRNA and metagenomic sequencing detected significantly more anaerobic bacteria, as compared to conventional cultures (59% and 76%, respectively vs. 26% in cultures, p=0.001). Culture-based results showed that Staphylococcus aureus was more prevalent among patients who were treated conservatively (p=0.048). In metagenomic analysis, the Bacteroides genus was more prevalent among patients who underwent 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 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 highlight possible associations among certain genera, species, and bacterial functional genes to clinical outcomes.

Keywords: 16S rRNA; amputation; diabetic foot ulcer; metagenomics; microbiome.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Mudrik-Zohar, Carasso, Gefen, Zalmanovich, Katzir, Cohen, Paitan, Geva-Zatorsky and Chowers.

Figures

Figure 1
Figure 1
Study design. Created with BioRender.com.
Figure 2
Figure 2
(A–D). Comparison of Gram staining characteristics and oxygen requirements of isolated and sequenced bacteria as reflected by traditional cultures, 16S rRNA and metagenomic sequencing. Numbers represent percentages (%); (A, B) Traditional cultures vs. 16S rRNA sequencing results (N=30): Gram staining, p=0.07; oxygen requirements (anaerobes and facultative anaerobes, p<0.001). (C, D) Traditional cultures, 16S rRNA sequencing and metagenomic sequencing results (N=13): Gram staining, p=0.01; oxygen requirements (anaerobes, p<0.001; facultative anaerobes, p=0.001).
Figure 3
Figure 3
(A–E). Occurrences of the most prevalent bacteria among the investigated IDFU. (A, B) Cultures results (genera and species level – N=30). (C) 16S rRNA sequencing results (genera level – N=30). (D, E) Metagenomic sequencing results (genera and species level – N=13).
Figure 4
Figure 4
Association between Bacteroides genus and amputation according to 16S rRNA sequencing and metagenomic sequencing. Bacteroides genus, p<0.001; Bacteroides fragilis, p=0.04; Bacteroides xylanisolvens, p=0.002.
Figure 5
Figure 5
(A, B). Associations between certain genera and species and ulcer size. (A) Beta diversity (Bray-Curtis dissimilarity index) of samples from patients with different ulcer sizes: PCoA plot of taxonomic features shows the clustering of samples according to ulcer sizes. Ellipsoids represent a 95% confidence interval surrounding each group. p<0.01 (PERMANOVA). (B) 16S rRNA results analysis: Shown are genera that were more common in ulcers smaller than 3cm, compared with ulcers 3-10cm, p<0.001.
Figure 6
Figure 6
(A, B). Analysis of functional genes. Yellow stars indicate genes related to bacterial virulence factors. (A) A cluster of five samples of patients with IDFU who underwent amputation. These patients had more severe peripheral vascular disease compared to other patients, p=0.003. (B) Functional genes differentiating patients who underwent amputation from patients who were treated conservatively.

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