The healing process of diabetic ulcers correlates with changes in the cutaneous microbiota

Abstract

Skin microbiota plays an essential role in the development and function of the cutaneous immune system, in the maintenance of the skin barrier through the release of antimicrobial peptides, and in the metabolism of some natural products. With the aim of characterizing changes in the cutaneous microbiota specifically associated with wound healing in the diabetic condition, we performed a 16 S rRNA gene Next Generation Sequencing of skin swabs taken within the ulcer bed of ten diabetic patients before (t0) and after 20 days of therapy (t20) with a fluorescein-based galenic treatment. Considering the twenty most representative genera, we found at t20 an increase of Corynebacterium, Peptostreptococcus, and Streptococcus, and a decrease of Enterococcus, Finegoldia, and Peptoniphilus genera. However, differences were not significant due to the high variability among samples and the small patient cohort. S. aureus was the most abundant species at t0 and was reduced by therapy in four patients. Comparing the microbiome in the ulcer bed and in the perilesional tissue of the same patient at t0, no major differences were observed. Taken together, our data indicate that in the absence of antibiotic-based therapy the healing process of diabetic ulcers is accompanied by changes in the microbiome composition.

Keywords: Diabetic ulcers; Fluorescein; Microbiome; Wound healing.

Figure 1

Ulcers characteristics. A. Photographs of diabetic ulcers at day 0 (T0; a, c, e), and after 20 days of treatment with Fluorexin (tT0; b, d, f). Representative images are shown of pt_001 (a, b); pt_006 (c, d); pt_007 (e, f). B. Stage, according to the University of Texas staging system for diabetic foot ulcers, where stage A comprises pre- or post-ulcerative lesions completely epithelialized (0); superficial ulcer, not involving tendon capsule or bone (1); ulcer penetrating to tendon or capsule (2). Stage B indicates the presence of infection in addition to what reported for stage A. Falanga’s score was calculated for each patient ulcer: (A) granulation tissue presence in the 100% of the ulcer; (B) granulation tissue presence in the 50-100% of the ulcer and presence of fibrinous tissue; (C) granulation tissue presence in <50% of the ulcer and presence of fibrinous tissue.

Figure 2

Ulcer bed microbiome mean data at t0 and t20. (A) Stacked bar plot of the most abundant 20 genera found in the lesional areas before (t0) and after the 20-day treatment (t20) with Fluorexin. Text layout in the plot was edited with GNU Image Manipulation Program (GIMP v2.10.34) (B) Beta diversity in lesional areas before (t0) and after the 20-day treatment (t20). Principal coordinate analysis (PCoA) plots were performed by the Bray-Curtis distance measures. Statistical significance was assessed by PERMANOVA.

Figure 3

Stacked bar plot of the most abundant 20 genera found in lesional areas before (t0) and after the 20-days treatment (t20) for each of the 10 patients enrolled in the study. Text layout in the plot was edited with GIMP.

Figure 4

Microbiome mean data of intra and perilesional tissue samples at t0. A. Stacked bar plot of the most abundant 20 genera found in the intralesional and perilesional areas before Fluorexin treatment (t0). B. Beta diversity in the intralesional and perilesional areas at t0. Principal coordinate analysis (PCoA) plots were performed by the Bray-Curtis distance measures. Statistical significance was assessed by PERMANOVA.

Figure 5

Stacked bar plot of the most abundant 20 genera found before treatment (t0) in 9 patients, evaluating the microbiome community in the ulcer bed (intralesional), and in the perilesional tissue. Perilesional swab was not available for pt_007 that was excluded. Text layout in the plot was edited with GIMP.

Figure 6

Microbiome data on ulcer body site. A. Stacked bar plot of the most abundant 20 genera found in the intralesional samples of patients with ulcer located at plantar, lower leg or heel before treatment initiation. B. Stacked bar plot of the most abundant 20 genera found after 20 days of treatment (t20) in intralesional samples collected from patients with ulcer located at plantar, lower leg or heel. C. Top 20 taxa at genus level of the 5 patient samples from plantar or lower leg ulcers at t0. The table below summarizes the result of post-hoc pairwise comparison (multi-group only) performing the regular Welch t-tests. The multi-testing adjustment is based on Benjamini-Hochberg procedure (FDR). No statistically significant difference was observed in the alpha diversity between groups (FDR always >0.05). D. Top 20 taxa at genus level of the 5 patient samples from plantar or lower leg ulcers at t20. The table below summarizes the result of post-hoc pairwise comparison (multi-group only) performing the regular Welch t-tests. Statistically significant difference was observed in the alpha diversity between the plantar and the lower leg group (FDR = 0.005).