Comparative investigation of the Diabetic foot ulcer microbiome

Abstract

BackgroundMany factors may affect wound healing in Diabetic foot ulcer, namely, microbial density, microorganisms, microbial synergy, the host immune response, and infected tissue quality.MethodologyThis study used a cross-sectional design. We assessed 38 Subjects with DFUs, 23 neuroischaimic, and 15 neuropathic, for microbiota colonizing the DFU utilizing traditional cultures and 16S gene sequencing methods. All the relevant clinical factors were collected. Wound swabs were collected for both traditional microbiological analysis, direct swab (DSM), and cultured (CM) microbiome analysis. DNA isolation, and 16SrRNA hypervariable regions were amplified. Bioinformatics analysis was performed using IonReporter Software, statistical analysis, and diversity indices were computed with vegan R-package.ResultsThe traditional microbiological method was able to detect a maximum of one or two pathogens, and, in some cases, no pathogen was detected. The total number of the observed species was 176. The number of identified species was higher in the cultured microbiome (155) than the direct swab microbiome (136). Diversity analysis indicated that biological diversity is higher in the cultured microbiome compared with the DSM. The Shannon H index was 2.75 for the cultured microbiome and 2.63 for DSM. We observed some differences in the major bacterial taxa amongst neuroischaimic and neuropathic DFU microbiomes.ConclusionsCultured microbiome is superior to both the traditional method and direct swab microbiome. The Neuroischaimic group showed higher values for the tested diversity indices than the Neuroischaimic group. Neither Cluster Analysis nor Principal Component Analysis showed apparent clustering amongst the two types of ulcers.

Direct metagenomics investigation of non-surgical hard-to-heal wounds: a review

“…A key point in the management of chronic wounds is the importance of multidrug resistance. Interestingly, information about antiseptic and antibiotic resistance could be predicted in silico by shotgun mNGS [37,45,59], as well as pathogen genotypes determined by microbial genome analysis [33,34,40]. In the future, continuous surveillance of wound evolution according to the microbial colonisation throughout treatment could help clinicians manage the wounds by revealing the role of bacteria in wound healing and patient outcomes [1,60,63,74,75].…”

“…Two studies were published in 2019 [30,31], and 11 between 2020 and September 2022. Regarding the geographical origin of publications, nine studies were from Asia, including two publications each from Chinese, Indian, and South Korean laboratories [31][32][33][34][35][36]; one each from Saudi Arabia, Israel, and Taiwan [37][38][39]; four in the United States [16,27,40,41]; four in Australia [30,[42][43][44] and only one from Europe, specifically France [45] (Supplementary Figure S1).…”

“…Chemical treatment by Tissue Lyser solution (Qiagen, Hilden, Germany) was used with or without prior incubation at 37 °C in five studies, followed by a vortexing step to destroy bacterial cells. A supplementary incubation with proteinase K at 56 °C was recommended before DNA isolation [16,27,37,42,45].…”

“…A DNA library was constructed using 16 S rDNA amplification following the Illumina Nextera-XT pairedend sequencing protocol (Illumina, San Diego, USA). An Ion-Torrent commercial multiplex amplification targeting most variable 16 S rDNA regions V2, V3, V4, V6, V7, V8, and V9 was used in two studies, followed by Ion Xpress Barcode Adapters library preparation protocol and Ion-Torrent sequencing [34,37]. Full length 16 S rDNA gene was sequenced in only one study using Rapid Barcoding Sequencing Kit (Oxford Nanopore technologies, Oxford Science Park, UK) [33].…”

“…Swabs were mainly used for superficial wound sampling, although this technique should be avoided, due to its sensitivity, which restricts the identification of anaerobic bacteria. More in-depth sampling methods should be considered [34,35,37,41,43]. Finally, DNA extracted from wound swabs and biopsies generated a high human genome level, which required microbial genome enrichment and/or human genome depletion to improve the microbial genome detection [31,38,39,41].…”