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Toward non-invasive collection methods for sampling the microbiome of diabetic foot ulcers.

ABSTRACT: Identifying the microbiome within chronic diabetic foot ulcers is essential if effective antimicrobial therapies are to be administered. Using culture and 16S rRNA gene sequencing, the aim of this study was to compare the microbiome of paired tissue scraping samples with swab samples, collected from participants during attendance at a high-risk foot clinic. The mean richness of cultured swab and tissue scraping samples was consistent, with anaerobes infrequently isolated from both sample types. Comparing percentage frequencies of detection of selected genera of known and potential pathogens namely Staphylococcus, Streptococcus, Enterococcus, Corynebacterium, Enterobacteriaceae and Pseudomonas from cultured and sequenced swab and tissue scrapings indicated that both collection methods captured varying percentages of all the selected genera. The mean abundance of sequenced samples was not significantly different between swabs and tissue scrapings. The mean richness or number of distinct operational taxonomic units (OTUs) and Shannon's H diversity index were not significantly different between the two collection methods. The mean relative abundance of the selected genera of known and potential pathogens, including anaerobes Anaerococcus and Finegoldia, was higher in swabs compared with tissue scrapings and significantly so in Staphylococcus and Pseudomonas genera. Multivariate analyses confirmed no significant differences between the bacterial community compositions of the paired samples. These results suggest that tissue scrapings and swabs can effectively capture the microbiome of chronic DFUs using culture and 16S rRNA gene sequencing.

SUBMITTER: Travis DJ

PROVIDER: S-EPMC10588311 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Toward non-invasive collection methods for sampling the microbiome of diabetic foot ulcers.

Travis Dorothy Jeanette DJ Bradbury Joanne J Benkendorff Kirsten K

International wound journal 20230727 9

Identifying the microbiome within chronic diabetic foot ulcers is essential if effective antimicrobial therapies are to be administered. Using culture and 16S rRNA gene sequencing, the aim of this study was to compare the microbiome of paired tissue scraping samples with swab samples, collected from participants during attendance at a high-risk foot clinic. The mean richness of cultured swab and tissue scraping samples was consistent, with anaerobes infrequently isolated from both sample types. ...[more]

PMID: 37501084

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Project description:Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.

Dataset Information

Toward non-invasive collection methods for sampling the microbiome of diabetic foot ulcers.

Publications

Toward non-invasive collection methods for sampling the microbiome of diabetic foot ulcers.

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