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      The microbiome of diabetic foot ulcers: a comparison of swab and tissue biopsy wound sampling techniques using 16S rRNA gene sequencing

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          Abstract

          Background

          Health-care professionals need to collect wound samples to identify potential pathogens that contribute to wound infection. Obtaining appropriate samples from diabetic foot ulcers (DFUs) where there is a suspicion of infection is of high importance. Paired swabs and tissue biopsies were collected from DFUs and both sampling techniques were compared using 16S rRNA gene sequencing.

          Results

          Mean bacterial abundance determined using quantitative polymerase chain reaction (qPCR) was significantly lower in tissue biopsies ( p = 0.03). The mean number of reads across all samples was significantly higher in wound swabs \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \Big(\overline{X} $$\end{document} = 32,014) compared to tissue ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \overline{X} $$\end{document} = 15,256, p = 0.001). Tissue biopsies exhibited greater overall diversity of bacteria relative to swabs (Shannon’s H diversity p = 0.009). However, based on a presence/absence analysis of all paired samples, the frequency of occurrence of bacteria from genera of known and potential pathogens was generally higher in wound swabs than tissue biopsies. Multivariate analysis identified significantly different bacterial communities in swabs compared to tissue ( p = 0.001). There was minimal correlation between paired wound swabs and tissue biopsies in the number and types of microorganisms. RELATE analysis revealed low concordance between paired DFU swab and tissue biopsy samples (Rho = 0.043, p = 0.34).

          Conclusions

          Using 16S rRNA gene sequencing this study identifies the potential for using less invasive swabs to recover high relative abundances of known and potential pathogen genera from DFUs when compared to the gold standard collection method of tissue biopsy.

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          Most cited references 30

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          Nonrandom distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds.

          The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.
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            IWGDF guidance on the diagnosis and management of foot infections in persons with diabetes.

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              The Neuropathic Diabetic Foot Ulcer Microbiome Is Associated With Clinical Factors

              Nonhealing diabetic foot ulcers (DFUs) are a common and costly complication of diabetes. Microbial burden, or “bioburden,” is believed to underlie delayed healing, although little is known of those clinical factors that may influence microbial load, diversity, and/or pathogenicity. We profiled the microbiomes of neuropathic nonischemic DFUs without clinical evidence of infection in 52 individuals using high-throughput sequencing of the bacterial 16S ribosomal RNA gene. Comparatively, wound cultures, the standard diagnostic in the clinic, vastly underrepresent microbial load, microbial diversity, and the presence of potential pathogens. DFU microbiomes were heterogeneous, even in our tightly restricted study population, but partitioned into three clusters distinguished primarily by dominant bacteria and diversity. Ulcer depth was associated with ulcer cluster, positively correlated with abundance of anaerobic bacteria, and negatively correlated with abundance of Staphylococcus. Ulcer duration was positively correlated with bacterial diversity, species richness, and relative abundance of Proteobacteria, but was negatively correlated with relative abundance of Staphylococcus. Finally, poor glycemic control was associated with ulcer cluster, with poorest median glycemic control concentrating to Staphylococcus-rich and Streptococcus-rich ulcer clusters. Analyses of microbial community membership and structure may provide the most useful metrics in prospective studies to delineate problematic bioburden from benign colonization that can then be used to drive clinical treatment.
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                Author and article information

                Contributors
                Kirsten.benkendorff@scu.edu.au
                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central (London )
                1471-2180
                16 June 2020
                16 June 2020
                2020
                : 20
                Affiliations
                [1 ]GRID grid.1031.3, ISNI 0000000121532610, School of Environment, Science and Engineering, , Southern Cross University, ; Lismore, NSW Australia
                [2 ]Limb Preservation and Wound Research Academic Unit, Western Sydney LHD, Liverpool, Sydney, NSW 2170 Australia
                [3 ]GRID grid.1029.a, ISNI 0000 0000 9939 5719, Infectious Diseases and Microbiology, School of Medicine, , Western Sydney University, ; Campbelltown Campus, Liverpool, Sydney, 2170 Australia
                [4 ]GRID grid.429098.e, Ingham Institute of Applied Medical Research, ; Liverpool, Sydney, NSW 2170 Australia
                [5 ]GRID grid.1004.5, ISNI 0000 0001 2158 5405, Surgical Infection Research Group Faculty of Medicine and Health Sciences, , Macquarie University, ; Sydney, Australia
                [6 ]GRID grid.417738.e, ISNI 0000 0001 2110 5328, Agresearch, Grasslands Research Centre, ; Palmerston North, New Zealand
                [7 ]GRID grid.415989.8, ISNI 0000 0000 9759 8141, Central Military Laboratories and Blood Bank, , Prince Sultan Military Medical City, ; Riyadh, Saudi Arabia
                [8 ]GRID grid.1024.7, ISNI 0000000089150953, Institute of Health and Biomedical Innovation, , Queensland University of Technology, ; Herston, QLD Australia
                [9 ]GRID grid.1024.7, ISNI 0000000089150953, School of Biomedical Science, , Queensland University of Technology, ; Brisbane, Australia
                [10 ]National Marine Science Centre, 2 Bay Drive, Coffs Harbour, NSW Australia
                Article
                1843
                10.1186/s12866-020-01843-2
                7296698
                32546123
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                Funding
                Funded by: Wound Management and Innovation Co-operative Research Centre
                Award ID: SP41-1
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2020

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