Complications of diabetes such as diabetic foot ulcers (DFUs), diabetic foot infections (DFIs) and lower extremity amputations (LEAs) continue to rise at an alarming rate, presenting a significant challenge for providers, patients and health systems around the world. In the United States, lower extremity care constitutes one-third of the annual direct costs for diabetes,1-3 and when compared to cancer, according the latest data, the five-year mortality rate following a major (proximal to ankle) lower extremity amputation (56.6 percent) is second only to lung cancer (80%).4,5
Chronic DFUs can be complicated by infection and poor adherence to offloading, and a better understanding of the full microbiological composition of these wounds may help improve outcomes. Thus, it is critical to define the role of bacteria and other microorganisms present in wounds that progress from chronic to closed, as well as those that become infected and do not heal.6
Next Generation Sequencing (NGS), or high throughput sequencing, refers to non-Sanger-based, high throughput, DNA sequencing methods.7-10 In NGS, millions or billions of DNA strands can be sequenced in parallel, thereby yielding substantially more throughput, and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing of genomes. Furthermore, NGS can accurately identify all microbes within each sample. Whereas traditional culture methods may successfully culture less than one percent of microbes present in a sample,11 by employing NGS one can detect and identify the species present in the entire microbial community of that sample. Thereby, DNA is extracted from each sample, sequenced, and the sequences matched to a database of over 165,000+ known microbes of bacterial, viral, fungal, and protist species. The use of rapid and accurate bioinformatics provides actionable identification and characterization to species and strain.
What Does The Literature Reveal About TCC And The Microbiome Of DFUs?
In a recent study conducted by our group, we examined the microbiome of a single chronic DFU undergoing weekly treatment with total contact casting (TCC).12 In this case, initial treatment consisted of sharp debridement, followed by weekly application of TCC. NGS swab testing of the ulcer took place at each subsequent cast change visit until achieving wound closure at four weeks. For each NGS swab taken, the distribution of organisms varied from week to week. Prior to the first treatment with TCC, the relative abundance of Staphylococcus aureus was highest, followed by Corynebacterium species and Acinetobacter species. However, as the ulcer progressed towards closure, the microbiome of the ulcer changed, as reflected by a decrease in the overall microbial community abundance present in the wound. In addition, genes coding for resistance to class A beta-lactamases, macrolide-lincosamine-streptogramin B and tetracyclines were detected initially, but following the first treatment with TCC, and thereafter, the resistance gene to tetracyclines was no longer present.12
Upon further review, in a study conducted by Loesche and colleagues, the authors analyzed the temporal dynamics of the DFU microbiota and its association with outcomes using high throughput sequencing of the 16S ribosomal RNA gene, and found a faster healing rate of ulcers with a more dynamic microbiota.13 In addition, Malone and colleagues researched infected DFUs (duration of less than six weeks) with NGS, and reported that Staphylococcus was the dominant species.14 Another study employing metagenomic shotgun sequencing of DFUs demonstrated that strain level variation in an abundance of Staphylococcus aureus and the genetic signatures of biofilm formation were associated with poor outcomes. A significant association between Staphylococcus aureus abundance in wounds and healing time was also noted.15
It has been well documented that the “gold-standard” for offloading DFUs is TCC, which can reduce pressure by up to 84 to 92 percent at the ulcer site, and heals most DFUs within six to eight weeks.16 Bus and coworkers conducted a systematic review and found that non-removable offloading devices (i.e. TCC) are more effective than removable devices at healing neuropathic foot ulcers.17 Yet, in a large multi-center study published by Wu and team among 895 centers involved in the treatment of DFUs across the United States, only 1.7 percent used TCC for the majority of cases.18 The authors noted several contributing factors to the minimal use of TCC, including patient tolerance, time needed to apply and remove the cast, materials cost and reimbursement challenges.18
TCC remains the gold-standard for offloading DFUs, yet little is known about the microbiome of the diabetic foot as it progresses from chronic ulcer to closed, or when infection develops. Our group’s study demonstrated dynamic changes associated with a chronic DFU undergoing treatment with TCC as it progressed to healing. Going forward, further studies are needed to improve our understanding of the composition and significance of the wound microbiome in patients with chronic DFUs and determine the optimal treatment for wound healing.
Dr. Isaac is the Director of Research with Foot & Ankle Specialists of the Mid-Atlantic (FASMA). He is a Diplomate of the American Board of Foot and Ankle Surgery.
Dr. Tritto is the Treasurer for Foot & Ankle Specialists of the Mid-Atlantic (FASMA). He is a Fellow of the American Society of Podiatric Surgeons.
1. Armstrong DG, Boulton AJ, Bus SA. Diabetic foot ulcers and their recurrence. N Engl J Med. 2017;376(24):2367-2375.
2. Driver VR, Fabbi M, Lavery LA, Gibbons G. The costs of diabetic foot: The economic case for the limb salvage team. J Vasc Surg. 2010;52(3):17S-22S.
3. American Diabetes Association. Economic costs of diabetes in the US in 2012. Diabetes Care 2013; 36: 1033–1046. Diabetes Care. 2013;36(6):1797.
4. Armstrong DG, Swerdlow MA, Armstrong AA, Conte MS, Padula WV, Bus SA. Five year mortality and direct costs of care for people with diabetic foot complications are comparable to cancer. J Foot Ankle Res. 2020;13(1):1-4.
5. Barshes NR, Sigireddi M, Wrobel JS, et al. The system of care for the diabetic foot: Objectives, outcomes, and opportunities. Diabet Foot Ankle. 2013;4(1):21847.
6. Gardner SE, Hillis SL, Heilmann K, Segre JA, Grice EA. The neuropathic diabetic foot ulcer microbiome is associated with clinical factors. Diabetes. 2013;623:923-930.
7. Spichler A, Hurwitz BL, Armstrong DG, Lipsky BA. Microbiology of diabetic foot infections: from Louis Pasteur to 'crime scene investigation'. BMC Med. 2015;13:2.
8. Watts GS, Thornton JE Jr, Youens-Clark K, et al. Identification and quantitation of clinically relevant microbes in patient samples: Comparison of three k-mer based classifiers for speed, accuracy, and sensitivity. PLoS Computation Biol. 2019;1511:e1006863.
9. Moffarah AS, Al Mohajer M, Hurwitz BL, Armstrong DG. Skin and soft tissue infections. Microbiol Spectr. 2016;44.
10. Armstrong DG, Lew EJ, Hurwitz B, Wild T. The quest for tissue repair's holy grail: The promise of wound diagnostics or just another fishing expedition? Wound Med. (2015); 8:1-5.
11. Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Ann Rev Microbiol. 1985;391:321-346.
12. Isaac AL, Tritto M, Colwell R, Armstrong DG, Metagenomics of diabetic foot ulcer undergoing treatment with total contact casting. J Wound Care. 2020:In Press.
13. Loesche M, Gardner SE, Kalan L, et al. Temporal stability in chronic wound microbiota is associated with poor healing. J Invest Dermatol. 2017;137(1):237-244.
14. Malone, M. Johani K, Jensen SO, et al. Next generation DNA sequencing of tissues from infected diabetic foot ulcers. EBioMedicine. 2017;21:142-149.
15. Kalan LR, Meisel JS, Loesche MA, et al. Strain-and species-level variation in the microbiome of diabetic wounds is associated with clinical outcomes and therapeutic efficacy. Cell Host Microbe. 2019;25(5):641-655.e5.
16. Lavery LA. Discussion: off-loading the diabetic foot for ulcer prevention and healing. Plast Reconstr Surg. 2011;127(suppl 1):257S-258S.
17. Bus SA, van Deursen RW, Armstrong DG. Footwear and offloading interventions to prevent and heal foot ulcers and reduce plantar pressure in patients with diabetes: a systematic review. Diabetes Metab Res. 2016;32(Suppl 1):99-118.
18. Wu SC, Jensen JL, Weber AK, Robinson DE, Armstrong DG. Use of pressure offloading devices in diabetic foot ulcers: do we practice what we preach? Diabetes Care. 2008;31(11):2118-2119.