Creager MA, Matsushita K, Arya S, Beckman JA, Duval S, Goodney PP, et al. Reducing nontraumatic lower-extremity amputations by 20% by 2030: time to get to our feet: a policy statement from the American Heart Association. Circulation. 2021;143(17):e875–91. https://doi.org/10.1161/cir.0000000000000967.

Article  PubMed  Google Scholar 

Prompers L, Huijberts M, Apelqvist J, Jude E, Piaggesi A, Bakker K, et al. High prevalence of ischaemia, infection and serious comorbidity in patients with diabetic foot disease in Europe. Baseline results from the Eurodiale study. Diabetologia. 2007;50(1):18–25. https://doi.org/10.1007/s00125-006-0491-1.

Centers for Disease Control and Prevention. National Diabetes Statistics Report. https://www.cdc.gov/diabetes/data/statistics-report/index.html. Accessed 16 Aug 2023.

Goodney PP, Tarulli M, Faerber AE, Schanzer A, Zwolak RM. Fifteen-year trends in lower limb amputation, revascularization, and preventive measures among medicare patients. JAMA Surg. 2015;150(1):84–6. https://doi.org/10.1001/jamasurg.2014.1007.

Article  PubMed  PubMed Central  Google Scholar 

Geiss LS, Li Y, Hora I, Albright A, Rolka D, Gregg EW. Resurgence of diabetes-related nontraumatic lower-extremity amputation in the young and middle-aged adult U.S. Population. Diabetes care. 2019;42(1):50–4. https://doi.org/10.2337/dc18-1380.

• Lipsky BA, Senneville É, Abbas ZG, Aragón-Sánchez J, Diggle M, Embil JM, et al. Guidelines on the diagnosis and treatment of foot infection in persons with diabetes (IWGDF 2019 update). Diabetes/metabolism research and reviews. 2020;36 Suppl 1:e3280. https://doi.org/10.1002/dmrr.3280. We structured this review around the six concerns about using molecular techniques in identifying infections from these guidelines.

Price LB, Liu CM, Melendez JH, Frankel YM, Engelthaler D, Aziz M, et al. Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota. PLoS ONE. 2009;4(7): e6462. https://doi.org/10.1371/journal.pone.0006462.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Schmidt BM. Emerging diabetic foot ulcer microbiome analysis using cutting edge technologies. J Diabetes Sci Technol. 2022;16(2):353–63. https://doi.org/10.1177/1932296821990097.

Article  PubMed  Google Scholar 

Mudrik-Zohar H, Carasso S, Gefen T, Zalmanovich A, Katzir M, Cohen Y, et al. Microbiome characterization of infected diabetic foot ulcers in association with clinical outcomes: traditional cultures versus molecular sequencing methods. Front Cell Infect Microbiol. 2022;12: 836699. https://doi.org/10.3389/fcimb.2022.836699.

Article  CAS  PubMed  PubMed Central  Google Scholar 

• Kalan LR, Meisel JS, Loesche MA, Horwinski J, Soaita I, Chen X, 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):641–55.e5. https://doi.org/10.1016/j.chom.2019.03.006. Kalan et al. used both 16S rRNA amplicon-based sequencing and metagenomics to identify strain-level taxa and functional genetic pathways.

Heravi FS, Zakrzewski M, Vickery K, Malone M, Hu H. Metatranscriptomic analysis reveals active bacterial communities in diabetic foot infections. Front Microbiol. 2020;11:1688. https://doi.org/10.3389/fmicb.2020.01688.

Article  PubMed  PubMed Central  Google Scholar 

Radzieta M, Peters TJ, Dickson HG, Cowin AJ, Lavery LA, Schwarzer S, et al. A metatranscriptomic approach to explore longitudinal tissue specimens from non-healing diabetes related foot ulcers. APMIS. 2022;130(7):383–96. https://doi.org/10.1111/apm.13226.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Loesche M, Gardner SE, Kalan L, Horwinski J, Zheng Q, Hodkinson BP, et al. Temporal stability in chronic wound microbiota is associated with poor healing. J Invest Dermatol. 2017;137(1):237–44. https://doi.org/10.1016/j.jid.2016.08.009.

Article  CAS  PubMed  Google Scholar 

Moon J, Kim N, Lee HS, Lee ST, Jung KH, Park KI, et al. Nanopore 16S amplicon sequencing enhances the understanding of pathogens in medically intractable diabetic foot infections. Diabetes. 2021;70(6):1357–71. https://doi.org/10.2337/db20-0907.

Article  CAS  PubMed  Google Scholar 

Kalan L, Loesche M, Hodkinson BP, Heilmann K, Ruthel G, Gardner SE, et al. Redefining the chronic-wound microbiome: fungal communities are prevalent, dynamic, and associated with delayed healing. mBio. 2016;7(5). https://doi.org/10.1128/mBio.01058-16.

Travis J, Malone M, Hu H, Baten A, Johani K, Huygens F, et al. The microbiome of diabetic foot ulcers: a comparison of swab and tissue biopsy wound sampling techniques using 16S rRNA gene sequencing. BMC Microbiol. 2020;20(1):163. https://doi.org/10.1186/s12866-020-01843-2.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Suryaletha K, John J, Radhakrishnan MP, George S, Thomas S. Metataxonomic approach to decipher the polymicrobial burden in diabetic foot ulcer and its biofilm mode of infection. Int Wound J. 2018;15(3):473–81. https://doi.org/10.1111/iwj.12888.

Article  PubMed  PubMed Central  Google Scholar 

van Asten SAV, La Fontaine J, Peters EKG, Bhavan K, Kim PJ, Lavery LA. The microbiome of diabetic foot osteomyelitis. Eur J Clin Microbiol Infect Dis. 2016;35(2):293–8. https://doi.org/10.1007/s10096-015-2544-1.

Article  CAS  PubMed  Google Scholar 

• Radzieta M, Sadeghpour-Heravi F, Peters TJ, Hu H, Vickery K, Jeffries T, et al. A multiomics approach to identify host-microbe alterations associated with infection severity in diabetic foot infections: a pilot study. NPJ biofilms and microbiomes. 2021;7(1):29. https://doi.org/10.1038/s41522-021-00202-x. Radzieta et al. used both metagenomics and metatranscriptomics to identify microbe community composition and determine relative activity of these communities in diabetic foot infections.

Zou M, Cai Y, Hu P, Cao Y, Luo X, Fan X, et al. Analysis of the composition and functions of the microbiome in diabetic foot osteomyelitis based on 16S rRNA and metagenome sequencing technology. Diabetes. 2020;69(11):2423–39. https://doi.org/10.2337/db20-0503.

Article  CAS  PubMed  Google Scholar 

Gontcharova V, Youn E, Sun Y, Wolcott RD, Dowd SE. A comparison of bacterial composition in diabetic ulcers and contralateral intact skin. The open microbiology journal. 2010;4:8–19. https://doi.org/10.2174/1874285801004010008.

Article  PubMed  PubMed Central  Google Scholar 

Gardiner M, Vicaretti M, Sparks J, Bansal S, Bush S, Liu M, et al. A longitudinal study of the diabetic skin and wound microbiome. PeerJ. 2017;5: e3543. https://doi.org/10.7717/peerj.3543.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Smith K, Collier A, Townsend EM, O’Donnell LE, Bal AM, Butcher J, et al. One step closer to understanding the role of bacteria in diabetic foot ulcers: characterising the microbiome of ulcers. BMC Microbiol. 2016;16:54. https://doi.org/10.1186/s12866-016-0665-z.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Jnana A, Muthuraman V, Varghese VK, Chakrabarty S, Murali TS, Ramachandra L, et al. Microbial community distribution and core microbiome in successive wound grades of individuals with diabetic foot ulcers. Applied and environmental microbiology. 2020;86(6). https://doi.org/10.1128/aem.02608-19.

Diabetic Foot Consortium. https://diabeticfootconsortium.org/. Accessed 13 Aug 2023.

Johani K, Malone M, Jensen S, Gosbell I, Dickson H, Hu H, et al. Microscopy visualisation confirms multi-species biofilms are ubiquitous in diabetic foot ulcers. Int Wound J. 2017;14(6):1160–9. https://doi.org/10.1111/iwj.12777.

Article  PubMed  PubMed Central  Google Scholar 

Liu F, Lu H, Dong B, Huang X, Cheng H, Qu R, et al. Systematic evaluation of the viable microbiome in the human oral and gut samples with spike-in gram+/- bacteria. mSystems. 2023;8(2):e0073822. https://doi.org/10.1128/msystems.00738-22.

Cheong JZA, Irvine JM, Roesemann S, Nora A, Morgan CE, Daniele C, et al. Ankle brachial indices and anaerobes: is peripheral arterial disease associated with anaerobic bacteria in diabetic foot ulcers? Therapeutic advances in endocrinology and metabolism. 2022;13:20420188221118748. https://doi.org/10.1177/20420188221118747.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Tudela H, Claus SP, Saleh M. Next generation microbiome research: identification of keystone species in the metabolic regulation of host-gut microbiota interplay. Frontiers in cell and developmental biology. 2021;9:719072. https://doi.org/10.3389/fcell.2021.719072.

Article  PubMed  PubMed Central  Google Scholar 

James GA, Swogger E, Wolcott R, Pulcini E, Secor P, Sestrich J, et al. Biofilms in chronic wounds. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2008;16(1):37–44. https://doi.org/10.1111/j.1524-475X.2007.00321.x.

Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001;45(4):999–1007. https://doi.org/10.1128/aac.45.4.999-1007.2001.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Pouget C, Pantel A, Dunyach-Remy C, Magnan C, Sotto A, Lavigne JP. Antimicrobial activity of antibiotics on biofilm formed by Staphylococcus aureus and Pseudomonas aeruginosa in an open microfluidic model mimicking the diabetic foot environment. J Antimicrob Chemother. 2023;78(2):540–5. https://doi.org/10.1093/jac/dkac438.

Article  CAS  PubMed  Google Scholar 

von Woedtke T, Kramer A. The limits of sterility assurance. GMS Krankenhaushygiene interdisziplinar. 2008;3(3):Doc19.

Min KR, Galvis A, Baquerizo Nole KL, Sinha R, Clarke J, Kirsner RS, et al. Association between baseline abundance of Peptoniphilus, a Gram-positive anaerobic coccus, and wound healing outcomes of DFUs. PLoS ONE. 2020;15(1): e0227006. https://doi.org/10.1371/journal.pone.0227006.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Hicks CW, Canner JK, Karagozlu H, Mathioudakis N, Sherman RL, Black JH 3rd, et al. Quantifying the costs and profitability of care for diabetic foot ulcers treated in a multidisciplinary setting. J Vasc Surg. 2019;70(1):233–40. https://doi.org/10.1016/j.jvs.2018.10.097.

Article  PubMed  Google Scholar 

Alamri AM, Alkhilaiwi FA, Ullah KN. Era of molecular diagnostics techniques before and after the COVID-19 pandemic. Curr Issues Mol Biol. 2022;44(10):4769–89. https://doi.org/10.3390/cimb44100325.

Article  CAS  PubMed  PubMed Central  Google Scholar 

White EK, Uberoi A, Pan JT-C, Ort JT, Campbell AE, Murga-Garrido SM, et al. Wound microbiota-mediated correction of matrix metalloproteinase expression promotes re-epithelialization of diabetic wounds. bioRxiv. 2023:2023.06.30.547263. https://doi.org/10.1101/2023.06.30.547263.

Kim JH, Ruegger PR, Lebig EG, VanSchalkwyk S, Jeske DR, Hsiao A, et al. High levels of oxidative stress create a microenvironment that significantly decreases the diversity of the microbiota in diabetic chronic wounds and promotes biofilm formation. Front Cell Infect Microbiol. 2020;10:259. https://doi.org/10.3389/fcimb.2020.00259.

Article  CAS  PubMed  PubMed Central  Google Scholar 

Biswas L, Götz F. Molecular mechanisms of Staphylococcus and Pseudomonas interactions in cystic fibrosis. Front Cell Infect Microbiol. 2021;11: 824042. https://doi.org/10.3389/fcimb.2021.824042.

Article  CAS  PubMed  Google Scholar 

Lee J, Mashayamombe M, Walsh TP, Kuang BKP, Pena GN, Vreugde S, et al. The bacteriology of diabetic foot ulcers and infections and incidence of Staphylococcus aureus Small Colony Variants. J Med Microbiol. 2023;72(6). https://doi.org/10.1099/jmm.0.001716.

Leal SM Jr, Jones M, Gilligan PH. Clinical significance of commensal gram-positive rods routinely isolated from patient samples. J Clin Microbiol. 2016;54(12):2928–36. https://doi.org/10.1128/jcm.01393-16.