Microbiome Characterization of Infected Diabetic Foot Ulcers in Association With Clinical Outcomes: Traditional Cultures Versus Molecular Sequencing Methods
Hadar Mudrik-Zohar, Shaqed Carasso, Tal Gefen, Anat Zalmanovich, Michal Katzir, Yael Cohen, Yossi Paitan, Naama Geva-Zatorsky, Michal Chowers, Hadar Mudrik-Zohar, Shaqed Carasso, Tal Gefen, Anat Zalmanovich, Michal Katzir, Yael Cohen, Yossi Paitan, Naama Geva-Zatorsky, Michal Chowers
Abstract
Background: Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. These potentially limb-threatening ulcers are challenging to treat due to impaired wound healing characterizing diabetic patients and the complex microbial environment of these ulcers.
Aim: To analyze the microbiome of IDFU in association with clinical outcomes.
Methods: Wound biopsies from IDFU were obtained from hospitalized patients and were analyzed using traditional microbiology cultures, 16S rRNA sequencing and metagenomic sequencing. Patients' characteristics, culture-based results and sequencing data were analyzed in association with clinical outcomes.
Results: A total of 31 patients were enrolled. Gram-negative bacteria dominated the IDFU samples (79%, 59% and 54% of metagenomics, 16S rRNA and cultures results, respectively, p<0.001). 16S rRNA and metagenomic sequencing detected significantly more anaerobic bacteria, as compared to conventional cultures (59% and 76%, respectively vs. 26% in cultures, p=0.001). Culture-based results showed that Staphylococcus aureus was more prevalent among patients who were treated conservatively (p=0.048). In metagenomic analysis, the Bacteroides genus was more prevalent among patients who underwent amputation (p<0.001). Analysis of metagenomic-based functional data showed that antibiotic resistance genes and genes related to biofilm production and to bacterial virulent factors were more prevalent in IDFU that resulted in amputation (p<0.001).
Conclusion: Sequencing tools uncover the complex biodiversity of IDFU and emphasize the high prevalence of anaerobes and Gram-negative bacteria in these ulcers. Furthermore, sequencing results highlight possible associations among certain genera, species, and bacterial functional genes to clinical outcomes.
Keywords: 16S rRNA; amputation; diabetic foot ulcer; metagenomics; microbiome.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Copyright © 2022 Mudrik-Zohar, Carasso, Gefen, Zalmanovich, Katzir, Cohen, Paitan, Geva-Zatorsky and Chowers.
Figures
References
- Abdulrazak A., Bitar Z. I., Al-Shamali A. A., Mobasher L. A. (2005). Bacteriological Study of Diabetic Foot Infections. J. Diabetes Its Complicat. 19, 138–141. doi: 10.1016/j.jdiacomp.2004.06.001
- Al Benwan K., Al Mulla A., Rotimi V. O. (2012). A Study of the Microbiology of Diabetic Foot Infections in a Teaching Hospital in Kuwait. J. Infect. Public Health 5, 1–8. doi: 10.1016/j.jiph.2011.07.004
- Armstrong D. G., Lavery L. A., Harkless L. B. (1998). Validation of a Diabetic Wound Classification System. The Contribution of Depth, Infection, and Ischemia to Risk of Amputation. Diabetes Care 21, 855–859. doi: 10.2337/diacare.21.5.855
- Boulton A. J. M., Vileikyte L., Ragnarson-Tennvall G., Apelqvist J. (2005). The Global Burden of Diabetic Foot Disease. Lancet 366, 1719–1724. doi: 10.1016/S0140-6736(05)67698-2
- Charles P. G. P., Uçkay I., Kressmann B., Emonet S., Lipsky B. A. (2015). The Role of Anaerobes in Diabetic Foot Infections. Anaerobe 34, 8. doi: 10.1016/j.anaerobe.2015.03.009
- Citron D. M., Goldstein E. J., Merriam C. V., Lipsky B. A., Abramson M. A. (2007). Bacteriology of Moderate-to-Severe Diabetic Foot Infections and In Vitro Activity of Antimicrobial Agents. J. Clin. Microbiol. 45, 2819–2828. doi: 10.1128/JCM.00551-07
- Dowd S. E., Sun Y., Secor P. R., Rhoads D. D., Wolcott B. M., James G. A., et al. . (2008). Survey of Bacterial Diversity in Chronic Wounds Using Pyrosequencing, DGGE, and Full Ribosome Shotgun Sequencing. BMC Microbiol. 8, 43. doi: 10.1186/1471-2180-8-43
- Dowd S. E., Wolcott R. D., Sun Y., McKeehan T., Smith E., Rhoads D. (2008). Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP). PLoS One 3 (10), e3326. doi: 10.1371/journal.pone.0003326
- Dunyach-Remy C., Ngba Essebe C., Sotto A., Lavigne J. P. (2016). Staphylococcus Aureus Toxins and Diabetic Foot Ulcers: Role in Pathogenesis and Interest in Diagnosis. Toxins (Basel) 8 (7), 209. doi: 10.3390/toxins8070209
- El-Tahawy A. (2000). Bacteriology of Diabetic Foot. Saudi Med. J. 21, 344–347.
- Gardner S. E., Haleem A., Jao Y. L., Hillis S. L., Femino J. E., Phisitkul P., et al. . (2014). Cultures of Diabetic Foot Ulcers Without Clinical Signs of Infection do Not Predict Outcomes. Diabetes Care 37, 10:2693–2701. doi: 10.2337/dc14-0051
- Gardner S. E., Hillis S. L., Heilmann K., Segre J. A., Grice E. A. (2013). The Neuropathic Diabetic Foot Ulcer Microbiome is Associated With Clinical Factors. Diabetes 62, 923. doi: 10.2337/db12-0771
- Hitam S., Hassan S. A., Maning N. (2019). The Significant Association Between Polymicrobial Diabetic Foot Infection and its Severity and Outcomes. Malaysian J. Med. Sci. 2, 1:107–1:114. doi: 10.21315/mjms2019.26.1.10
- Hunt J. A. (1992). Foot Infections in Diabetes are Rarely Due to a Single Microorganism. Diabetes Med. J. Br. Diabetes Assoc. 9, 749–752. doi: 10.1111/j.1464-5491.1992.tb01885
- Kalan L., Meisel J. S., Loesche M. A., Horwinski J., Soaita I., Chen X., et al. . (2018). The Microbial Basis of Impaired Wound Healing: Differential Roles for Pathogens, "Bystanders", and Strain-Level Diversification in Clinical Outcomes. bioRxiv, 427567. doi: 10.1101/427567
- Kalan L. R., Meisel J. S., Loesche M. A., Horwinski J., Soaita L., Chen X., et al. . (2019). Strain and Species Level Variations in the Microbiome of Diabetic Wounds is Associated With Clinical Outcomes and Therapeutic Efficacy. Cell Host Microbe 25, 641–655. doi: 10.1016/j.chom.2019.03.006
- Kallstrom G. (2014). Are Quantitative Bacterial Wound Cultures Useful? J. Clin. Microbiol. 52, 8:2753–8:2756. doi: 10.1128/JCM.00522-14
- Lavery L. A., Armstrong D. G., Harkless L. B. (1996). Classification of Diabetic Foot Wounds. J. Foot Ankle Surg. 35, 528–531. doi: 10.1016/S1067-2516(96)80125-6
- Lavery L. A., Armstrong D. G., Murdoch D. P., Peters E. J. P., Lipsky B. A. (2017). Validation of the Infectious Diseases Society of America's Diabetic Foot Infection Classification System. Clin. Infect. Dis. 44, 562–565. doi: 10.1086/511036
- Lavery L. A., Armstrong D. G., Wunderlich R. P., Mohler M. J., Wendel C. S., Lipsky B. A. (2006). Risk Factors for Foot Infections in Persons With Diabetes Mellitus. Diabetes Care 29, 1288–1293. doi: 10.2337/dc05-2425
- Lipsky B. A., Aragón-Sánchez J., Diggle M., Embil J., Kono S., Lavery L., et al. . (2016). IWGDF Guidance on the Diagnosis and Management of Foot Infections in Persons With Diabetes. Diabetes Metab. Res. Rev. 32, 45–74. doi: 10.1002/dmrr.2699
- Lipsky B. A., Armstrong D. G., Citron D. M., Tice A. D., Morgenstern D. E., Abramson M. A. (2005). Ertapenem Versus Piperacil-Lin/Tazobactam for Diabetic Foot Infections (SIDESTEP): Prospective, Randomised, Controlled, Double-Blinded, Multicentre Trial. Lancet 366, 1695–1703. doi: 10.1016/S0140-6736(05)67694-5
- Lipsky B. A., Berendt A. R., Cornia P. B., Pile J. C., Peters E. J., Armstrong D. G., et al. . (2012). 2012 Infectious Diseases Society of America Clinical Practice Guideline for the Diagnosis and Treatment of Diabetic Foot Infections. Clin. Infect. Dis. 54, e132–e173. doi: 10.1093/cid/cis346
- Lipsky B. A., Berendt A. R., Deery H. G., Embil J. M., Joseph W. S., Karchmer A. W., et al. . (2006). Infectious Diseases Society of America. Diagnosis and Treatment of Diabetic Foot Infections. Plast. Reconstr. Surg. 117, 212S–238S. doi: 10.1097/01.prs.0000222737.09322.77
- Lipsky B. A., Senneville É, Abbas Z. G., et al. . (2020). Guidelines on the Diagnosis and Treatment of Foot Infection in Persons With Diabetes (IWGDF 2019 Update). Diabetes Metab. Res. Rev. 36 (S1), 1–24. doi: 10.1002/dmrr.3280
- Lu Q., Wang J., Wei X., Wang G., Xu Y. (2021). Risk Factors for Major Amputation in Diabetic Foot Ulcer Patients. Diabetes Metab. Syndr. Obes. 14, 2019–2027. doi: 10.2147/DMSO.S307815
- Miyan Z., Fawwad A., Sabir R., Basit A. (2017). Microbiological Pattern of Diabetic Foot Infections at a Tertiary Care Center in a Developing Country. J. Pak Med. Assoc. 67 (5), 665–669.
- Oyibo S. O., Jude E. B., Tarawneh I., Nguyen H. C., Armstrong D. G., Harkless L. B., et al. . (2001). The Effects of Ulcer Size and Site, Patient's Age, Sex and Type and Duration of Diabetes on the Outcome of Diabetic Foot Ulcers. Diabetes Med. 18 (2), 133–138. doi: 10.1046/j.1464-5491.2001.00422.x
- Pellizzer G., Strazzabosco M., Presi S., Furlan F., Lora L., Benedetti P., et al. . (2001). Deep Tissue Biopsy vs. Superficial Swab Culture Monitoring in the Microbiological Assessment of Limb-Threatening Diabetic Foot Infection. Diabet. Med. 18, 822–827. doi: 10.1046/j.1464-5491.2001.00584.x
- Rhoads D. D., Cox S. B., Rees E. J., Sun Y., Wolcott R. D. (2012. a). Clinical Identification of Bacteria in Human Chronic Wound Infections: Culturing vs. 16S Ribosomal DNA Sequencing. BMC Infect. Dis. 12, 321. doi: 10.1186/1471-2334-12-321
- Rhoads D. D., Wolcott R. D., Sun Y., Dowd S. E. (2012. b). Comparison of Culture and Molecular Identification of Bacteria in Chronic Wounds. Int. J. Mol. Sci. 13, 2535–2550. doi: 10.3390/ijms13032535
- Saltoglu N., Ergonul O., Tulek N., Yemisen M., Kadanali A., Karagoz G., et al. . (2018). Influence of Multidrug Resistant Organisms on the Outcome of Diabetic Foot Infection. Int. J. Infect. Dis. 70, 10–14. doi: 10.1016/j.ijid.2018.02.013
- Shin J. Y., Roh S. G., Sharaf B., Lee N. H. (2017). Risk of Major Limb Amputation in Diabetic Foot Ulcer and Accompanying Disease: A Meta-Analysis. J. Plast. Reconstr. Aesthet. Surg. 70, 12:1681–1688. doi: 10.1016/j.bjps.2017.07.015
- Smith K., Collier A., Townsend E. M., O’Donnell L. E., Bal A. M., Butcher J., et al. . (2016). One Step Closer to Understanding the Role of Bacteria in Diabetic Foot Ulcers: Characterizing the Microbiome of Ulcers. BMC Microbiol. 16, 54. doi: 10.1186/s12866-016-0665-z
- Suryaletha K., John J., Radhakrishnan M. P., George S., Thomas S. (2018). Metataxonomic Approach to Decipher the Polymicrobial Burden in Diabetic Foot Ulcer and its Biofilm Mode of Infection. Int. Wound J. 15, 473–481. doi: 10.1111/iwj.12888
- Treece K. A., Macfarlane R. M., Pound N., Game F. L., Jeffcoate W. J. (2004). Validation of a System of Foot Ulcer Classification in Diabetes Mellitus. Diabetes Med. 21, 987–991. doi: 10.1111/j.1464-5491.2004.01275.x
- Van Asten S. A. V., La Fontaine J., Peters E. J. G., Bhavan K., Kim P. J., Lavery L. A. (2016). The Microbiome of Diabetic Foot Osteomyelitis. Eur. J. Clin. Microbiol. Infect. Dis. 35, 293–298. doi: 10.1007/s10096-015-2544-1
- Volmer Thole M., Lobmann R. (2016). Neuropathy and Diabetic Foot Syndrome. Int. J. Mol. Sci. 17, 6. doi: 10.3390/ijms17060917
- Wolcott R. D., Hanson J. D., Rees E. J., Koenig L. D., Phillips C. D., Wolcott R. A. (2016). Analysis of the Chronic Wound Microbiota of 2,963 Patients by 16S rDNA Pyrosequencing. Wound Repair Regener. 24, 163–174. doi: 10.1111/wrr.12370
- Zakrzewski M., Vickery K., Malone M., Hu H. (2020). Metatranscriptomic Analysis Reveals Active Bacterial Communities in Diabetic Foot Infections. Front. Microbiol. 11. doi: 10.3389/fmicb.2020.01688
Source: PubMed