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.

Microbial burden of chronic wounds is believed to play an important role in impaired healing and development of infection-related complications. However, clinical cultures have little predictive value of wound outcomes, and culture-independent studies have been limited by cross-sectional design and small cohort size. We systematically evaluated the temporal dynamics of the microbiota colonizing diabetic foot ulcers (DFU), a common and costly complication of diabetes, and its association with healing and clinical complications. Dirichlet multinomial mixture modeling, Markov chain analysis, and mixed-effect models were used to investigate shifts in the microbiota over time and its associations with healing. Here we show to our knowledge previously unreported temporal dynamics of the chronic wound microbiome. Microbiota community instability was associated with faster healing and improved outcomes. DFU microbiota were found to exist in one of four community types that experienced frequent and non-random transitions. Transition patterns and frequencies associated with healing time. Exposure to systemic antibiotics destabilized the wound microbiota, rather than altering overall diversity or relative abundance of specific taxa. This study provides to our knowledge previously unreported evidence that the dynamic wound microbiome is indicative of clinical outcomes and may be a valuable guide for personalized management and treatment of chronic wounds.

It is uncertain how accurately classic signs of acute infection identify infection in chronic wounds, or if the signs of infection specific to secondary wounds are better indicators of infection in these wounds. The purpose of this study was to examine the validity of the "classic" signs (i.e., pain, erythema, edema, heat, and purulence) and the signs specific to secondary wounds (i.e., serous exudate, delayed healing, discoloration of granulation tissue, friable granulation tissue, pocketing at the base of the wound, foul odor, and wound breakdown). Thirty-six chronic wounds were assessed for these signs and symptoms of infection with interobserver reliability ranging from 0.53 to 1.00. The wounds were then quantitatively cultured, and 11 (31%) were found to be infected. Increasing pain, friable granulation tissue, foul odor, and wound breakdown showed validity based on sensitivity, specificity, discriminatory power, and positive predictive values. The signs specific to secondary wounds were better indicators of chronic wound infection than the classic signs with a mean sensitivity of 0.62 and 0.38, respectively. None of the signs or symptoms was a necessary indicator of infection, but increasing pain and wound breakdown were both sufficient indicators with specificity of 100%.

Chronic nonhealing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial biofilms in the wound bed are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic-wound microbiome have almost exclusively focused on bacteria, omitting what we hypothesize are important fungal contributions to impaired healing and the development of complications. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with poor outcomes, and form mixed fungal-bacterial biofilms. We longitudinally profiled 100, nonhealing diabetic-foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The “mycobiome” was highly heterogeneous over time and between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. The proportions of the phylum Ascomycota were significantly greater (P = 0.015) at the beginning of the study in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while taxa identified as allergenic filamentous fungi were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that interkingdom biofilms formed between yeasts and coisolated bacteria. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clinical outcomes.

The incidence of chronic wounds is increased among older adults, and the impact of chronic wounds on quality of life is particularly profound in this population. It is well established that wound healing slows with age. However, the basic biology underlying chronic wounds and the influence of age-associated changes on wound healing are poorly understood. Most studies have used in vitro approaches and various animal models, but observed changes translate poorly to human healing conditions. The impact of age and accompanying multi-morbidity on the effectiveness of existing and emerging treatment approaches for chronic wounds is also unknown, and older adults tend to be excluded from randomized clinical trials. Poorly defined outcomes and variables, lack of standardization in data collection, and variations in the definition, measurement, and treatment of wounds also hamper clinical studies. The Association of Specialty Professors, in conjunction with the National Institute on Aging and the Wound Healing Society, held a workshop, summarized in this paper, to explore the current state of knowledge and research challenges, engage investigators across disciplines, and identify key research questions to guide future study of age-associated changes in chronic wound healing.