Alcaligenes faecalis corrects aberrant matrix metalloproteinase expression to promote reepithelialization of diabetic wounds.

Chronic wounds are a common and costly complication of diabetes, where multifactorial defects contribute to dysregulated skin repair, inflammation, tissue damage, and infection. We previously showed that aspects of the diabetic foot ulcer microbiota were correlated with poor healing outcomes, but many microbial species recovered remain uninvestigated with respect to wound healing. Here, we focused on Alcaligenes faecalis, a Gram-negative bacterium that is frequently recovered from chronic wounds but rarely causes infection. Treatment of diabetic wounds with A. faecalis accelerated healing during early stages. We investigated the underlying mechanisms and found that A. faecalis treatment promotes reepithelialization of diabetic keratinocytes, a process that is necessary for healing but deficient in chronic wounds. Overexpression of matrix metalloproteinases in diabetes contributes to failed epithelialization, and we found that A. faecalis treatment balances this overexpression to allow proper healing. This work uncovers a mechanism of bacterial-driven wound repair and provides a foundation for the development of microbiota-based wound interventions.

The heterogeneous wound microbiome varies with wound care pain, dressing type, and inflammatory gene expression.

Wound dressing changes are essential procedures for wound management. However, ~50% of patients experience severe pain during these procedures despite the availability of analgesic medications, indicating a need for novel therapeutics that address underlying causes of pain. Along with other clinical factors, wound pathogens and inflammatory immune responses have previously been implicated in wound pain. To test whether these factors could contribute to severe pain during wound dressing changes, we conducted an exploratory, cross-sectional analysis of patient-reported pain, inflammatory immune responses, and wound microbiome composition in 445 wounds at the time of a study dressing change. We profiled the bacterial composition of 406 wounds using 16S ribosomal RNA amplicon sequencing and quantified gene expression of 13 inflammatory markers in wound fluid using quantitative real-time polymerase chain reaction (qPCR). Neither inflammatory gene expression nor clinically observed inflammation were associated with severe pain, but Corynebacterium and Streptococcus were of lower relative abundance in wounds of patients reporting severe pain than those reporting little or no pain. Wound microbiome composition differed by wound location, and correlated with six of the inflammatory markers, including complement receptor C5AR1, pro-inflammatory cytokine interleukin (IL)1ß, chemokine IL-8, matrix metalloproteinase MMP2, and the antimicrobial peptide encoding cathelicidin antimicrobial peptide. Interestingly, we found a relationship between the wound microbiome and vacuum-assisted wound closure (VAC). These findings identify preliminary, associative relationships between wound microbiota and host factors which motivate future investigation into the directional relationships between wound care pain, wound closure technologies, and the wound microbiome.

Variable staphyloxanthin production by Staphylococcus aureus drives strain-dependent effects on diabetic wound-healing outcomes.

Strain-level variation in Staphylococcus aureus is a factor that contributes to disease burden and clinical outcomes in skin disorders and chronic wounds. However, the microbial mechanisms that drive these variable host responses are poorly understood. To identify mechanisms underlying strain-specific outcomes, we perform high-throughput phenotyping screens on S. aureus isolates cultured from diabetic foot ulcers. Isolates from non-healing wounds produce more staphyloxanthin, a cell membrane pigment. In murine diabetic wounds, staphyloxanthin-producing isolates delay wound closure significantly compared with staphyloxanthin-deficient isolates. Staphyloxanthin promotes resistance to oxidative stress and enhances bacterial survival in neutrophils. Comparative genomic and transcriptomic analysis of genetically similar clinical isolates with disparate staphyloxanthin phenotypes reveals a mutation in the sigma B operon, resulting in marked differences in stress response gene expression. Our work illustrates a framework to identify traits that underlie strain-level variation in disease burden and suggests more precise targets for therapeutic intervention in S. aureus-positive wounds.

Wound microbiota-mediated correction of matrix metalloproteinase expression promotes re-epithelialization of diabetic wounds.

Chronic wounds are a common and costly complication of diabetes, where multifactorial defects contribute to dysregulated skin repair, inflammation, tissue damage, and infection. We previously showed that aspects of the diabetic foot ulcer microbiota were correlated with poor healing outcomes, but many microbial species recovered remain uninvestigated with respect to wound healing. Here we focused on Alcaligenes faecalis , a Gram-negative bacterium that is frequently recovered from chronic wounds but rarely causes infection. Treatment of diabetic wounds with A. faecalis accelerated healing during early stages. We investigated the underlying mechanisms and found that A. faecalis treatment promotes re-epithelialization of diabetic keratinocytes, a process which is necessary for healing but deficient in chronic wounds. Overexpression of matrix metalloproteinases in diabetes contributes to failed epithelialization, and we found that A. faecalis treatment balances this overexpression to allow proper healing. This work uncovers a mechanism of bacterial-driven wound repair and provides a foundation for the development of microbiota-based wound interventions.

A clinical tool to predict severe pain during wound dressing changes.

ABSTRACT: Dressing changes cause severe pain (ie, 8-10 on a 10-point scale) for approximately one-third (36%) of patients with open skin wounds. No tool exists that allows nurses to predict which patients are likely to experience severe pain during dressing changes. The aim of this study was to develop a clinical tool to predict severe pain during dressing changes using clinically accessible wound and pain predictors and to evaluate the diagnostic validity of this model. Using a cross-sectional design, a one-time study dressing change was conducted by the same wound care nurse on 445 subjects while concurrently measuring patient and wound predictors and pain intensity during the dressing change. Three predictors came out of the study as most useful for a clinical prediction tool: type of dressing, resting wound pain, and expected pain. Algorithms based on these predictors are presented, which can be applied in other settings to predict patients likely to experience severe pain during a dressing change. This is the first study to systematically examine a comprehensive set of wound and patient predictors for their individual and collective associations with pain during dressing changes using precisely defined and rigorously measured study variables. The ability to predict which patients are likely to have severe pain during dressing changes is critically needed so that they can be targeted for preventive pain control strategies.

Preliminary Experience with Conservative Sharp Wound Debridement by Nurses in the Outpatient Management of Diabetic Foot Ulcers: Safety, Efficacy, and Economic Analysis.

Background: Treatment of diabetes costs the United States an estimated $245 billion annually; one-third of which is related to the treatment of diabetic foot ulcers (DFUs). We present a safe, efficacious, and economically prudent model for the outpatient treatment of uncomplicated DFUs. Methods: 77 patients (mean age = 54 years, range 31 to 83) with uncomplicated DFUs prospectively enrolled from September 2008 through February 2012. All patients received an initial sharp debridement by one of two orthopaedic foot and ankle fellowship trained surgeons. Ulcer dressings, offloading devices, and debridement procedures were standardized. Patients were evaluated every two weeks by research nurses who utilized a clinical management algorithm and performed conservative sharp wound debridement (CSWD). Results: Average time to clinical healing was 6.0 weeks. There were no complications of CSWD performed by nurses. The sensitivity for the timely identification of wound deterioration was 100%, specificity = 86.49%, PPV = 68.75% and NPV = 100% with an overall accuracy of 89.58%. The estimated cost savings in this model by having nurses perform CSWD was $223.26 per encounter, which, when extrapolated to national estimates, amounts to $1.56 billion to $2.49 billion in potential annual savings across six to ten-week treatment periods, respectively. Conclusion: CSWD of DFUs by nurses in a vertically integrated multidisciplinary team is a safe, effective, and fiscally responsible clinical practice. This clinical model on a national scale could result in significant healthcare savings. Surgeons and other licensed independent practitioners would have more time for evaluating and treating more complex and operative patients; nurses would be practicing closer to the full extent of their education and training as allowed in most states.Level of Evidence: III.

An integrated model of multimorbidity and symptom science.

BACKGROUND: Prevalence and complexity of persons with multiple chronic conditions (MCC), also known as multimorbidity, are shifting clinical practice from a single disease focus to one considering MCC and symptoms. Although symptoms are intricately bound to concepts inherent in MCC science, symptoms are largely ignored in multimorbidity research and literature. PURPOSE: Introduce an Integrated Model of Multimorbidity and Symptom Science. METHODS: Critical integrative review and synthesis process. FINDINGS: The model comprises three primary domains: 1. Contributing/ Risk Factors; 2. Symptom/Disease/Treatment Interactions; and 3. Patient Outcomes. DISCUSSION: The model highlights the multilevel nature of contributing factors and the recursive interactions among multiple etiologies, conditions, symptoms, therapies, and outcomes.

Human macrophage response to microbial supernatants from diabetic foot ulcers.

Diabetic foot ulcers (DFUs) are a major clinical problem exacerbated by prolonged bacterial infection. Macrophages, the primary innate immune cells, are multifunctional cells that regulate diverse processes throughout multiple phases of wound healing. To better understand the influence of microbial species on macrophage behavior, we cultured primary human monocyte-derived macrophages from four donors for 24 hours in media conditioned by bacteria and fungi (Pseudomonas aeruginosa, Corynebacterium amycolatum, Corynebacterium striatum, Staphylococcus aureus, Staphylococcus simulans, and Candida albicans) isolated from the DFUs of six patients. The effects of these microbe-derived signals on macrophage behavior were assessed by measuring the gene expression of a panel of 25 genes related to macrophage phenotype, angiogenesis, bacterial recognition, and cell survival, as well as secretion of two inflammatory cytokines using NanoString multiplex analysis. Principal component analysis showed that macrophage gene expression and protein secretion were affected by both microbial species as well as human donor. S. simulans and C. albicans caused up-regulation of genes associated with a proinflammatory (M1) phenotype, and P. aeruginosa caused an increase in the secretion of the proinflammatory cytokine and M1 marker tumor necrosis factor-alpha (TNFα). Together, these results suggest that macrophages respond to secreted factors from microbes by up-regulating inflammatory markers, and that the effects are strongly dependent on the monocyte donor. Ultimately, increased understanding of macrophage-microbe interactions will lead to the development of more targeted therapies for DFU healing.

Strain- and Species-Level Variation in the Microbiome of Diabetic Wounds Is Associated with Clinical Outcomes and Therapeutic Efficacy.

Chronic wounds are a major complication of diabetes associated with high morbidity and health care expenditures. To investigate the role of colonizing microbiota in diabetic wound healing, clinical outcomes, and response to interventions, we conducted a longitudinal, prospective study of patients with neuropathic diabetic foot ulcers (DFU). Metagenomic shotgun sequencing revealed that strain-level variation of Staphylococcus aureus and genetic signatures of biofilm formation were associated with poor outcomes. Cultured wound isolates of S. aureus elicited differential phenotypes in mouse models that corresponded with patient outcomes, while wound "bystanders" such as Corynebacterium striatum and Alcaligenes faecalis, typically considered commensals or contaminants, also significantly impacted wound severity and healing. Antibiotic resistance genes were widespread, and debridement, rather than antibiotic treatment, significantly shifted the DFU microbiota in patients with more favorable outcomes. These findings suggest that the DFU microbiota may be a marker for clinical outcomes and response to therapeutic interventions.

Severe pain during wound care procedures: A cross-sectional study protocol.

AIM: The aim of this study is to: (a) develop and evaluate a model to predict severe pain during wound care procedures (WCPs) so that high-risk patients can be targeted for specialized dressings and preventive pain control; and (b) identify biological factors associated with severe pain during WCPs so that novel pain control strategies can be developed. BACKGROUND: Wound care procedures such as dressing changes can cause moderate to severe pain in 74% of patients, with nearly half (36%) of all patients experiencing severe pain (rated as 8-10 on a 10-point numeric rating scale) during dressing change. Additionally, clinicians have little direction with current guidelines regarding pain control during WCPs including the selection of the appropriate advanced wound dressings and the appropriate use of analgesics. DESIGN: This is a cross-sectional study. METHODS: The National Institute of Nursing Research approved and funded the study June of 2015 and the appropriate Institutional Review Board approved all study protocols prior to funding. Study enrolment is underway at the University of Iowa Hospitals and Clinics with a target of 525 participants. Potential participants must be adults (21+ years) and have a nonburn, nondiabetic foot, full-thickness wound. The research team performs a one-time study dressing change on enrolled participants and collects all study data. DISCUSSION: This study will allow the development of a tool for clinicians to use to predict severe pain during WCPs and identify biological factors significantly associated with severe pain during WCPs.

Exploring inappropriate certified nursing assistant glove use in long-term care.

BACKGROUND: Certified Nursing Assistants (CNAs) frequently wear gloves when they care for patients in standard precautions. If CNAs use gloves inappropriately, they may spread pathogens to patients and the environment, potentially leading to health care-associated infections (HAIs). METHODS: Using a descriptive structured observational design, we examined the degree of inappropriate health care personnel glove use in a random sample of 74 CNAs performing toileting and perineal care at 1 long-term care facility. RESULTS: During the 74 patient care events, CNAs wore gloves for 80.2% (1,774/2,213) of the touch points, failing to change gloves at 66.4% (225/339) of glove change points. CNAs changed gloves a median of 2.0 times per patient care event. A median of 1.0 change occurred at a change point. CNAs failed to change their gloves at a glove change point a median of 2.5 times per patient care event. Most (61/74; 82.4%) patient care events had >1 contaminated touch point. Over 44% (782/1,774) of the gloved touch points were defined as contaminated for a median of 8.0 contaminated glove touch points per patient care event. All contaminated touches were with gloved hands (P <.001). CONCLUSIONS: Inappropriate glove use was frequently observed in this study. Contaminated gloves may be a significant cause of cross-contamination of pathogens in health care environments. Future research studies should evaluate strategies to improve glove use to reduce HAIs.

Factors associated with high pain intensity during wound care procedures: A model.

The most common wound care procedures (WPCs) performed on open wounds are dressing changes and wound cleansing. Dressing changes cause moderate to severe pain in 74% of patients, nearly half (36%) of whom experience severe pain (rated as 8-10 on a 10-point numeric rating scale). The purpose of this paper is to propose a model of clinically accessible factors that can be tested in order to develop a clinical tool to identify which patients are likely to experience high intensity pain during nonoperative WCPs, such as dressing changes. Although multiple factors are known to be associated with pain, the factors selected for this model were limited to those that (1) are supported based on evidence and/or pain mechanisms and (2) are readily accessible to clinicians/practitioners and can be tested as a prediction tool to be used prior to WCPs. This model may be helpful to identify those likely to experience high intensity pain during WCPs. In this way, use of aggressive pain management strategies, including specialty dressings, pharmacologic analgesics, and/or non-pharmacological strategies, such as high intensity transcutaneous electrical stimulation.

Temporal Stability in Chronic Wound Microbiota Is Associated With Poor Healing.

Microbial burden of chronic wounds is believed to play an important role in impaired healing and the 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, 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 their 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. Diabetic foot ulcer microbiota were found to exist in one of four community types that experienced frequent and nonrandom transitions. Transition patterns and frequencies were 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 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.

Measuring Weight-Bearing Activities in Patients With Previous Diabetic Foot Ulcers.

PURPOSE: The purpose of this article was to evaluate the accuracy of 2 physical activity monitors, monitors 1 and 2, for measuring weight-bearing activity in persons with prior diabetic foot ulcers. DESIGN: Cross-sectional design. INSTRUMENTS: Two recently developed monitors were used to differentiate anatomical postures such as lying, sitting, and standing upright. One monitor was designed to distinguish between duration of standing and walking and the other combines duration of standing and walking into 1 measure. SUBJECTS AND SETTING: Thirty-one subjects were recruited; all participants had experienced a diabetic foot ulcer and completed participation in a previous cohort study. The study setting was 2 medical centers in the Midwestern United States. METHODS: Subjects simultaneously wore the 2 monitors while performing 14 weight-bearing (ie, walking and standing) and non-weight-bearing (ie, sitting and lying) activities. The duration spent on each activity and the total number of steps taken for each walking activity were directly observed and recorded with each monitor. The accuracy of monitors 1 and 2 was assessed via direct observation as a reference standard. Paired-samples t tests were used to examine the difference in accuracy between the 2 monitors. RESULTS: For measuring duration of activity, the accuracy of monitor 1 ranged from 73% to 100% for walking, 50% for standing, and from 42% to 100% for sitting/lying. In contrast, the accuracy of monitor 2 ranged from 98% to 100% for walking, 100% for standing, and from 97% to 100% for sitting/lying. The accuracy of monitor 1 for counting the number of steps ranged from 43% to 81%, while the accuracy of monitor 2 ranged from 91% to 99%. Monitor 2 was significantly more accurate than monitor 1 in measuring duration of standing still, slow walking, pedaling while sitting, lying on the left, and lying on the right, as well as measuring steps across different kinds of walking activities. Differences in monitor accuracy between subjects with and without foot pain and between subjects with and without foot amputation were not statistically significant. CONCLUSION: These findings suggest that monitor 2 is a more accurate measure of weight-bearing activities than monitor 1 among patients with previous diabetic foot ulcers. Additionally, the 2 monitors differ in terms of function; monitor 2 distinguishes standing from walking, whereas monitor 1 combines standing and walking into 1 measure. We recommend monitor 2 to examine the impact of weight-bearing activity on foot ulceration in patients with diabetic neuropathy.

Redefining the Chronic-Wound Microbiome: Fungal Communities Are Prevalent, Dynamic, and Associated with Delayed Healing.

UNLABELLED: 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. IMPORTANCE: Wounds are an underappreciated but serious complication for a diverse spectrum of diseases. High-risk groups, such as persons with diabetes, have a 25% lifetime risk of developing a wound that can become chronic. The majority of microbiome research related to chronic wounds is focused on bacteria, but the association of fungi with clinical outcomes remains to be elucidated. Here we describe the dynamic fungal communities in 100 diabetic patients with foot ulcers. We found that communities are unstable over time, but at the first clinical presentation, the relative proportions of different phyla predict healing times. Pathogenic fungi not identified by culture reside in necrotic wounds and are associated with a poor prognosis. In wounds stably colonized by fungi, we identified yeasts capable of forming biofilms in concert with bacteria. Our findings illuminate the associations of the fungal mycobiome with wound prognosis and healing.

A Tool to Assess the Signs and Symptoms of Catheter-Associated Urinary Tract Infection: Development and Reliability.

The purpose of this pilot study was to determine the inter-rater reliability of four clinical manifestations of catheter-associated urinary tract infections (CAUTI) among hospitalized adults with short-term indwelling urinary catheters using a tool developed for this purpose: the CAUTI Assessment Profile (CAP). Study participants included 30 non-pregnant English-speaking adults, recruited from two community hospitals. Three nurses assessed each participant for fever, suprapubic tenderness, flank tenderness, and delirium using standardized techniques. Based on the generalized Kappa statistic and 95% confidence intervals, there was evidence of strong inter-rater reliability for fever (K = 1.00, 0.793-1.207), suprapubic tenderness (K = 0.39, 0.185-0.598), and delirium (K = 0.58, 0.379-0.792), but not for flank tenderness (K = 0.29, -0.036 to 0.617). This study provides preliminary evidence that the CAP can be used to consistently identify these clinical signs and symptoms of CAUTI in hospitalized adults.

The healthy newborn hydration model: a new model for understanding newborn hydration immediately after birth.

The normal small volume of breast milk produced in the first 2 days following birth may raise concerns about adequate hydration in breast-fed newborns. These concerns are further magnified when breast-fed infants lose ≥7% of their birth weight within 2 days postnatally. Weight loss following birth is presumably mostly water loss that could result in hypohydration and subsequent hypernatremic dehydration. However, excess fluid loss immediately following birth is a normal and necessary process. Furthermore, newborns exposed to excess fluid intake during labor may need to lose ≥7% of birth weight in the first 2 days following birth in order to achieve euhydration. Normal newborn fluid loss following birth confounds the use of weight loss as the sole measure of newborn hydration. We thus propose the healthy newborn hydration model that highlights the normalcy of newborn weight loss immediately following birth and the healthy newborn's compensatory mechanisms for preserving adequate hydration. We also recommend the use of serum sodium to measure intravascular osmolarity in addition to monitoring weight loss to obtain a more comprehensive newborn hydration assessment. Research is necessary in healthy newborns to identify relationships among fluids received in utero, newborn weight loss, and hydration, as evaluated with laboratory measures, in the first 2 days following birth. This information will guide clinicians in correctly identifying newborns with inadequate hydration who are in need of supplementary fluids versus newborns with adequate hydration for whom exclusive breast-feeding can be supported and encouraged.

The Wound Microbiome: Modern Approaches to Examining the Role of Microorganisms in Impaired Chronic Wound Healing.

Significance: Bacterial burden is believed to play a significant role in impaired wound healing of chronic wounds and the development of infection-related complications. The standard of care in the clinic relies upon cultivation-dependent methods to identify microorganisms. These assays are biased toward microorganisms that thrive in isolation under laboratory conditions. Recent Advances: Significant advances in genomic technologies have enabled less-biased, culture-independent approaches to characterize microbial communities, or microbiomes. The aggregate sequencing and analysis of 16S ribosomal RNA genes has demonstrated that cultures under-represent true microbial diversity and load. Critical Issues: Despite recent advances that enable culture-independent analyses of microbiomes, those organisms that are important in impaired healing remain ambiguous. Inconsistent findings across various studies highlight the need to characterize microbiomes of chronic wounds with homogenous etiology to determine differences in microbiomes that may be driven by the wound environment and that may affect wound outcomes. Rigorous analyses of wound microbiomes in light of the three dimensions of bioburden (microbial diversity, microbial load, and pathogenic organisms), clinical metadata, and wound outcomes will be a significant step forward in our quest to understand the role of microorganisms in impaired healing. Future Directions: Longitudinal studies employing serial sampling are needed to appreciate the role of the dynamic microbial community in chronic wound healing. The value of clinical metadata needs to be examined as potential biomarkers of problematic microbiota and wound outcomes. Lastly, better characterization and understanding of wound microbiomes will open avenues for improved diagnostic and therapeutic tools for the nonhealing wound.

Cultures of diabetic foot ulcers without clinical signs of infection do not predict outcomes.

OBJECTIVE: We examined associations between ulcer bioburden and ulcer outcomes in neuropathic diabetic foot ulcers (DFUs) that lacked clinical signs of infection. RESEARCH DESIGN AND METHODS: Three dimensions of bioburden (i.e., microbial load, microbial diversity, and the presence of likely pathogens) were measured at baseline using swab cultures obtained by Levine's technique. Subjects were assessed every 2 weeks for 26 weeks to determine the rate of healing and development of infection-related complications. Foot ulcers were off-loaded using total-contact casts and routinely debrided. To establish associations between bioburden and rate of healing, Cox proportional hazards and least squares regression were used after adjusting for ulcer depth, surface area, and duration. RESULTS: A total of 77 subjects completed the study. Sixty-five (84.4%) had ulcers that healed during follow-up; weeks-to-closure ranged from 2 to 26 (median 4.0). Mean (± SD) percent reduction in surface area/week was 25.0% (± 23.33). Five (6.5%) of the DFUs developed an infection-related complication. None of the bioburden dimensions (i.e., microbial load, microbial diversity, or presence of likely pathogens) was significantly associated with weeks-to-closure or percent reduction in surface area per week. Weeks-to-closure was best predicted by ulcer duration, depth, and surface area (c-statistic = 0.75). CONCLUSIONS: Culturing DFUs that showed no clinical signs of infection had no predictive value for outcomes of DFUs managed with total-contact casts and routine debridement. These findings support recommendations of the Infectious Disease Society of America that culturing and antibiotics should be avoided in treating DFUs that show no clinical signs of infection.