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BackgroundBacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota.Methodology/Principal FindingsWe prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds—approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics.Conclusions/SignificanceThe 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure—reducing some bacteria while selecting for others.

Chronic wounds represent a worldwide problem. For laboratory and clinical research to adequately address this problem, a common language needs to exist. This language should include a system of wound classification, a lexicon of wound descriptors, and a description of the processes that are likely to affect wound healing and would healing end points. The report that follows defines wound, acute wound, chronic wound, healing and forms of healing, wound assessment, wound extent, wound burden, and wound severity. The utility of these definitions is demonstrated as they relate to the healing of a skin wound, but these definitions are broadly applicable to all wounds.

Chronic wounds represent a worldwide problem. For laboratory and clinical research to adequately address this problem, a common language needs to exist. This language should include a system of wound classification, a lexicon of wound descriptors, and a description of the processes that are likely to affect wound healing and would healing end points. The report that follows defines wound, acute wound, chronic wound, healing and forms of healing, wound assessment, wound extent, woundburden, and wound severity. The utility of these definitions is demonstrated as they relate to the healing of a skin wound, but these definitions are broadly applicable to all wounds. (WOUND REP REG 4994 ;2 :965-70

SllmruarySecretory granules of human dermal mast cells contain a chymotrypsin-like serine proteinase called chymase. In this study, we demonstrate that the inactive cytokine, 31 kD interleukin 18 (IL-lfl), can be converted rapidly to an 18 kD biologically active species by human mast cell chymase. The product formed is three amino acids longer at the amino terminus than the mature IL-lfl produced by peripheral blood mononuclear cells and has comparable biological activity. Because chymase is a secretory granule constituent, it is likely to be released into the surrounding tissue when mast cells degranulate. It is also known that non-bone marrow derived cells resident in skin (keratinocytes, fibroblasts) produce but do not process 31 kD IL-I~. In this context, chymase may be a potent activator of locally produced 31 kD IL-lfl. Mast cells lie in close apposition to blood vessels in dermis; therefore, chymase mediated conversion of 31 kD IL-1/~ might be expected to have a critical role in the initiation of the inflammatory response in skin.

Lower-extremity wounds are a major complication of diabetes. Hemoglobin A1c (HbA1c) reflects glycemia over 2–3 months and is the standard measure used to monitor glycemia in diabetic patients, but results from studies have not shown a consistent association of HbA1c with wound healing. We hypothesized that elevated HbA1c would be most associated with poor wound healing. To test this hypothesis we conducted a retrospective cohort study of 183 diabetic individuals treated at the Johns Hopkins Wound Center. Our primary outcome was wound-area healing rate (cm2/day). Calibrated tracings of digital images were used to measure wound area. We estimated coefficients for healing rate using a multiple linear regression model controlling for clustering of wounds within individuals and other common clinic variables. The study population was 45% female and 41% black with mean age of 61 years. Mean HbA1c was 8.0% and there were 2.3 wounds per individual (310 wounds total). Of all measures assessed, only HbA1c was significantly associated with wound-area healing rate. Specifically, for each 1.0% point increase in HbA1c, the daily wound-area healing rate decreased by 0.028 cm2/day (95% CI: 0.003, 0.0054, p=0.027). Our results suggest that glycemia, as assessed by HbA1c, may be an important biomarker in predicting wound healing rate in diabetic patients.

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.

Chronic wounds contain complex polymicrobial communities of sessile organisms that have been underappreciated because of limitations of standard culture techniques. The aim of this work is to combine recently developed next-generation investigative techniques to comprehensively describe the microbial characteristics of chronic wounds. Tissue samples were obtained from 15 patients with chronic wounds presenting to the Johns Hopkins Wound Center. Standard bacteriological cultures demonstrated an average of 3 common bacterial species in wound samples. By contrast, high-throughput pyrosequencing revealed increased bacterial diversity with an average of 17 genera in each wound. Data from microbial community profiling of chronic wounds was compared to published sequenced analyses of bacteria from normal skin. Increased proportions of anaerobes, Gram-negative rods and Gram-positive cocci were found in chronic wounds. In addition, chronic wounds had significantly lower populations of Propionibacterium compared to normal skin. Using epifluorescence microscopy, wound bacteria were visualized in highly organized thick confluent biofilms or as scattered individual bacterial cells. Fluorescent in-situ hybridization allowed for the visualization of Staphylococcus aureus cells in a wound sample. Quorum sensing molecules were measured by bioassay to evaluate signaling patterns amongst bacteria in the wounds. A range of autoinducer-2 activities were detected in the wound samples. Collectively, these data provide new insights into the identity, organization, and behavior of bacteria in chronic wounds. Such information may provide important clues to effective future strategies in wound healing.