[Show abstract][Hide abstract] ABSTRACT: Infection with
leads to impairment of healing and many deaths in severe burn patients. The phenotypic diversity of
strains makes it difficult to define a therapeutic strategy. Here we report the genome sequence of a highly virulent strain of
, VA-134, isolated from a burn patient.
Preview · Article · Feb 2016 · Genome Announcements
[Show abstract][Hide abstract] ABSTRACT: The mechanisms by which the epidermis responds to disturbances in barrier function and restores homeostasis are unknown. With a perturbation of the epidermal barrier, water is lost, resulting in an increase in extracellular sodium concentration. We demonstrate that the sodium channel Nax functions as a sodium sensor. With increased extracellular sodium, Nax up-regulates prostasin, which results in activation of the sodium channel ENaC, resulting in increased sodium flux and increased downstream mRNA synthesis of inflammatory mediators. Nax is present in multiple epithelial tissues, and up-regulation of its downstream genes is found in hypertrophic scars. In animal models, blocking Nax expression results in improvement in scarring and atopic dermatitis-like symptoms, both of which are pathological conditions characterized by perturbations in barrier function. These findings support an important role for Nax in maintaining epithelial homeostasis.
No preview · Article · Nov 2015 · Science translational medicine
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Cutaneous allograft is commonly used in the early coverage of excised burns when autograft is unavailable. However, allograft is also applied in intermediate-extent burns (25-50%), during cases in which it is possible to autograft. In this population, there is a paucity of data on the indications for allograft use. This study explores the indications for allograft usage in moderate size burns.
Under an IRB-approved protocol, patients admitted to our burn unit between March 2003 and December 2010 were identified through a review of the burn registry. Data on allograft use, total burn surface area, operation performed, operative intent, number of operations, intensive care unit length of stay, and overall length of stay were collected and analyzed. Data are presented as means±standard deviations, except where noted.
In the study period, 146 patients received allograft during their acute hospitalization. Twenty-five percent of allograft recipients sustained intermediate-extent burns. Patients with intermediate-extent burns received allograft later in their hospitalization than those with large-extent (50-75% TBSA) burns (6.8 days vs. 3.4 days, p=0.01). Allografted patients with intermediate-extent burns underwent more operations (10.8 vs. 6.1, p=0.002) and had longer hospitalizations (78.3 days vs. 40.9 days, p<0.001) than non-allografted patients, when controlled for TBSA. Clinical rationale for placement of allograft in this population included autograft failure, uncertain depth of excision, lack of autograft donor site, and wound complexity. When uncertain depth of excision was the indication, allograft was universally applied onto the face. In half of allografted intermediate-extent burn patients the inability to identify a viable recipient bed was the ultimate reason for allograft use.
Unlike large body surface area burns, allograft skin use in intermediate-extent injury occurs later in the hospitalization and is driven by the inability to determine wound bed suitability for autograft application. Allograft application can be utilized to test recipient site viability in cases of autograft failure or uncertain depth of excision.
No preview · Article · Oct 2015 · Burns: journal of the International Society for Burn Injuries
[Show abstract][Hide abstract] ABSTRACT: Texture, color, and durability are important characteristics to consider for skin replacement in conspicuous and/or mobile regions of the body such as the face, neck, and hands. Although autograft thickness is a known determinant of skin quality, few studies have correlated the subjective and objective characters of skin graft healing with their associated morphologic and cellular profiles. Defining these relationships may help guide development and evaluation of future skin replacement strategies.
Six-centimeter-diameter full-thickness wounds were created on the back of female Yorkshire pigs and covered by autografts of variable thicknesses. Skin quality was assessed on day 120 using an observer scar assessment score and objective determinations for scar contraction, erythema, pigmentation, and surface irregularities. Histological, histochemical, and immunohistochemical assessments were performed.
Thick grafts demonstrated lower observer scar assessment score (better quality) and decreased erythema, pigmentation, and surface irregularities. Histologically, thin grafts resulted in scar-like collagen proliferation while thick grafts preserves the dermal architecture. Increased vascularity and prolonged and increased cellular infiltration were observed among thin grafts. In addition, thin grafts contained predominately dense collagen fibers, whereas thick grafts had loosely arranged collagen. α-Smooth muscle actin staining for myofibroblasts was observed earlier and persisted longer among thinner grafts.
Graft thickness is an important determinant of skin quality. High-quality skin replacements are associated with preserved collagen architecture, decreased neovascularization, and decreased inflammatory cellular infiltration. This model, using autologous skin as a metric of quality, may give a more informative analysis of emerging skin replacement strategies.
[Show abstract][Hide abstract] ABSTRACT: Objective: The contemporary treatment of a full-thickness burn consists of early eschar excision followed by immediate closure of the open wound using autologous skin. However, most animal models study burn wound healing with the persistence of the burn eschar. Our goal is to characterize a murine model of burn eschar excision to study wound closure kinetics. Approach: C57BL/6 male mice were divided into three groups: contact burn, scald burn, or unburned control. Mice were burned at 80°C for 5, 10, or 20 s. After 2 days, the eschar was excised and wound closure was documented until postexcision day 13. Biopsies were examined for structural morphology and α-smooth muscle actin. In a subsequent interval-excision experiment (80°C scald for 10 s), the burn eschar was excised after 5 or 10 days postburn to determine the effect of a prolonged inflammatory focus. Results: Histology of both contact and scald burns revealed characteristics of a full-thickness injury marked by collagen coagulation and tissue necrosis. Excision at 2 days after a 20-s burn from either scald or contact showed significant delay in wound closure. Interval excision of the eschar, 5 or 10 days postburn, also showed significant delay in wound closure. Both interval-excision groups showed prolonged inflammation and increased myofibroblasts. Innovation and Conclusions: We have described the kinetics of wound closure in a murine model of a full-thickness burn excision. Both contact and scald full-thickness burn resulted in significantly delayed wound closure. In addition, prolonged interval-excision of the eschar appeared to increase and prolong inflammation.
[Show abstract][Hide abstract] ABSTRACT: Klebsiella pneumoniae is an important infectious agent of surgical sites and combat wounds. Antibiotic resistance and tolerance are common impediments
to the healing of chronic infections. Here, we report the genome sequence of a highly multidrug-resistant strain of K. pneumoniae, BAMC 07-18, isolated from a combat wound of a soldier.
Full-text · Article · Nov 2014 · Genome Announcements
[Show abstract][Hide abstract] ABSTRACT: The importance of bacterial biofilms to chronic wound pathogenesis is well-established. Different treatment modalities, including topical dressings, have yet to demonstrate consistent efficacy against wound biofilm. This study evaluates the impact of a novel, anti-microbial Test Dressing on Pseudomonas aeruginosa biofilm-infected wounds. Six-mm dermal punch wounds in rabbit ears were inoculated with 106 colony-forming units of P. aeruginosa. Biofilm was established in vivo using our published model. Dressings changes were performed every other day with either Active Control or Test Dressings. Treated and untreated wounds were harvested for several quantitative endpoints. Confirmatory studies were performed to measure treatment impact on in vitro P. aeruginosa and in vivo polybacterial wounds containing P. aeruginosa and Staphylococcus aureus. The Test Dressing consistently decreased P. aeruginosa bacterial counts and improved wound healing relative to Inactive Vehicle and Active Control wounds (p<0.05). In vitro bacterial counts were also significantly reduced following Test Dressing therapy (p<0.05). Similarly, improvements in bacterial burden and wound healing were also achieved in polybacterial wounds (p<0.05). This study represents the first quantifiable and consistent in vivo evidence of a topical antimicrobial dressing's impact against established wound biofilm. Development of clinically applicable therapies against biofilm such as this is critical to improving chronic wound care.
No preview · Article · Sep 2014 · Wound Repair and Regeneration
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.
No preview · Article · Jul 2014 · Cell and Tissue Research
[Show abstract][Hide abstract] ABSTRACT: Background
Bacterial infections of wounds impair healing and worsen scarring. We hypothesized that transcriptome analysis of wounds infected with Klebsiella pneumoniae (K.p.) or Pseudomonas aeruginosa (P.a.) would indicate host-responses associated with the worse healing of P.a.- than K.p.-infected wounds.
Wounds created on post-operative day (POD) 0 were infected during the inflammatory phase of healing on POD3 and were harvested on POD4 for microarray and transcriptome analysis. Other wounds received topical antibiotic after infection for 24 hours to promote biofilm development, and were harvested on POD6 or POD12.
Wounds infected for 24 hours, relative to uninfected wounds, elevated transcripts of immune-response functions characteristic of infiltrating leukocytes. But P.a.-infected wounds elevated many more transcripts and to higher levels than K.p.-infected wounds. Coincidently, suppressed transcripts of both wounds enriched into stress-response pathways, including EIF2 signaling; however, this was more extensive for P.a.-infected wounds, including many-fold more transcripts enriching in the ‘cell death’ annotation, suggesting resident cutaneous cell toxicity in response to a more damaging P.a. inflammatory milieu. The POD6 wounds were colonized with biofilm but expressed magnitudes fewer immune-response transcripts with no stress-response enrichments. However, elevated transcripts of P.a.-infected wounds were inferred to be regulated by type I interferons, similar to a network unique to P.a.-infected wounds on POD4. On POD12, transcripts that were more elevated in K.p.-infected wounds suggested healing, while transcripts more elevated in P.a.-infected wounds indicated inflammation.
An extensive inflammatory response of wounds was evident from upregulated transcripts 24 hours after infection with either bacterium, but the response was more intense for P.a.- than K.p.-infected wounds. Coincidently, more extensive down-regulated transcripts of P.a.-infected wounds indicated a stronger “integrated stress response” to the inflammatory milieu that tipped more toward cutaneous cell death. Unique to P.a.-infected wounds on POD4 and POD6 were networks inferred to be regulated by interferons, which may result from intracellular replication of P.a. These data point to specific downregulated transcripts of cells resident to the wound as well as upregulated transcripts characteristic of infiltrating leukocytes that could be useful markers of poorly healing wounds and indicators of wound-specific treatments for improving outcomes.
Full-text · Article · May 2014 · BMC Clinical Pathology
[Show abstract][Hide abstract] ABSTRACT: The medical importance of bacterial biofilms has increased with the recognition of biofilms as one of the major contributors to the slow or non-healing chronic wounds such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers. Being a protected community of microorganisms, biofilms are notoriously refractory to antibiotic treatments. As the conventional treatment modalities have proven ineffective, this study provides the in vitro evidence to support the use of a novel combination of DispersinB(®) antibiofilm enzyme that inhibits biofilm formation and disperses preformed biofilm, and thus making the biofilm bacteria more susceptible to a broad-spectrum KSL-W antimicrobial peptide. The combination of DispersinB(®) and KSL-W peptide showed synergistic antibiofilm and antimicrobial activity against chronic wound infection associated biofilm-embedded bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Coagulase-negative Staphylococci (CoNS), and Acinetobacter baumannii. In addition, the wound gel formulation comprising DispersinB(®), KSL-W peptide, and a gelling agent Pluronic F-127 showed a broad-spectrum and enduring antimicrobial activity against test organisms. Furthermore, as compared to commercial wound gel Silver-Sept™, DispersinB(®)-KSL-W peptide-based wound gel was significantly more effective in inhibiting the biofilm-embedded MRSA, S. epidermidis, CoNS, Vancomycin-resistant Enterococci, A. baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa (P < 0.05). Thus, this study provides promising evidence for the potential application of antibiofilm-antimicrobial DispersinB(®)-KSL-W wound gel in chronic wound management.
No preview · Article · Jan 2014 · Current Microbiology
[Show abstract][Hide abstract] ABSTRACT: Encapsulated Klebsiella pneumoniae has emerged as one of the most clinically relevant and more frequently encountered opportunistic pathogens in combat wounds
as the result of nosocomial infection. In this report, we show that imipenem displayed potent activity against established
K. pneumoniae biofilms under both static and flow conditions in vitro. Using a rabbit ear model, we also demonstrated that imipenem was highly effective against preformed K. pneumoniae biofilms in wounds.
Preview · Article · Nov 2013 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial peptides have been the focus of much research over the last decade because of their effectiveness and broad-spectrum activity against microbial pathogens. These peptides also participate in inflammation and the innate host defense system by modulating the immune function that promotes immune cell adhesion and migration as well as the respiratory burst, which makes them even more attractive as therapeutic agents. This has led to the synthesis of various antimicrobial peptides, including KSL-W (KKVVFWVKFK-NH2), for potential clinical use. Because this peptide displays antimicrobial activity against bacteria, we sought to determine its antifungal effect on C. albicans. Growth, hyphal form, biofilm formation, and degradation were thus examined along with EFG1, NRG1, EAP1, HWP1, and SAP 2-4-5-6 gene expression by quantitative RT-PCR.
This study demonstrates that KSL-W markedly reduced C. albicans growth at both early and late incubation times. The significant effect of KSL-W on C. albicans growth was observed beginning at 10 mug/ml after 5 h of contact by reducing C. albicans transition and at 25 mug/ml by completely inhibiting C. albicans transition. Cultured C. albicans under biofilm-inducing conditions revealed that both KSL-W and amphotericin B significantly decreased biofilm formation at 2, 4, and 6 days of culture. KSL-W also disrupted mature C. albicans biofilms. The effect of KSL-W on C. albicans growth, transition, and biofilm formation/disruption may thus occur through gene modulation, as the expression of various genes involved in C. albicans growth, transition and biofilm formation were all downregulated when C. albicans was treated with KSL-W. The effect was greater when C. albicans was cultured under hyphae-inducing conditions.
These data provide new insight into the efficacy of KSL-W against C. albicans and its potential use as an antifungal therapy.
[Show abstract][Hide abstract] ABSTRACT: Diabetic patients exhibit dysregulated inflammatory and immune responses that predispose them to chronic wound infections and the threat of limb loss. The molecular underpinnings responsible for this have not been well elucidated, particularly in the setting of wound biofilms. This study evaluates host responses in biofilm-impaired wounds using the TallyHo mouse, a clinically relevant polygenic model of type 2 diabetes. No differences in cytokine or Toll-like receptor (TLR) expression were noted in unwounded skin or noninoculated wounds of diabetic and wild-type mice. However, diabetic biofilm-containing wounds had significantly less TLR 2, TLR 4, interleukin-1β, and tumor necrosis factor-α expression than wild-type wounds with biofilm (all p < 0.001). Both groups had similar bacterial burden and neutrophil infiltration after development of biofilms at 3 days postwounding, but diabetic wounds had significantly less neutrophil oxidative burst activity. This translated into a log-fold greater bacterial burden and significant delay of wound epithelization for biofilm-impaired diabetic wounds at 10 days postwounding. These results suggest that impaired recognition of bacterial infection via the TLR pathway leading to inadequate cytokine stimulation of antimicrobial host responses may represent a potential mechanism underlying diabetic susceptibility to wound infection and ulceration.
No preview · Article · Oct 2013 · Wound Repair and Regeneration
[Show abstract][Hide abstract] ABSTRACT: Biofilm formation and persistence are essential components for the continued survival of pathogens inside the host and constitute
a major contributor to the development of chronic wounds with resistance to antimicrobial compounds. Understanding these processes
is crucial for control of biofilm-mediated disease. Though chronic wound infections are often polymicrobial in nature, much
of the research on chronic wound-related microbes has focused on single-species models. Klebsiella pneumoniae and Pseudomonas aeruginosa are microbes that are often found together in wound isolates and are able to form stable in vitro biofilms, despite the antagonistic nature of P. aeruginosa with other organisms. Mutants of the K. pneumoniae strain IA565 lacking the plasmid-borne mrkD1P gene were less competitive than the wild type in an in vitro dual-species biofilm model with P. aeruginosa (PAO1). PAO1 spent medium inhibited the formation of biofilm of mrkD1P-deficient mutants and disrupted preestablished biofilms, with no effect on IA565 and no effect on the growth of the wild
type or mutants. A screen using a two-allele PAO1 transposon library identified the LasB elastase as the secreted effector
involved in biofilm disruption, and a purified version of the protein produced results similar to those with PAO1 spent medium.
Various other proteases had a similar effect, suggesting that the disruption of the mrkD1P gene causes sensitivity to general proteolytic effects and indicating a role for MrkD1P in protection against host antibiofilm effectors. Our results suggest that MrkD1P allows for competition of K. pneumoniae with P. aeruginosa in a mixed-species biofilm and provides defense against microbial and host-derived proteases.
Full-text · Article · Aug 2013 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Micro- and nanoscale technologies have radically transformed biological research from genomics to tissue engineering, with the relative exception of microbial cell culture, which is still largely performed in microtiter plates and petri dishes. Here, we present nanoscale culture of the opportunistic fungal pathogen Candida albicans on a microarray platform. The microarray consists of 1,200 individual cultures of 30 nl of C. albicans biofilms ("nano-biofilms") encapsulated in an inert alginate matrix. We demonstrate that these nano-biofilms are similar to conventional macroscopic biofilms in their morphological, architectural, growth, and phenotypic characteristics. We also demonstrate that the nano-biofilm microarray is a robust and efficient tool for accelerating the drug discovery process: (i) combinatorial screening against a collection of 28 antifungal compounds in the presence of immunosuppressant FK506 (tacrolimus) identified six drugs that showed synergistic antifungal activity, and (ii) screening against the NCI challenge set small-molecule library identified three heretofore-unknown hits. This cell-based microarray platform allows for miniaturization of microbial cell culture and is fully compatible with other high-throughput screening technologies.
Microorganisms are typically still grown in petri dishes, test tubes, and Erlenmeyer flasks in spite of the latest advances in miniaturization that have benefitted other allied research fields, including genomics and proteomics. Culturing microorganisms in small scale can be particularly valuable in cutting down time, cost, and reagent usage. This paper describes the development, characterization, and application of nanoscale culture of an opportunistic fungal pathogen, Candida albicans. Despite a more than 2,000-fold reduction in volume, the growth characteristics and drug response profiles obtained from the nanoscale cultures were comparable to the industry standards. The platform also enabled rapid identification of new drug candidates that were effective against C. albicans biofilms, which are a major cause of mortality in hospital-acquired infections.