Polymicrobial biofilms are a hallmark of chronic wound infection. The forces governing assembly and maturation of these microbial ecosystems are largely unexplored but the consequences on host response and clinical outcome can be significant. In the context of wound healing, formation of a biofilm and a stable microbial community structure is associated with impaired tissue repair resulting in a non-healing chronic wound. These types of wounds can persist for years simmering below the threshold of classically defined clinical infection (which includes heat, pain, redness, and swelling) and cycling through phases of recurrent infection. In the most severe outcome, amputation of lower extremities may occur if spreading infection ensues. Here we take an ecological perspective to study priority effects and competitive exclusion on overall biofilm community structure in a three-membered community comprised of strains of Staphylococcus aureus, Citrobacter freundii, and Candida albicans derived from a chronic wound. We show that both priority effects and inter-bacterial competition for binding to C. albicans biofilms significantly shape community structure on both abiotic and biotic substrates, such as ex vivo human skin wounds. We further show attachment of C. freundii to C. albicans is mediated by mannose-binding lectins. Co-cultures of C. freundii and C. albicans trigger the yeast-to-hyphae transition, resulting in a significant increase in neutrophil death and inflammation compared to either species alone. Collectively, the results presented here facilitate our understanding of fungal-bacterial interactions and their effects on host-microbe interactions, pathogenesis, and ultimately, wound healing.
Wound cleansing agents are routine in wound care and preoperative preparation.Antiseptic activity intends to prevent contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. We evaluated the cytotoxicity of five clinically used wound cleaning agents (saline, povidone iodine, Dove ® and Dial ® soaps, and chlorhexidine gluconate [CHG]) using both an ex vivo and in vivo human skin xenograft mouse model, in contrast to classical in vitro models that lack the structural and compositional heterogeneity of human skin. We further established an ex vivo wound contamination model inoculated with $100 cells of Pseudomonas aeruginosa or Staphylococcus aureus to evaluate antimicrobial efficacy. Scanning electron microscopy and confocal microscopy were used to evaluate phenotypic and spatial characteristics of bacterial cells in wound tissue.CHG significantly reduced metabolic activity of the skin explants, while all treatments except saline affected local cellular viability. CHG cytotoxicity persisted and progressed over 14 days, impairing wound healing in vivo. Within the contamination model, CHG treatment resulted in a significant reduction of P. aeruginosa wound surface counts at 24 h post-treatment. However, this effect was transient and serial application of CHG had no effect on both P. aeruginosa or S. aureus microbial growth.Microscopy revealed that viable cells of P. aeruginosa reside deep within wound tissue post-CHG application, likely serving as a reservoir to re-populate the tissue to a high bioburden. We reveal concerning cytotoxicity and limited antimicrobial activity of CHG in human skin using clinically relevant models, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.
A hallmark of chronic infections are polymicrobial biofilms. The forces governing assembly and maturation of these microbial ecosystems are largely unexplored but the consequences on host response and clinical outcome can be significant. In the context of wound healing, formation of a biofilm and a stable microbial community structure is associated with impaired tissue repair resulting in a non-healing chronic wound. These types of wounds can persist for years simmering below the threshold of classical clinical infection or cycling through phases of recurrent infection. In the most severe outcome amputation of lower extremities may occur if spreading infection ensues. Here we take an ecological perspective to study priority effects and competitive exclusion on overall biofilm community structure in a three-membered community of microbes derived from a chronic wound. We find that priority effects occur across both biotic and abiotic substrates, and ecological interactions can alter both fungal physiology and host inflammatory response. We show that bacterial-competition occurs for binding to fungal structures, and some species trigger the yeast-hyphae switch, resulting in enhanced neutrophil killing and inflammation. Collectively, the results presented here facilitate our understanding of fungal-bacterial microbial community dynamics and their effects on, host-microbe interactions, pathogenesis, and ultimately, wound healing.
Wound cleansing agents are routine in wound care, even in the absence of signs of infection. Antiseptic activity prevents contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. Here, we used an ex vivo human skin excisional wound model to evaluate the cytotoxicity of five clinically-used wound cleaning agents (saline, povidone iodine, Dove® soap, Dial® soap, and chlorhexidine gluconate). We established a wound contamination model using ~100 cells of Pseudomonas aeruginosa per wound to evaluate antiseptic efficacy and microbial biofilm spatial organization. We found that Dial® soap and chlorhexidine gluconate significantly reduced metabolic activity of the biopsies, while all treatments except saline affected local cellular viability. Within the contamination model, only chlorhexidine gluconate treatment resulted in significantly lower P. aeruginosa counts at 24 hours post-treatment, driven by sub-limit-of-detection counts immediately post-treatment. Later applications of chlorhexidine gluconate had no effects on microbial growth, with microscopy showing extensive surface colonization of the wound bed. We present a clinically-relevant model for evaluating antiseptic cytotoxicity and efficacy, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.
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