Understanding the pathology resulting from Staphylococcus aureus and Pseudomonas aeruginosa polymicrobial wound infections is of great importance due to their ubiquitous nature, increasing prevalence, growing resistance to antimicrobial agents, and ability to delay healing. Methicillin-resistant S. aureus USA300 is the leading cause of community-associated bacterial infections resulting in increased morbidity and mortality. We utilized a well-established porcine partial thickness wound healing model to study the synergistic effects of USA300 and P. aeruginosa on wound healing. Wound re-epithelialization was significantly delayed by mixed-species biofilms through suppression of keratinocyte growth factor 1. Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo. Polymicrobial wound infection in the presence of P. aeruginosa resulted in induced expression of USA300 virulence factors Panton-Valentine leukocidin and α-hemolysin. These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.

Diabetic foot ulcers (DFUs) are a debilitating complication of diabetes in which bacterial presence, including the frequent colonizer Staphylococcus aureus, contributes to inhibition of healing. MicroRNAs (miRs) play a role in healing and host response to bacterial pathogens. However, the mechanisms by which miR response to cutaneous S. aureus contributes to DFU pathophysiology are unknown. Here, we show that S. aureus inhibits wound closure and induces miR-15b-5p in acute human and porcine wound models and in chronic DFUs. Transcriptome analyses of DFU tissue showed induction of miR-15b-5p to be critical, regulating many cellular processes, including DNA repair and inflammatory response, by suppressing downstream targets IKBKB, WEE1, FGF2, RAD50, MSH2, and KIT. Using a human wound model, we confirmed that S. aureus-triggered miR-15b-5p induction results in suppression of the inflammatory- and DNA repair-related genes IKBKB and WEE1. Inhibition of DNA repair and accumulation of DNA breaks was functionally confirmed by the presence of the pH2AX within colonized DFUs. We conclude that S. aureus induces miR-15b-5p, subsequently repressing DNA repair and inflammatory response, showing a mechanism of inhibition of healing in DFUs previously unreported, to our knowledge. This underscores a previously unknown role of DNA damage repair in the pathophysiology of DFUs colonized with S. aureus.

Diabetic foot ulcers (DFUs), a life-threatening complication of diabetes mellitus, have limited treatment options, often resulting in amputations. HMG-CoA reductase inhibitors such as statins are cholesterol-reducing agents that may provide a new therapeutic option. Statins target the cholesterol pathway and block the synthesis of the wound-healing inhibitors farnesyl pyrophosphate (FPP) and cortisol, ligands for the glucocorticoid receptor (GR). Here we demonstrate that the naturally occurring statin mevastatin reverses FPP's effects and promotes healing by using wound healing assays, human and porcine wound models, and DFU tissue. Moreover, we measured cortisol levels by ELISA and found that mevastatin inhibited cortisol synthesis in keratinocytes and biopsies from patients with DFU. Of note, topical mevastatin stimulated epithelialization and angiogenesis Mevastatin also reversed FPP-mediated induction of the GR target, the transcription factor c-Myc (a biomarker of non-healing wounds), in porcine and human wound models. Importantly, mevastatin reversed c-Myc overexpression in DFUs. It induced expression of the long noncoding RNA Gas5 that blocks c-Myc expression, which was confirmed by overexpression studies. We conclude that topical mevastatin accelerates wound closure by promoting epithelialization via multiple mechanisms: modulation of GR ligands and induction of the long noncoding RNA Gas5, leading to c-Myc inhibition. In light of these findings, we propose that repurposing statin drugs for topical treatment of DFUs may offer another option for managing this serious condition.

The formation of biofilms is a developmental process initiated by planktonic cells transitioning to the surface, which comes full circle when cells disperse from the biofilm and transition to the planktonic mode of growth. Considering that pyruvate has been previously demonstrated to be required for the formation of P. aeruginosa biofilms, we asked whether pyruvate likewise contributes to the maintenance of the biofilm structure, with depletion of pyruvate resulting in dispersion. Here, we demonstrate that the enzymatic depletion of pyruvate coincided with the dispersion of established biofilms by S. aureus and laboratory and clinical P. aeruginosa isolates. The dispersion response was dependent on pyruvate fermentation pathway components but independent of proteins previously described to contribute to P. aeruginosa biofilm dispersion. Using porcine second-degree burn wounds infected with P. aeruginosa biofilm cells, we furthermore demonstrated that pyruvate depletion resulted in a reduction of biofilm biomass in vivo . Pyruvate-depleting conditions enhanced the efficacy of tobramycin killing of the resident wound biofilms by up to 5-logs. Our findings strongly suggest the management of pyruvate availability to be a promising strategy to combat biofilm-related infections by two principal pathogens associated with wound and cystic fibrosis lung infections.

Keratin gene expression is regarded as a hallmark of epidermal biology. It demarcates the three keratinocyte phenotypes: basal (expressing KRT5 and KRT14), differentiating (expressing KRT1 and KRT10), and activated (wound healing), which is characterized by expression of KRT6, KRT16, and KRT17. Activated keratinocytes are among the first signals of epidermal wound healing. In addition, they are found deregulated in nonhealing chronic wounds. To examine keratins as a potential modality for wound-healing disorders, we evaluated two different keratin dressings, liquid or solid, and assessed their effects of epithelialization and closure using porcine partial-thickness wound-healing model in vivo. We found that both forms of keratin dressings accelerated closure and epithelialization, achieving statistically significant differences on day 5. Evidence suggesting early onset of epithelialization was corroborated further by gene expression analyses revealing induction of KRT6A, KRT16, and KRT17 by day 2 postwounding. The data suggest that keratin dressings may stimulate epithelialization by enhancing the activation of keratinocytes. We conclude that keratin-containing dressings can accelerate wound healing and closure. Further studies are needed to determine the molecular mechanisms of this activation.