Abstract
Chronic wounds such as diabetic ulcers account for a large portion of health-related problems. The annual burden to the US healthcare system alone is estimated to be $15 billion [1] and this cost is estimated to rise as incidence in diabetes has nearly doubled in the past 25 years. Normal healing is classically grouped into four distinct phases: hemostasis, inflammation, proliferation and tissue remodeling. [2] Chronic wounds are characterized by a lack of progression through these normal phases of healing, and most commonly persist in the inflammatory phase. [2, 3] Commonly isolated pro-inflammatory mediators including IL-1 and IL-6 are necessary to control microbial infection are not properly regulated in diabetes patients. [3, 4] One major contributing factor exacerbating chronicity of wounds is persistent polymicrobial infection by members of the normal skin microbiota or environmental opportunistic pathogens, including Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). The most common infectious architectural state is commonly referred to as biofilm. [3, 5] Biofilms are characterized by the adhesion of bacterial cells to each other and to a substrate followed by secretion of an extracellular matrix (ECM) consisting of DNA, proteins and polysaccharides. [6, 7] Counterintuitively, the presence of ligands attributed to biofilm-embedded bacterial cells prolong the inflammatory response, yet the ECM architecture facilitates evasion of degradation by inflammatory mediators. [8] Furthermore, ECM constituents mediate resistance to intervention strategies including detergents, shearing forces, and antibiotics. [8, 9]Mechanisms mediating biofilm formation and ECM production in wound microenvironments remain largely uncharacterized, leaving a paucity of novel non-antibiotic treatments to eliminate microbes and promote healing.
Further complicating the interplay between microbes and wound tissue is the presence of neuroendocrine hormone signalers called catecholamines (CATs). CATs such as epinephrine (EPI) and norepinephrine (NE), have been shown to regulate pathways that impair healing upon binding to adrenergic receptors (ARs). [9-11] Conversely, clinical data shows that oral administration of AR antagonists, including timolol (TIM) and propranolol (PROP), ameliorate CAT activity to enhance re-epithelialization and wound healing. [12] Interestingly, bacterial pathways of gene expression modulated by quorum sensing and associated virulence properties have been shown to be regulated by the presence of exogenous EPI. [13-15] This is the case for the gastrointestinal pathogen E. coli, in which researchers have demonstrated altered expression of specific microbial receptors and metabolic pathways leading to increased toxin production and biofilm formation. [16] Equivalent CAT-microbial receptor interactions for skin-associated microbes remain largely uncharacterized. Therefore, we hypothesize that CATs modulate growth and biofilm formation of skin- and wound- associated microbiota and that these CAT mediated responses can be ameliorated by AR antagonists such as TIM and PROP.
The field of wound healing is left with several important unanswered questions including:
▪ How do CATs modulate virulence of skin- and wound-associated bacterial species?
▪ Do exogenous CATs induce reciprocal CAT production by wound-associated bacteria?
▪ Are the potential aforementioned effects reversible by AR antagonist addition?
To answer the questions above and address our hypothesis, we have developed culture assays for monitoring bacterial growth and biofilm formation using a combination of spectrophotometry and microscopy and created an ex vivo model using human skin tissue. We investigated these questions by the following specific aims:
A. Characterize growth and biofilm modulations mediated by CATs.
• Growth assays show growth rates of P. aeruginosa (PA) and S. aureus (SA) increased significantly in the presence of 50 μM EPI 40%, respectively when monitored by optical density in physiologically relevant media (p < 0.01).
• Crystal violet (CV) staining demonstrate that CATs modulate biofilm formation during early binding events and later biofilm formation after 24 hours.
• Confocal laser scanning microscopy (CLSM) demonstrate that CATs modulate biofilm formation during early binding events and later biofilm formation (50 μM EPI: 18 to 27.5 μm, PA; 10 to 30 μm, SA; p < 0.01) when incubated up to 6 days (p < 0.01).
• RT-qPCR for targeted biofilm related genes shows that 50 μM EPI increases gene expression for factors associated with attachment for both P. aeruginosa (psl) and S. aureus (sarA) 3 days of biofilm development (p < 0.01).
B. Determine whether AR antagonists including TIM and PROP affect CAT-induced alterations to microbial virulence.
• Growth assays show increased growth rates of P. aeruginosa (PA) and S. aureus (SA) by EPI were ameliorated significantly (p < 0.01) by the addition of 10 mM TIM and 1mM PROP (0.8 fold; PA, 0.9-fold; SA) when monitored by optical density in physiologically relevant media.
• Crystal violet (CV) staining demonstrate CAT mediated biofilm modulation was ameliorated significantly by addition of 10 mM TIM when incubated up for 24 hours (p < 0.01)
• Confocal laser scanning microscopy (CLSM) demonstrate CAT mediated biofilm modulation was ameliorated significantly by addition of 10 mM TIM (10mM TIM + EPI: 18 to 27.5 μm, PA; 10 to 30 μm, SA; p < 0.01) when incubated up to 6 days.
• RT-qPCR for targeted biofilm related genes shows that EPI increased gene expression for factors associated with attachment was ameliorated by addition of TIM or PROP for both P. aeruginosa (psl) and S. aureus (sarA) after 3 days of biofilm development (p < 0.01).
C. Characterize wound biofilms in an ex vivo model using human skin explants.
• Established an ex vivo model for biofilm formation assessed by gram staining.
• Designed and created probes for EUB, S. aureus, P. aeruginosa species-specific probes for fluorescence in-situ hybridization (FISH) in single and mix culture.