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Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm communities; however, there are few studies showing the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for studying the effect of biofilms on the cell types involved in wound healing. Cocultures of Staphylococcus aureus biofilms and human keratinocytes (HK) were created by initially growing S. aureus biofilms on tissue culture inserts then transferring the inserts to existing HK cultures. Biofilm-conditioned medium (BCM) was prepared by culturing the insertsupported biofilm in cell culture medium. As a control planktonic-conditioned medium (PCM) was also prepared. Biofilm, BCM, and PCM were used in migration, cell viability, and apoptosis assays. Changes in HK morphology were followed by brightfield and confocal microscopy. After only 3 hours exposure to BCM, but not PCM, HK formed dendrite-like extensions and displayed reduced viability. After 9 hours, there was an increase in apoptosis (p≤0.0004). At 24 hours, biofilm, BCM, and PCM exposed HK all showed reduced scratch closure (p≤0.0001). The results showed that soluble products of both S. aureus planktonic cells and biofilms inhibit scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability and significantly increased HK apoptosis compared with planktonic S. aureus.Keratinocytes are the major cell type of the epidermis, which serves as the primary barrier between the external environment and the internal tissues. In this capacity, the epidermis also functions as a barricade to microorganisms, toxins, and various antigens. When the barrier is breached due to wounding, basal keratinocytes from the wound edges or dermal appendages migrate over the open wound to reestablish the barricade in a process called reepithelialization. Chronic wounds, such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers, are characterized by prolonged inflammation, an altered wound matrix, and the failure to reepithelialize.Chronic wounds are also characterized as supporting a diverse microbial flora. A literature review by Bowler examined culture data from 62 published studies dating between 1969 and 1997. 1 The most predominant wound isolate in both chronic and acute wounds was Staphylococcus aureus (reported in 63% of the studies), followed by coliforms (45%), It has been speculated that bacteria colonizing chronic wounds exist as a biofilm. [3][4][5][6][7] Biofilms represent bacterial communities surrounded by extracellular polysaccharide matrix. Such communities are often polymicrobial and resistant to antimicrobials. Chronic wounds are an ideal environment for bacterial infection and biofilm formation. The wound remains open for a prolonged period of time, increasing the odds of bacterial infection. The wound bed provides a surface for growth, and poor blood flow and hypoxia discourage native defenses. 8 Studies have shown that wounds inoculated with bacteria form biofilms. 6,9 Furthermore, in a recent study by Ja...

Wound bioburden in the form of colonizing biofilms is a major contributor to nonhealing wounds. Staphylococcus aureus is a Gram-positive, facultative anaerobe commonly found in chronic wounds; however, much remains unknown about the basic physiology of this opportunistic pathogen, especially with regard to the biofilm phenotype. Transcriptomic and proteomic analysis of S. aureus biofilms have suggested that S. aureus biofilms exhibit an altered metabolic state relative to the planktonic phenotype. Herein, comparisons of extracellular and intracellular metabolite profiles detected by 1H NMR were conducted for methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains grown as biofilm and planktonic cultures. Principal component analysis distinguished the biofilm phenotype from the planktonic phenotype, and factor loadings analysis identified metabolites that contributed to the statistical separation of the biofilm from the planktonic phenotype, suggesting that key features distinguishing biofilm from planktonic growth include selective amino acid uptake, lipid catabolism, butanediol fermentation, and a shift in metabolism from energy production to assembly of cell-wall components and matrix deposition. These metabolite profiles provide a basis for the development of metabolite biomarkers that distinguish between biofilm and planktonic phenotypes in S. aureus and have the potential for improved diagnostic and therapeutic use in chronic wounds.

Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus, and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor-necrosis factor-α production in fibroblasts compared to planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared to controls.