Secreted biofilm factors adversely affect cellular wound healing responses in vitro.
Although most chronic wounds possess an underlying pathology, infectious agents also contribute. In many instances, pathogens exist as biofilms forming clusters surrounded by a secreted extracellular substance. We hypothesized that compounds secreted by biofilm bacteria may inhibit normal wound healing events including cell proliferation and migration. Conditioned media from two common bacterial species associated with chronic skin wounds and chronic tympanic membrane perforations, Staphylococcus aureus and Pseudomonas aeruginosa, were evaluated for their capacity to affect keratinocyte proliferation and migration. Additionally, proteomic analysis was performed to identify proteins within the biofilm conditioned media that may contribute to these observed effects. Biofilm conditioned media from both species inhibited proliferation in human tympanic membrane derived keratinocytes, whereas only biofilm conditioned media from S. aureus inhibited migration. Human epidermal keratinocytes were found to be more sensitive to the effects of the conditioned media resulting in high levels of cell death. Heat treatment and microfiltration suggested that S. aureus activity was due to a protein, while P. aeruginosa activity was more likely due to a small molecule. Proteomic analysis identified several proteins with putative links to delayed wound healing. These include alpha hemolysin, alcohol dehydrogenase, fructose-bisphosphate aldolase, lactate dehydrogenase and epidermal cell differentiation inhibitor.
1] Ear Science Institute, Suite 1, Level 2, 1 Salvado Road, Subiaco, WA 6008, Australia [2] Ear Sciences Centre, School of Surgery, The University of Western Australia, Crawley, Western Australia.
Robert
R
Jeffery Marano
1] Burn Injury Research Unit, School of Surgery, The University of Western Australia, Western Australia, Australia [2] Fiona Wood Foundation, Western Australia, Murdoch 6150, Australia.
Hilary
H
Jane Wallace
1] Burn Injury Research Unit, School of Surgery, The University of Western Australia, Western Australia, Australia [2] Fiona Wood Foundation, Western Australia, Murdoch 6150, Australia.
Dulharie
D
Wijeratne
1] Burn Injury Research Unit, School of Surgery, The University of Western Australia, Western Australia, Australia [2] Fiona Wood Foundation, Western Australia, Murdoch 6150, Australia.
Mark
M
William Fear
School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy Campus, Adelaide, South Australia, Australia.
Hui
H
San Wong
School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy Campus, Adelaide, South Australia, Australia.
Ryan
R
O'Handley
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D040901:Proteomics; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000378:metabolism*; D013696:Temperature; D014432:Tympanic Membrane / Q000166:cytology; D014945:Wound Healing / Q000187:drug effects*
D017077:Culture Media, Conditioned; D006460:Hemolysin Proteins; D000426:Alcohol Dehydrogenase; D007770:L-Lactate Dehydrogenase; D005634:Fructose-Bisphosphate Aldolase