|LONE, ABDUL - Washington State University|
|ATCI, ERHAN - Washington State University|
|RENSLOW, RYAN - Pacific Northwest National Laboratory|
|FRANSSON, BOEL - Washington State University|
|BEYENAL, HALUK - Washington State University|
|ABU-LAIL, NEHAL - Washington State University|
|JEONG-JIN, PARK - Washington State University|
|GANG, DAVID - Washington State University|
|CALL, DOUGLAS - Washington State University|
Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2015
Publication Date: 4/6/2015
Citation: Lone, A., Noh, S.M., Atci, E., Renslow, R., Fransson, B., Beyenal, H., Abu-Lail, N., Jeong-Jin, P., Gang, D.R., Call, D. 2015. Staphylococcus aureus induces hypoxia and cellular damage in porcine dermal explants. PLoS Pathogens. 83(6):2531-2541.
Interpretive Summary: Multidrug resistant Staphylococcus aureus biofilms are a major problem in acute and chronic wounds, but the mechanisms responsible for impaired healing are not well defined. In addition to cell survival, optimum levels of oxygen are needed for wound repair and a more acidic environment aids in wound healing by inhibiting proteolytic enzymes. Our objective was to determine if multidrug-resistant S. aureus biofilms impact these parameters in the context of a tissue matrix. We developed a dermal explant model that resembles a skin injury where MRSA biofilms were grown on the superficial surface. We characterized changes in cell structure, tissue viability, changes in effective diffusion coefficients, and changes in full-depth oxygen and pH profiles. Irrespective of the biofilm mass, the biofilms completely depleted oxygen and increased pH within the dermal tissue. Cell nuclei of the dermal tissue were nearly completely absent, indicating widespread cell death. Infected explants released substantial quantities of pro-apoptotic proteins, indicating programmed cell death or apoptosis had occurred in the explants. MRSA biofilms impact underlying tissue by scavenging oxygen, increasing pH, and killing the host cells. All of these factors are consistent with impaired wound healing.
Technical Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) can infect wounds and produce difficult-to- treat biofilms. To determine the extent that MRSA biofilms can deplete oxygen, change pH and damage host tissue, we developed a porcine dermal explant model on which we cultured GFP-labeled MRSA biofilms. Effective diffusion coefficients were measured for explants using magnetic resonance imaging. Explants were examined histopathologically for cytotoxic effects. Dissolved oxygen and pH profiles within the dermal tissue were determined using microelectrodes attached to step motors. The biofilm- and porcine-origin proteins that accumulated in the culture media were identified using MSE mass spectrometry. Biofilm-free and biofilm-infected dermal tissue showed statistically significant differences in relative effective diffusion coefficients (0.25 ± 0.88 to 0.29 ± 0.12 vs. 0.40 ± 0.11 to 0.47 ± 0.11, respectively; P < 0.001). Oxygen content in biofilm-free dermal tissue was lower than in dead tissue (4.45 ± 1.17 mg/l and 5.87 ± 0.73, respectively). Oxygen levels for biofilm-infected tissue declined sharply with no measurable oxygen detectable in the underlying dermal tissue and this loss of oxygen is directly attributable to biofilm-induced oxygen demand. The pH within biofilm-infected explants was more alkaline than in biofilm-free explants (8.0 ± 0.17 vs. 7.5 ± 0.15, respectively; P < 0.05). Cellular and nuclear details were lost in the infected explants, indicating cell death. Substantially higher amounts of both pro-apoptotic programmed cell death protein 5, PDCD5, (1351-fold) and anti-apoptotic macrophage migration inhibitory factor, MIF (58-fold) accumulated in the infected-explant spent media as compared with uninfected-explant spent media, consistent with the co-occurrence of apoptosis and necrosis in the explants. Biofilm-origin proteins reflected an extracellular matrix adapted life-style of MRSA. MRSA biofilms deplete oxygen, increase pH, increase relative effective diffusion coefficients, induce cell death; all factors that likely contribute to impeded wound healing.