Colonization of Epidermal Tissue by Staphylococcus aureus Produces Localized Hypoxia and Stimulates Secretion of Antioxidant and Caspase-14 Proteins

Abstract

A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H 2 O 2 ) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS E ). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2-to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H 2 O 2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H 2 O 2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

Trade-offs between microbiome diversity and productivity in a stratified microbial mat

“…Samples were positioned on agar in 15 mm nuclear magnetic resonance tubes, and degassed under dark conditions to remove bubbles prior to interrogation. ParaVision v5.1 was used to run chemical shift selective imaging (mic chess) (Ha et al, 2015;Lone et al, 2015a) and 2D diffusion-mapping experiments (DtiStandard) (Lone et al, 2015b), both with a resolution of 78 × 156 μm. Scalar irradiance (photosynthetically active radiation) profiles were measured in quadruplicate with isotropic photosynthetically active radiation microsensors with spherical 70 μm diameter tips (Zensor, Åls-gårde, DK) fiber optically connected to a spectrophotometer (USB2000+-ES, Ocean Optics, Dunedin, FL, USA) and inter-calibrated against a radiometer (LI-250A and LI-COR Quantum sensor, LI-COR Biosciences, Lincoln, NE, USA) (Lassen et al, 1992).…”

In vitro studies evaluating the effects of biofilms on wound‐healing cells: a review

“…For example, Lone et al. examined the growth of S. aureus biofilms on partial‐thickness epidermal explants. Epidermal explants from porcine ears were harvested, cleaned, and cut into 12‐mm disks.…”

“…Another method of examining the effects of biofilms on keratinocytes is to grow biofilms directly on whole epithelial structures. For example, Lone et al (24) examined the growth of S. aureus biofilms on partial-thickness epidermal explants. Epidermal explants from porcine ears were harvested, cleaned, and cut into 12-mm disks.…”

“…The disks were then inoculated with 500-800 colony-forming units (CFU) of S. aureus and allowed to grow for 4-7 days. Microscopic evaluation found that bacterial biofilms readily formed around hair follicles and their associated interstices (24). The authors also found that all epidermal cell layers displayed some cellular damage, although only the surface of the epithelium was inoculated and that biofilm was only visible in the stratum corneum layers (24).…”

“…Microscopic evaluation found that bacterial biofilms readily formed around hair follicles and their associated interstices (24). The authors also found that all epidermal cell layers displayed some cellular damage, although only the surface of the epithelium was inoculated and that biofilm was only visible in the stratum corneum layers (24). Basal cells were vacuolated and their nuclei were often shrunken or absent, which indicated cellular damage and death.…”