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Kimberly Brothers, Nicholas Stella, Jes Klarlund, Robert Shanks; Impediment of corneal cell migration by a Serratia marcescens factor in an in vitro wound healing model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5220. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the effects of S. marcescens secreted factors on corneal wound healing.
Human corneal limbal epithelial (HCLE) cells were grown to confluence and subsequently stratified in 12-well plates with agarose strips. Bacterial secreted fractions (BSF) of S. marcescens, Esherichia coli and Pseudomonas aeruginosa were grown over-night in LB medium, normalized by optical density, and filter sterilized. Upon removal of agarose strips to simulate a wound, HCLEs were washed in PBS and provided with fresh media. BSFs were heat treated (10 minutes at 95 C) or added directly to plates of HCLEs. Epidermal growth factor (EGF) and serum concentrations were also tested in this model. After 20-24 hours of incubation at 37 C + 5% CO2, the cell layers were fixed and stained with gentian violet. The remaining wound area for each treatment condition was assessed.
Stratified HCLE cell layers treated with BSF from S. marcescens exhibited dose dependent inhibition of wound healing in this in vitro model. BSF from E. coli or P. aeruginosa did not have the same effect. The impaired healing effect was unaltered by the presence or absence of epidermal growth factor (EGF), and only partially reduced by heat-treatment of BSF. This indicates an uncharacterized bacterial factor necessary for the impaired wound healing phenotype. Mutant strains of S. marcescens were tested and BSF from one strain was found insufficient to inhibit healing.
Serratia species are commonly isolated from contact lens cases putting individuals at risk for corneal infections. We have identified a potentially important host-pathogen interaction involved in bacterial infection and corneal wound healing. This model promises to provide insight into corneal wound healing in response to bacterial contamination by providing a means to visually analyze the effects of BSFs on corneal wound healing.
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