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2014 Agenda and Abstracts | < Previous | Next >

2014 OMIG Abstract 5

Impediment of Corneal Cell Migration by a Bacterial Factor
K.M. Brothers; N.A. Stella; K.M. Hunt; E.G. Romanowski; J.K. Klarlund; R.M.Q. Shanks

The Charles T. Campbell Laboratory, UPMC Eye Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA

Purpose: To test the hypothesis that bacterial secreted factors mediate corneal wound healing. Serratia species are commonly isolated from chronic wounds, contact lenses (CL) and CL cases and are a major cause of corneal infections. Epithelial cell defects are associated with corneal ulcers.  

Methods: Bacteria free secreted fractions (BFSFs) were derived from S. marcescens, Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and other bacteria grown in lysogeny broth, normalized by turbidity, centrifuged, and filtered (0.22 µm) to remove bacteria. Human corneal limbal epithelial (HCLE) cells were grown to confluence and subsequently stratified in 96-well plates containing a silicone “stopper”. The silicone stoppers were removed and BFSFs were added directly to plates of HCLEs.  After 24 hours of incubation at 37oC + 5% CO2, the cell layers were stained with Calcein AM fluorescent viability stain and imaged with confocal microscopy.

Results: HCLE cell layers were treated with BFSFs from S. marcescens and 10 out of 17 S. marcescens clinical isolates had dose dependent inhibition of wound healing in this in vitro model. These results have also been recapitulated using an ex vivo porcine corneal wound healing model. BFSFs from other tested bacteria did not have the same effect. Strains of S. marcescens with mutations in the LPS biosynthetic locus were tested, with some resulting in rescued healing in this model. S. marcescens BFSFs were treated with polymyxin B agarose to deplete LPS. Depletion of LPS resulted in BFSFs unable to inhibit corneal cell migration. S. marcescens and E. aerogenes were the only tested Gram-negative bacteria that inhibited epithelial wound healing in this study.

Conclusions: Our preliminary data indicate S. marcescens LPS may be necessary for the impaired wound healing phenotype. This study provides insight into the impact of bacteria on wound healing.

Disclosure: S=Research to Prevent Blindness, Eye and Ear Institute of Pittsburgh, NIH grant EY017271-06A1, NIH grant AI085570, NIH Core Grant EY08098

2014 Agenda and Abstracts | < Previous | Next >