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E. C. Ledbetter, D. Kowbel, J. J. Mun, T. J. Kern, R. C. Riis, J. M. Scarlett, F. W. Wallace-Gadsden, S. M. J. Fleiszig; Pathogenic Phenotype and Genotype of Canine Ocular Pseudomonas aeruginosa Isolates. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4726.
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Some strains of Pseudomonas aeruginosa can invade or kill corneal epithelial cells. The purpose of this study was to determine whether the ability to adversely affect epithelial cell health was a factor common to ulcerative keratitis strains.
Pseudomonas aeruginosa isolates were collected from the conjunctiva of dogs without extraocular disease, dogs with ocular infections, and the conjunctiva of dogs 2-3 months post-resolution of Pseudomonas aeruginosa ulcerative keratitis. Phenotype was determined in vitro by quantifying corneal epithelial cell invasion by gentamicin survival assays, and cytotoxic activity by LDH release and trypan blue exclusion assays. Genotyping was performed to determine the presence or absence of genes encoding the type III secreted effectors ExoS, ExoU, ExoT, and ExoY.
Normal conjunctival flora strains were genotypically invasive (n=5) and ExoS+/ExoU+/ExoY+/ExoT+ (n=1). Ulcerative keratitis strains were genotypically invasive (n=5), cytotoxic (n=2), ExoS-/ExoU+/ExoY-/ExoT+ (n=1), and ExoS-/ExoU-/ExoY-/ExoT- (n=1). Conjunctivitis strains were genotypically invasive (n=1) and ExoS+/ExoU-/ExoY+/ExoT+ (n=1). A single endophthalmitis strain was genotypically invasive. Pseudomonas aeruginosa was reisolated from the conjunctival flora of 4 dogs post-resolution of ulcerative keratitis (all 4 were genotypically invasive), but only 1 of these isolates shared the same phenotype/genotype as the original isolate. Visible adverse effects on epithelial cell health (e.g., rounding, sloughing from plates, loss) were significantly (p<0.01) more frequent for keratitis strains (6/9) than other strains (1/13). Of the 6 keratitis strains adversely affecting cell health, 3 were invasive, 2 were cytotoxic, and 1 was neither genotype (lacked all 4 known effectors). The 1 non-keratitis strain affecting cell health was cytotoxic and unusual in that it encoded all 4 known effectors. Therefore, only keratitis strains had visible adverse effects on live host cells (4/9 keratitis strains vs. 0/13 non-keratitis strains).
The invasive phenotype and genotype of Pseudomonas aeruginosa predominates in dogs with and without ocular disease. Only ulcerative keratitis strains had visible adverse effects on epithelial cell health in the absence of overt cytotoxicity, suggesting that virulence strategies that affect live epithelial cell health are selected for among ulcerative keratitis strains.
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