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D. K. Augustin, D. J. Evans, S. M. J. Fleiszig; Pseudomonas Aeruginosa Translocation of Corneal Epithelia Is Enhanced by Bacterial Adaptation, but Is Reduced by Epithelial Pre-exposure to Bacterial Antigens. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3107.
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Contact lens wear is known to predispose the cornea to P. aeruginosa keratitis with enhanced risk of infection occurring during extended wear. We have previously reported that lens-induced infections in animal models show delayed disease onset. Here, we tested the hypothesis that P. aeruginosa has the capacity to adapt to corneal host defenses that otherwise limit their ability to penetrate (translocate) the corneal epithelium.
In vitro translocation assays were used to quantify the ability of P. aeruginosa to cross multilayered human corneal epithelial cells (telomerase-immortalized) grown on 3 µm pore-size Transwell filters. GFP-expressing P. aeruginosa (PA01, ~106 cfu) were added to apical compartments and aliquots collected from apical and basal compartments at 8 h post-infection to monitor bacterial growth and to determine translocation efficiency, respectively. Intracellular bacteria were quantified using gentamicin survival assays. Translocation of naïve bacteria was compared to bacteria that had previously translocated corneal epithelia (pre-conditioned bacteria). In other experiments, cells were pre-treated with bacterial culture supernatant (bacteria removed by filtration) that contains antigenic components (e.g. LPS, flagella, pili) known to activate (putative) innate defense systems.
Measurable translocation required at least 4 h and did not increase substantially with increasing inoculum size. Preconditioned bacteria (that had previously translocated corneal epithelial cells) were significantly more efficient at translocation (~3 log increase, p = 0.0003, t-Test), and cellular invasion (~ 1 log increase, p = 0.008), than naïve bacteria. In contrast, corneal epithelial cells pre-treated with bacterial supernatant prior to inoculation with whole bacteria showed an increased resistance to bacterial translocation (~1 log reduction, p = 0.0001, t-Test).
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