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MS F Kwong, EJ Lee, SM J Fleiszig; Human Tears Protect Cells From Pseudomonas aeruginosa Virulence Mechanisms While Supporting Bacterial Growth . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1666.
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Purpose: Both invasive and cytotoxic P. aeruginosa strains can damage corneal epithelial cells in vitro. Nevertheless, neither can infect healthy cornea in vivo. Differences between in vivo and in vitro include shear stresses during blinking and the presence of the tear film. Having already demonstrated a role for shear, in this study we tested the hypothesis that the tear film also plays a protective role. Methods: Whole tear film was collected from the conjunctival sac of human volunteers. Cultured corneal epithelium was inoculated with 106 cfu/ml cytotoxic (6206) or invasive (6294) P. aeruginosa in the presence of tears or cell culture media (MEM). Cytotoxicity was quantified by lactate dehydrogenase (LDH) release assays; invasion by gentamicin exclusion. Growth/survival of bacteria during the assays was determined by viable counts at various time points. Video microscopy was used to visualize bacteria in tears or MEM in the presence and absence of corneal epithelium. Results: Human tear film inhibited both cytotoxicity and invasion by P. aeruginosa. The invasive strain grew unhindered by the presence of human tears (3-fold replication in 3 h), despite a 97% reduction in ability to invade corneal cells (p= <0.0001). In contrast, the tears reduced the growth rate of the cytotoxic strain as compared to MEM (by 75%). Suppression of cytotoxicity was only partially explained by the bacteriostatic activity. Matched bacteriostatic activity using 1 mg/ml sulfacetamide did not reproduce the magnitude of protection caused by tears (80% tears, 48% sulfacetamide). Furthermore, protective activity, but not bacteriostatic activity, was rapidly lost by dilution of tears by more than 2-fold. Video microscopy showed that in tears and in sulfacetamide bacteria could reproduce, but formed chains. Singular bacteria were motile in sulfacetamide, but not in tears. Conclusion: Human tears can protect corneal epithelial cells against damage caused by both cytotoxic and invasive P. aeruginosa. Tears slowed growth of cytotoxic strain 6206; unknown factors accounted for an additional reduction in cytotoxicity. Human tears were not bacteriostatic against invasive strain 6294, yet efficiently blocked bacterial invasion of cells. Whether the mechanism(s) involve effects on the bacterium or on the host cell, is yet to be determined.
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