Resistance of the murine cornea to bacterial colonization during experimental dry eye

The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.