Clearance of Pseudomonas aeruginosa From the Healthy Ocular Surface in a "Null Infection" Model

Abstract

Purpose: : Bacterial inoculation of the healthy ocular surface does not result in infection. Little is known about the mechanisms involved in maintaining this resistant state. Here we used mouse and rat "null infection models" to study the rate at which large bacterial inocula are cleared from the healthy ocular surface, and whether the timing is modified by prior challenge. The role of surfactant protein-D (SP-D), required for protecting against bacterial colonization after scratch injury, was also examined.

Methods: : Healthy eyes of female Black Swiss mice (6-8 weeks old) were inoculated with GFP expressing P. aeruginosa strain PA01 (10⁷ cfu in 5 µl). Eyes were washed with PBS at 6 or 12 h to enumerate live bacteria by viable counts. In other experiments, wildtype or SP-D knockout mice were inoculated with bacteria for 12 h or 1 week before rechallenging the eyes for an additional 3 h. Female Lewis rats (3 month of age) were also examined. One group was pre-exposed to bacteria 2 weeks before rechallenging for 6 h (PA01, 10⁹ cfu in 10 µl). The other group was not pre-exposed. Bacteria recovered from contact lenses worn in rat eyes for 3 weeks were compared to bacteria that had not been exposed to the ocular surface. These animals were rechallenged twice, once immediately after washing, and again after an additional 12 hours. 3 h later viable counts were again performed.

Results: : Both mouse and rat eyes cleared 99.99% of bacteria within 3 h post-inoculation and all challenged eyes remained healthy. Irrespective of the timing of rechallenge, pre-exposure of the ocular surface to bacterial challenge, or of bacteria to the ocular surface had no influence on the rate at which eyes cleared bacteria. SP-D knockout mice cleared bacteria as efficiently as wildtype mice.

Conclusions: : Normal healthy eyes clear viable bacteria within a few hours. The timing is not modified by prior challenge of the eye to bacteria or vice versa. These data suggest the involvement of preformed innate defenses in resistance to infection by the ocular surface. While SP-D has been shown to play a role in clearing bacteria during infectious keratitis, it is not required for bacterial clearance by the healthy ocular surface.

Keywords: cornea: basic science • pseudomonas • immunomodulation/immunoregulation