Abstract
Purpose: :
Extended contact lens wear is a major risk factor for P. aeruginosa keratitis, but underlying mechanisms of this disease are poorly understood. We previously reported that a single high-inoculum (109 cfu) of P. aeruginosa administered when fitting a low Dk contact lens enabled corneal infection in Lewis rats. Disease occurred within 5-14 days and did not require other manipulations to the cornea. Here we used lower inocula to more closely mimic "real world" environments.
Methods: :
Low Dk hydrogel contact lenses (custom fit for rat eyes) were placed in lens cases and soaked for 24 h in a minimal medium inoculated with 103 cfu/ml P. aeruginosa strain PAO1 expressing GFP. This resulted in 105 cfu bacteria adherent to each lens. Other lenses were inoculated with a suction pen (previously used for high inoculum experiments, but ethanol rinsed then dry-stored for 6 months). A third group contained lenses soaked in 1011 cfu/ml bacteria for 3 h (high-inoculum control). Contralateral eyes were untreated. Within 24 h of detecting corneal opacity, lenses were removed and transferred to the left eyes of naïve rats or were examined by confocal microscopy before homogenization to quantify viable bacteria. Infected corneas were washed with PBS to collect non-adherent bacteria then analyzed by immunohistochemistry.
Results: :
In each of the three situations (including inadvertent inoculation with a previously "sterilized" suction pen), all eyes wearing contaminated contact lenses developed corneal disease (onset 3-14 days). Eyes wearing lenses transferred from infected animals also became infected, some within 24 h. Phagocyte infiltration was found in corneas showing opacity. Once eyes were infected, consistent numbers of GFP-expressing bacteria were detected in the ocular wash (1.8±3.4 x105 cfu) and on worn lenses (7.8±6.8 x107 cfu), irrespective of the duration of lens wear. Confocal microscopy of worn lenses from infected eyes showed that classical bacterial biofilms had formed on the posterior, but not anterior, lens surfaces.
Conclusions: :
A single low bacterial inoculum (105 cfu) placed on the intact rat cornea with a contact lens enables infection. Disease onset was comparable for high and low inocula. Bacteria persisting at the ocular surface can form biofilms on the posterior lens surface and ultimately cause disease within 1-2 weeks. Lenses that have already developed biofilms in vivo can induce infection after only one day. These data suggest that biofilm formation and/or bacterial adaptation to the in vivo environment contribute to the pathogenesis of contact lens related keratitis.
Keywords: contact lens • bacterial disease • cornea: epithelium