Pretreatment of Ocular Surface Cell Lines With Cell-Associated Antibiotics Protects Against Clinical Ocular Staphylococcus aureus Challenge

Abstract

Purpose: : Standard MIC determination may not optimally evaluate the effectiveness of certain antibiotics in protecting the ocular surface against bacterial infection. In particular, antibiotic association with epithelial cells may play a key role. We used a previously described assay to evaluate the cell-associated efficacy of azithromycin (AZ), erythromycin (ER), tetracycline (TET), and bacitracin (BAC). Antibiotic toxicity was also evaluated.

Methods: : Chang conjunctival and human corneal limbal epithelial (HCLE) cells were grown to confluence in 96-well plates using antibiotic-free media. Cells were washed and incubated in triplicate in media containing AZ, ER, TET, and BAC (0-512 µg/ml). After 24 hours, cells were washed 2X and challenged with 6 clinical conjunctivitis/blepharitis S. aureus isolates (5x10⁵ CFU) without antibiotics in the culture media. After another 24 hours, bacterial growth and epithelial cell layer survival were assessed. Bacterial viability was determined by culture turbidity (A=600 nm) and growth on blood agar plates. Epithelial cells were stained with gentian violet, with positive staining representing intact monolayers. After imaging each plate, dye was solubilized and measured at A=590 nm. Antibiotic toxicity was determined with alamar blue. Experiments were repeated at least twice per cell line.

Results: : Incubation of Chang and HCLE cells with AZ, ER, and TET at ≥64 µg/ml provided protection against challenge with AZ-susceptible S. aureus strains, with increasing protection at higher concentrations. This was shown in turbidity assays and confirmed by bacterial outgrowth from supernatants as well as visual observation and quantitative analysis of gentian violet staining. TET toxicity was demonstrated at 64 µg/ml, whereas no other antibiotic demonstrated consistent toxicity even at 512 µg/ml.

Conclusions: : A spectrum of protective efficacy against S. aureus challenge was displayed by the four antibiotics tested, with consistent results across all assays. AZ, ER, and TET were all protective, but TET also demonstrated toxicity. BAC did not demonstrate protection. In vivo effectiveness of antibiotic therapy for conjunctivitis and blepharitis may depend on the ability of the antibiotic to associate with epithelial cells to provide continued protection.

Keywords: bacterial disease • antibiotics/antifungals/antiparasitics • cornea: basic science