2010 OMIG, Abstract 20

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Cell-associated protection assay (CAPA) demonstrates antibiotic protection of ocular surface cell lines against clinical S. aureus even after all free antibiotic is removed from the culture medium
J.B. Wingard, E.G. Romanowski, R.P. Kowalski, F.S. Mah, Y.J. Gordon, R.M.Q. Shanks, The Charles T. Campbell Laboratory, UPMC Eye Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA

Purpose: Standard antibiotic testing only evaluates the interaction between the bacteria and the drug, ignoring the interplay with the treated tissue.  We developed a novel cell-associated protection assay (CAPA) to evaluate the role of antibiotic-epithelial cell association in the effectiveness of antibiotic protection.  This assay was employed to test the cell-associated efficacy of azithromycin (AZ), erythromycin (ER), tetracycline (TET), and bacitracin (BAC).
Methods: Chang conjunctival and human corneal limbal epithelial (HCLE) cells were grown to confluence in 96-well plates using antibiotic-free media. Cells were incubated in triplicate with AZ, ER, TET, and BAC (0-512 μg/ml). After 24 hours, cells were washed and challenged with 6 S. aureus conjunctivitis 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 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. Experiments were repeated at least twice per cell line.  Maximum likelihood estimation was used to model the antibiotic dose effect on epithelial cell layer survival, and we defined the 50% effect dose as a statistic to compare antibiotics, cell lines, and bacterial strains. Antibiotic toxicity was determined with Alamar blue.
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. TET toxicity was demonstrated at 64 mg/ml, whereas no other antibiotic demonstrated consistent toxicity except at the highest studied doses.
Conclusions: The CAPA assay demonstrated that AZ, ER, and TET protected epithelial cells from bacterial challenge even when all free antibiotic was removed from the culture, although TET although displayed toxicity. BAC did not demonstrate protection.This assay can be used to assess a variety of antibiotic-epithelial cell line-bacteria interactions.
Support:  Inspire Pharmaceuticals, NIH EY08098; Disclosure code: F,C