New assay helps determine antibiotic efficacy for ocular surface infections

A newly developed assay can measure the ability of antibiotics to associate with mammalian cells and protect the cells from bacterial destruction. Of 4 antibiotics tested in this assay, the data showed a range of therapeutic effectiveness in 2 cell lines that clinicians can consider when establishing dosing regimens, according to Jeremy Wingard, MD, who reported his group’s findings at the American Society of Cataract and Refractive Surgery annual meeting, San Diego.

“Standard antibiotic testing evaluates only the interaction between the bacteria and the drug. However, the tissue likely contributes to the effectiveness of antibiotic therapy,” Dr Wingard said.

Dr Wingard and his colleagues at The Charles T. Campbell Ophthalmic Microbiology Laboratory, UPMC Eye Center at the University of Pittsburgh, suggested that the association of antibiotics with epithelial cells may play a role in the therapeutic efficacy of treatments used to treat ocular surface infections. With that in mind, they set out to develop an assay that measures the ability of antibiotics to associate with ocular tissues. Their dual hypothesis was that different antibiotics have different affinities for epithelial cells and that cell-associated antibiotics can protect ocular surface cell lines from bacterial challenge even after antibiotics are removed from the culture medium.

The investigators grew a monolayer of mammalian cells in antibiotic-free media and incubated the cells in media containing one antibiotic. The cells then were challenged with Staphylococcus aureus ocular isolates without antibiotics in the media. The integrity of the epithelial cells was assessed by gentian violet staining, and the minimal cell-layer protective concentration of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined.

“Pre-incubation of the cell lines, Chang conjunctival and human corneal-limbal epithelial cells, with azithromycin, erythromycin, and tetracycline at 64 µg/ml or greater, provided protection against azithromycin-susceptible S. aureus strains, and protection increased with higher concentrations. Tetracycline was toxic at concentrations exceeding 64 µg/ml, and azithromycin was toxic to one cell line at 512 µg/ml. Bacitracin protection was inconsistent at any dose, despite bacterial susceptibility to bacitracin as determined by traditional antibiotic susceptibility testing,” Dr Wingard reported.

The investigators concluded that their new assay can measure the impact of an antibiotic’s association with epithelial cells on its efficacy in vitro. Azithromycin and erythromycin afforded epithelial cells protection, bacitracin did not, and tetracycline was cytotoxic to the two ocular epithelial cell lines. The research was funded by Inspire Pharmaceuticals, National Institutes of Health CORE Grant P30 EY008098, Eye and Ear Foundation of Pittsburgh, and an unrestricted Grant from Research to Prevent Blindness, New York. The test is not yet commercially available.