The Development of a Novel in vitro Efficacy Testing Method for a Cell-Associated Antibiotic

Abstract

Purpose: : Some antibiotics are cell-associated and their efficacy may not be optimally evaluated using standard MIC methods. We developed and evaluated a novel in vitro method of antibiotic efficacy testing for cell-associated antibiotics. Specifically we determined the ability of cell-associated azithromycin (AZ) and ofloxacin (OFX) to protect conjunctival cells from infection with Staphylococcus aureus (SA).

Methods: : Chang conjunctival epithelial cells were grown to confluence in 96 well plates. Triplicate wells were incubated with medium containing a range of concentrations (4-512 µg/ml) of AZ, OFX, or no-antibiotics. After 24 hrs, the antibiotic and control medium-treated cells were washed to remove non-cell-associated antibiotics. The cells were then challenged with 4 isolates of AZ-, OFX-susceptible (AZ MICs ≤1.5 µg/ml; OFX MICs ≤0.75 µg/ml) SA(5 x 10⁵ CFU/ml) for 24 hrs. Subsequently, the cells were stained with gentian violet. Positive deep-blue staining indicated that the cell monolayer was intact and was deemed protected from bacterial challenge and free of any antibiotic toxicity. Unstained wells were deemed unprotected from bacterial challenge and/or demonstrated antibiotic toxicity. Microscopy verified the presence or lack of cells.

Results: : Incubation of conjunctival epithelial cells with ≥32 µg/ml of AZ fully protected all cells from challenge with all 4 SAisolates, while incubation with 256 µg/ml of OFX was required for comparable protection for some isolates but was inadequate for others. Toxicity was observed for OFX at 512 µg/ml whereas AZ exhibited no toxicity.

Conclusions: : AZ protected conjunctival cells from SA challenge at a dose at least 8-fold lower than OFX, suggesting that AZ is more cell associated than OFX. The susceptibility of bacteria to an antibiotic in vivo may be, in part, a function of the affinity of the antibiotic to host cellular components. Antibacterial activity and cell protection may not be accurately measured by standard susceptibility testing alone, and further study is warranted.

Keywords: antibiotics/antifungals/antiparasitics • conjunctivitis • Staphylococcus