2008 OMIG, Abstract 15

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In Vitro Susceptibility Testing of Staphylococcus aureus to a Cell-Associated Antibiotic
R.P. Kowalski, E.G. Romanowski, F.S.Mah, Y.J. Gordon, R.M.Q. Shanks.
The Charles T. Campbell Laboratory, University of Pittsburgh, Pittsburgh, PA

Purpose:Some antibiotics are cell-associated and their antibiotic efficacy may not be optimally evaluated with standard MIC determination methods. We developed and evaluated a cell-associated method of antibiotic efficacy testing to determine the susceptibility of Staphylococcus aureus strains to azithromycin (AZ) and ofloxacin (OFX). 
Methods:Chang conjunctival cells were grown to confluence in four 96 flat-well plates. The cells were washed, and incubated with medium containing decreasing concentrations (512 to 4 mg/ml) of AZ, OFX, or no-antibiotics. After 24 hours, the antibiotic-treated and control medium-treated cells were washed to remove non-cell-associated antibiotics, and challenged with four isolates of AZ-, OFX-susceptible Staphylococcus aureus (5 x 105 cfu/ml) for another 24 hours. The plates were washed and stained with gentian violet. Positive staining for deep-blue denoted that the conjunctival cell monolayer remained intact.  Positively stained wells were deemed protected from bacterial challenge and free of any antibiotic toxicity effects.
Results:Initial incubation of Chang conjunctival cells with >16 mg/ml of AZ fully protected all cells from challenge with all four Staphylococcus aureus isolates, while incubation with 128-256 mg/ml OFX was required for comparable protection. Toxicity was observed for OFX at 512 mg/ml while AZ exhibited no toxicity at this concentration.
Conclusions:Azithromycin protected Chang conjunctival cells from S. aureus challenge at lower doses than ofloxacin, suggesting that AZ is more cell associated than OFX. The susceptibility of bacteria to an antibiotic may be, in part, a function of the affinity of the antibiotic to host cellular components. Anti-bacterial activity and cell protection may not be accurately measured by standard susceptibility testing alone, and further study is warranted.

Support: Studies supported by Inspire Pharmaceuticals.
Disclosure Code: F,C,R