Treatment of Staphylococcus Aureus with Antibiotic and Quorum-Sensing Inhibitor Combinations Reduces Severity of Keratitis

Purpose: To examine whether treatment with ciprofloxacin and fimbrolides improves the outcome of Staphylococcus aureus keratitis. Methods: A S. aureus strain was tested for the minimum inhibitory concentration (MIC) of ciprofloxacin and fimbrolides, and whether they had synergistic antimicrobial activity. The effect of fimbrolides on production of α-toxin was measured after growth at sub-MIC and testing supernatants for hemolysis. Cytotoxicity of the fimbrolides was measured by adding increasing concentrations to corneal epithelial cells and measuring release of lactate dehydrogenase. Ten hours after scratching corneas of mice and applying bacteria, treatment was initiated with vehicle, ciprofloxacin, fimbrolide, or a combination of ciprofloxacin and fimbrolide. The number of infecting S. aureus cells was measured after 6 hours treatment, and clinical responses were recorded (maximum score 20). Results: The MIC of the fimbrolides was 6.25-12.5 μM and growth in fimbrolides reduced production of α-toxin by > 90%. Fimbrolides were only cytotoxic at ≥ 25 μM. Treatment of keratitis with ciprofloxacin significantly (p < 0.05) reduced numbers of S. aureus in corneas. Although there was minimal synergism between the two antimicrobials in vitro and treatment with the fimbrolide/ciprofloxacin combination in vivo did not significantly reduce numbers of S. aureus, there was a significantly reduced clinical score (by > 45%; p < 0.05) of the combination compared to single application of antimicrobials or vehicle control (average 4.25 ± 0.35). Conclusion: Application of non-toxic concentrations of quorum-sensing inhibitor fimbrolides in combination with ciprofloxacin reduce the numbers infecting bacteria to the same extent as ciprofloxacin alone, but also significantly improved the clinical outcome of S. aureus keratitis. Fimbrolides may be useful adjunct agents to treat keratitis. Citation: Willcox MDP, Hume EBH, Schubert TL, Kumar N. Treatment of Staphylococcus Aureus with Antibiotic and Quorum-Sensing Inhibitor Combinations Reduces Severity of Keratitis. J Ocular Biol. 2017;5(1): 5. J Ocular Biol 5(1): 5 (2017) Page 02 ISSN: 2334-2838 and ciprofloxacin was determined. Bacteria were cultured in tryptone soya broth (TSB; Oxoid, Basingstoke UK) overnight at 37 °C and then resuspended in TSB. Two microliters of stock fimbrolide (in absolute ethanol) or ciprofloxacin hydrochloride (ICN Biomedicals Australasia, Australia; diluted to stock concentration of 10 mg/ml in sterile water) was added to a final concentration of 0.31 μM to 50 μM for fimbrolide or 0.002 μg/ml to 1 mg/ml for ciprofloxacin in TSB containing 1 x 105 S. aureus cfu/ml in a chequerboard design [28]. Absolute ethanol (2 μl) in TSB was used as a control. The MIC was determined as the lowest concentration of fimbrolide or ciprofloxacin that inhibited bacterial growth after incubation for 18 hours at 37 °C. Fractional inhibitory concentration of ciprofloxacin in the presence or absence of fimbrolides Synergy of the antimicrobials was determined using the sum of the fractional inhibitory concentrations (FIC) of antibiotic and fimbrolide. FIC index was used to determine synergy and was calculated using the following equation: FIC index = ((MIC of antibiotic + fimbrolide)/MIC of fimbrolide)/(MIC of antibiotic + fimbrolide)/MIC of antibiotic)). Synergism was defined as an FIC index of ≤ 0.5 indifference as an FIC index of > 0.5 but ≤ 4 and antagonism as an FIC index of > 4 [29]. Effect of fimbrolide on production of α-toxin The effect of the fimbrolide on the production of α-toxin was measured Briefly; S. aureus 38 was grown in TSB overnight at 37 °C in the presence or absence of a sub-inhibitory concentration of each furanone (2.5 μM). After growth, cells were removed by centrifugation (5,000 g 10 mins) and the supernatants removed and filtered through 0.22 μm filters. Citrated rabbit erythrocytes were purchased from Bioreclamation IVT (NY, USA), washed in PBS (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4, pH 7.3) and resuspended in PBS and added to wells of a 96 well micro titer plate at approximately 107 erythrocytes/well [30]. Bacterial supernatants were added and the mixtures allowed incubating at 37 °C for 1 hour. After that time, plates were centrifuged, the supernatants removed and the level of hemolysis was estimated by measuring the absorbance of the supernatants at OD 545 nm.