Christine Hesje, BSc; Joseph M. Blondeau, PhD Low Propensity of Gatifloxacin-BAK Combination to Select for Fluoroquinolone Resistance Among Methicillin-Resistant Staphylococcus aureus Christine Hesje, BSc; Joseph M. Blondeau, PhD Department of Clinical Microbiology, Royal University Hospital and the Departments of Microbiology and Immunology and Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Financial Disclosures Study supported by an unrestricted educational grant from Allergan, Inc. Christine Hesje and Joseph Blondeau have no financial interests in any product mentioned in this study

INTRODUCTION Staphylococcus aureus is a common cause of ocular infections Methicillin-resistant S aureus (MRSA) strains are now prevalent in both the hospital and community settings1 Co-resistance of MRSA to third-generation fluoroquinolones is well known2 The fourth-generation fluoroquinolone gatifloxacin has a reduced probability of resistance because 2 mutations are necessary for resistance to develop3 Older fluoroquinolones develop resistance with only 1 mutation

Purpose The ability of an antibiotic to overcome antimicrobial resistance can be evaluated using the mutant prevention concentration (MPC)4,5 MPC is defined as the drug concentration that prevents the growth of the most resistant first-step mutants in a large heterogeneous bacterial population In vitro potency of the gatifloxacin commercial formulation has been evaluated with the active ingredient alone Commercial formulation of gatifloxacin (Zymar®; Allergan Inc.; Irvine, CA) contains 0.005% benzalkonium chloride (BAK) as a preservative6 Recent studies from our laboratory demonstrated that the presence of BAK increases antimicrobial activity of gatifloxacin7 The purpose of this study was to determine the minimal inhibitory concentration (MIC) and MPC values of gatifloxacin, BAK, and the gatifloxacin-BAK combination against clinical isolates of MRSA

METHODS Seventeen clinical isolates of MRSA were tested MIC testing Bacteria (105 colony-forming units [CFU]/mL) were inoculated in Mueller-Hinton broth containing 2-fold concentration increments of the test agents The lowest concentration that prevented growth of 90% of bacteria was recorded as the MIC90 MPC testing Bacteria (1010 CFU/mL) were inoculated onto agar plates in the presence of increasing concentrations of the test agents The lowest drug concentration preventing bacterial growth was recorded as the MPC The range of gatifloxacin concentrations tested was from 8 µg/mL to < 0.004 µg/mL

Gatifloxacin-BAK Combination RESULTS The MIC90 of Gatifloxacin, BAK, and the Gatifloxacin-BAK Combination Against Clinical Isolates of MRSA 4.0 3.1 3.0 0.125 MIC90 (µg/mL) < 0.004 Gatifloxacin BAK Gatifloxacin-BAK Combination

Gatifloxacin-BAK Combination RESULTS The MPC of Gatifloxacin, BAK, and the Gatifloxacin-BAK Combination Against Clinical Isolates of MRSA 6a ≥ 4 MPC (µg/mL) < 0.004b Gatifloxacin BAK Gatifloxacin-BAK Combination aRanged from 6 to 10 µg/mL. bThe concentration of BAK was 10 µg/mL.

CONCLUSIONS The combination of gatifloxacin and BAK was highly active against MRSA in vitro The MIC of the gatifloxacin-BAK combination was over 30-fold and 775-fold lower than the MIC of gatifloxacin and BAK alone, respectively The MPC of the gatifloxacin-BAK combination was at least 1000-fold and 1500-fold lower than the MPC of gatifloxacin and BAK alone, respectively These findings suggest that Zymar® may have low propensity to select for fluoroquinolone-resistant MRSA

REFERENCES Blomquist PH. Methicillin-resistant Staphylococcus aureus infections of the eye and orbit. Trans Am Ophthalmol Soc. 2006;104:322-345. Marangon FB, Miller D, Muallem MS, Romano AC, Alfonso EC. Ciprofloxacin and levofloxacin resistance among methicillin-sensitive Staphylococcus aureus isolates from keratitis and conjunctivitis. Am J Ophthalmol. 2004;137(3):453-458. Hooper DC. Mechanisms of action and resistance of older and newer fluoroquinolones. Clin Infect Dis. 2000;31 (suppl 2):S24-S28. Hansen G, Blondeau JM. Mutant prevention concentrations as a strategy to minimize antimicrobial resistance: a timely concept but will its acceptance be too late? Therapy. 2005;2:61-66. Blondeau JM, Zhao X, Hansen GT, Drlica K. Mutant prevention concentrations (MPC) of fluoroquinolones for clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother. 2001;45(2):433-438. ZYMAR® [package insert]. Irvine, CA: Allergan, Inc.; 2004. Blondeau JM, Borsos S, Hesje CK. Antimicrobial efficacy of gatifloxacin and moxifloxacin with and without benzalkonium chloride compared with ciprofloxacin and levofloxacin against methicillin-resistant Staphylococcus aureus. J Chemother. 2007;19(2):146-151.