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Purpose: Fluoroquinolones, rapidly gaining prominence in treatment of Stenotrophomonasmaltophilia (SMP), are noted for their potency and tolerability. However, SMP may rapidly acquire resistance to fluoroquinolones. We evaluated associations of clinical factors with acquisition of levofloxacin resistance (LFr) in SMP. Materialsand Methods: Our retrospective cohort study was based on patient data collectedbetween January 2008 and June 2010. Through screening of 1275 patients, we identified 122 patients with data for SMP antibiotic susceptibility testing in ≥3 serial SMP isolates. Results: We assigned the 122 patients to either the SS group (n=54) in which levofloxacin susceptibility was maintained or the SR group (n=31) in which susceptible SMP acquired resistance. In multivariate regression analysis, exposure to levofloxacin for more than 3 weeks [odds ratio (OR) 15.39, 95% confidential interval(CI) 3.08--76.93, p=0.001] and co-infection or co-colonization with Klebsiella pneumoniae resistant to levofloxacin (OR 4.85, 95% CI 1.16--20.24, p=0.030) were independently associated with LFr acquisition in SMP. Conclusion: Acquisition of LFr during serial sampling of SMP was related to the levofloxacin exposure.