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We conducted a 12-year study to identify and compare trends in annual prevalence of fluoroquinolone (FQ) resistance among Enterobacteriaceae isolates obtained from inpatients and outpatients in our health care system. A total of 46,070 clinical Enterobacteriaceae isolates underwent susceptibility testing. Although there were significant increases in inpatient FQ resistance for all Enterobacteriaceae, FQ resistance trends differed significantly across Enterobacteriaceae (P < .001). For isolates obtained from outpatients, only Escherichia coli and Proteus mirabilis demonstrated significant increases in FQ resistance (P < .001 for each). Trends in outpatient FQ resistance also differed significantly across Enterobacteriaceae (P < .001). There were significant differences between inpatient and outpatient FQ resistance trends for all Enterobacteriaceae except P. mirabilis and Enterobacter cloacae. Although hospital-wide use of certain antibiotics correlated significantly with inpatient FQ resistance, these correlations differed substantially across organisms. Efforts to elucidate the epidemiology of FQ resistance and identify targets for intervention must recognize and account for the variability of FQ resistance across organisms and clinical settings.