Objective: Many clinical microbiology laboratories procure antimicrobial susceptibility testing data using guidelines established by Clinical and Laboratory Standards Institute (CLSI). When necessary, CLSI revises interpretive breakpoints in efforts to improve clinical correlation, with two revisions relative to fluoroquinolone agents occurring in 2019. The purpose of this investigation was to determine the impact of fluoroquinolone breakpoint revisions on Wisconsin clinical isolates of Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa.Design: Multi-center laboratory surveillance, with testing at a single location utilizing standardized media and susceptibility testing protocols.Methods: From the Surveillance of Wisconsin Organisms for Trends in Antimicrobial Resistance and Epidemiology (SWOTARE) program, levofloxacin and ciprofloxacin minimum inhibitory concentration (MIC) values for 1911, 1521, and 1463 Wisconsin isolates of E. coli, P. mirabilis, and P. aeruginosa, respectively, were determined by broth microdilution testing. In separate data analyses, all MIC data were interpreted using CLSI breakpoints published prior to 2019, then secondarily by using CLSI breakpoints published since 2019 (which reflect lower breakpoints for both levofloxacin and ciprofloxacin resistance). Findings were further stratified by Wisconsin Department of Health Services region.Results: Up to 3.2% decreased statewide fluoroquinolone susceptibility was observed for E. coli isolates, while 5.1% and 6.3% decreases in levofloxacin susceptibility were noted for P. aeruginosa and P. mirabilis isolates, respectively, when revised breakpoints were applied. E. coli isolates from the Western region and P. mirabilis isolates from the Southeastern region demonstrated significant shifts toward decreased fluoroquinolone susceptibility upon application of revised breakpoints. Northern region P. mirabilis isolates exhibited consistently decreased fluoroquinolone susceptibility.Conclusions: Fluoroquinolone resistance has been underreported in Wisconsin as a whole, yet geographic variability continues to exist. Targeted annual surveillance is important to identify and monitor resistance trending. Compilations of SWOTARE surveillance data can be utilized to predict the impact of future CLSI interpretive breakpoint revisions in Wisconsin.