OBJECTIVE: To determine whether plasmid-mediated quinolone resistance (PMQR) determinants play a role in the increasing resistance to fluoroquinolones among Escherichia coli isolates in Canadian hospitals, and to determine the mechanisms of reduced susceptibility to ciprofloxacin in a recent collection of 190 clinical E coli isolates.METHODS: E coli isolates (n=1702) were collected as part of the 2007 Canadian Hospital Ward Antibiotic Resistance Surveillance (CANWARD) study. Antimicrobial susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. Using a representative subset of isolates (n=190), the mechanisms of reduced susceptibility to ciprofloxacin were detected by polymerase chain reaction and sequencing of the quinolone resistance-determining regions (QRDR) of chromosomal gyrA and parC genes, and by polymerase chain reaction for the PMQR genes: qnr, aac(6) Ib-cr and qepA.RESULTS: 2.1% and 1.1% of E coli harboured aac(6)Ib-cr and qnrB, respectively. Single amino acid substitutions in the QRDR of gyrA were observed among isolates with ciprofloxacin minimum inhibitory concentrations as low as 0.12 μg/mL. As the ciprofloxacin minimum inhibitory concentration increased to 1 μg/mL (which is still considered to be susceptible by the CLSI), the vast majority of isolates demonstrated both gyrA and parC mutations.CONCLUSION: PMQR determinants and QRDR mutants among clinical E coli isolates with reduced susceptibility to ciprofloxacin demonstrates the need for increased surveillance and the need to re-evaluate the current CLSI breakpoints to prevent further development of fluoroquinolone resistance.