To substantiate a common genetic background of ciprofloxacin-resistant Enterococcus faecium, 32 ciprofloxacin-resistant (Cip(r)) and 31 ciprofloxacin-susceptible (Cip(s)) isolates from outbreaks, clinical infections, surveillances, and animals from 10 different countries were genotyped by multilocus sequence typing. Additionally, susceptibilities to ampicillin and vancomycin and the presence of esp were determined and the quinolone resistance-determining regions of parC, gyrA, parB, and gyrE were sequenced. High-level Cip(r) (MIC > or = 64 microg/ml) due to point mutations in the quinolone resistance-determining region was unique to a distinct hospital-adapted genetic complex in E. faecium, previously designated CC17. Low-level Cip(r) (MIC = 4 microg/ml) in non-CC17 strains is not attributable to point mutations in any subunit of the topoisomerase genes, and the mechanism of resistance remains unclear. Acquisition of mutations in parC and gyrA, leading to high-level Cip(r), is, in addition to ampicillin resistance and the presence of a putative pathogenicity island, another cumulative step in hospital adaptation of CC17.