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To determine the prevalence and characteristics of bloodstream isolates of Escherichia coli and Klebsiella pneumoniae with qnr genes in a Taiwanese hospital.A total of 2035 E. coli and 1147 K. pneumoniae isolates collected between 1999 and 2005 were screened for qnrA, qnrB and qnrS by PCR and colony hybridization. β-Lactamase genes, genetic relatedness and transferability were examined by PCR, PFGE and conjugation, respectively.The prevalence of qnr genes was 7.8% and 0.6% for K. pneumoniae and E. coli, respectively. The prevalence rates of qnrB2, qnrB4 and qnrS1 genes for K. pneumoniae were 2.3%, 3.6% and 2.8%, respectively, and for E. coli were 0.2%, 0% and 0.4%, respectively. The prevalence of qnrB4 in K. pneumoniae increased remarkably from 0% to 7.6% over the 7 study years. qnrA was not detected. Overall, the SHV-5-related, CTX-M-14, CTX-M-3, CMY-2, DHA-1 and IMP-8 β-lactamases were detected alone or in combination in 82.0% of qnr-positive K. pneumoniae isolates and 41.7% of qnr-positive E. coli isolates. Notably, all qnrB4-positive isolates possessed the DHA-1 enzyme, and the majority of the qnrB2-positive isolates (E. coli, 100%; K. pneumoniae, 80.8%) produced SHV-5-related β-lactamases. Genetic diversity was demonstrated by PFGE. Conjugation experiments revealed co-transfer of blaSHV-12, blaDHA-1 and blaSHV-5 with qnrB2, qnrB4 and qnrS1, respectively.qnr genes remained rare in E. coli but appeared to be increasing in K. pneumoniae in our hospital. Horizontal transfer may play a major role in the intra-hospital spread of qnr.