A novel plasmid-mediated quinolone resistance gene, qnrB, has been discovered in a plasmid encoding the CTX-M-15 beta-lactamase from a Klebsiella pneumoniae strain isolated in South India. It has less than 40% amino acid identity with the original qnr (now qnrA) gene or with the recently described qnrS but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with qnrB demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to >or=95% homogeneity. As little as 5 pM of QnrB-His6 protected purified DNA gyrase against inhibition by 2 microg/ml (6 microM) ciprofloxacin. With a PCR assay qnrB has been detected in Citrobacter koseri, Enterobacter cloacae, and Escherichia coli isolates from the United States, linked to SHV-12 beta-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The qnrB gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.