qnrB, another plasmid-mediated gene for quinolone resistance

Abstract

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.

FIG. 1.

CLUSTAL W alignment of QnrA, QnrB1, QnrB2, and QnrS.

FIG. 2.

Maps of cloned qnrB genes. The sizes of the cloned fragments vary, as indicated by the 1-kb reference bars. Orf1 resembles pspF, Orf2 is related to genes of unknown function in various gram-negative bacteria, and Orf3 resembles sapA.

FIG. 3.

QnrB1 protection of DNA gyrase from ciprofloxacin inhibition of supercoiling. Reaction mixtures of 30 μl were analyzed by agarose gel electrophoresis. Reaction mixtures contained 0.2 μg relaxed pBR322 DNA (lanes 1 to 14), 6.7 nM gyrase (lanes 2 to 14), 2 μg/ml ciprofloxacin (lanes 3 to 14), and QnrB-His₆ fusion protein at 25 μM (lane 4), 5 μM (lane 5), 2.5 μM (lane 6), 0.5 μM (lane 7), 50 nM (lane 8), 5 nM (lane 9), 0.5 nM (lane 10), 50 pM (lane 11), 5 pM (lane 12), or 0.5 pM (lane 13).