Mechanism of plasmid-mediated quinolone resistance

Infectious Disease Department, Lahey Clinic, Burlington, MA 01805

^{To whom reprint requests should be addressed at: Lahey Clinic, 41 Mall Road, Burlington, MA 01805. E-mail:}gro.yehal@ybocaj.a.egroeg.

Communicated by Paul C. Zamecnik, Massachusetts General Hospital, Charlestown, MA

Received 2001 Dec 20; Accepted 2002 Feb 15.

Abstract

Quinolones are potent antibacterial agents that specifically target bacterial DNA gyrase and topoisomerase IV. Widespread use of these agents has contributed to the rise of bacterial quinolone resistance. Previous studies have shown that quinolone resistance arises by mutations in chromosomal genes. Recently, a multiresistance plasmid was discovered that encodes transferable resistance to quinolones. We have cloned the plasmid-quinolone resistance gene, termed qnr, and found it in an integron-like environment upstream from qacEΔ1 and sulI. The gene product Qnr was a 218-aa protein belonging to the pentapeptide repeat family and shared sequence homology with the immunity protein McbG, which is thought to protect DNA gyrase from the action of microcin B17. Qnr had pentapeptide repeat domains of 11 and 28 tandem copies, separated by a single glycine with a consensus sequence of A/C D/N L/F X X. Because the primary target of quinolones is DNA gyrase in Gram-negative strains, we tested the ability of Qnr to reverse the inhibition of gyrase activity by quinolones. Purified Qnr-His₆ protected Escherichia coli DNA gyrase from inhibition by ciprofloxacin. Gyrase protection was proportional to the concentration of Qnr-His₆ and inversely proportional to the concentration of ciprofloxacin. The protective activity of Qnr-His₆ was lost by boiling the protein and involved neither quinolone inactivation nor independent gyrase activity. Protection of topoisomerase IV, a secondary target of quinolone action in E. coli, was not evident. How Qnr protects DNA gyrase and the prevalence of this resistance mechanism in clinical isolates remains to be determined.

Abstract

Since the introduction of nalidixic acid in the 1960s, quinolones have been increasingly used to treat a variety of infectious diseases. This widespread use has been followed by increasing bacterial resistance (1). Previous studies have shown that quinolone resistance arises by mutations in the chromosomal genes for type II topoisomerases, the targets of quinolone action (2), and by changes in expression of efflux pumps and porins that control the accumulation of these agents inside the bacterial cell (3).

In bacteria, the type II topoisomerases DNA gyrase and topoisomerase IV change the topology of DNA. These enzymes cleave both strands of DNA to allow one double-stranded DNA molecule to pass through another and then reseal the break. DNA gyrase wraps DNA around itself, and so introduces negative superhelical twists into DNA, whereas topoisomerase IV does not, and consequently relaxes DNA. Quinolones inhibit these enzymes by stabilizing the complex between DNA and DNA gyrase or topoisomerase IV and thus block progression of polymerase and DNA replication (4, 5).

Both enzymes are tetramers. DNA gyrase comprises two GyrA and two GyrB subunits. Mutations in the N-terminal domain of GyrA or the C-terminal portion of GyrB augment quinolone resistance, typically 4- to 8-fold (6). In Escherichia coli, mutations in topoisomerase IV do not confer resistance by themselves but enhance resistance attributable to GyrA, increasing it another 4- to 8-fold (2).

Transmissible resistance to quinolones, long thought not to exist (7), has recently been discovered on a plasmid from a resistant clinical isolate (8). The plasmid augments resistance to nalidixic acid, ciprofloxacin, and other fluoroquinolones 4- to 8-fold and supplements resistance due to gyrA, gyrB, and porin or efflux pump mutations (L. Martínez-Martínez, A. Pascual, I. García, J.H.T. and G.A.J., unpublished data). In plasmid-containing strains no change in quinolone accumulation, outer membrane porins, or drug inactivation could be detected, implying a resistance mechanism different from those currently known. Here we report the cloning of qnr, the novel plasmid-encoded quinolone resistance gene, the amplification and purification of Qnr, its gene product, and the demonstration that Qnr directly protects E. coli DNA gyrase from quinolone inhibition.

Acknowledgments

This work was supported by Public Health Service Grant AI43312 from the National Institute of Allergy and Infectious Diseases (to G.A.J.).

Acknowledgments

Footnotes

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY070235","term_id":"19568070","term_text":"AY070235"}}AY070235).

Footnotes

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