Fluoroquinolones, such as ciprofloxacin, have been very useful antimicrobial agents because they are highly potent, active against a wide range of bacteria, and relatively non-toxic. Their broad use, however, has been followed by rising rates of resistance. Quinolone resistance has traditionally been understood to arise either by mutations that alter DNA gyrase and topoisomerase IV, enzymes that are the targets for quinolone action, or by mutations that increase expression of efflux pumps that actively eliminate the agents from the cell. Neither type of resistance has been transmissible since both are due to mutations on the bacterial chromosome. Hence, it came as a surprise when plasmid-mediated quinolone resistance was discovered in 1998. Three distinct mechanisms for such resistance are known: target protection by pentapeptide repeat proteins of the Qnr family that bind gyrase and may act in part as DNA mimics, quinolone inactivation by a mutant aminoglycoside 6'N-acetyltransferase [Aac(6')-Ib-cr], and provision of new systems for quinolone efflux. Each mechanism confers low-level resistance but facilitates selection of higher level, clinically significant resistance. The Qnr family is largest group with QnrA, QnrB, QnrS, QnrC, and QnrD subgroups and is now distributed worldwide with the qnr genes generally present within integrons on multidrug resistance plasmids. This renewal application builds on our prior studies to obtain a deeper and more detailed understanding of the resistance due to Qnr proteins.
Under Specific Aim 1, based on our mutant Qnr studies we propose to perform site-specific cross linking of key residues of Qnr B with gyrase and analysis of sites of linkage by mass spectrometry. Bacterial 2-hybrid studies will also be used to evaluate binding of specific QnrB and gyrase mutants for their interactions in intact cells.
Under Specific Aim 2, we propose to evaluate the native functions of qnr genes and a novel pathway of regulation of qnrS expression. Having excluded Qnr protection of natural gyrase toxins CcdB and ParE, we will next evaluate its protection from natural gyrase toxin MccB17 using plasmid constructs with graded expression of toxin and Qnr. We will also construct deletions of the native homologs qnrA and qnrS in Shewanella algae and Vibrio splendidus, the respective reservoir organisms and test for differences in quinolone susceptibility and growth under environmental conditions relevant for their native habitats. We will also construct a qnrS-lacZ transcriptional fusion to screen an E. coli transposon mutant library for genes necessary for the SOS-independent induction of qnrS by ciprofloxacin.
Under Specific Aim 3, we propose to assess Qnr interactions with other proteins by testing the effects of qnr on expression of other E. coli genes in a transcripitional microarray with confirmation in the native organisms and by coimmunoprecipitation experiments for direct identification of binding partners with mass spectrometry analysis.

Public Health Relevance

Quinolones are widely used antimicrobial agents that have been compromised by bacterial resistance, which was originally thought only to occur from chromosomal mutation. Plasmid-encoded transferable resistance has now been shown to have emerged linked to multidrug resistance and to have spread worldwide to many gram-negative human pathogens. The most widespread mechanism involves Qnr proteins that interact with topoisomerases, the quinolone target enzymes. Thus, understanding of these mechanisms of resistance and how they protect the target enzymes from quinolone action and their natural reservoirs and possible other natural functions is of importance for public health and for understanding of the range of bacterial adaptation strategies.

National Institute of Health (NIH)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Huntley, Clayton C
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Massachusetts General Hospital
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Tavío, María M; Jacoby, George A; Hooper, David C (2014) QnrS1 structure-activity relationships. J Antimicrob Chemother 69:2102-9
Liao, Chun-Hsing; Hsueh, Po-Ren; Jacoby, George A et al. (2013) Risk factors and clinical characteristics of patients with qnr-positive Klebsiella pneumoniae bacteraemia. J Antimicrob Chemother 68:2907-14
Jacoby, George A; Corcoran, Marian A; Mills, Debra M et al. (2013) Mutational analysis of quinolone resistance protein QnrB1. Antimicrob Agents Chemother 57:5733-6
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Kwak, Yee Gyung; Jacoby, George A; Hooper, David C (2013) Induction of plasmid-carried qnrS1 in Escherichia coli by naturally occurring quinolones and quorum-sensing signal molecules. Antimicrob Agents Chemother 57:4031-4
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Kim, Hong Bin; Park, Chi Hye; Gavin, Mariah et al. (2011) Cold shock induces qnrA expression in Shewanella algae. Antimicrob Agents Chemother 55:414-6
Vetting, Matthew W; Hegde, Subray S; Wang, Minghua et al. (2011) Structure of QnrB1, a plasmid-mediated fluoroquinolone resistance factor. J Biol Chem 286:25265-73
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Kim, Hong Bin; Wang, Minghua; Ahmed, Sabeena et al. (2010) Transferable quinolone resistance in Vibrio cholerae. Antimicrob Agents Chemother 54:799-803

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