Qnr-mediated Bacterial Quinolone Resistance Mechanisms

Fluoroquinolone resistance has been increasing in Gram-negative pathogens worldwide. The traditional understanding that quinolone resistance is acquired only through mutation and transmitted only vertically does not entirely account for the relative ease with which resistance develops in exquisitely susceptible organisms, or for the very strong association between resistance to quinolones and to other agents. The recent discovery of plasmid-mediated horizontally transferable genes encoding quinolone resistance might shed light on these phenomena. The Qnr proteins, capable of protecting DNA gyrase from quinolones, have homologues in water-dwelling bacteria, and seem to have been in circulation for some time, having achieved global distribution in a variety of plasmid environments and bacterial genera. AAC(6')-Ib-cr, a variant aminoglycoside acetyltransferase capable of modifying ciprofl oxacin and reducing its activity, seems to have emerged more recently, but might be even more prevalent than the Qnr proteins. Two plasmid-mediated quinolone transporters have now been found:OqxAB and QepA. Plasmid-medicated quinolone resistance mechanisms provide low-level quinolone resistance that facilitates the emergence of higher-level resistance in the presence of quinolones at therapeutic levels. Much remains to be understood about these genes, but their insidious promotion of substantial resistance, their horizontal spread, and their co-selection with other resistance elementsindicate that a more cautious approach to quinolone use and a reconsideration of clinical breakpoints are needed.Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6_)-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25μg/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindⅢ, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP 10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%,42%,59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.Plasmid-mediated Qnr proteins provide low-level quinolone resistance and protect bacterial DNA gyrase and topoisomeraseⅣfrom quinolone inhibition. QnrA, QnrB, and QnrS are currently known. All are pentapeptide repeat proteins differing from each other by 40% or more in amino acid sequence, while within each type minor variations in sequence define alleles such as QnrB1 and QnrB2. In addition to protecting DNA gyrase, QnrB1 (but not QnrA1) at high concentrations has been shown to inhibit the enzyme in vitro, which may explain the bacterial growth inhibition observed when the gene is maximally expressed. We have discovered that qnrB is regulated by the SOS system so that quinolone exposure augments its expression. To determine whether expression of qnrB alleles is under SOS control, plasmids were introduced into Escherichia coli GW1000 with recA441, which encodes a RecA protease that is more easily activated. GW1000 derivatives containing plasmids with qnrB alleles demonstrated two-to eightfold decreases in ciprofloxacin susceptibility as the growth temperature increased from 21℃to 43℃. A two-to threefold decrease in susceptibility was also seen in strains with plasmids carrying qnrA1 or qnrS1 alleles. In E. coli J53 with unmodified SOS regulation, temperature had only a twofold effect on the level of qnrB1-mediated ciprofloxacin resistance. While the trend observed suggested that qnrB alleles are specifically regulated by the SOS system, the MIC results were not clear-cut because of a background effect of temperature on quinolone susceptibility. To document SOS regulation directly, the expression of qnr genes was measured by real-time quantitative PCR after a 15-to 30-min exposure to agents known to trigger the SOS response. In E. coli J53 with intact lexA and recA genes, expression of qnrB alleles increased between 2.1-and 9.9-fold in response to the inducing agents while expression of qnrA1 was unchanged. Proof that this increase in qnrB expression required an intact SOS system was obtained with a set of related strains. Expression of qnrB4 increased in response to ciprofloxacin or mitomycin C in E. coli AB1157 with wild-type lexA and recA genes but not in two strains derived from it:strain AB1157 LexA300::spec, which has a defective LexA protein so that LexA-regulated genes are constitutively expressed, or strain DM49, which has a protease-resistant LexA product and consequently is defective in SOS induction. SOS regulation of QnrB could be a carryover reflecting a role for this topoisomerase-interacting protein in response to DNA damage. Alternatively, SOS regulation serves to protect the host cell from the potentially toxic effects of QnrB while allowing augmented production upon exposure to quinolone antimicrobial agents.Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of the high resistance rates to other agents, but its utility has been compromised by decreasing ciprofloxacin susceptibility of Vibrio cholerae over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in gyrA, encoding a subunit of DNA gyrase, followed by mutation in parC, encoding a subunit of DNA topoisomeraseⅣ. We found that ciprofloxacin activity was more recently further compromised in strains containing qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that qnrVC3 confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae and on the chromosome of Escherichia coli transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in V. cholerae are linked to cumulative mutations in quinolone targets and most recently to a qnr gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.