Review
The worldwide emergence of plasmid-mediated quinolone resistance

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Summary

Fluoroquinolone resistance is emerging 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 ciprofloxacin and reducing its activity, seems to have emerged more recently, but might be even more prevalent than the Qnr proteins. Both 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 elements indicate that a more cautious approach to quinolone use and a reconsideration of clinical breakpoints are needed.

Introduction

The development of quinolone resistance by Gram-negative pathogens constitutes a remarkable bacterial success story. Quinolones were introduced into clinical use in 1962 in the form of nalidixic acid,1 a fully synthetic agent with bactericidal effects on most Enterobacteriaceae at clinical concentrations. A pharmacological innovation—addition of a fluorine at the C-6 position and piperazinyl or related ring at position C-7 of the quinolone molecule—yielded the fluoroquinolones, first available clinically in the 1980s.2 These agents achieve higher serum levels than those of nalidixic acid and are more potent against Enterobacteriaceae; drug concentrations 1000-fold those required to inhibit growth are routinely achieved. Thus, these agents entered into use endowed with two advantages over the bacteria. First, although organisms could develop mutations that reduced quinolone susceptibility, the potency of these agents was such that a wild-type Escherichia coli would need to acquire spontaneously two or more resistance mutations to survive at clinical drug concentrations. Since independent mutations generally arise once per 107 cell divisions or less, the likelihood that multiple mutations would occur in a single clone seemed negligible. Second, many resistance genes have co-evolved in nature with the antibiotics that they counteract, especially those that modify or inactivate the drug. Since the quinolones are fully synthetic, it seemed unlikely that resistance genes would be available for recruitment onto mobile elements. Thus, the quinolones seemed to confound resistance; they were a class of agents to which mutational resistance was unlikely to develop and against which resistance genes could not be acquired.

Over the 20 years that have elapsed since the introduction of fluoroquinolones, resistance to these agents by Enterobacteriaceae has become common and widespread, and, remarkably, is generally not clonal. This finding implies that fluoroquinolone resistance has arisen many times in organisms that were once exquisitely susceptible. A recent survey of enteric bacteria in US intensive care units found that more than 10% of these organisms were resistant to ciprofloxacin.3 Levels of quinolone resistance in clinical E coli isolates have been reported at 40% in Hong Kong,4 and about 25% of healthy individuals living in Barcelona were found to be intestinally colonised with quinolone-resistant E coli.5

Until recently, two mechanisms of resistance had been found to determine resistance to fluoroquinolones (and quinolones, since in almost all cases organisms resistant to fluoroquinolones are resistant to nalidixic acid as well). The most important of these mechanisms in Enterobacteriaceae is the accumulation of mutations in the bacterial enzymes targeted by fluoroquinolones: DNA gyrase and DNA topoisomerase IV.6 When bound to DNA, these enzymes transiently break the closed circular DNA molecule, pass another strand through the break, and then reseal the DNA. This process effects changes in DNA topology that are essential in DNA replication, transcription, recombination, and repair. Quinolones bind to these enzymes and stabilise a drug-enzyme-cleaved DNA complex, allowing lethal double-stranded DNA breaks to accumulate unrepaired.7 Each of the target enzymes has a quinolone-resistance determining region (QRDR), a portion of the DNA-binding surface of the enzyme8 at which aminoacid substitutions can diminish quinolone binding. Generally, multiple such mutations are required to achieve clinically important resistance in Enterobacteriaceae; when such organisms are quinolone resistant they are nearly always found to have one or more QRDR mutations. The other classically described mechanism of resistance operates by decreasing intracellular drug accumulation by upregulation of native efflux pumps9 either alone or together with decreased expression of outer membrane porins.10

Both mechanisms of resistance are mutational, arising in an individual organism and then passing vertically to surviving progeny. Neither mechanism seems to transfer effectively on mobile genetic elements. A laboratory-generated plasmid overexpressing a quinolone-resistant mutant DNA gyrase gene caused only a modest increase in quinolone minimum inhibitory concentration (MIC), since the host gyrase remained quinolone susceptible.11 Accordingly, plasmids encoding mutant gyrases have not been found in nature. The development of plasmid-mediated quinolone resistance (PMQR) through decreased drug accumulation has also not been described. The impression that PMQR did not exist was bolstered by surveys in the 1970s that did not uncover any plasmids capable of transferring quinolone resistance.12 Although a 1987 report described the identification of PMQR in an outbreak strain of Shigella dysenteriae,13 the quinolone resistance was later attributed to chromosomal mutation and not a plasmid-encoded gene.14

Section snippets

Plasmid-encoded Qnr protein

The discovery of PMQR in the late 1990s was made inadvertently by Luis Martinez-Martinez and colleagues.15 A quinolone was included as a control in a study of the ability of a plasmid called pMG252 to increase resistance to multiple antibiotics in a porin-deficient strain of Klebsiella pneumoniae. Unexpectedly, a large increase in quinolone MIC was found. The effect of the plasmid was magnified in this porin-deficient isolate, but even in an E coli strain with intact porins, pMG252 increased

Mechanism of Qnr action

The QnrA protein belongs to the pentapeptide-repeat family, which is defined by a tandem five aminoacid repeat with the recurrent motif [Ser, Thr, Ala or Val] [Asp or Asn] [Leu or Phe] [Ser, Thr or Arg] [Gly].17 To date, more than 500 proteins are known to contain such pentapeptide-repeat motifs, but the function of nearly all of these proteins is unknown. Two pentapeptide-repeat proteins are of particular interest. A naturally occurring peptide, microcin B17, is a bacterial poison with a

Resistance activity of Qnr

The extent to which QnrA protects Enterobacteriaceae against fluoroquinolones has usually been examined by measuring the difference in quinolone MIC for an E coli strain with and without a qnrA-bearing plasmid. The first report of a qnrA plasmid found that the MIC of ciprofloxacin increased from 0·008 μg/mL to 0.25 μg/mL in an E coli J53 transconjugant, with a range from 0·125 μg/mL26 to 2·0 μg/mL27 for other qnr plasmid transconjugants of this strain. One study assessed the quinolone

Epidemiology of qnrA

After the initial discovery of qnrA in a K pneumoniae isolate obtained in 1994 from the urine of a patient in Alabama, USA, efforts were made to find this gene elsewhere. A survey for qnrA by PCR of more than 350 Gram-negative isolates collected mainly in the 1990s and chosen to include a broad geographic range and a variety of genera of Gram-negative bacteria found qnrA in only six isolates (four E coli and two Klebsiella spp), all from the same centre in Alabama where the original strain had

Newly identified qnr genes

Until recently, the sequence of qnrA was believed to be highly conserved. Initial reports of qnrA from the USA, Europe, and China reported sequences that varied in a single silent polymorphism (CTA→CTG at position 537).16, 26, 30, 34, 35 Subsequently, a K oxytoca isolate from Anhui Province, China (where the rate of ciprofloxacin resistance in E coli is 70%57) was reported to carry a variant of qnrA differing in four codons from the originally detected gene. This variant was designated qnrA2,36

Qnr plasmids

Plasmids carrying qnr genes vary widely in size and associated resistances but almost all carry multiple resistance determinants. Genes for qnrA and sometimes qnrB are found as part of complex sul1-type integrons containing a presumed recombinase, Orf513 (figure 5). Typically, resistance genes within an integron are associated with 59-base element recombination sites and are situated immediately 3L' to an integrase.61 The absence of these features for qnrA suggests that the mechanism through

Origins of qnr genes

The recent profusion of qnr variants, along with the evidently extensive penetration of these genes into populations of Enterobacteriaceae worldwide strongly suggests that these genes have considerably predated our knowledge of them. This finding raises the interesting questions of where these genes came from, and what they were doing there before clinical use of quinolones selected for their dissemination. Provisional answers to the first question are provided by a number of recent reports.

AAC(6′)-Ib-cr, another PMQR protein

Shortly after the discovery of QnrA, it was observed that not all qnr-bearing plasmids transferred the same level of quinolone resistance. Wild-type E coli have an MIC of ciprofloxacin of about 0·008 μg/mL. Most qnr plasmids determine an MIC of ciprofloxacin of 0·25 μg/mL in E coli. We saw, however, that certain plasmids from clinical E coli collected in Shanghai provided about four-fold higher levels of ciprofloxacin resistance (1·0 μg/mL). Although apparent differences in levels of expression

Clinical importance of PMQR

The discovery of PMQR could help explain how numerous independent clones of initially exquisitely susceptible wild-type Enterobacteriaceae have managed to develop high-level quinolone resistance. In the face of clinical concentrations of quinolone, organisms with even a single resistance mutation can survive—and give rise to increasingly resistant mutants. In our recent survey of 313 ceftazidime-resistant US Enterobacteriaceae, qnr-bearing strains were as likely to be susceptible by the

Conclusions

Faced with the challenge of potent fluoroquinolones, bacteria have not devised a high-level defense mechanism, such as the mecA gene that protects staphylococci from β-lactam antibiotics. Instead, Enterobacteriaceae have improvised multiple mechanisms for low-level resistance, assembling them together to chisel away at quinolone effectiveness. Chromosomal mutations accrue, progressively barring quinolone entry, diminishing quinolone accumulation in the cytoplasm, and discouraging quinolone

Search strategy and selection criteria

An English language literature search without time restrictions was done using the PubMed database for studies examining PMQR. The keywords used were “qnr”, “plasmid”, “quinolone”, “fluoroquinolone”, “resistance”, “pentapeptide repeat”, and “extended spectrum beta lactamase”. Reference lists of related articles were searched for relevant studies, as were the abstracts of recent conferences.

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