Plasmid-mediated quinolone resistance – current knowledge and future perspectives

Abstract

Quinolones are a group of antimicrobial agents that were serendipitously discovered as byproducts of the synthesis of chloroquine. Chemical modifications, such as the addition of fluorine or piperazine, resulted in the synthesis of third- and fourth-generation fluoroquinolones, with broad-spectrum antimicrobial actions against aerobic or anaerobic, Gram-positive or Gram-negative bacteria. The efficacy and consequent widespread use of quinolones and fluoroquinolones has led to a steady global increase in resistance, mediated via gene mutations, alterations in efflux or cell membranes and plasmid-conferred resistance. The first plasmid-mediated quinolone resistance gene, qnrA1, was detected in 1998. Since then, many other genes have been identified and the underlying mechanisms of resistance have been elucidated. This review provides an overview of quinolone resistance, with particular emphasis on plasmid-mediated resistance.

Introduction

Quinolones are a group of antimicrobial agents with a 4-quinolone nucleus that were serendipitously discovered as byproducts of the synthesis of chloroquine. ¹ The active 1,8-naphthyridine derivative of quinolones, nalidixic acid, was widely used for the treatment of Gram-negative urinary tract infections from the early 1960s. Chemical modifications of the 4-quinolone nucleus resulted in the first-generation quinolones pipemidic acid, oxolinic acid and cinoxacin, but these drugs were limited by their lack of efficacy against Gram-positive and anaerobic Gram-negative bacteria due to their low tissue and serum kinetics. ² Modifications at the C6 or C7 positions of the 4-quinolone nucleus, such as addition of a fluorine atom at the C6 position, significantly increased efficacy and penetrance. ²

The addition of a fluorine atom at C6 and a cyclic diamine piperazine at C7 resulted in the generation of the first fluoroquinolone, norfloxacin, which was effective against aerobic Gram-positive bacteria but remained ineffective against anaerobic micro-organisms. ³ Subsequently, third- and fourth-generation quinolones were developed by state-of-the-art pharmaceutical design and organic synthesis, and were effective against anaerobic micro-organisms. ⁴ Strategies for further improvement of this class of antimicrobials are limited by the lack of options for chemical modification, with the exception of removing the fluorine atom at C6 to generate 6-desfluoro compounds. ⁵ A prominent example of a 6-desfluoro compound is garenoxacin, which shows promiscuous antimicrobial effects against a broad spectrum of aerobic and anaerobic Gram-positive and Gram-negative bacteria.^6–8

Mechanism of action

Quinolones target the bacterial enzymes DNA gyrase and topoisomerase IV, which are essential for cell growth and proliferation. ⁹ DNA gyrase and topoisomerase IV are both tetrameric enzymes, with DNA gyrase comprising two subunits each of gyrA and gyrB, and topoisomerase IV comprising two subunits each of parC and parE. DNA gyrase regulates DNA supercoiling and relieves topological stress arising from translocation and replication complexes along the bacterial DNA; topoisomerase IV is a decatenating enzyme that unwinds interlinked daughter chromosomes following DNA replication. ⁹ In Gram-positive organisms, topoisomerase IV and DNA gyrase are the primary and secondary targets of quinolones, respectively, with the target preference reversed in Gram-negative organisms. ¹⁰

The inhibitory function of quinolones is initiated via binding to complexes that form between DNA and gyrase or topoisomerase IV, resulting in conformational change in the enzyme and subsequent cleavage of the bacterial DNA. Importantly, quinolones not only induce nicks in the bacterial genomic DNA but also prevent such nicks from re-ligating, thus perpetuating the inhibitory action. ¹¹ Inhibition occurs more quickly for DNA gyrase than topoisomerase IV, because the former is located at the replication fork and the latter is located behind the replication fork. ¹² DNA damage initiates the apoptotic pathway, and high doses of fluoroquinolone can also disrupt the gyrase–DNA complex. ⁹

Commonly treated infections

The broad-spectrum antimicrobial nature of fluoroquinolones has led to their use in a wide variety of microbiological infections, including gonorrhoea and uncomplicated urinary tract infections, and in areas where β-lactamase resistance is prevalent. ¹³ Gemifloxacin and moxifloxacin show outstanding clearance of respiratory tract infections, methicillin-susceptible Staphylococcus infections, and Gram-negative bacterial infections. ⁵

This class of drug is also used to treat invasive gastrointestinal tract infections and moderate-to-severe enteric infections. ¹⁴ Norfloxacin and ciprofloxacin show comparable inhibitory activity to trimethoprim–sulphamethoxazole in the treatment of diarrhoea caused by enterotoxigenic Escherichia coli, Campylobacter jejuni and Shigella spp., and more potent activity than doxycycline and trimethoprim–sulphamethoxazole in the treatment of Vibrio cholerae infection. ¹⁵ The quinolones are also routinely used, with good outcome, against typhoid fever ¹⁶ and nontyphoidal Salmonella gastroenteritis. ¹⁷

Resistance

The efficacy and consequent widespread use of quinolones and fluoroquinolones has led to a steady global increase in resistance.^18,19 In the USA, a 40% increase in the use of fluoroquinolones led to a concurrent doubling in the rate of resistance to ciprofloxacin among Gram-negative bacilli isolated from hospital intensive care units. ²⁰ Resistance to fluoroquinolones is widespread in Spain, with the result that they have not been the first-choice treatment for urinary tract infections since the mid-1990s. ²¹ In China, between 1997 and 1999, ∼50% of community isolated and 60% of hospital acquired E. coli strains were resistant to ciprofloxacin. ²² Resistance against fluoroquinolone has been observed in the treatment of certain strains of Salmonella typhi in Africa, ²³ India ²⁴ and Vietnam. ²⁵ High-level resistance to fluoroquinolones is uncommon among isolates of nontyphoidal Salmonella, ²⁶ although a study in Finland reported an increase in the minimum inhibitory concentration (MIC) of ciprofloxacin in Salmonella enterica isolates from travellers returning from southeast Asia. ²⁶ Increased quinolone resistance in S. enterica isolates from Denmark has been associated with drug use in food animals. ²⁷ Physicians therefore recommend cautious (and even restricted) use. ²⁸ The rate of quinolone resistance in Norway is somewhat lower than that in other Scandinavian countries, however. ²⁹

Mechanisms of quinolone resistance

There are four known mechanisms of quinolone resistance that work discretely or in combination and confer varying degrees of resistance, ranging from reduced susceptibility (not always detected by prevalent antimicrobial susceptibility tests) to clinically relevant resistance. ³⁰ These four mechanisms are detailed below.

Gyrase and Topoisomerase Gene Mutations

Chromosomal point mutations in the genes encoding DNA gyrase and topoisomerase IV are major mechanisms of quinolone resistance. In Gram-negative bacteria, the primary target of quinolones is the gyrA subunit of DNA gyrase, and point mutations are mostly observed within a highly conserved domain of the gyrA N-terminus, known as the quinolone resistance determining region (QRDR). ³¹ This is in close proximity to the DNA-binding region near the putative active site tyrosine-122. ³¹ These point mutations result in amino acid substitutions, with mutations at serine-83 and asparagine-87 being most often observed in E. coli. ³¹ Mutations have also been reported outside the QRDR, including an alanine to valine substitution at position 51 in E. coli, ³² and mutations at alanine-131, glutamic acid-139 and aspartic acid-144 in S. enterica. ³³ Mutations have been mapped to the N-terminal QRDR of the gyrB gene (located between aspartic acid-426 and lysine-447) and it is likely that these mutations result in amino acid substitutions. ³² It is possible that the cumulative effect of several mutations can equip a micro-organism with increased resistance capacity, and mutations in gyrB may occur independently of those in gyrA. ³⁴

Point mutations in the topoisomerase IV subunit genes parC and parE are also present in Gram-negative bacteria, but at a significantly lower frequency than gyrA mutations. Interestingly, Ling et al. ³⁵ identified a parC mutation in the absence of a gyrA mutation in a Salmonella strain. This is surprising because it is generally believed that parC mutations arise after gyrA mutations, as DNA gyrase (rather than topoisomerase IV) is the preferred target of quinolones in Gram-negative bacteria.

Alterations in Efflux

Bacteria are able to increase the expression of nonspecific, energy-dependent efflux systems that prevent the accumulation of effective intracellular concentrations of quinolones by active pumping of drug across the cell membrane. ³⁶ The multidrug efflux system AcrA-AcrB-TolC has been shown to be a critical mediator of quinolone efflux in E. coli, and may be the primary mechanism of fluoroquinolone resistance in Salmonella. ³⁶ It is thought that efflux systems are capable of producing only low-level resistance to quinolones, and become clinically relevant only when combined with mutations in target enzymes or membrane alterations. ³⁶

Cell Membrane Alternations

Gram-negative bacteria can regulate membrane permeability by altering the levels of outer membrane porins that form the channels responsible for passive diffusion. Loss of or reduction in outer membrane porin 1a (OmpF) has been shown to confer antimicrobial resistance, ³⁷ but this observation could not be validated by two subsequent studies.^38,39

Plasmid-mediated Resistance

A natural transferable plasmid (pMG252) encoding low-level resistance to quinolones was isolated from a clinical strain of Klebsiella pneumoniae. ⁴⁰ This 56 kb plasmid had a broad host range that included other members of the Enterobacteriaceae and Pseudomonas aeruginosa, ⁴⁰ and contained a qnr gene that encoded a 218 amino acid protein. A qnr is a naturally occurring gene encoding a pentapeptide repeat protein that confers reduced susceptibility to nalidixic acid or fluoroquinolone.^41,42 The first qnr gene, now known as qnrA, was found to protect E. coli DNA gyrase from inhibition by ciprofloxacin. ⁴³ Subsequent research demonstrated that qnr genes co-occur with other resistance determinants on sul1-type integrons. ⁴³ In fact, qnr determinants have been reported in bacterial strains producing extended spectrum β-lactamases. ⁴⁴

Other qnr genes have been isolated, including qnrS1 (from Shigella flexneri 2b) ⁴³ and qnrB1 (from K. pneumoniae). ¹⁹ These genes encode proteins with 41% and 59% amino acid homology to the original qnr protein (qnrA1), respectively.^45,46 A qnrS1 variant sharing 91% homology with the parent gene was detected in the US and designated qnrS2. ⁴⁷ Similarly, qnrB variants have been identified including qnrB2 and qnrB5 isolated from nontyphoidal S. enterica, ⁴⁷ and qnrB12 from poultry isolates of Citrobacter werkmanii. ⁴⁸ A search of the GenBank® sequence database (performed on 1 September 2012) identified six qnrA variants, two qnrS variants and 17 qnrB variants.

It is important to note that plasmid-encoded qnr genes do not confer quinolone resistance by themselves, but facilitate the selection of bacteria bearing higher-level resistance, thereby augmenting the effect of other resistance mutations. ⁴⁰

Origin and transfer of qnr determinants

Studies have identified qnr determinants worldwide, in the US, Europe, Asia and Africa.^45–49 They have been found in many enterobacterial species (E. coli, Enterobacter spp., Klebsiella spp., Salmonella spp., Proteus mirabilis, Serratia marcescens, Citrobacter freudii, Providencia stuartii),^50–57 as well as Acinetobacter baumannii, a nonenterobacterial Gram-negative species. ⁴⁹ Interestingly, qnr determinants have not been detected in P. aeruginosa, a clinically important nonenterobacterial Gram-negative bacterium, but this could be due to a lack of extensive screening. ⁵⁸

The wide geographical and species distribution of qnr genes suggests that they have been in existence for some time. The qnrA gene is thought to have originated in Shewanella algae, an environmental species from marine and fresh water. ⁵⁹ Qnr-like proteins have been detected in water-borne Vibrionaceae bacterial isolates and may be the origin of the more clinically relevant qnrA, qnrS, and qnrB determinants, with which they share 40–67% homology.^59,60 These qnr-like genes conferred reduced susceptibility to quinolones when cloned into E. coli. ⁶⁰ In addition, qnrVS1 and qnrVS2, isolated from Vibro splendidus and another Vibro species, may be the natural reservoir of qnrS genes. ⁶⁰ Taken together, this evidence suggests that Gram-negative aquatic bacteria such as Vibrionaceae and Shewanellaceae may serve as the reservoir of qnr genes.

Resistance conferred by qnr determinants

When cloned into E. coli J53, the qnrA determinant increased the MICs of nalidixic acid and fluoroquinolones from 4 to 32 µg/ml and from 0.008 to 0.25 µg/ml, respectively. ⁶¹ Comparison with other studies suggests that similar increases in quinolone resistance are found with qnrA, qnrS and qnrB.^58–61 It is interesting to note that qnr proteins have a significantly greater inhibitory effect on ciprofloxacin than nalidixic acid. Resistance to nalidixic acid was increased by two- to 12-fold by qnrA, and ciprofloxacin resistance was increased by 12.5–250-fold.^45,62–64 Similar findings were seen with qnrS, which increased the MIC of nalidixic acid by three- to eight-fold and ciprofloxacin by 16–62.5-fold.^{47,55,65–68} In addition, qnrB resulted in a four- to eight-fold increase in nalidixic acid MIC and an eight- to 62.5-fold increase in that of ciprofloxacin.^19,47,48,68

As expected, donor bacteria (generally clinical isolates) usually exhibit higher levels of quinolone resistance than the transconjugants. ⁴⁶ This can be explained by the presence of additional chromosomally encoded quinolone resistance determinants, particularly mutations in the gyrA and parC QRDRs. ⁴⁵ In other cases, the differences in resistance levels among transconjugants may be due to the transfer of other plasmid-mediated mechanisms, such as aac(6′)-Ib-cr (a variant of aminoglycoside acetyltransferase). ⁶⁴

Genetic environment of qnrA

Plasmids carrying qnrA variants often carry other antibiotic resistance genes, which may or may not be transferable. ⁴⁵ Interestingly, hybridization studies for qnrA in an E. coli clinical isolate revealed integration with chromosomal DNA. ⁶⁹ It is known that qnrA genes are often embedded in complex sul1-type integrons. ⁴⁵ These integrons include a single 5′-conserved segment that contains the integrase gene (intl1), and duplicated 3′-conserved segments, each of which contain quaternary ammonium compound-resistance and sulphonamide resistance protein (sul1) genes. The two 3′-conserved segments surround a common region that contains the putative transposase orf513, which may act as a recombinase for mobilization of downstream-located antibiotic resistance genes. ⁴⁵

Genetic environment of qnrS

The qnrS genes are also typically located on plasmids but, in contrast to qnrA, they are not part of a sul1-type integron structure. ⁴⁶ Studies have identified an association between qnrS1 and a Tn3-like-blaTEM-1-containing transposon,^46,66,70 which increases recombination and insertion efficiency. In some cases, the Tn3 element is substituted with an Ambler class A β-lactamase gene, blaLAP-1.^65,71,72 The plasmid pGNB2 was the original source of qnrS2, which shares 92% amino acid homology with qnrS1. ⁷³ QnrS2 is not part of an integron but is linked to orf1, a remnant of a Tn1721-like transposon. Interestingly, the G+C content of the orfI-qnrS2 gene region differs considerably from the rest of the plasmid, suggesting that pGNB2 acquired qnrS2 together with Tn1721-orf1. ⁷³

Genetic environment of qnrB

Although qnrB variants are generally carried by plasmids, the results of plasmid transformation-conjugation experiments and Southern blotting suggest a chromosomal location for qnrB12. ⁷⁴ The qnrB1 gene was shown to be associated with orf1005, which encodes a putative transposase. ¹⁹ QnrB2 was reported to be located in a complex sul1-type integron, comprising two class 1 integrons surrounding two common regions, separated by a partial 3′-conserved segment. ⁷⁵ The qnrB2 gene was adjacent to the first common region and in the opposite orientation. In Spanish clinical isolates of Citrobacter spp., the qnrB2 and qnrB6 determinants were carried in complex sul1-type integrons. ⁷⁶ The qnrB4 gene is bracketed between a phage shock protein (psp) operon at its 5′ extremity and a sensitivity to antimicrobial peptide (sap) operon encoding a putative peptide transport system permease at its 3′ extremity. ⁶⁰ However, the qnrB4 gene on plasmid pHS7 obtained from a clinical isolate of K. pneumonia was localized in a sul1-type integron with (upstream) ampicillin resistance (ampR), orf1, plasmid-mediated inducible β-lactamase (blaDHA-1), psp and partial qacE1 upstream, and (downstream) insertion sequences IS26, acid phosphatase (aphA), and antimicrobial sensitivity genes sapA and partial sapB. ⁶⁷

Aac(6′)-Ib-cr

Bacterial enzymes that modify antimicrobial agents have principally coevolved with the antimicrobial agent on which they act. ⁵⁸ Selective enzymatic modification of quinolones by bacteria has therefore been thought not to exist in nature (since these drugs are fully synthetic), and the time available for coevolving such quinolone modifying enzymes is limited. A plasmid-mediated enzyme with quinolone modifying capabilities has been identified, however. ⁵⁸ Aac(6′)-Ib-cr is a variant of aac(6′)-Ib (a common aminoglycoside acetyltransferase), that reduces the activity of both the aminoglycosides kanamycin, tobramycin and amikacin and the fluoroquinolones ciprofloxacin and norfloxacin. ⁵⁸ The -cr variant of aac(6′)-Ib has two amino acid changes, Trp102Arg and Asp179Tyr, both of which are necessary for the N-acetylation of the amino nitrogen of a piperazinyl group. ⁵⁸ Ciprofloxacin and norfloxacin are the only fluoroquinolones inhibited by aac(6′)-Ib-cr as they are the only compounds with an unsubstituted piperazinyl group. Despite the limited targets of aac(6′)-Ib-cr, this enzyme plays an important role in fluoroquinolone-resistance for several reasons. First, aac(6′)-Ib-cr is common in clinical isolates of Gram-negative bacteria, ⁴⁵ including 51% of ciprofloxacin-resistant clinical E. coli isolates collected from China. ⁵⁸ Secondly, although the degree of resistance conferred by aac(6′)-Ib-cr is low, when both qnr and aac(6′)-Ib-cr are present in the same cell, the level of resistance reaches clinical significance. ⁵⁸ Thirdly, the presence of aac(6′)-Ib-cr resulted in substantial increases in the selection of chromosomal mutants on exposure to ciprofloxacin. ⁵⁸

Conclusions and future perspectives

This review highlights the recent advances in knowledge of plasmid-mediated quinolone resistance. It is important to investigate whether the horizontal transmission of quinolone resistance is following a unique pattern, or whether it resembles general horizontal transmission patterns of genetic elements. More detailed knowledge is also required regarding the origin of qnr determinants, in order to determine their usefulness as targets for drug design. It is also vital to determine the clinical cut-off point for fluoroquinolone usage, which is only possible through robust definition of phenotypic changes associated with such resistance patterns.