Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection

Joaquim Ruiz*

Department of Microbiology, Institut Clínic Infeccions i Immunologia, Hospital Clínic, C/.Villarroel 170, 08036-Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
Quinolones are broad-spectrum antibacterial agents, commonly used in both clinical and veterinary medicine. Their extensive use has resulted in bacteria rapidly developing resistance to these agents. Two mechanisms of quinolone resistance have been established to date: alterations in the targets of quinolones, and decreased accumulation due to impermeability of the membrane and/or an overexpression of efflux pump systems. Recently, mobile elements have also been described, carrying the qnr gene, which confers resistance to quinolones.

Keywords: quinolone resistance, DNA gyrase, topoisomerase IV, efflux pumps, qnr


    Introduction
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
Fleming’s description of penicillin in the late 1930s heralded the beginning of the antibacterial era. During the following years, research in the antibacterial field resulted in the synthesis or isolation of a great number of antimicrobial agents with different mechanisms of action and a broad spectrum of activity against a number of microorganisms. In 1962, during the process of synthesis and purification of chloroquine (an antimalarial agent), a quinolone derivative, nalidixic acid, was discovered which possessed bactericidal activity.1 However, its clinical use was limited to the treatment of urinary tract infections (UTIs). Thereafter, novel compounds of this family, such as pipemidic acid and oxolinic acid, were synthesized and introduced into clinical practice, although the clinical indication for these quinolones still remained only for UTIs. The addition of a fluorine atom at position 6 of the quinolone molecules greatly enhanced their activity, facilitating their usage beyond UTIs.

During the 1980s, a great number of fluoroquinolones were developed. These agents showed potent activity against Gram-negative bacteria, but not against the Gram-positive bacteria or anaerobes. In the 1990s, further alterations of the quinolones resulted in the discovery of novel compounds that not only showed potent activity against Gram-negative bacteria but also against the Gram-positives. In addition, some of the new compounds, such as trovafloxacin, also showed promising activity against the anaerobes.2 Recently, non-fluorinated quinolones (such as PGE9262932 or PGE9509924) have been developed, further opening novel avenues in the development of quinolone research.3

The fluoroquinolones have been used to treat a great variety of infections, including gonococcal infections, osteomyelitis, enteric infections or respiratory tract infections,46 and as prophylaxis in neutropenic patients, surgery or to prevent spontaneous bacterial peritonitis in cirrhotic patients, among others.5,7 Moreover, quinolones, along with other antibacterial agents, have been extensively used in veterinary practice, either for medical reasons or as growth promoters.6

As a result of their wide spectrum of activity, quinolones have been extensively used. Recently, ciprofloxacin was pointed out as the most consumed antibacterial agent world- wide.6 This high level of use, and to some degree of misuse in the sense of unnecessary use,8 or use of quinolones with poor activity in some developing countries,9 has been blamed for the rapid development of bacterial resistance to these agents.

To date, two main mechanisms of quinolone resistance have been established: alterations in the targets of quinolones, and decreased accumulation inside the bacteria due to impermeability of the membrane and/or an overexpression of efflux pump systems. Both of these mechanisms are chromosomally mediated. Furthermore, mobile elements have been described carrying the qnr gene which confers resistance to quinolones. These mobile elements have the potential for horizontal transfer of quinolone resistance genes.


    Target alterations
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
Quinolones act by inhibiting the action of type II topoisomerases, DNA gyrase and topoisomerase IV.1012

DNA gyrase is a tetrameric enzyme composed of two A subunits and two B subunits, encoded by gyrA and gyrB, respectively. The main function of this enzyme is to catalyse the negative supercoiling of DNA.13 Topoisomerase IV is an A2B2 enzyme as well, encoded by parC and parE (referred to as grlA and grlB in Staphylococcus aureus). These subunits (ParC and ParE) are highly homologous to GyrA and GyrB, respectively. The main role of topoisomerase IV seems to be associated with decatenating the daughter replicons.14

The quinolone targets are basically different in Gram-negative and Gram-positive microorganisms. For Gram-negative bacteria it is the DNA gyrase, whereas in the Gram-positives it is the topoisomerase IV. However, some studies indicate that the DNA gyrase may act as the primary target in Gram-positive microorganisms for some quinolones, such as sparfloxacin and nadifloxacin.1517 Moreover, some recently developed quinolones, such as clinafloxacin and moxifloxacin, have similar affinity for both targets.17

The majority of the literature regarding the mechanisms of action and resistance to the quinolones refers to studies done on the Enterobacteriaceae, especially Escherichia coli. Amino acid substitutions involved in the development of quinolone resistance in this microorganism have been described for GyrA/GyrB and ParC/ParE (Tables Go and Go). The prevalence of mutations in their respective encoding genes is associated with the in vitro or in vivo origin of the strains. Thus, when comparing the presence of mutations in the DNA gyrase of quinolone-resistant E. coli strains obtained in vitro, results showed a similar proportion of mutations in gyrA and gyrB,18 whereas, in studies using clinical isolates, the results showed an exclusive prevalence of mutations in gyrA.19,20


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Table 1.  Mutations described in GyrA and GyrB subunits of quinolone-resistant strains of E. coli
 

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Table 2.  Mutations described in ParC and ParE of the quinolone-resistant strains of E. coli
 

    Alterations in the DNA gyrase
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
Alterations described in the GyrA of E. coli are predominantly in the so-called quinolone-resistance determining region (QRDR),21 between positions 67 and 106 (Table Go). Mutations in codons 67, 81, 82, 83, 84, 87 and 106 of gyrA have been observed to be responsible for the development of quinolone resistance in E. coli.1927 However some of these mutations within the QRDR (e.g. in E. coli mutations at positions 67, 82 and 106), have only been described in laboratory-obtained quinolone-resistant mutants.21,23,26,27 Recently, position 51, a region outside the QRDR, has been proposed as a novel point mutation resulting in decreased susceptibility to the quinolones.28

The presence of a single mutation in the above-mentioned positions of the QRDR of gyrA usually results in high-level resistance to nalidixic acid, but to obtain high levels of resistance to fluoroquinolones, the presence of additional mutation(s) in gyrA and/or in another target such as parC is required.20,29 Thus, it has been proposed that the MIC of nalidixic acid could be used as a generic marker of resistance for the quinolone family in Gram-negative bacteria.29,30 Yet, nalidixic acid-susceptible, ciprofloxacin-resistant (NalS CipR) phenotypes have been described in two laboratory mutants of E. coli. In E. coli this phenotype is associated with the presence of the substitutions Gly-81 to Asp or Asp-82 to Gly.22,26 However, in spontaneous mutants of Salmonella typhimurium, a mutation from Gly-81 to Ser does not affect the MIC of any of the six tested quinolones (including nalidixic acid and ciprofloxacin).31 The NalS CipR phenotype has also been described in Campylobacter jejuni, although the molecular basis underlying it remains unknown.32 In fact, the only NalS CipR C. jejuni isolate in which the presence of mutations in gyrA and gyrB were analysed showed a single mutation in codon 86 (equivalent to Ser-83 of E. coli) resulting in the substitution Thr to Ile, the most frequently found alteration among quinolone-resistant isolates of C. jejuni,32,33 The possible involvement of compensatory mutations in other gyrA codons was suggested to explain this isolate’s phenotype. However, the possible hypersusceptibility to nalidixic acid of the parental strain due to increased uptake should also be taken into account. Susceptibility to nalidixic acid, but resistance to different fluoroquinolones (such as ciprofloxacin or norfloxacin) seems to be usual for Stenotrophomonas maltophilia.34,35 In a study involving over 109 isolates of S. maltophilia, 88% were susceptible to nalidixic acid, whereas only 20.2% were susceptible to norfloxacin.35 Interestingly, it has been shown that the development of quinolone resistance in this microorganism is not related to the presence of mutations in the gyrA or parC genes.36,37 This fact suggests the possibility of potent efflux pumps playing a role in the resistance to quinolones for this microorganism. In this line of thought, a study by Alonso et al.38 showed the in vitro obtention of a quinolone-resistant mutant selected with tetracycline. Recently, the SmeDEF efflux system (which is capable of pumping quinolones out of the bacteria) has been characterized in S. maltophilia.39

The most frequent mutation observed in quinolone-resistant E. coli is at codon 83 of gyrA.1921,24,25,29 Moreover, it seems to be the most frequently found in most clinical and laboratory quinolone-resistant isolates of other Enterobacteria, such as Citrobacter freundii or Shigella spp. or in pathogens such as Neisseria gonorrhoeae or Acinetobacter baumannii.40,41 In E. coli, and other microorganisms such as S. typhimurium or A. baumannii, codon 83 is located in a Hinf I restriction site, enabling mutations at this position to be easily detected with a combination of PCR and RFLP analysis.29,41,42

In clinical isolates, the second most commonly observed mutation is at codon 87 of gyrA.19,20 Strains with a double mutation at codons 83 and 87 have higher MICs of quinolones.19,20 This fact is true for other Gram-negative microorganisms, such as C. freundii, Pseudomonas aeruginosa or N. gonorrhoeae.40

Substitutions in the positions equivalent to the aforementioned amino acids 83 and 87 of E. coli have also been the most frequently described in quinolone-resistant Gram-positive microorganisms.15,16,43,44

In quinolone-resistant S. typhimurium strains, a mutation has been described in codon 119, resulting in the substitution of Ala to Glu or Val. This codon, outside the QRDR, has been implicated in the development of nalidixic acid resistance.45 A mutation in this codon generating the substitution Ala-119 to Ser has also been described for A. baumannii. However, in A. baumannii this mutation was found in both quinolone-resistant and quinolone-susceptible isolates, suggesting that other mechanisms may be responsible for the changes in quinolone susceptibility observed.41

Different amino acid substitutions at the same position result in different quinolone susceptibility levels,25,40 indicating that the final MIC is a function of the specific substitution.46 This fact is probably due to the mechanism of interaction between the quinolones and their targets. It has been suggested that amino acid 83 (numeration for E. coli) of GyrA interacts with the radical in position 1 of quinolones, whereas amino acid 87 of GyrA interacts with the radical in position 7.20 This model also applies for amino acids 80 and 84 (numeration for E. coli) of ParC. Thus, different amino acid substitutions at these points would affect in different ways the affinity for the quinolone molecule. In addition, mutations in other positions might affect the whole protein structure, affecting the interaction with quinolones.

In GyrB of E. coli, substitutions resulting in resistance to quinolones have been described at positions 426 (Asp-426 to Asn) and 447 (Lys-447 to Glu).47 Substitutions at position 426 seem to confer resistance to all quinolones, whereas those at position 447 result in an increased level of resistance to nalidixic acid, but a greater susceptibility to fluorinated quinolones. Mutations in equivalent positions have been described for Gram-positive microorganisms.48 In S. typhimurium, the amino acid substitution Ser to Tyr at position 463 has been related to the development of quinolone resistance.49


    Alterations in topoisomerase IV
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
In the parC gene of E. coli, among other microorganisms, the most common substitutions occur at codons 80 and 84.4,15,16,19,40,43,44,5053 In E. coli, another substitution (Gly-78 to Asp) has been described both in clinical isolates and laboratory-obtained quinolone-resistant mutants (Table Go).50,51 A substitution described in the parC gene of in vitro mutants of Shigella flexneri54 affects position 79 (Asp to Ala). Other substitutions in the same position have been found in other microorganisms both Gram-negatives [such as Haemophilus influenzae (Asp to Asn)] and Gram-positives [such as Streptococcus pneumoniae (Asp to Asn)].4,43 Although, in every case they were found concomitantly with other mutations either in gyrA or parC.

A mutation, to date only described in grlA of S. aureus, affects codon 116, producing a change from Ala to Glu or Pro.52,55 This codon is an analogue of codon 119 of GyrA in S. typhimurium.45 Similarly, mutations in other codons such as 23 (Lys to Asn), 69 (Asp to Tyr), 176 (Ala to Gly) or 451 (Pro to Gln) have been described in S. aureus. However, what effect they have on quinolone susceptibility, has yet to be determined.55

The role of amino acid substitutions in ParE, resulting in the development of quinolone resistance in clinical isolates of Gram-negative microorganisms appears to be irrelevant.19,56 In fact, only one substitution (Leu-445 to His) has been described in parE of a single quinolone-resistant in vitro mutant of E. coli. Moreover, this mutation only seems to affect the MIC of quinolones in the presence of a concomitant mutation in gyrA.57 Alterations in this subunit have also been described both in clinical and laboratory-obtained quinolone-resistant Gram-positive microorganisms. In S. pneumoniae58,59 for example, the mutations found produced changes from Asp-435 to Asn or from His-102 to Tyr, whereas in S. aureus the amino acid changes Pro-25 to His, Glu-422 to Asp, Asp-432 to Asn or Gly, Pro-451 to Ser or Gln and Asn-470 to Asp have been described.44,55,60 However, it is possible that some or all of these substitutions may not play any role in the development of quinolone resistance, as has been suggested in S. pneumoniae by some authors.43


    Decreased uptake
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
Decreased quinolone uptake may be associated with two factors: an increase in the bacterial impermeability to these antibacterial agents or the overexpression of efflux pumps.

Quinolones may cross the outer membrane in two different ways: through specific porins or by diffusion through the phospholipid bilayer. The degree of diffusion of a quinolone is greatly associated with, and dependent on, its level of hydrophobicity. All quinolones may cross the outer membrane through the porins, but only those with a greater level of hydrophobicity may diffuse through the phospholipid bilayer.61 Thus alterations in the composition of porins and/or in the lipopolysaccharides may alter susceptibility profiles. In lipopolysaccharide-defective mutants, increased susceptibility to hydrophobic quinolones has been described, without alterations in the level of resistance to the hydrophilic quinolones.62,63

Alterations in membrane permeability are usually associated with decreased expression of porins. This has been described both in E. coli and other Gram-negative bacteria.40,62,64,65

The outer membrane of E. coli possesses three main porins (OmpA, OmpC and OmpF). A decrease in the level of expression of OmpF is related to an increase in the resistance to some quinolones,62,64,66 but does not affect the MIC of others, such as tosufloxacin or sparfloxacin.65 Moreover, a decreased expression of OmpF results in a decrease in susceptibility to a variety of antibacterial agents such as ß-lactams, tetracyclines and chloramphenicol.66

Some chromosomal loci such as MarRAB (constituted by three genes: marR that encodes a repressor protein, marA, encoding a transcriptional activator and marB which encodes a protein with an unknown function) or SoxRS (this operon encodes for two proteins, SoxR, a regulator protein, and SoxS, a transcriptional activator) regulate both the levels of expression of OmpF and some efflux pumps in E. coli.6770

It has been shown that chloramphenicol, tetracycline and other substrates such as salicylate, may induce the expression of MarA, producing an increase in the expression of micF, an antisense regulator that induces a post-transcriptional repression of the synthesis of OmpF. The expression of micF may also be regulated by the SoxRS operon.68

In E. coli, the MarRAB and SoxRS operons also regulate the level of expression of efflux pumps systems such as AcrAB.69,70 Mutations affecting MarR induce the constitutive expression of this operon, leading to the development of a multiresistance phenotype.67

Recently, Baucheron et al.,71 working with strains of S. typhimurium carrying amino acid substitutions either in GyrA (Ser-83 to Ala and Asp-87 to Asn), ParC (Ser-80 to Ile) and GyrB (Ser-464 to Phe), have shown the high relevance of the AcrAB efflux pump in the development of quinolone resistance in S. typhimurium.71 This study showed that disruption or inhibition (with Phe-Arg-ß-naphthylamide) of the AcrAB operon results in a decrease in the MIC of all tested quinolones (e.g. MIC of ciprofloxacin decreased from 32 mg/L to 2–4 mg/L; MIC of enrofloxacin decreased from 64 mg/L to 2 mg/L; MIC of marbofloxacin decreased from 32 mg/L to 2–4 mg/L).

The outer membrane composition of some microorganisms such as A. baumannii or P. aeruginosa, has been associated with their intrinsic resistance. Wild-type strains of A. baumannii show MICs of ciprofloxacin ranging between 0.125 and 1 mg/L.40,41 In contrast, wild-type E. coli strains show MICs of ciprofloxacin ranging between 0.007 and 0.25 mg/L.20 This result has been interpreted as intrinsic resistance or due to the overexpression of an efflux pump(s). Interestingly, this proportion is not conserved when analysing the MIC of nalidixic acid.40,41 The outer membrane of P. aeruginosa has very low non-specific permeability to small hydrophobic molecules,72,73 which may account for the intrinsic resistance of this microorganism against quinolones. In fact the outer membrane of P. aeruginosa is 10- to 100-fold less permeable to antibiotics than that of E. coli.73

Different efflux systems shown to pump out quinolones such as MexAB-OprM, MexCD-OprJ or MexEF-OprN have been described in P. aeruginosa.69 A fourth efflux system named MexXY capable of pumping out quinolones has also been described, but no open reading frame corresponding to an outer membrane protein has been found downstream of mexXY. In fact, it may be that OprM (which is encoded downstream of MexAB) might act as the outer membrane protein of this efflux system.74,75 It has been reported that the disruption of OprM produces a greater effect in the susceptibility levels to some antimicrobial agents, than the disruption of MexA or MexB. This may be due to the presence of a weak promoter in the mexB gene upstream of the oprM gene, which facilitates the expression of oprM in the absence of expression of the other components of the MexAB–OprM operon. This would imply that OprM may contribute to the intrinsic resistance levels to antimicrobial agents by cooperation with other inner and periplasmic membrane components.76 Other efflux pumps associated with increasing levels of quinolone resistance have also been characterized in E. coli and other Gram-negative microorganisms (Table Go).39,69,7779 In addition, recent studies analysing whole genomes have reported the high number of putative efflux pumps which might be able to pump out antibacterial agents that are present in microorganisms. For example, in E. coli, 37 different putative drug transporters have been found.80


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Table 3.  Characterized efflux pumps responsible for quinolone resistance in Gram-negative microorganisms
 
Efflux pumps have also been described in Gram-positive microorganisms,79 the best characterized being NorA, from S. aureus. NorA is an ATP-dependent efflux pump capable of pumping out hydrophilic quinolones like enoxacin or norfloxacin, but not affecting the hydrophobic quinolones such as sparfloxacin.16,81 This efflux pump can also extrude other molecules like basic dyes, puromycin or chloramphenicol.69 Two different DNA sequences encoding closely related NorA efflux pumps have been described, but to date no strain carrying the two sequences together has been found, suggesting that these sequences might be two different alleles of the same gene.82 Two NorA-related efflux pumps, Bmr and Blt, have been described in Bacillus subtilis. Their overexpression provides a similar resistance spectrum to that of NorA.81,83 The presence of NorA-like efflux systems has also been described or suggested in other Gram-positive microorganisms such as S. pneumoniae or Streptococcus group viridans (Table Go).84,85


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Table 4.  Characterized efflux pumps responsible for quinolone resistance in Gram-positive microorganisms
 
To date, different substances capable of inhibiting the action of some efflux pumps such as reserpine or CCCP have been described.16,25 Unfortunately, such compounds cannot be used in clinical practice due to their high toxicity. Currently, novel compounds, such as Phe-Arg-ß-naphthylamide, are under investigation.71,86,87


    Transferability of quinolone resistance
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
There have been reports describing the presence of quinolone resistance genes on plasmids.88,89 However, in the strains described by Munshi et al.,88 the possible presence of mutations in the gyrA gene was suspected.90 The possibility of the presence of a plasmid capable of carrying quinolone resistance genes in Shigella spp. in an epidemic outbreak in Rwanda was proposed89 although the presence of mutations in gyrA was not even looked at.

Recently, a plasmid in Klebsiella pneumoniae has been described, capable of conferring quinolone resistance when transferred to a recipient strain.91 Tran & Jacoby92 have demonstrated that the plasmid contains a novel gene, which they named qnr, that encodes a protein of 218 amino acids belonging to the pentapeptide repeat family. The product of this gene protects the DNA gyrase from quinolone inhibition, although its effect on topoisomerase IV is unclear. This gene is flanked by ORF513, an ORF previously identified in some integrons, suggesting that the qnr gene may be located within an integron. In February 2003, Jacoby et al.93 described the extremely low prevalence of this gene analysing a long series of Gram-negative microorganisms (mainly K. pneumoniae and E. coli) from different geographical origins (19 countries around the world). The qnr gene was only found in six strains (five K. pneumoniae and one E. coli) isolated in 1994 (four K. pneumoniae and the E. coli) and 1995 (the remaining K. pneumoniae), all of them having the same geographical origin (University of Alabama in USA), although no studies of clonality among the K. pneumoniae strains were carried out. The exact mechanism of DNA gyrase protection conferred by Qnr has yet to be established.

In addition to these reports on mobile elements,94 the ability of both in vitro95 and clinical isolates of S. pneumoniae and viridans streptococci to incorporate via transformation fragments of gyrA as well as parC genes, including those carrying the QRDR has been described.74,94 The studies developed in vitro showed that resistance could be transferred with DNA from viridans streptococci to S. pneumoniae or from S. pneumoniae to viridans streptococci. The frequencies of transformation ranged from 10–3 to <10–7 in correlation with the homologies of their QRDRs.


    Conclusions
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
The continuous rise in the prevalence of quinolone-resistant isolates can be attributed to the extensive use and misuse of these antibacterial agents, both in clinical and veterinary medicine. This resistance is mainly due to the presence of mutations in the quinolone targets (DNA gyrase and topoisomerase IV), or the presence of decreased uptake. The first description of a transferable quinolone resistance mechanism is of great concern. Horizontal transfer of quinolone resistance would facilitate the rapid dissemination of the quinolone resistance genes, even between animal and human pathogens, further compromising the use of these antimicrobial agents.


    Acknowledgements
 
I am indebted to Drs Margarita M. Navia and Anna Ribera for their helpful comments and suggestions, and would like to express my gratitude to the editor and referees for their valuable support.


    Footnotes
 
* Tel: +34-93-227-5522; Fax: +34-93-227-5454; E-mail: joruiz{at}clinic.ub.es Back


    References
 Top
 Abstract
 Introduction
 Target alterations
 Alterations in the DNA...
 Alterations in topoisomerase IV
 Decreased uptake
 Transferability of quinolone...
 Conclusions
 References
 
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