Journal of Antimicrobial Chemotherapy (2003) 51, 11091117DOI: 10.1093/jac/dkg222Advance Access publication 14 April 2003

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2003 The British Society for Antimicrobial Chemotherapy

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

Joaquim Ruiz*

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

Quinolones are broad-spectrum antibacterial agents, commonly used in both clinical andveterinary medicine. Their extensive use has resulted in bacteria rapidly developing resistanceto these agents. Two mechanisms of quinolone resistance have been established to date: alter-ations in the targets of quinolones, and decreased accumulation due to impermeability of themembrane and/or an overexpression of efflux pump systems. Recently, mobile elements havealso been described, carrying the qnr gene, which confers resistance to quinolones.

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

Introduction

Flemings description of penicillin in the late 1930s heraldedthe beginning of the antibacterial era. During the followingyears, research in the antibacterial field resulted in the synthe-sis or isolation of a great number of antimicrobial agents withdifferent mechanisms of action and a broad spectrum of activ-ity against a number of microorganisms. In 1962, during theprocess of synthesis and purification of chloroquine (an anti-malarial agent), a quinolone derivative, nalidixic acid, wasdiscovered which possessed bactericidal activity.1 However,its clinical use was limited to the treatment of urinary tractinfections (UTIs). Thereafter, novel compounds of this family,such as pipemidic acid and oxolinic acid, were synthesizedand introduced into clinical practice, although the clinicalindication for these quinolones still remained only for UTIs.The addition of a fluorine atom at position 6 of the quinolonemolecules greatly enhanced their activity, facilitating theirusage beyond UTIs.

During the 1980s, a great number of fluoroquinolones weredeveloped. These agents showed potent activity againstGram-negative bacteria, but not against the Gram-positivebacteria or anaerobes. In the 1990s, further alterations of thequinolones resulted in the discovery of novel compounds thatnot only showed potent activity against Gram-negative bac-teria but also against the Gram-positives. In addition, some ofthe new compounds, such as trovafloxacin, also showed promis-ing activity against the anaerobes.2 Recently, non-fluorinated

quinolones (such as PGE9262932 or PGE9509924) havebeen developed, further opening novel avenues in thedevelopment of quinolone research.3

The fluoroquinolones have been used to treat a great vari-ety of infections, including gonococcal infections, osteo-myelitis, enteric infections or respiratory tract infections,46and as prophylaxis in neutropenic patients, surgery or to pre-vent spontaneous bacterial peritonitis in cirrhotic patients,among others.5,7 Moreover, quinolones, along with other anti-bacterial agents, have been extensively used in veterinarypractice, either for medical reasons or as growth promoters.6

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

To date, two main mechanisms of quinolone resistancehave been established: alterations in the targets of quinolones,and decreased accumulation inside the bacteria due to imper-meability of the membrane and/or an overexpression of effluxpump systems. Both of these mechanisms are chromosomallymediated. Furthermore, mobile elements have been describedcarrying the qnr gene which confers resistance to quinolones.These mobile elements have the potential for horizontal trans-fer of quinolone resistance genes.

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*Tel: +34-93-227-5522; Fax: +34-93-227-5454; E-mail: joruiz@clinic.ub.es

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Target alterations

Quinolones act by inhibiting the action of type II topoisomer-ases, DNA gyrase and topoisomerase IV.1012

DNA gyrase is a tetrameric enzyme composed of two Asubunits and two B subunits, encoded by gyrA and gyrB,respectively. The main function of this enzyme is to catalysethe negative supercoiling of DNA.13 Topoisomerase IV is anA2B2 enzyme as well, encoded by parC and parE (referred toas 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 beassociated 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 theGram-positives it is the topoisomerase IV. However, somestudies indicate that the DNA gyrase may act as the primarytarget in Gram-positive microorganisms for some quino-lones, such as sparfloxacin and nadifloxacin.1517 Moreover,some recently developed quinolones, such as clinafloxacinand moxifloxacin, have similar affinity for both targets.17

The majority of the literature regarding the mechanismsof action and resistance to the quinolones refers to studiesdone on the Enterobacteriaceae, especially Escherichia coli.Amino acid substitutions involved in the development ofquinolone resistance in this microorganism have beendescribed for GyrA/GyrB and ParC/ParE (Tables 1 and 2).The prevalence of mutations in their respective encodinggenes is associated with the in vitro or in vivo origin of the

strains. Thus, when comparing the presence of mutations inthe DNA gyrase of quinolone-resistant E. coli strains obtainedin vitro, results showed a similar proportion of mutations ingyrA and gyrB,18 whereas, in studies using clinical isolates,the results showed an exclusive prevalence of mutations ingyrA.19,20

Alterations in the DNA gyrase

Alterations described in the GyrA of E. coli are predomin-antly in the so-called quinolone-resistance determiningregion (QRDR),21 between positions 67 and 106 (Table 1).Mutations in codons 67, 81, 82, 83, 84, 87 and 106 of gyrAhave been observed to be responsible for the development ofquinolone resistance in E. coli.1927 However some of thesemutations within the QRDR (e.g. in E. coli mutations at posi-tions 67, 82 and 106), have only been described in laboratory-obtained quinolone-resistant mutants.21,23,26,27 Recently, posi-tion 51, a region outside the QRDR, has been proposed as anovel point mutation resulting in decreased susceptibility tothe quinolones.28

The presence of a single mutation in the above-mentionedpositions of the QRDR of gyrA usually results in high-levelresistance to nalidixic acid, but to obtain high levels of resist-ance to fluoroquinolones, the presence of additional muta-tion(s) in gyrA and/or in another target such as parC isrequired.20,29 Thus, it has been proposed that the MIC of nali-dixic acid could be used as a generic marker of resistance forthe quinolone family in Gram-negative bacteria.29,30 Yet, nali-dixic acid-susceptible, ciprofloxacin-resistant (NalS CipR)phenotypes have been described in two laboratory mutants ofE. coli. In E. coli this phenotype is associated with the pres-ence of the substitutions Gly-81 to Asp or Asp-82 to Gly.22,26However, in spontaneous mutants of Salmonella typhi-murium, a mutation from Gly-81 to Ser does not affect theMIC of any of the six tested quinolones (including nalidixicacid and ciprofloxacin).31 The NalS CipR phenotype has alsobeen described in Campylobacter jejuni, although themolecular basis underlying it remains unknown.32 In fact, theonly NalS CipR C. jejuni isolate in which the presence of

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

aMutations in other codons, such as codon 93, have been described,but their role in development of resistance to quinolones remainsunclear.bOnly described in mutants obtained in vitro.

Codona Wild amino acid Mutations described

GyrA51b Ala Val67b Ala Ser81 Gly Cys, Asp82b Asp Gly83 Ser Leu, Trp, Ala, Val 84 Ala Pro, Val87 Asp Asn, Gly, Val, Tyr, His

106b Gln Arg, HisGyrB

426 Asp Asn447 Lys Glu

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

Codon Wild amino acid Mutations described

ParC78 Gly Asp80 Ser Ile, Arg84 Glu Lys, Val, Gly

ParE445 Leu His

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mutations in gyrA and gyrB were analysed showed a singlemutation in codon 86 (equivalent to Ser-83 of E. coli) result-ing in the substitution Thr to Ile, the most frequently foundalteration among quinolone-resistant isolates of C. jejuni,32,33The possible involvement of compensatory mutations inother gyrA codons was suggested to explain this isolatesphenotype. However, the possible hypersusceptibility tonalidixic acid of the parental strain due to increased uptakeshould also be taken into account. Susceptibility to nalidixicacid, but resistance to different fluoroquinolones (such asciprofloxacin or norfloxacin) seems to be usual for Stenotro-phomonas maltophilia.34,35 In a study involving over 109isolates of S. maltophilia, 88% were susceptible to nalidixicacid, whereas only 20.2% were susceptible to norfloxacin.35Interestingly, it has been shown that the development ofquinolone resistance in this microorganism is not related tothe presence of mutations in the gyrA or parC genes.36,37 Thisfact suggests the possibility of potent efflux pumps playing arole in the resistance to quinolones for this microorganism. Inthis line of thought, a study by Alonso et al.38 showed the invitro obtention of a quinolone-resistant mutant selected withtetracycline. Recently, the SmeDEF efflux system (which iscapable of pumping quinolones out of the bacteria) has beencharacterized 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, itseems to be the most frequently found in most clinical andlaboratory quinolone-resistant isolates of other Entero-bacteria, such as Citrobacter freundii or Shigella spp. or inpathogens such as Neisseria gonorrhoeae or Acinetobacterbaumannii.40,41 In E. coli, and other microorganisms such asS. typhimurium or A. baumannii, codon 83 is located in aHinf I restriction site, enabling mutations at this position to beeasily detected with a combination of PCR and RFLPanalysis.29,41,42

In clinical isolates, the second most commonly observedmutation is at codon 87 of gyrA.19,20 Strains with a doublemutation at codons 83 and 87 have higher MICs of quino-lones.19,20 This fact is true for other Gram-negative micro-organisms, such as C. freundii, Pseudomonas aeruginosa orN. gonorrhoeae.40

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

In quinolone-resistant S. typhimurium strains, a mutationhas been described in codon 119, resulting in the substitutionof Ala to Glu or Val. This codon, outside the QRDR, has beenimplicated in the development of nalidixic acid resistance.45A mutation in this codon generating the substitution Ala-119to Ser has also been described for A. baumannii. However, inA. baumannii this mutation was found in both quinolone-resistant and quinolone-susceptible isolates, suggesting that

other mechanisms may be responsible for the changes inquinolone susceptibility observed.41

Different amino acid substitutions at the same positionresult in different quinolone susceptibility levels,25,40 indi-cating that the final MIC is a function of the specific subs-titution.46 This fact is probably due to the mechanism ofinteraction between the quinolones and their targets. It hasbeen suggested that amino acid 83 (numeration for E. coli) ofGyrA interacts with the radical in position 1 of quinolones,whereas amino acid 87 of GyrA interacts with the radical inposition 7.20 This model also applies for amino acids 80 and84 (numeration for E. coli) of ParC. Thus, different aminoacid substitutions at these points would affect in differentways the affinity for the quinolone molecule. In addition,mutations in other positions might affect the whole proteinstructure, affecting the interaction with quinolones.

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

Alterations in topoisomerase IV

In the parC gene of E. coli, among other microorganisms,the most common substitutions occur at codons 80 and84.4,15,16,19,40,43,44,5053 In E. coli, another substitution (Gly-78to Asp) has been described both in clinical isolates and labora-tory-obtained quinolone-resistant mutants (Table 2).50,51 Asubstitution described in the parC gene of in vitro mutantsof Shigella flexneri54 affects position 79 (Asp to Ala). Othersubstitutions in the same position have been found in othermicroorganisms both Gram-negatives [such as Haemophilusinfluenzae (Asp to Asn)] and Gram-positives [such as Strepto-coccus pneumoniae (Asp to Asn)].4,43 Although, in every casethey were found concomitantly with other mutations either ingyrA or parC.

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

The role of amino acid substitutions in ParE, resulting inthe development of quinolone resistance in clinical isolates ofGram-negative microorganisms appears to be irrelevant.19,56

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In fact, only one substitution (Leu-445 to His) has beendescribed in parE of a single quinolone-resistant in vitromutant of E. coli. Moreover, this mutation only seems toaffect the MIC of quinolones in the presence of a concomitantmutation in gyrA.57 Alterations in this subunit have alsobeen described both in clinical and laboratory-obtainedquinolone-resistant Gram-positive microorganisms. InS. pneumoniae58,59 for example, the mutations found pro-duced 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 Glnand Asn-470 to Asp have been described.44,55,60 However, it ispossible that some or all of these substitutions may not playany role in the development of quinolone resistance, as hasbeen suggested in S. pneumoniae by some authors.43

Decreased uptake

Decreased quinolone uptake may be associated with twofactors: an increase in the bacterial impermeability to theseantibacterial agents or the overexpression of efflux pumps.

Quinolones may cross the outer membrane in two differentways: through specific porins or by diffusion through thephospholipid bilayer. The degree of diffusion of a quinoloneis greatly associated with, and dependent on, its level of hydro-phobicity. All quinolones may cross the outer membranethrough the porins, but only those with a greater level ofhydrophobicity may diffuse through the phospholipidbilayer.61 Thus alterations in the composition of porins and/orin the lipopolysaccharides may alter susceptibility profiles.In lipopolysaccharide-defective mutants, increased suscep-tibility to hydrophobic quinolones has been described, with-out alterations in the level of resistance to the hydrophilicquinolones.62,63

Alterations in membrane permeability are usually associ-ated with decreased expression of porins. This has beendescribed both in E. coli and other Gram-negative bac-teria.40,62,64,65

The outer membrane of E. coli possesses three main porins(OmpA, OmpC and OmpF). A decrease in the level of expres-sion of OmpF is related to an increase in the resistance to somequinolones,62,64,66 but does not affect the MIC of others, suchas tosufloxacin or sparfloxacin.65 Moreover, a decreasedexpression of OmpF results in a decrease in susceptibility to avariety of antibacterial agents such as -lactams, tetracyclinesand chloramphenicol.66

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

It has been shown that chloramphenicol, tetracycline andother substrates such as salicylate, may induce the expressionof MarA, producing an increase in the expression of micF, anantisense regulator that induces a post-transcriptional repres-sion of the synthesis of OmpF. The expression of micF mayalso be regulated by the SoxRS operon.68

In E. coli, the MarRAB and SoxRS operons also regulatethe level of expression of efflux pumps systems such asAcrAB.69,70 Mutations affecting MarR induce the constitutiveexpression of this operon, leading to the development of amultiresistance phenotype.67

Recently, Baucheron et al.,71 working with strains ofS. typhimurium carrying amino acid substitutions either inGyrA (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 ofthe AcrAB efflux pump in the development of quinoloneresistance in S. typhimurium.71 This study showed that disrup-tion or inhibition (with Phe-Arg--naphthylamide) of theAcrAB operon results in a decrease in the MIC of all testedquinolones (e.g. MIC of ciprofloxacin decreased from 32 mg/Lto 24 mg/L; MIC of enrofloxacin decreased from 64 mg/L to2 mg/L; MIC of marbofloxacin decreased from 32 mg/L to24 mg/L).

The outer membrane composition of some microorgan-isms such as A. baumannii or P. aeruginosa, has beenassociated with their intrinsic resistance. Wild-type strains ofA. baumannii show MICs of ciprofloxacin ranging between0.125 and 1 mg/L.40,41 In contrast, wild-type E. coli strainsshow MICs of ciprofloxacin ranging between 0.007 and0.25 mg/L.20 This result has been interpreted as intrinsicresistance or due to the overexpression of an efflux pump(s).Interestingly, this proportion is not conserved when analysingthe MIC of nalidixic acid.40,41 The outer membrane ofP. aeruginosa has very low non-specific permeability tosmall hydrophobic molecules,72,73 which may account for theintrinsic resistance of this microorganism against quinolones.In fact the outer membrane of P. aeruginosa is 10- to 100-foldless permeable to antibiotics than that of E. coli.73

Different efflux systems shown to pump out quinolonessuch as MexAB-OprM, MexCD-OprJ or MexEF-OprN havebeen described in P. aeruginosa.69 A fourth efflux systemnamed MexXY capable of pumping out quinolones has alsobeen described, but no open reading frame corresponding toan outer membrane protein has been found downstream ofmexXY. In fact, it may be that OprM (which is encoded down-stream of MexAB) might act as the outer membrane protein ofthis efflux system.74,75 It has been reported that the disruptionof OprM produces a greater effect in the susceptibility levelsto some antimicrobial agents, than the disruption of MexA orMexB. This may be due to the presence of a weak promoter inthe mexB gene upstream of the oprM gene, which facilitatesthe expression of oprM in the absence of expression of theother components of the MexABOprM operon. This would

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imply that OprM may contribute to the intrinsic resistancelevels to antimicrobial agents by cooperation with other innerand periplasmic membrane components.76 Other efflux pumpsassociated with increasing levels of quinolone resistance havealso been characterized in E. coli and other Gram-negativemicroorganisms (Table 3).39,69,7779 In addition, recent studiesanalysing whole genomes have reported the high number ofputative efflux pumps which might be able to pump out anti-bacterial agents that are present in microorganisms. Forexample, in E. coli, 37 different putative drug transportershave been found.80

Efflux pumps have also been described in Gram-positivemicroorganisms,79 the best characterized being NorA, fromS. aureus. NorA is an ATP-dependent efflux pump capable ofpumping out hydrophilic quinolones like enoxacin or nor-floxacin, but not affecting the hydrophobic quinolones suchas sparfloxacin.16,81 This efflux pump can also extrude othermolecules like basic dyes, puromycin or chloramphenicol.69Two different DNA sequences encoding closely related NorAefflux pumps have been described, but to date no strain carry-ing the two sequences together has been found, suggestingthat these sequences might be two different alleles of the samegene.82 Two NorA-related efflux pumps, Bmr and Blt, havebeen described in Bacillus subtilis. Their overexpression pro-vides a similar resistance spectrum to that of NorA.81,83 Thepresence of NorA-like efflux systems has also been describedor suggested in other Gram-positive microorganisms such asS. pneumoniae or Streptococcus group viridans (Table 4).84,85

To date, different substances capable of inhibiting theaction of some efflux pumps such as reserpine or CCCP havebeen described.16,25 Unfortunately, such compounds cannotbe used in clinical practice due to their high toxicity. Cur-rently, novel compounds, such as Phe-Arg--naphthylamide,are under investigation.71,86,87

Transferability of quinolone resistance

There have been reports describing the presence of quinoloneresistance genes on plasmids.88,89 However, in the strainsdescribed by Munshi et al.,88 the possible presence of muta-tions in the gyrA gene was suspected.90 The possibility of thepresence of a plasmid capable of carrying quinolone resist-ance genes in Shigella spp. in an epidemic outbreak inRwanda was proposed89 although the presence of mutationsin gyrA was not even looked at.

Recently, a plasmid in Klebsiella pneumoniae has beendescribed, capable of conferring quinolone resistance whentransferred to a recipient strain.91 Tran & Jacoby92 havedemonstrated that the plasmid contains a novel gene, whichthey named qnr, that encodes a protein of 218 amino acidsbelonging to the pentapeptide repeat family. The product ofthis gene protects the DNA gyrase from quinolone inhibition,although its effect on topoisomerase IV is unclear. This geneis flanked by ORF513, an ORF previously identified in someintegrons, suggesting that the qnr gene may be located withinan integron. In February 2003, Jacoby et al.93 described theextremely low prevalence of this gene analysing a long seriesof Gram-negative microorganisms (mainly K. pneumoniaeand E. coli) from different geographical origins (19 countriesaround the world). The qnr gene was only found in six strains(five K. pneumoniae and one E. coli) isolated in 1994 (fourK. pneumoniae and the E. coli) and 1995 (the remainingK. pneumoniae), all of them having the same geographicalorigin (University of Alabama in USA), although no studiesof clonality among the K. pneumoniae strains were carriedout. The exact mechanism of DNA gyrase protection con-ferred by Qnr has yet to be established.

Table 3. Characterized efflux pumps responsible for quinolone resistance in Gram-negative microorganisms

aAcrAB-related efflux systems have been described indifferent Enterobacteriaceae.

Microorganism Efflux system

A. baumannii AdeABCC. jejuni CmeABCE. coli AcrABa

AcrEFEmrABMdfAYdhE

P. aeruginosa MexAB-OprMMexCD-OprJMexEF-OprNMexXY-OprM

S. maltophilia SmeDEFVibrio cholerae VceABVibrio parahaemolyticus NorM

Table 4. Characterized efflux pumps responsible for quinolone resistance in Gram-positive microorganisms

Microorganism Efflux system

B. subtilis BltBmrABmr3

S. aureus NorAS. pneumoniae PmrA

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In addition to these reports on mobile elements,94 the abilityof both in vitro95 and clinical isolates of S. pneumoniae andviridans streptococci to incorporate via transformation frag-ments of gyrA as well as parC genes, including those carryingthe QRDR has been described.74,94 The studies developedin vitro showed that resistance could be transferred with DNAfrom viridans streptococci to S. pneumoniae or from S. pneu-moniae to viridans streptococci. The frequencies of transfor-mation ranged from 103 to

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23. Hallett, P. & Maxwell, A. (1991). Novel quinolone-resistantmutations of the Escherichia coli DNA gyrase A protein: enzymaticanalysis of the mutant proteins. Antimicrobial Agents and Chemo-therapy 35, 33540.

24. Oram, M. & Fisher, L. M. (1991). 4-Quinolone resistance muta-tions in the DNA Gyrase of Escherichia coli clinical isolates identi-fied by using the polymerase chain reaction. Antimicrobial Agentsand Chemotherapy 35, 3879.

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