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Journal of Antimicrobial Chemotherapy (2003) 51, 11091117

DOI: 10.1093/jac/dkg222
Advance Access publication 14 April 2003

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, 08036Barcelona, Spain
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

Flemings 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 worldwide.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.


*Tel: +34-93-227-5522; Fax: +34-93-227-5454; E-mail:


2003 The British Society for Antimicrobial Chemotherapy

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

Table 2. Mutations described in ParC and ParE of

the quinolone-resistant strains of E. coli




Wild amino acid

Mutations described


Cys, Asp
Leu, Trp, Ala, Val
Pro, Val
Asn, Gly, Val, Tyr, His
Arg, His




Wild amino acid

Mutations described


Ile, Arg
Lys, Val, Gly



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

aMutations in other codons, such as codon 93, have been described,

but their role in development of resistance to quinolones remains
bOnly described in mutants obtained in vitro.

Alterations in the DNA gyrase

Target alterations
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 Gramnegative and Gram-positive microorganisms. For Gramnegative 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 1 and 2).
The prevalence of mutations in their respective encoding
genes is associated with the in vitro or in vivo origin of the

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 1).
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 laboratoryobtained 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


Resistance to quinolones
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 isolates
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 quinoloneresistant 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
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 Grampositive 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 quinoloneresistant 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
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 2).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
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


J. Ruiz
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
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
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 -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--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 24 mg/L; MIC of enrofloxacin decreased from 64 mg/L to
2 mg/L; MIC of marbofloxacin decreased from 32 mg/L to
24 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 MexABOprM operon. This would


Resistance to quinolones
Table 3. Characterized efflux pumps
responsible for quinolone resistance in Gramnegative microorganisms

Efflux system

A. baumannii
C. jejuni
E. coli


P. aeruginosa

S. maltophilia
Vibrio cholerae
Vibrio parahaemolyticus

Table 4. Characterized efflux

pumps responsible for quinolone
resistance in Gram-positive

Efflux system

B. subtilis


S. aureus
S. pneumoniae

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--naphthylamide,
are under investigation.71,86,87

aAcrAB-related efflux systems have been described in

different Enterobacteriaceae.

Transferability of quinolone resistance

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 3).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
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 4).84,85

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.


J. Ruiz
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 103 to <107 in correlation with the
homologies of their QRDRs.

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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.

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I am indebted to Drs Margarita M. Navia and Anna Ribera for
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