Review
Fluoroquinolone resistance:
mechanisms, impact on bacteria,
and role in evolutionary success
Liam S. Redgrave*, Sam B. Sutton*, Mark A. Webber, and Laura J.V. Piddock*,y
School of Immunity and Infection, Institute of Microbiology and Infection, Biosciences Building, University Road West, University
of Birmingham, Birmingham B15 2TT, UK
Quinolone and fluoroquinolone antibiotics are potent,
broad-spectrum agents commonly used to treat a range
of infections. Resistance to these agents is multifactorial
and can be via one or a combination of target-site gene
mutations, increased production of multidrug-resis-
tance (MDR) efflux pumps, modifying enzymes, and/or
target-protection proteins. Fluoroquinolone-resistant
clinical isolates of bacteria have emerged readily and
recent data have shown that resistance to this class of
antibiotics can have diverse, species-dependent impacts
on host-strain fitness. Here we outline the impacts of
quinolone-resistance mutations in relation to the fitness
and evolutionary success of mutant strains.
The challenge of fluoroquinolone resistance
Antibiotic resistance is one of the most pressing global
concerns in medicine, with highly resistant pathogens of
many species proving difficult to treat. Against this back-
drop there are few new drugs in development, so maintain-
ing the utility of the currently available agents is of crucial
importance. To make this possible, a thorough knowledge of
the mechanisms of resistance and the fate of resistant
strains is needed to understand the conditions where resis-
tance is selected and persists. Here we give an overview of
how bacteria can become resistant to fluoroquinolone anti-
biotics and describe some recent advances in our under-
standing of the ecology of resistance to these agents.
Quinolones and fluoroquinolones
The fluoroquinolones are potent, broad-spectrum antibiotics
that have been used in medical practice for the treatment of
severe or resistant infections since the late 1980s. As their
name suggests, they are derived from the quinolone family
of antibiotics; quinolones themselves are synthetic con-
structs, developed by modification of 1-alkyl-1,8-naphthyr-
idin-4-one-3-carboxylic acid [1]. Fluoroquinolones differ
from quinolones by the replacement of the eighth carbon
atom of the backbone with a nitrogen atom and the addition
Corresponding author: Piddock, L.J.V. (l.j.v.piddock@bham.ac.uk).
Keywords: chromosome structure; fitness.
*
These authors should be considered as joint first authors.
y
Twitter: @laurapiddock
0966-842X/
ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.04.007
438 Trends in Microbiology, August 2014, Vol. 22, No. 8
of a fluorine atom at the sixth position, giving them more
potent antibiotic action and a broader spectrum of activity
[2]. Their spectrum of efficacy against a wide range of Gram-
positive and Gram-negative pathogenic bacteria has led to
widespread use worldwide, although, in an attempt to
maintain their effectiveness, current UK prescribing guide-
lines largely recommend this class as second-line agents for
use when narrow-spectrum antibiotics have failed (http://
publications.nice.org.uk/antibiotic-prescribing-especially-
quinolones-and-cephalosporins-ktt9/evidence-context).
Even with current guidelines aiming to preserve the efficacy
of these drugs, resistance to fluoroquinolones is still occur-
ring at an increasing rate in numerous bacterial species and
their usage varies around the world. Due to an absence of
active surveillance, data on fluoroquinolone consumption
are lacking for many countries, making comparison of con-
sumption and rates of resistance between different parts of
the world difficult. However, within Europe there are suffi-
cient data collected by the European Centre for Disease
Prevention and Control (ECDC) that allow comparison
between European countries (http://www.ecdc.europa.eu/
en/healthtopics/antimicrobial_resistance/database/Pages/
database.aspx). For instance, fluoroquinolone consumption
and rates of resistance can be reviewed using Greece,
France, and Sweden as examples of countries where usage
and resistance rates vary. Greece is the highest user of
fluoroquinolones and has the highest incidence of fluoro-
quinolone-resistant Escherichia coli isolates (Figure 1). Con-
versely, Sweden has the lowest consumption rate and the
lowest incidence of resistance. There are now four genera-
tions of quinolone/fluoroquinolone antibiotics in clinical use
(Table 1); the most commonly prescribed fluoroquinolones in
current medical practice are ciprofloxacin, levofloxacin, and
moxifloxacin.
Supercoiling and type II topoisomerases
Fluoroquinolones are potent inhibitors of bacterial type II
topoisomerases, which are essential enzymes involved in
key cellular processes including DNA replication [3–5]. In
both prokaryotes and eukaryotes, DNA exists as double
strands that intertwine around each other to form a dou-
ble-helix structure. However, in bacteria further twisting
of the double-strand structure can occur whereby torsional
stresses force the double helix to cross over on itself to
produce a plectonemic arrangement [6] (Figure 2). This
 Review Trends in Microbiology August 2014, Vol. 22, No. 8

Correlaon of fluoroquinolone resistance and consumpon in E. coli in Greece, France, and

Sweden
85.0% 3.2
Key: Greece
80.0% France
Sweden
75.0%

Fluoroquinolone consumpon (DDD per 1000 paents per day)

Percentage of E. coli isolates resistant to fluoroquinolones (%)

2.7
70.0%
65.0%
60.0% 2.2

55.0%
50.0% 1.7
45.0%
40.0%
1.2
35.0%
30.0%
25.0% 0.7
20.0%
15.0%
0.2
10.0%
5.0%
0.0% –0.3
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Year
TRENDS in Microbiology

Figure 1. Correlation between fluoroquinolone (FQ) usage and resistance rates in countries with high, medium, and low use of FQs. Percentage of FQ-resistant Escherichia
coli isolates (bars) and FQ consumption in defined daily dose (DDD) per 1000 patients per day (lines) in Greece, France, and Sweden. Greece, France, and Sweden are high,
moderate, and low users of FQs respectively (relative to other European countries) and this correlates with high, moderate, and low rates of resistance in E. coli. Data from
http://www.ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/database/Pages/database.aspx.

process, also known as supercoiling (Box 1), enables bacte- response to environmental stress, growth stage, and cellu-
rial DNA to exist in a complicated condensed state in which lar processes such as transcription, DNA replication, and
the DNA can be condensed into compact supercoils allow- recombination [6–9]. Topoisomerase I and topoisomerase
ing large amounts of DNA to be packed into the cell [7]. The II enzymes work in opposition to control the level of
degree of supercoiling of DNA is not fixed and there is twisting within DNA. Topoisomerase I reduces the number
continuous remodelling of DNA topology within bacteria in of negative supercoils [6]. By contrast, topoisomerase II
introduces negative supercoils, which unwind over-twisted
Table 1. Fluoroquinolones licensed for clinical use and their DNA into a relaxed state and can further change the DNA
current status topology into an under-twisted plectoneme [6]. DNA gyrase
Generation Drug Use in clinical practice and DNA topoisomerase IV are both heterotetrameric type
First generation Nalidixic acid Generic form available II topoisomerase enzymes comprising two copies of each of
Cinoxacin Discontinued either a GyrA and GyrB subunit or a ParC and ParE (GrlA
Second generation Norfloxacin Available as Noroxin and GrlB in Staphylococcus aureus) subunit, respectively.
Ciprofloxacin Available as Cipro The enzymes have homologous action but with subtle
and generic form
differences; although both DNA gyrase and topoisomerase
Lomefloxacin Discontinued
IV relax positively supercoiled DNA, only DNA gyrase can
Ofloxacin Available as Floxin
and generic form
go on to introduce negative supercoils into relaxed DNA [9].
Levofloxacin Available as Levaquin Topoisomerase IV has decatenating (unlinking) activity,
and generic form allowing the segregation of catenated daughter chromo-
Third generation Sparfloxacin Discontinued somes at cell division [8–10]. The careful maintenance of
Gatifloxacin Discontinued supercoiling is essential in key genetic processes that can
Grepafloxacin Discontinued control gene expression and thereby determine the pheno-
Fourth generation Trovafloxacin Discontinued type of a cell [7,11–13]. Such is the importance of super-
Moxifloxacin Available as Avelox coiling that strict homeostatic control is fundamental for
Gemifloxacin Available as Factive cell survival, because changes in the global degree of
439
Review
Bacterial type II topoisomerases
Negavely supercoiled Relaxed Posively supercoiled
Gyrase Gyrase
Topo IV Topo IV
Topo IV
Interlinked chromosomes
TRENDS in Microbiology
Figure 2. Action of type II topoisomerases on DNA. The control of supercoiling by
type II topoisomerases in bacteria. Supercoiling is a measure of how over- or
under-wound the double helix is and is required for packaging of the bacterial
chromosome within the cell and to relieve the torsional stress resulting from DNA
replication. DNA gyrase introduces negative supercoils into DNA; bacterial
chromosomes favour a negatively supercoiled state. Topoisomerase IV
positively supercoils DNA, can also introduce negative supercoils, and has a role
in separating the interlinked daughter chromosomes (themselves usually
supercoiled) resulting from replication of the chromosome.
supercoiling alter the expression of multiple genes includ-
ing many involved in responses to stress and pathogenesis
[6,14,7].
Mechanisms of fluoroquinolone action
Fluoroquinolones target DNA gyrase and topoisomerase
IV with varying efficiency in different bacteria and inhibit
their control of supercoiling within the cell, resulting in
impaired DNA replication (at lower concentrations) and
cell death (at lethal concentrations) [10,15]. The targeting
of either DNA gyrase or topoisomerase IV as the primary
target by fluoroquinolones varies with bacterial species
and specific fluoroquinolone; however, as a broad general-
isation, the key target in Gram-negative bacteria is
DNA gyrase, whereas in Gram-positive microorganisms
Box 1. Supercoiling: what is it and why is it important?
In bacteria, chromosomal DNA exists in a very compact condensed
state. This is due to twisting of the DNA in a process known as
supercoiling. Supercoiling allows the chromosome to be greatly
condensed, and a large amount of genetic information can fit into a
bacterial cell. Supercoiling can be either positive or negative and
this describes the direction in which twisting has occurred,
homologous to being turned in a clockwise or anticlockwise
direction. Within a cell the degree of supercoiling is controlled by
enzymes that work in opposition to supercoil DNA either positively
or negatively. The level of supercoiling (how twisted the DNA is) is
not fixed and changes constantly in response to key cellular
processes and the environment. Changes in the level of supercoiling
of the DNA within a cell alter a great deal of cellular processes and
many genes are differentially expressed in response to changes in
supercoiling. As a result perturbations in supercoiling can have
large phenotypic consequences.
440
Trends in Microbiology August 2014, Vol. 22, No. 8
topoisomerase IV is preferentially targeted [16]. When
either DNA gyrase or topoisomerase IV induces transient
double-strand DNA breaks, they first bind covalently to
the DNA to form enzyme–DNA complexes before breaking
the bound DNA, passing another segment of DNA through
this break, and rejoining the original DNA segment [16].
When a fluoroquinolone is present, the complex is altered
into a drug–enzyme–DNA complex (known as a ternary
complex) in which the type II topoisomerase is trapped
with the bound DNA [16]. The basis of the interaction of
the quinolone with topoisomerase IV is the formation of a
water–metal ion bridge between the oxygen molecules in
the amine group of the drug and the hydroxyl residues in
conserved serine or acidic residues in the enzyme, medi-
ated by a Mg2+ ion [16–19]. The crystal structure of target
enzymes, both with and without the bound drug, has been
solved in Streptococcus pneumoniae and Acinetobacter
baumannii [8,20–22]. The binding of fluoroquinolones
occurs within the enzyme at the target site of helix-4 in
either GyrA or ParC. Fluoroquinolones bind to DNA gyr-
ase or topoisomerase IV, which is then unable to re-ligate
the DNA substrate; the broken segments of DNA bound to
the enzyme are referred to as cleaved complexes [16]. The
formation of cleaved complexes is a reversible process in
vitro and a pivotal point in the fluoroquinolone killing
pathway, which can follow one of two irreversible courses
depending on the molecule. The two main routes of lethal
fluoroquinolone action are the protein synthesis-depen-
dent pathway (also known as the chloramphenicol-sensi-
tive pathway, due to chloramphenicol’s ability to inhibit
fluoroquinolone-mediated cell death) and the protein syn-
thesis-independent pathway (the chloramphenicol-insen-
sitive pathway) [15,16]. Older first-generation quinolones
such as nalidixic acid work via the former mechanism,
whereas newer fluoroquinolones largely work via the lat-
ter pathway, although both result in fragmentation of
chromosomal DNA and ultimately cell death [10,15,16,
23–25]. In addition to these rapid killing routes, cleaved
complexes reversibly block DNA replication and induce
the SOS stress response, which in E. coli results in upre-
gulation of various stress-response genes that enhance
DNA-repair capability, leading to the formation of fila-
mentous cells due to the inhibition of cell division [23–25].
Fluoroquinolone-resistance mechanisms
Fluoroquinolones have been extensively used in human
and veterinary medicine due to their effectiveness against
both Gram-positive and Gram-negative bacteria [17]. De-
spite prescribing guidelines now recommending reserving
fluoroquinolone use, resistance continues to rise and is a
major problem encountered in the clinical setting. The
percentage of E. coli isolates in the UK resistant to fluor-
oquinolones rose from 6% to 20% from 2001 to 2006 and
remained at about 17% for the rest of the decade [26].
Similar rises have been seen in other species; for example,
the proportion of fluoroquinolone-resistant Klebsiella
pneumoniae isolates in Italy has consistently increased
yearly, with an almost fivefold increase from 11% in
2005 to 50% in 2012 [27]. Resistance to the fluoroquino-
lones is evidently common and can occur via a range of
mechanisms (Table 2).
Review Trends in Microbiology August 2014, Vol. 22, No. 8

Table 2. Summary of the impact of different resistance infection caused by S. pneumoniae. This condition was
mechanisms on susceptibility to ciprofloxacin predominantly treated with the older second-generation
Resistance mechanism Fold change in Refs fluoroquinolones ciprofloxacin and levofloxacin, which pri-
ciprofloxacin MIC marily target ParC of topoisomerase IV in this species.
Gram-negative species a Resistance to ciprofloxacin in pneumococci occurred rapid-
Topoisomerase substitutions ly and when moxifloxacin was introduced prescribing pat-
gyrA 10–16 [31,33,79,80]
terns shifted to this newer, more effective drug that targets
parC 0 [31,33,79,80]
GyrA and ParC with equal affinity in this species [34,35].
gyrA ( 2) + parC 60 [31,33,79,80]
This (dual-targeting) property of moxifloxacin allowed it to
Permeability changes
be effective against ciprofloxacin-resistant S. pneumoniae
Efflux upregulation 4–8 [79]
[36]. Before the use of moxifloxacin, the vast majority of
Porin loss 4 [45]
fluoroquinolone-resistant S. pneumoniae had parC muta-
PMQRs
Carriage of qnr alleles >30 [37,43]
tions [34]. The increased use of moxifloxacin changed the
Carriage of qepA 32 [81] selection pressures on S. pneumoniae and there has been a
Carriage of oxqAB 16 [82] consequent increase in the proportion of isolates with both
Carriage of aac(60 )Ib-cr 4 [83] parC and gyrA mutations [34].
Gram-positive species b
Topoisomerase substitutions Transmissible quinolone-resistance mechanisms
grlA 4–8 [30,32] Various genes encoding different resistance mechanisms
grlB 4–8 [30,32] and on mobile genetic elements can decrease susceptibility
gyrA 0 [30,32] to quinolone or fluoroquinolone antibiotics; these are often
grlA + gyrB 64–128 [30,32] encoded on plasmids and known as plasmid-mediated
Permeability changes quinolone resistance (PMQR) genes. The archetypal
Efflux upregulation 4 [30,32,53] PMQR gene, qnr, was first found on a plasmid in a clinical
a
Based on data from Escherichia coli. isolate of K. pneumoniae [37]. The protein it encodes is
b
Based on data from Staphylococcus aureus. characterised by a pentapeptide-repeat motif and has
similarities to immunity proteins such as McbG [38], a
protein that confers immunity to the DNA replication
inhibitor microcin B17 [39]. Data from a recent structural
Target-site mutation analysis of a Qnr protein suggest that resistance to fluor-
The most common mechanism of high-level fluoroquino- oquinolones is achieved by the binding of the Qnr protein to
lone resistance is due to mutation in one or more of the the topoisomerase, which physically prevents the interca-
genes that encode the primary and secondary targets of lation of the antibiotic with the target enzyme [40]. The qnr
these drugs, the type II topoisomerases (gyrA, gyrB, parC, genes generally confer modest protection against fluoro-
and parE). The region where mutations arise in these quinolones; for example, when the original qnr plasmid
genes that encode fluoroquinolone resistance is a short was transferred into E. coli J53, a 16-fold increase in the
DNA sequence known as the quinolone resistance-deter- minimum inhibitory concentration (MIC) of ciprofloxacin
mining region (QRDR) [28,29]. Mutations in the QRDR of was observed [37]. Subsequently, qnr was later renamed
these genes, resulting in amino acid substitutions, alter the qnrA and families of qnr genes (qnrB, qnrS, qnrC, and
target protein structure and subsequently the fluoroquin- qnrD) have been described [41,42]. However, there is a
olone-binding affinity of the enzyme, leading to drug resis- wide range of MIC changes reported as a result of carriage
tance [30,31]. Although fluoroquinolones preferentially of qnr genes; this has been attributed to differences in
target either DNA gyrase or topoisomerase IV, they will plasmid copy number and gene expression. The greatest
bind to the secondary target and exert an antibacterial changes are often seen when comparing the impact of
effect even if the primary target has been mutated to a carriage by highly antibiotic-susceptible laboratory strains
resistant allele. For example, in E. coli DNA gyrase is the (Table 2) [43]. Another PMQR gene is a variant of an
primary target of fluoroquinolones, although topoisomer- aminoglycoside acetyl transferase, aac(60 )-lb-cr, which is
ase IV also becomes a target once gyrA is mutated [31,32]. able to degrade certain fluoroquinolones [37]. This is also
Alteration of the primary target site can be followed by common on plasmids and confers decreased susceptibility
secondary mutations in lower-affinity binding sites and to ciprofloxacin and norfloxacin by acetylating the amino
highly resistant organisms will typically carry a combina- nitrogen on the piperazinyl substituent present in these
tion of mutations within gyrA and parC in Gram-negative drugs [42]. The third class of mobile fluoroquinolone-resis-
organisms [30,33]. Primary mutations in the QRDR often tance genes includes the oqxAB and qepA efflux systems,
involve a substitution at serine 83 (E. coli numbering) which encode transporters that can export fluoroquinolone
within GyrA for Gram-negative organisms or analogous molecules. Carriage of these genes again confers modest
sites within ParC/GrlA for Gram-positive organisms; these increases in the MIC of fluoroquinolones [44].
mutations alter the target site structure and reduce the
binding efficiency of the fluoroquinolones. Membrane permeability
The specificity of different fluoroquinolones in the selec- The permeability of the cell membrane and therefore the
tion of topoisomerase mutations has been evident in clini- ability of an antibiotic to enter the cell is a key determinant
cal practice, with the example of lower respiratory tract of the efficacy of drugs such as the fluoroquinolones that
441
Review
have intracellular targets [45]. Due to the double-mem-
brane structure, the Gram-negative cell wall presents a
particular barrier for hydrophilic molecules where the
ability to cross the outer membrane is governed by the
presence of outer membrane porin proteins. Mutations
resulting in downregulation of these proteins increase
the MIC of fluoroquinolones and other drugs [46,47] and
are commonly seen in fluoroquinolone-resistant isolates of
diverse species [33,48].
Efflux
In addition to the plasmidic efflux systems, chromosomal
multidrug efflux pumps are capable of actively removing
fluoroquinolones and other drugs from the bacterial cell.
These include transporters of various classes; for example,
the major facilitator superfamily (MFS) pump NorA of S.
aureus and the resistance nodulation division (RND) fami-
ly of tripartite transporters of Gram-negative pathogens
[49,50]. These efflux systems are largely responsible for the
intrinsic susceptibility of a species to fluoroquinolones and
other drugs but are also responsible for increased MICs
resulting from de-repression of the transporter. Exposure
of many bacteria to fluoroquinolones can select mutants
that overexpress efflux systems (e.g., patAB in S. pneumo-
niae [51], acrAB-tolC in Salmonella enterica and E. coli
[52]) as a result of mutations within the normal regulatory
networks that control the expression levels of these sys-
tems. Efflux mutants are commonly recovered in clinical
isolates of S. aureus, S. pneumoniae, and E. coli [51,53–55].
Efflux acts in conjunction with other mechanisms; for
example, highly fluoroquinolone-resistant S. pneumoniae
isolates with parC mutations in conjunction with over-
expression of patAB are inhibited by fluoroquinolone MICs
between 8 and 64 mg/ml [51]. These values were reduced
when antibiotics were combined with an efflux inhibitor
and reduced further still when the patA and patB genes
were inactivated, demonstrating an important contribu-
tion of efflux to resistance in these isolates. Efflux has been
shown to be crucial for the development of high-level
fluoroquinolone resistance, because the inactivation of
major efflux systems prevents the selection of fluoroquino-
lone-resistant mutants and strains carrying specific target-
site mutations are no longer clinically resistant if efflux
pumps are inactivated [56–58].
The evolutionary consequences of fluoroquinolone
resistance
Various theories have been postulated to try to explain the
large numbers of fluoroquinolone-resistant isolates that
have emerged in various species in relation to the biologi-
cal impact of fluoroquinolone resistance. Long-term evo-
lution experiments with E. coli have implicated mutations
within genes that control supercoiling as being subject to
selection for fitness under some conditions in the absence
of any antibiotic selective pressure. For example, muta-
tions in topA and fis that increased supercoiling levels
within the cell have occurred in multiple lineages sub-
jected to long-term passage [59]. The reason for this
is unclear but may be related to changes in the evolution-
ary flexibility of the cell as a result of changes in chromo-
some structure. A similar effect may be expected from
442
Trends in Microbiology August 2014, Vol. 22, No. 8
mutations within genes encoding topoisomerase II
enzymes that compromise the efficiency of negative super-
coiling, thus resulting in a similar net overall effect on
chromosome structure [60]. In practice, fluoroquinolone-
resistant mutants have emerged in many different species
but at different rates; the differential impact of carriage of
resistance mutations in different species and in diverse
environments may explain this.
The measurement of fitness costs of mutations usually
relies on measurements of growth rates or competition
experiments where mutants can be coinoculated in vitro
or in an in vivo model with a wild type strain and the
relative survival of mutant to parental strain can be com-
pared. There are examples where single mutations in
topoisomerase genes confer a fitness cost; for example,
in E. coli, where a gyrA mutation individually reduced
fitness by approximately 6%, and in S. pneumoniae, where
a parC mutation resulted in an 8% reduction in fitness
[61,62].
However, there is evidence from some species that,
under some conditions, mutation in gyrA influences fitness
positively and mutant strains are not only fluoroquinolone
resistant but can out-compete wild type strains in drug-
free competitive index experiments. This phenomenon has
been observed in both Campylobacter jejuni and S. enterica
serovar Typhi. In both examples this phenotype was asso-
ciated with changes to supercoiling [63,64]. Selection of
fluoroquinolone-resistant Campylobacter in pigs and poul-
try has also been shown to occur rapidly in multiple
lineages and these strains persist long after removal of
pressure on farms [65,66].
Combinations of mutations within topoisomerase genes
often have smaller impacts on fitness than single point
mutations [61]. This was predicted to be a result of com-
pensation for functional defects arising from the primary
mutation. The selective pressure to restore the function of
the target enzymes to close to optimal will promote selec-
tion of mutations that interact in an epistatic manner.
These are often in closely located sites or within genes that
are within the same pathway and that are likely to in-
crease fluoroquinolone resistance as well; therefore, once a
single resistance mutation is selected there is an increased
likelihood of selection of high-level resistance driven by
both fitness and drug pressure. The limited or lack of a
fitness cost from fluoroquinolone-resistance mutations and
the ability of further mutations to alleviate any cost sug-
gests that these strains will be at no disadvantage in the
absence of drug pressure and will therefore persist indefi-
nitely [64]. The specific combinations of mutations is im-
portant in relation to fitness; for example, fluoroquinolone-
resistant Neisseria gonnorheae strains have emerged rap-
idly and it has been discovered that two mutations within
gyrA result in strains with decreased susceptibility to
fluoroquinolones but high fitness; an additional parC mu-
tation results in high-level resistance but also reduces
fitness [67].
Further evidence for a beneficial role for gyrA mutations
was recently seen in S. enterica serovar Typhimurium,
where a change within DNA gyrase at codon 87 altered
susceptibility to a broad range of non-quinolone antibiotics.
This mutant also had altered global supercoiling and this
Review
was associated with numerous changes to the transcrip-
tome. These gene-expression changes included upregula-
tion of several stress responses, which can help the cell
tolerate the effects of antibiotic exposure [7]. The generic
nature of the stress responses and protective effect seen in
this mutant would provide a survival advantage in a wide
range of environments where low concentrations of anti-
microbials are present and may help explain why gyrA
mutants are successful.
Traditionally it has been assumed that selection of
antibiotic-resistant mutants occurs when bacteria are ex-
posed to lethal concentrations of drugs and mutants able to
survive proliferate. However, recent work has demonstrat-
ed that very low concentrations of antibiotics also exert a
selective pressure and select for the evolution of mutant
strains over time [68–70]. This has been shown for cipro-
floxacin, where exposure of E. coli to very low concentra-
tions of the drug over time exerted a selective pressure
favouring the growth of fluoroquinolone-resistant
mutants. This demonstrates that gyrA mutants are bene-
ficial and can become fixed even under sublethal exposure
to fluoroquinolones [69]. The carriage of PMQR genes has
also been shown to influence the development of fluoro-
quinolone resistance, even in the absence of fluoroquino-
lone drug pressure. PMQR genes are often carried on
plasmids with other antibiotic-resistance genes; notably,
genes encoding extended-spectrum beta-lactamases
(ESBLs) [71]. Exposure to non-quinolone antibiotics can
favour persistence of PMQRs and consequent decreased
susceptibility to fluoroquinolones. Recently, Vien le et al.
demonstrated that, after treatment for respiratory disease
in Vietnam with non-quinolone antibiotics, there was sig-
nificant enrichment for carriage of PMQRs in the gut flora
of patients and this occurred within days of treatment
commencing. This was postulated to be due to coselection
of mobile elements carrying multiple antibiotic-resistance
genes [72].
These examples show that different mechanisms of
fluoroquinolone resistance can result in very different
levels of bacterial fitness and this is not the same for all
species and all resistance mutations.
Fluoroquinolone resistance and the expansion of
successful clones
Given the high prevalence in many species and ease of
selection of mutants in vitro, development of fluoroquino-
lone resistance is clearly not incompatible with success of
pathogens. There are numerous examples where fluoroquin-
olone-resistant clones of pathogenic species have become
globally successful and established themselves in many
environments. These include the ST131-H30 clone of E. coli
responsible for extraintestinal infection, an epidemic meth-
icillin-resistant S. aureus (MRSA) strain (EMRSA-15), Clos-
tridium difficile O27 FQR1 and FQR2 lineages, and S.
enterica serovar Kentucky ST198 [73–78]. The EMRSA-15
strain belongs to sequence type 22 (ST22) and in 2000 was
responsible for over 60% of MRSA nosocomial bacteraemias
in England [78]. Recent genomic analysis has documented
the evolution and spread of EMRSA-15 over the past 20
years in response to different drug regimens. Holden et al.
[78] showed how fluoroquinolone use, introduced in the
Trends in Microbiology August 2014, Vol. 22, No. 8
1980s, promoted the spread of ST22-A2 worldwide.
ST22-A2 is a fluoroquinolone-resistant subclone that orig-
inated from the West Midlands in the UK and before the
use of fluoroquinolones was limited to this region [78].
With the introduction of fluoroquinolones in the 1980s,
ST22-A2 began to spread throughout the UK, with isolates
detected in Scotland by 1993; by 2014 it had spread
globally [78]. Fluoroquinolones are excreted in sweat
and onto the skin and therefore inhibit the growth of
normal skin flora, including S. aureus. Therefore, it was
postulated that the fluoroquinolone resistance of ST22-A2
allowed it to proliferate and spread. This resulted after
fluoroquinolones were widely used to treat numerous
infections despite these drugs not being used to treat
staphylococcal infections.
Another example where fluoroquinolone use has trig-
gered the emergence of a clone can be seen with C. difficile
O27, a globally epidemic clone that is the major cause of
antibiotic-associated diarrhoea worldwide [77]. Recent ge-
nomic analysis of O27 isolates has identified the selection
of two independent fluoroquinolone-resistant lineages,
FQR1 and FQR2, in North America in the early 1990s,
when fluoroquinolone usage was high. Both lineages have
proved successful, with FQR1 being the major cause of C.
difficile infections in North America and FQR2 being
transplanted across the world. The genetic basis for the
evident fitness of both clones was evaluated by comparing
the genomes of multiple isolates to identify mutations
responsible for success. The mutation in gyrA in both
lineages was the only common mutation and no other
changes that may contribute to fitness were identified as
a common cause [77].
The impact of the host strain in determining the fitness
of fluoroquinolone-resistant mutants can be great and the
examples of epidemic spread of clones represent those
where a resistance mutation has been well tolerated or
even beneficial to its host strain, allowing wide dissemina-
tion.
Concluding remarks
Fluoroquinolone resistance is not simple and our under-
standing of the multiple mechanisms of resistance, the
biological impact of each on the host strain, and the inter-
play between different mechanisms has increased dramat-
ically in recent years. Similarly, the long-term fate of
different strains of different species with diverse combina-
tions of fluoroquinolone-resistance mutations is extremely
hard to predict. Clearly there is no ‘one-size-fits-all’ answer
to how fluoroquinolone resistance will influence the evo-
lution of pathogens and broad conclusions based on one
organism may not be relevant to all (Box 2). What is
becoming increasingly clear is that resistance to fluoroqui-
nolones can be selected under a wide range of selective
conditions and in certain circumstances resistant strains
may demonstrate a fitness advantage over antibiotic-sen-
sitive strains, even in the absence of fluoroquinolone pres-
sure. Fluoroquinolone resistance is common and this is
likely to be related to the biology of resistance as well as a
direct response to drug pressure. Therefore, minimising
resistance will not be as simple as restricting the use of
these agents.
443
Review
Box 2. Outstanding questions
 A complete understanding of how fluoroquinolone resistance
influences the biology of host cells is required.
 How do differential levels of supercoiling in different species
influence fluoroquinolone susceptibility and the impacts of
resistance mutations?
 What is the impact of different dosing regimens on the selection
of fluoroquinolone-resistant bacteria in patients?
 How do we best use fluoroquinolones to minimise selection of
resistance in the clinic and beyond?
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