Microbiology (2011), 157, 937944

Mini-Review

DOI 10.1099/mic.0.046870-0

Parallel evolution and local differentiation in

quinolone resistance in Pseudomonas aeruginosa
Alex Wong and Rees Kassen

Correspondence
Alex Wong

Center for Advanced Research in Environmental Genomics, Department of Biology, University of

Ottawa, Ottawa, ON, Canada

awong@uottawa.ca

The emergence and spread of antibiotic resistance in pathogens is a major impediment to the
control of microbial disease. Here, we review mechanisms of quinolone resistance in
Pseudomonas aeruginosa, an important nosocomial pathogen and a major cause of morbidity in
cystic fibrosis (CF) patients. In this quantitative literature review, we find that mutations in DNA
gyrase A, the primary target of quinolones in Gram-negative bacteria, are the most common
resistance mutations identified in clinical samples of all origins, in keeping with previous
observations. However, the identities of non-gyrase resistance mutations vary systematically
between samples isolated from CF patients and those isolated from acute infections. CF-derived
strains tend to harbour mutations in the efflux pump regulator nfxB, while non-CF strains tend to
bear mutations in the efflux regulator mexR or in parC, which encodes one of two subunits of DNA
topoisomerase IV. We suggest that differences in resistance mechanisms between CF and nonCF strains result either from local adaptation to different sites of infection or from differences in
mutational processes between different environments. We further discuss the therapeutic
implications of local differentiation in resistance mechanisms to a common antibiotic.

Introduction
Antibiotic resistance is a serious and growing obstacle to
the treatment and management of microbial disease. Since
the introduction of mass-produced antibiotics in the 1940s,
single- and multi-drug resistant strains of most major
pathogens have steadily increased in frequency (CDC,
2010; Government of Canada, 2007). Infection with a
resistant pathogen increases the risk of negative patient
outcomes, including mortality (Shorr, 2009), and often
necessitates aggressive treatment strategies. Understanding
and ultimately controlling resistance will require insights
from a range of disciplines including epidemiology,
molecular biology and evolutionary biology. Since resistance is, in effect, an example of adaptation in response to
strong natural selection imposed by the use of antibiotics,
evolutionary biology is key to addressing the problem of
resistance. Under discussion here are some of the most
central and long-standing problems in evolution: understanding the rate and extent of adaptation, the variety of
genetic targets underlying adaptation, and the specificity of
adaptation.
In this review, we discuss the evolution of quinolone
resistance, focusing on the opportunistic pathogen Pseudomonas aeruginosa. We summarize published surveys on the
mechanisms of resistance identified in clinical samples, and
A supplementary table giving details of the studies that investigated the
molecular mechanism(s) of resistance in clinical isolates of P. aeruginosa
is available with the online version of this paper.

046870 G 2011 SGM

Printed in Great Britain

find that mutations in gyrA, which encodes the primary

target of quinolones, are most common. This observation
has been made previously by a number of other studies in
P. aeruginosa and other bacterial species, and so constitutes
an example of parallel evolution at the molecular level.
Notably, though, we also find evidence that additional
resistance mutations differ between Pseudomonas strains
sampled from the lungs of cystic fibrosis (CF) patients and
those sampled from acute, non-CF infections. We discuss
both of these findings highly parallel evolution and local
differentiation in the light of evolutionary theory, and
suggest further studies and therapeutic implications.
Quinolone targets and mechanisms of resistance
Quinolone antibiotics constitute a widely used class of
broad spectrum antibiotics first discovered in 1962 and
introduced for clinical use in 1967 as a treatment for
urinary tract infections caused by enterococcal bacteria
(Emmerson & Jones, 2003). Following the introduction of
the first quinolone antibiotic, nalidixic acid, subsequent
work led to the development of fluoroquinolones (e.g.
ciprofloxacin), a large and effective family of compounds.
Quinolones target two paralogous enzyme complexes:
DNA gyrase and DNA topoisomerase IV. Each complex
is made up of two subunits: GyrA and GyrB in the case of
gyrase, and ParC and ParE in the case of topoisomerase IV.
The gene duplication events that gave rise to these two
complexes are very ancient, predating the split between
937

A. Wong and R. Kassen

Gram-negative and Gram-positive bacteria (e.g. Sissi &

Palumbo, 2010). DNA gyrase and topoisomerase IV play
important roles in DNA supercoiling and in the decatenation of closed circular DNA molecules. As such, they
function in a number of cellular processes, including DNA
replication, transcription and recombination (reviewed by
Nollmann et al., 2007; Schoeffler & Berger, 2005).
Quinolones trap DNA gyrase or topoisomerase IV in a
complex with cleaved DNA, blocking DNA replication and
eventually leading to cell death. Quinolone resistance can be
acquired via mutations that reduce drug binding without
abolishing normal enzymic functions (Chen & Lo, 2003;
Kureishi et al., 1994). A number of mutations in the
quinolone-resistance-determining regions (QRDR) of gyrase
and topoisomerase IV can grant resistance in this manner;
the most important of these mutations occurs at position 83
of GyrA, which corresponds to residue 80 in ParC (Fig. 1a; all
residue numbering follows the P. aeruginosa positions, which
match the canonical Escherichia coli positions). These
residues are highly conserved throughout prokaryotes (Fig.
1b), with serine and threonine as the most common amino
acids. Mutations to leucine (from serine) or isoleucine (from
threonine), and less frequently to alanine, have been reported
in clinical and in vitro studies as conferring resistance
(reviewed by Piddock, 1999). Additional mutations at
residue 87 of GyrA (Fig. 1a; residue 84 of ParC), as well as
in GyrB and ParE, also contribute to resistance, and are
frequently observed in combination with S83L or T83I
mutations (Chen & Lo, 2003).
In addition to mutations in target enzymes, quinolone
resistance can be gained via a reduction in intracellular
drug concentration, either by reducing influx or by
increasing efflux. In Gram-negative bacteria, a reduction
in influx can be achieved by mutations in porin structural
genes. Porins form channels in the bacterial membrane,

through which exogenous molecules can enter the

cell. Thus, a decrease in the number of porin channels
serves to reduce entry of antibiotic into the cell (Jacoby,
2005).
Increased efflux of antibiotic in P. aeruginosa is typically
achieved by the upregulation of chromosomally encoded
efflux pumps. Efflux pump repertoires vary widely among
different bacterial species, such that different mutations are
responsible for quinolone resistance in different taxa. In P.
aeruginosa, at least two efflux pumps are thought to play
important roles in quinolone resistance: MexABOprM
and MexCDOprJ (Poole, 2005). Each pump is encoded by
a single operon, and this operon is repressed by a single
regulatory protein. Loss-of-function mutations in the gene
encoding the regulatory protein thus serve to increase
efflux pump expression, leading to decreased drug susceptibility. In P. aeruginosa, the mexR and nfxB genes encode
repressors of MexABOprM and MexCDOprJ, respectively, and mutations in these genes can increase pump
expression (e.g. Poole, 2005).
The resistance mechanisms described thus far are all
examples of chromosomal resistance mutations. In the last
13 years, however, a number of plasmid-borne quinolone
resistance genes have been identified, primarily in E. coli
and in Klebsiella pneuomoniae (reviewed by Strahilevitz
et al., 2009). Plasmid-borne resistance genes are transferred
horizontally between bacteria, and thus represent the
potential for the rapid spread of resistance, both within
and among species. None of these genes has been reported
in a clinical isolate of P. aeruginosa, although MartnezMartnez et al. (1998) demonstrated that the qnrA gene
does confer resistance in this species if introduced via
conjugation. As such, for the remainder of this paper,
we restrict our discussion to chromosomal resistance
determinants.

Fig. 1. (a) The structure of E. coli DNA gyrase, with S83 highlighted orange and D87 highlighted red. (b) Conservation of the
DNA binding pocket in GyrA. Residues 83 and 87 are highlighted in orange and red, respectively. The phylogeny was
reconstructed from the full amino acid sequence of GyrA.
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Microbiology 157

Quinonolone resistance in Pseudomonas aeruginosa

A survey of clinically relevant quinolone

resistance mutations
The widespread use of quinolones has led to a rapid
increase in the incidence of resistance in many pathogenic
bacteria. Prevalence of resistance to ciprofloxacin, a commonly used fluoroquinolone, ranges from 5 % to over 80 %
amongst hospital isolates of important nosocomial pathogens (Jacoby, 2005). Given the prevalence of quinolone
resistance, considerable effort has been devoted to
identifying resistance mutations in clinical isolates of many
pathogens.
In order to quantitatively ascertain the frequency and
importance of different resistance mutations, we have
undertaken a survey of known resistance mutations
reported in quinolone- and/or fluoroquinolone-resistant
isolates of P. aeruginosa. P. aeruginosa is a Gram-negative
bacterium that occupies a broad range of environmental
and pathogenic habitats. Importantly, it has the capacity to
cause radically different acute and chronic infections. P.
aeruginosa infects a number of sites, including the urinary
tract, eye, burn wounds, lung and blood. It is responsible
for about 10 % of acute hospital-acquired infections, where
it can cause acute pneumonia in hospitalized patients
(Hancock, 1998). In addition, P. aeruginosa is a major
pathogen of individuals with CF. In Canada, for example, it
occurs in 6070 % of adults with CF (Stephenson, 2008).
Both population-based and case-control studies have
demonstrated that infection with P. aeruginosa appears to
be an independent prognostic factor that carries with it an
increased risk of death in CF patients, irrespective of lung
function (Corey & Farewell, 1996; Rosenfeld et al., 1997).
We identified 16 publications that investigated the
molecular mechanism(s) of resistance in one or more
clinical isolates of P. aeruginosa. Of these 16 studies, 13
examined only isolates from non-CF patients, two
examined only CF isolates and one examined isolates from
both CF and non-CF patients. Most studies identified
resistance mutations by direct sequencing of candidate loci,
or by genotyping specific SNPs; a few studies examined
efflux pump mRNA levels or used complementation tests
to identify gyrA mutants. The full datasets are given in
Supplementary Table S1, available with the online version
of this paper. Interestingly, we find evidence of both
widespread parallel evolution and local differentiation
amongst documented mechanisms of antibiotic resistance.
These patterns give hints regarding the processes underlying the evolution of antibiotic resistance, and suggest
further avenues of research.
Parallel evolution in quinolone resistance
Parallel evolution is the evolution of the same genotypes
and/or phenotypes repeatedly and independently. The
observation of parallel evolution as a response to similar
ecological pressures is a powerful indication that natural
selection is responsible for the evolution of a given trait or
http://mic.sgmjournals.org

genotype. Parallel evolution has historically been discussed

in the context of whole organism studies of adaptive
evolution where it is used to infer patterns of past selection.
For example, populations of three-spine sticklebacks (a
widespread species of fish) have independently colonized
freshwater lakes from the ocean hundreds or thousands
of times since the last ice age. Colonization events are
typically accompanied by a suite of parallel morphological
changes, including reductions in body armour and spine
length. The consistency with which these changes occur
upon freshwater colonization is strong evidence that they
are driven by natural selection, rather than another process,
e.g. genetic drift (e.g. Bell & Foster, 1994). In microbes,
parallel evolution is less well studied, although it is known
to occur in the context of metronidazole resistance in
Helicobacter pylori (Albert et al., 2005). However, the
simple fact that we know the genetic targets of resistance
for the most commonly used antibiotics, and that these are
highly conserved across species (for example, gyrA mutations confer resistance to quinolones, rpoB mutations
confer resistance to rifampicin and so on), suggests that
parallel evolution is a hallmark of resistance evolution.
We find a marked pattern of parallel evolution with respect
to quinolone resistance in P. aeruginosa. We find that
mutations in gyrA are very common amongst resistant
strains, regardless of the type of infection (CF vs nonCF) (Fig. 2), in accordance with previous suggestions
(Henrichfreise et al., 2007). Similar patterns have been
observed in numerous other species, with gyrA mutations
common in Gram-negative bacteria (Piddock, 1999).
Recent genetic screens in E. coli (Tamae et al., 2008) and P.
aeruginosa (Breidenstein et al., 2008) have identified genes
whose knockouts either reduce or increase sensitivity to
ciprofloxacin, with .100 and 40 genes, respectively, contributing to resistance. Most of these mutations have not
been genotyped in clinical samples, so that their contributions to clinical resistance cannot yet be fully determined.
Nonetheless, the overwhelming prevalence of gyrA mutations suggests that regardless of how many other potential
mutations could contribute to resistance, the gyrA mutants
are favoured. Part of the reason for the selection of gyrA
mutations is probably the greater reduction in susceptibility that they afford. In P. aeruginosa, a 1670-fold increase
in MIC can be attributed to the T83I mutation (Cambau
et al., 1995; Diver et al., 1991; Kureishi et al., 1994). By
contrast, the transposable element mutations described by
Breidenstein et al. (2008) result in much more modest (2
46) increases in MIC. Thus, even though a gyrA T83I
variant is no more likely to arise in a population than any
other resistance (point) mutation, the fitness benefit that it
affords in the presence of a high concentration of antibiotic
virtually guarantees its success once it does arise. At lower
antibiotic concentrations, however, other mutations could
play important roles (Zhou et al., 2000).
Other factors may also help to explain the prevalence of
gyrA mutations. The costs of resistance may be particularly
939

A. Wong and R. Kassen

Fig. 2. Frequencies of major quinolone resistance mutations in P. aeruginosa samples from

CF (light grey) and non-CF (dark grey)
patients. *Significant difference in mutation
counts (given above the bars), P,0.001, by
Fishers exact test.

important in understanding why some resistance mutations are prevalent, and why they persist following the
cessation of antibiotic treatment. Resistance mutations,
while beneficial in the presence of antibiotic, may inflict
fitness costs in the absence of drug (Andersson, 2006;
Perron et al., 2010; Ward et al., 2009). Such costs arise
through pleiotropic effects of the mutation on other traits
important to fitness, such as growth rate. Once antibiotic
treatment has finished, costly resistance mutations should
be rapidly eliminated from a population by genotypes
bearing the wild-type, non-costly allele. Such genotypes
could be introduced anew into the population from
environmental sources, or by back-mutation from the
mutant type. However, resistance is often maintained even
after treatment has run its course and antibiotic use has
been terminated (Andersson, 2006). The maintenance of
resistance could be explained by no-cost resistance
mutations that have wild-type (or better) fitness in the
absence of antibiotic. Alternatively, second-site mutations may compensate for the costs of resistance, while
maintaining the resistance afforded by the original
mutation.
The available data are mixed with respect to the costs of
resistance to fluoroquinolones, although there is a general
pattern that commonly found S83L/T83I mutations do
bear a cost. Bagel et al. (1999) found that E. coli strains
bearing single S83L or D87G mutations in gyrA had a 33 %
longer doubling time than did isogenic wild-type strains,
and that double mutants carrying both mutations had a
104 % increase in doubling time. Similarly, Kugelberg et al.
(2005) found a reduced growth rate for P. aeruginosa
bearing the T83I mutation. Interestingly, however, other
nalidixic-acid-resistant mutants isolated in the Kugelberg
study, including D87G and T83A, suffered no substantial
cost (these mutants also had higher susceptibility than T83I
mutants). Moreover, the fitness cost of the T83I mutation could be compensated after only 2030 generations
of serial passaging in permissive conditions. Kassen &
Bataillon (2006) identified between 10 and 28 nalidixicacid resistant mutants (out of 665 resistant mutants
940

screened) of Pseudomonas fluorescens that were fitter than

the wild-type strain in permissive media. The uncertainty
associated with the number of mutants reflects how
statistically stringent one wishes to be in defining a mutant
as fitter than the wild-type. Preliminary work has shown
that mutations at codons 83 and 87 of gyrA predominate
among these mutants, although a comprehensive screen
has yet to be conducted. In general, if S83L/T83I mutations
are costly, then compensatory second-site mutations may
play an important role in their prevalence and persistence.
Environment-specific mechanisms of resistance
in P. aeruginosa
In our literature survey, mechanisms of quinolone
resistance differed between P. aeruginosa samples taken
from CF and non-CF patients. For both groups, mutations
in gyrA are the most common amongst resistant clinical
strains. However, frequencies of additional resistance
mutations vary significantly between the two types of
sample (Fig. 2): amongst samples from CF patients, 57 % of
resistant strains bear mutations in nfxB, which acts as a
negative regulator of the MexCDOprJ efflux pump. None
of the CF strains carried mutations in parC or in mexR.
Amongst non-CF samples, however, the situation is
reversed: 48 and 33 % bear mutations in parC and mexR,
respectively, while only 16 % carry nfxB mutations. Thus,
these data suggest an important role for nfxB-mediated
resistance in samples from CF patients, with a major role
for parC mutations, and for different efflux mechanisms in
non-CF samples. These patterns have been hinted at in
earlier studies (Henrichfreise et al., 2007; Jakics et al.,
1992), but this is the first systematic analysis, to our
knowledge, of quinolone resistance mutations between CF
and non-CF isolates.
These observations raise the question as to why different
resistance mutations are seen in different types of infection,
that is, how evolutionary outcomes are dictated by
environmental differences between the CF lung and sites
of acute infection. It is becoming increasingly clear that the
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Quinonolone resistance in Pseudomonas aeruginosa

unique environment of the CF lung plays an important role

in determining the physiological state and evolutionary
trajectory of P. aeruginosa during chronic infection. Lungs
of CF patients become filled with viscous sputum, and the
viscosity, nutritional composition and low aerobicity of
CF sputum are important and peculiar features of this
microbial habitat (Brown et al., 2008; Hassett et al., 2009;
Worlitzsch et al., 2002). In the present context, such
features of the CF lung must influence either (or both) the
processes of mutation and selection in order to account for
differences in drug resistance mechanisms.
Differences in mutational patterns between environments
could account for the different resistance mutations if, for
example, mutations at nfxB occur more often in the CF lung
than elsewhere. While descriptions of similar environmentspecific variation in mutational patterns are rare, Bjorkman
et al. (2000) argued for their occurrence during compensatory evolution in Salmonella typhimurium. They found
different mutations in streptomycin-resistant strains that
had been allowed to undergo compensatory evolution in LB
or in mice. The fitness effects of the different mutations did
not differ between the two environments, leaving differential
mutational processes as the best explanation for the
observation. Similarly, it is possible that differences in the
spectrum of quinolone resistance mutations among strains
isolated from CF and non-CF patients are the result of
systematic variation in mutational processes.
Alternatively, the fitness effects of different mutations may
be environment-specific, such that the unique culture
conditions presented by the CF lung favour nfxB mutations
over parC or mexR mutations, owing to the level of
resistance granted and/or any negative, indirect consequences of resistance (e.g. reduced growth rate). This
alternative hypothesis thus invokes selection rather than
mutation. A difference in the rank order of fitness effects of
mutations by environment is often referred to as a
genotype-by-environment (GxE) interaction in evolutionary genetics. GxE interactions have been the subject of
much theoretical and empirical work in evolutionary
biology (e.g. Dean, 1995; Hedrick, 2006; Remold &
Lenski, 2001), and the existence of GxE interactions has
important implications for the maintenance of polymorphism and for local adaptation. Genetic variation can be
supported if different mutations are favoured in different
environments, and this situation will lead to populations
that are well adapted to their local environments, but not
necessarily to other conditions.
Work on GxE interactions draws attention to three distinct
mechanisms by which differential fitnesses could be
generated for P. aeruginosa drug resistance mutations.
Broadly, differences in the strength of selection, or in either
the physiological state or the genetic background of P.
aeruginosa in CF and non-CF infections, may underlie
differences in fitness ranking. We will refer to these three
mechanisms as the effective dose, plasticity and epistasis hypotheses.
http://mic.sgmjournals.org

Hypothesis 1: effective dose

Drug concentrations can vary dramatically amongst tissues

and, as previously noted, different resistance mutations can
be selected at different antibiotic concentrations (Zhou
et al., 2000). Studies of in vivo ciprofloxacin concentrations
in CF patients have found systematically lower drug levels
in sputum than in blood (sputum concentrations of 0.2
1.9 mg l21 versus blood concentrations of 2.44 mg l21,
depending on dose and study; Pedersen et al., 1987, and
references therein). Thus, variations in drug resistance
mechanisms may reflect these local differences in concentration, if, for example, nfxB mutations confer a modest
decrease in susceptibility with few negative consequences.
Ciprofloxacin concentrations in other tissues in non-CF
patients also vary widely, however (e.g. 1.1 mg l21 in
adipose tissue, 2 mg l21 in lung epithelial lining fluid,
3 mg l21 in the prostate; Hoogkamp-Korstanje et al., 1984;
Joukhadar et al., 2005; Winter & Sweeney, 1991). Thus, it is
not clear whether the prevalence of nfxB mutations in CF
samples are attributable to differences in drug concentration alone.
Hypothesis 2: plasticity

P. aeruginosa undergoes a number of physiological changes

in the CF lung, such as growth as an unattached biofilm or
microcolony, in contrast with its planktonic lifestyle in
other types of infection (Worlitzsch et al., 2002). Such
physiological changes can be referred to as plastic insofar
as they do not result from genetic mutations, but rather
from cellular responses to the environment. The carbohydrate matrix surrounding microcolonies may decrease drug
penetration, and biofilm-specific efflux mechanisms (e.g.
Zhang & Mah, 2008) involving additional efflux pumps can
reduce intracellular drug concentrations. If the level of
resistance afforded by nfxB mutations is lower than that
afforded by parC and/or mexR mutations, but with fewer
indirect effects for nfxB mutations, then nfxB mutants
would be expected to benefit from the biofilm growth
typical of CF infections. Other physiological changes
associated with the CF lung may also play important roles,
such as decreased metabolic activity in biofilm colonies
(Hiby et al., 2010).
Hypothesis 3: epistasis

While some of the physiological changes associated with

the CF lung habitat are likely to be purely plastic, other
alterations are known to have a genetic basis. Following
colonization of the CF lung, P. aeruginosa undergoes a
number of repeatable genetic changes, such as mutations to
lasR and mucA (DArgenio et al., 2007; Mathee et al., 2008;
Rau et al., 2010; Rowen & Deretic, 2000), that have not
been reported in other types of infection. Epistatic
interactions between quinolone-resistance mutations and
these presumably CF-adaptive mutations may therefore
underlie differences in patterns of clinical resistance
mutations. For example, nfxB mutations may have an
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A. Wong and R. Kassen

advantage when present on a lasR mutant background,

with mexR and/or parC mutants most fit on a wild-type
lasR background.
Testing the hypotheses
We have suggested four hypotheses (mutation, effective
dose, plasticity and epistasis) to account for different
mechanisms of quinolone resistance between CF and nonCF strains of P. aeruginosa. Currently, data are not
available to directly test these hypotheses, but several
experimental approaches could be useful in distinguishing
amongst them.
Selective and mutational explanations for differences in
quinolone resistance mechanisms offer differing predictions with respect to the fitness effects of resistance
mutations in CF and non-CF culture conditions. If
differences in mutational spectra are driven by selection,
then one would expect nfxB mutations to be favoured
under conditions resembling the CF lung, with mexR and/
or parC favoured under conditions resembling acute
infections. In contrast, a mutational mechanism would be
suggested if no differences in fitness were observed.
Further testing of the mutational hypothesis could be
obtained by characterizing the mutational profile of
P. aeruginosa under different environmental conditions.
Spontaneous mutants can be isolated by using fluctuation
assays (Kassen & Bataillon, 2006; Luria & Delbruck, 1943)
or by mutation accumulation (Halligan & Keightley, 2009).
Identification of mutations in a large number of mutants
isolated under CF-like and non-CF-like conditions would
serve to test the hypothesis that mutational processes differ
between these two environments.
Several approaches can contribute to distinguishing between
the three possible selective hypotheses. For example,
selection of mutants at different quinolone concentrations
chosen to reflect known concentrations in patients
would test the hypothesis that nfxB mutants are more
prevalent at lower drug concentrations. A previous study in
Mycobacterium has shown that gyrA mutations are more
likely to be selected at high quinolone concentrations, with
non-gyrA mutations prevalent at low concentrations (Zhou
et al., 2000). However, this study did not identify the nongyrA mutations, and so does not bear directly on the
differences in non-gyrase mutations described here.
With respect to plasticity, genetic approaches should help
to identify genes that are important for quinolone
resistance in CF-like conditions; such genes may encode
inducible efflux pumps [such as those described by Zhang
& Mah (2008) for tobramycin resistance], proteins
important for biofilm formation and other physiologically
regulated molecules (e.g. Fung-Tomc et al., 1993; Palmer
et al., 2007). For any gene contributing to GxE interactions,
the change in rank order of fitness of nfxB, mexR and parC
mutations should be dependent on its expression. Thus, for
example, rank order of fitness would not be expected to
942

change in a genotype where an inducible efflux pump had

been knocked out or repressed, if upregulation of this
pump were responsible for the GxE interaction.
Finally, studies of adaptation to the CF lung have identified
a number of loci that are often mutated in CF infections
(e.g. lasR, mucA), and these loci are good candidate genes
that could have epistatic relationships with nfxB, mexR or
parC. Epistasis between quinolone resistance mutations
and mutations beneficial in the CF lung can be evaluated
by generating all possible mutant combinations (for
example, all four possible genotypes bearing wild-type or
mutant lasR and wild-type or mutant nfxB) on an isogenic
background, and assaying fitness using standard competitive or non-competitive techniques. If epistasis causes GxE
interactions, one would expect (for example) lasR, nfxB
double mutants to have the highest fitness under CF-like
conditions, and wild-type lasR, mexR mutant genotypes to
have the highest fitness under conditions characteristic of
acute infections.
Conclusions
The increasing prevalence of single- and multi-drug resistant
pathogens necessitates the development of carefully planned
antibiotic treatment regimens. Importantly, the occurrence
of environment-specific mechanisms of resistance complicates treatment and mitigation strategies. Approaches such
as switching to new antibiotics or using multi-drug cocktails
might be effective under one set of conditions but not
another, depending on the identity of the second site
mutations. In P. aeruginosa, the MexABOprM efflux pump
(regulated by mexR) extrudes a number of antibiotics,
including quinolones, macrolides and b-lactams (Masuda
et al., 2000). The MexCDOprJ efflux pump (regulated
by nfxB) also extrudes a variety of antibiotics, but it is
ineffective against some b-lactams, such as cephems. Thus,
these b-lactams may be unsuitable for use against quinolone-resistant P. aeruginosa in acute infections but may be
suitable for CF patients. The phenomenon of local differences in resistance mechanisms (whether driven by
mutation or selection) should be addressed during the
design of treatment regimens: if different host environments
tend to favour different resistance mutations, then the
choice of drug combinations should reflect this fact.
Other bacterial species may also evolve environmentspecific resistance mechanisms. Staphylococcus aureus, like
P. aeruginosa, can cause acute infections as well as chronic
biofilm infections in CF patients. It would be very interesting to determine whether S. aureus strains isolated from CF
and non-CF patients show analogous similarities and
differences in mechanisms of quinolone resistance. At this
time, however, there have been insufficient studies on
mechanisms of quinolone resistance in S. aureus to make
this comparison.
The management of antibiotic resistance at both the
patient and population level is a complex and difficult
Microbiology 157

Quinonolone resistance in Pseudomonas aeruginosa

problem, necessarily drawing on a wide range of disciplines. Because pathogen populations are subject to
intense natural selection by antibiotics, it is important to
understand the evolutionary patterns and processes underlying resistance. For example, we have highlighted here the
potential roles of biased mutation and GxE interactions.
Ultimately, predicting the evolutionary responses of microbial populations to antibiotic treatment will be a necessary
component of controlling the emergence, spread and
persistence of resistant pathogens.
Acknowledgements

Diver, J. M., Schollaardt, T., Rabin, H. R., Thorson, C. & Bryan, L. E.

(1991). Persistence mechanisms in Pseudomonas aeruginosa from

cystic fibrosis patients undergoing ciprofloxacin therapy. Antimicrob

Agents Chemother 35, 15381546.
Emmerson, A. M. & Jones, A. M. (2003). The quinolones: decades of

development and use. J Antimicrob Chemother 51 (Suppl. 1), 1320.

Fung-Tomc, J., Kolek, B. & Bonner, D. P. (1993). Ciprofloxacin-

induced, low-level resistance to structurally unrelated antibiotics in

Pseudomonas aeruginosa and methicillin-resistant Staphylococcus
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Antimicrobial Resistance Surveillance (CIPARS). Guelph, ON: Public

Health Agency of Canada.
Halligan, D. L. & Keightley, P. D. (2009). Spontaneous mutation

We thank the Canadian Institutes for Health Research and the

Natural Sciences and Engineering Research Council for funding, and
anonymous reviewers for their helpful and insightful comments.

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