International Journal of Antimicrobial Agents 24 (2004) 529–535

Review

Role of efflux mechanisms on fluoroquinolone resistance in

Streptococcus pneumoniae and Pseudomonas aeruginosa
George G. Zhanela,b,c,∗ , Daryl J. Hobana,c , Kristen Schureka , James A. Karlowskyd
aDepartment of Medical Microbiology, Faculty of Medicine, University of Manitoba, Manitoba, Canada
b Department of Medicine, Health Sciences Centre, Winnipeg, Manitoba, Canada
c Department of Clinical Microbiology, MS673, Health Sciences Centre, 820 Sherbook St., Winnipeg, Manitoba, Canada R3A 1R9
d Focus Technologies, Herndon, VA, USA

Abstract

Prokaryotic efflux mechanisms can effectively increase the intrinsic resistance of bacteria by actively transporting antibiotics out of cells,
thus reducing the effective concentration of these agents. The fluoroquinolones, similar to most other antimicrobial classes, are susceptible to
efflux mechanisms, particularly in Gram-negative organisms, such as Pseudomonas aeruginosa. Resistant P. aeruginosa clones isolated after
fluoroquinolone therapy frequently over express at least one of the multiple efflux pump mechanisms found in this organism. Gram-positive
bacteria, such as Streptococcus pneumoniae, also possess efflux mechanisms, though their effect on fluoroquinolone resistance seems to be
more limited and selective. In the future, efflux pump inhibitors may offer effective adjunctive therapy to antibiotics for the treatment of
difficult infections by efflux mutants. In the meantime, appropriate antibiotic selection and optimal dosing strategies should aim to eradicate
the causative pathogen before a resistant efflux mutant can emerge.
© 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Keywords: Efflux; Fluoroquinolone; Resistance; Streptococcus pneumoniae; Pseudomonas aeruginosa

1. Introduction
Since the introduction of the fluoroquinolones in the
1980s, the use of this class of drugs has rapidly increased to
become one of the more common choices of antimicrobials
for various types of bacterial infection [1]. This widespread
use has had its consequences, as selective pressure has in-
creased resistance for some species over the years [1]. Though
resistance to common respiratory pathogens, including Strep-
tococcus pneumoniae, remains ≤1% in the US, other parts of
the world are not so fortunate [2–4]. In addition, resistance in
Gram-negative organisms, such as Pseudomonas aeruginosa,
has increased significantly [5,6]. Fluoroquinolone resistance
can involve two well-documented and separate mechanisms:
(1) point mutations in the genes of the quinolone target
enzymes, topoisomerase and gyrase, or (2) expression of
drug efflux pumps that reduce the accumulation of antibi-
∗ Corresponding author. Tel.: +1 204 787 4902; fax: +1 204 787 4699.
E-mail address: ggzhanel@pcs.mb.ca (G.G. Zhanel).
otic in the cell [1,7]. This review will concentrate on the
latter mechanism, particularly for two bacteria, S. pneumo-
niae and P. aeruginosa. These two clinically important respi-
ratory pathogens will provide examples of the differences in
efflux mechanisms associated with Gram-positive and Gram-
negative bacteria. The clinical implications of efflux mecha-
nisms will be discussed as well as possible future strategies
that may be used to combat efflux-mediated resistance and
limit resistance emergence.
2. Efflux pumps
Efflux pumps (or drug transporters) are essential mecha-
nisms used by prokaryotes to transport various substances,
including toxic compounds such as antibiotics, out of cells to
protect themselves from the adverse effects. The importance
of efflux pumps is reflected in the large portion of a bac-
terium’s genetic makeup dedicated to this function, where an
estimated 5–10% of bacterial genes are involved in transport

0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/j.ijantimicag.2004.08.003
530 G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535
[8]. The first efflux pump was identified in studies analysing
tetracycline resistance [9,10]. Through the years, the role of
efflux mechanisms in eliciting resistance to other antimicro-
bial classes was identified and it was soon established that
efflux was a common mechanism for bacterial resistance.
To varying extents, the fluoroquinolones have been shown
to be vulnerable to efflux pumps, and may depend on cer-
tain structural characteristics, such as size and hydrophobic-
ity of the fluoroquinolone molecule [11]. Numerous stud-
ies have assessed the role of efflux in fluoroquinolone resis-
tance [7,12,13]. One concern of these studies is determining
if efflux is truly involved in resistance in a particular isolate
or if an alternate mechanism is involved. Certain applica-
tions have been used to inhibit the efflux pump mechanism,
such as reserpine or the energy inhibitor carbonyl cyanide m-
chlorophenylhydrazone (CCCP) for Gram-positive bacteria
[14]. Gram-negative bacteria employ multiple efflux systems,
so it is more difficult to identify inhibitors that specifically
affect one or all of these systems. Because of this, genetic
techniques are employed to study the role of efflux in these
organisms.
Strains exhibiting efflux-mediated fluoroquinolone resis-
tance frequently demonstrate cross-resistance to other struc-
turally unrelated antimicrobials [15,16]. This may be due
to the broad substrate specificity of fluoroquinolone efflux
pumps and raises concern over the use of fluoroquinolones
and the impending increase in the prevalence of multidrug
resistant strains. Therefore, a thorough understanding of the
use of fluoroquinolones and the impact of efflux pumps may
help limit resistance development and preserve the clinical
value of this class of antibiotics.
2.1. Streptococcus pneumoniae
S. pneumoniae is a predominant pathogen in community-
acquired respiratory tract infections and its resistance to an-
tibiotics has presented an increasing global concern over
the past decade. Surveillance studies in the US have shown
high-level penicillin resistance approaching 20% and resis-
tance to the macrolides at over 25% [2,17]. Fortunately, flu-
oroquinolone resistance (to the respiratory fluoroquinolones
such as gatifloxacin, levofloxacin and moxifloxacin) has re-
mained low throughout the United States and Canada (at or
below 1%) but signs of increasing resistance are apparent
[18,19]. S. pneumoniae resistance to the fluoroquinolones
is caused by mutations in the genes of the target enzymes,
topoisomerase and gyrase. A single mutation (frequently in
the quinolone resistance determining region—QRDR) usu-
ally results in low-level resistance, while high-level fluo-
roquinolone resistance requires at least two mutations in
these genes [7,20,21]. However, efflux mechanisms have
been shown to play a role in conferring quinolone resistance,
though at lower levels [13,14].
Identification of a putative efflux pump gene, pmrA, re-
sponsible for fluoroquinolone resistance in S. pneumoniae
was first reported in 1999 [22]. Inactivation of this gene in a
strain demonstrating an efflux phenotype resulted in restora-
tion of drug susceptibility to norfloxacin. With the complete
sequence of the S. pneumoniae genome now available, [23]
other putative efflux pump genes have been identified. Yet,
at present no other pumps have been identified that actively
efflux fluoroquinolones, though alternative pumps are sus-
pected [24]. Efflux pump mutants in S. pneumoniae are usu-
ally identified by comparing the MIC value of an isolate ei-
ther with or without the presence of a known efflux pump in-
hibitor, such as reserpine. Chemically unrelated efflux pump
substrates, such as ethidium bromide and acriflavine, are also
used as controls to distinguish further the role of efflux pumps
in a particular isolate.
It is important to note that all fluoroquinolones do
not demonstrate equivalent susceptibility to efflux mecha-
nisms in S. pneumoniae. Accumulation of fluoroquinolones
within wild-type strains has been shown to be dependent on
size and hydrophobicity of the agent [11]. Smaller agents
(ciprofloxacin, norfloxacin, and ofloxacin) and more hy-
drophobic compounds (ciprofloxacin and norfloxacin) show
greater accumulation in the cell than other agents. However,
in studies with an efflux pump mutant strain, the greatest de-
crease in drug accumulation was observed with ciprofloxacin,
ofloxacin and gatifloxacin, while there was no significant
change in the accumulation of levofloxacin, moxifloxacin,
and sitafloxacin, among others [11].
Fluoroquinolone-resistant strains developed from in vitro
selection commonly demonstrate efflux phenotypes along
with point mutation in the target genes. One study showed
that 23 efflux mutants selected in the laboratory exhib-
ited 4–8-fold increases in MIC values for norfloxacin and
ciprofloxacin, without significantly changing the MICs of
moxifloxacin or sparfloxacin [25]. A larger in vitro study
of 80 fluoroquinolone-resistant strains reported that the pres-
ence of reserpine decreased the MIC values 4–32-fold for
ciprofloxacin in 53 mutants, and for gemifloxacin in 39 mu-
tants, while decreasing the MIC values for 13 mutants when
tested against gatifloxacin [26]. Reserpine had little effect on
the MICs of moxifloxacin and trovafloxacin.
Several studies have investigated the prevalence of efflux
pump mutants in clinical isolates of S. pneumoniae [27,28].
Of 1037 clinical isolates collected in the United Kingdom,
273 (26.3%) showed reduced susceptibility to norfloxacin
and ciprofloxacin, while 124 (12.0%) demonstrated an efflux
phenotype [25]. In a Canadian study of fluoroquinolone-
resistant clinical isolates of S. pneumoniae, 12 of 34 (35.3%)
clinical isolates demonstrated reserpine-sensitive efflux of
ciprofloxacin, decreasing the MIC in the presence of reser-
pine by 4–8-fold [21]. However, reserpine did not affect the
MIC values of any isolate for the newer fluoroquinolones,
including gatifloxacin, levofloxacin, moxifloxacin, and
gemifloxacin. Other studies have reported a high prevalence
of reserpine-inhibited efflux of ciprofloxacin in quinolone-
resistant clinical isolates of S. pneumoniae [7,29] while
newer fluoroquinolones tend to be less affected by efflux
mechanisms [30].
 G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535
The high susceptibility of ciprofloxacin to efflux mech-
anisms can have significant implications in the emergence
of fluoroquinolone-resistance. Though efflux mutants only
moderately increase S. pneumoniae resistance in most cases
(four-fold increase in MIC), this may be adequate to en-
sure the survival of a single-step mutant (in topoiso-
merase or gyrase), and increase the probability of multi-
step fluoroquinolone-resistant strains. In vitro pharmacody-
namic models show ineffective killing of S. pneumoniae
by ciprofloxacin at normal pharmacological concentrations
[31–33]. Initial killing by ciprofloxacin is usually followed
by regrowth and the emergence of resistant strains with MIC
values 2–8-fold higher than the initial MIC [31,32]. Similar
studies with gatifloxacin, levofloxacin and moxifloxacin re-
sult in effective killing of these strains and no emergence of
resistance as long as the pharmacokinetic/pharmacodynamic
target of AUC/MIC >30 is met. Newer fluoroquinolones (gat-
ifloxacin, levofloxacin, moxifloxacin, and gemifloxacin) have
been reported to effectively kill efflux mutants that exhibited
ciprofloxacin resistance [12,34,35].
Though ciprofloxacin is not recommended for respiratory
infections caused by S. pneumoniae, use of ciprofloxacin for
other indications, such as urinary tract infections, skin infec-
tions, and nosocomial pneumonia, may potentially impact the
spread of fluoroquinolone resistance by this pathogen. It is
presently unclear whether S. pneumoniae (present in the com-
mensal flora of the nasopharynx) exposed to subinhibitory
doses of ciprofloxacin will result in a single-step mutant that
is now non-susceptible to ciprofloxacin. However, once these
strains are present, subsequent exposure to ciprofloxacin
or another fluoroquinolone may favour the selection of a
multi-step mutant that exhibits high-level ciprofloxacin re-
sistance and cross-resistance to the newer fluoroquinolones.
Use of newer fluoroquinolones that are less vulnerable to
efflux mechanisms and are effective against efflux mutants
or single-step mutants may limit the emergence of fluoro-
quinolone resistance, especially when the pharmacodynamic
targets are achieved [33,36].
2.2. Pseudomonas aeruginosa
Gram-negative bacteria typically present higher intrinsic
resistance than Gram-positive organisms. This is a result of
a selectively permeable outer membrane that can act as a
barrier to antibiotics, in addition to the presence of multi-
ple multidrug efflux systems that continually extrude toxic
compounds out of the cell [15,37]. This is particularly true
for P. aeruginosa, which can cause difficult-to-treat respi-
ratory infections because of its intrinsic resistance to many
available antibiotics. The outer membrane of P. aeruginosa
has very low nonspecific permeability to small hydrophobic
molecules, which may contribute to the high intrinsic resis-
tance to fluoroquinolones [37]. Sequencing the P. aeruginosa
genome revealed that this organism contains a high propor-
tion of regulatory genes and a large number of genes involved
in the catabolism, transport and efflux of organic compounds
531
[38]. The size and complexity of the genome may explain its
ability to adapt to diverse environments and resist a variety
of antimicrobial agents.
P. aeruginosa resistance to the fluoroquinolones began in-
creasing with widespread use of ciprofloxacin. One surveil-
lance study of US intensive care units reported a decrease
in P. aeruginosa susceptibility to ciprofloxacin from 89% in
1990–1993, to 86% in 1994, to 76% in 2000 [5]. Data from the
National Nosocomial Infections Surveillance (NNIS) System
reported fluoroquinolone resistance by P. aeruginosa in noso-
comial infections to be 23% in 1999 [39]. Due to the preva-
lence of P. aeruginosa resistance to the fluoroquinolones, an
adjunctive agent with antipseudomonal activity is commonly
recommended for infections proven or suspected to be caused
by this pathogen.
Unlike efflux pumps in Gram-positive bacteria, the ef-
flux mechanisms in Gram-negative organisms contain many
components that coordinate the expulsion of compounds
from within the cytosole across the cytoplasmic membrane,
through the periplasmic space and past the outer mem-
brane. P. aeruginosa transporters belong to the resistance-
nodulation-division (RND) family and are composed of a
transporter, linker, and outer membrane (OM) pore [15]. This
system ensures that efflux of molecules through the cyto-
plasmic membrane will not accumulate in the periplasmic
space, which may lead to rapid diffusion back into the cy-
toplasm. The various efflux systems of P. aeruginosa have
been well studied over the past decade. Currently, there are
five multicomponent efflux pumps (MexAB-OprM, MexCD-
Opr-J, MexEF-OprN, MexXY-OprM, and the recently iden-
tified MexVW-OprM) that have been shown to efflux fluo-
roquinolones [16,40–42]. Of the known efflux pumps in P.
aeruginosa, only MexAB-OprM is expressed constitutively
at sufficient levels to result in intrinsic fluoroquinolone resis-
tance in wild-type cells.
Genetic approaches are commonly used to decipher the
role of efflux pumps and resistance in P. aeruginosa, either
by disrupting or over expressing genes coding for efflux pump
proteins. In one study, deletion of the genes coding for the
MexAB-OprM pump resulted in decreased resistance to flu-
oroquinolones (16-fold reduction in the levofloxacin MIC),
while over expression of any of the three pumps caused a
4–8-fold increase in levofloxacin MIC values [43]. Another
study compared the effect of efflux mutants to various flu-
oroquinolones, including ciprofloxacin, gemifloxacin, mox-
ifloxacin, and trovafloxacin [44]. Mutant strains that either
over express efflux pump genes or contain deletions of these
genes showed that all fluoroquinolones were substrates for
efflux mechanisms in P. aeruginosa. Overproduction of the
MexAB-OprM caused the greatest effect, raising the MIC
values of most fluoroquinolones tested by 16-fold. An in-
depth study that compared substrate specificities of three ef-
flux pump systems (by systematically over expressing one
pump system and deleting the other two systems) showed
that the fluoroquinolones (as well as the macrolides, tetra-
cyclines, chloramphenicol, and most penicillins) were sub-
532 G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535
strates for three pump systems tested [40]. Only imipenem
and polymyxin B were unaffected by overproduction of any
of the efflux pumps.
Clinical isolates provide additional evidence regarding the
importance of efflux mechanisms in emerging resistance of
P. aeruginosa to the fluoroquinolones. Exposure to the fluo-
roquinolones typically produces resistant strains with mu-
tations in the topoisomerase genes and efflux genes [45].
Target site mutations in tandem with mutations in the ef-
flux regulatory genes tend to result in magnified levels of
fluoroquinolone resistance [46,47]. One case reported the
emergence of a resistant P. aeruginosa strain after 4 days
of ciprofloxacin therapy [48]. The isolate had mutations in
the gyrB gene and over expressed the MexAB-OprM sys-
tem, causing a 128-fold increase in MIC to ciprofloxacin.
A larger study of cystic fibrosis patients that underwent re-
peated ciprofloxacin regimens reported an accumulation of
resistance mutations in gyrA and parC genes, as well as in
the efflux mechanisms during the 3 years of the study [49].
Alterations in two efflux systems, MexCD-OprJ and MexEF-
OprN, were the most frequent mechanisms of resistance in
isolates from these patients and caused a two-fold increase
in the median MIC to norfloxacin and ciprofloxacin over the
study period. Overall, 80% (16 of 20) of isolates contained
efflux mutations, suggesting that this mechanism was respon-
sible for at least partial resistance in a large proportion of re-
sistant clinical isolates. Clinical isolates that simultaneously
overproduce multiple efflux pumps have been identified, and
these strains tend to have broader resistance profiles and have
additive effects on the MICs of effluxed substrates [49–51].
All fluoroquinolones seem to be affected by P. aeruginosa
efflux mechanisms [40,44]. One study compared resistance
selection of P. aeruginosa by exposing the bacteria to each of
12 quinolones at 1–8 times the MIC value [52]. For a given
antibiotic concentration, the frequencies of resistant colonies
were comparable for most of the agents (between 1.2 × 10−6
and 1.3 × 10−7 at twice the MIC). Doubling the drug concen-
tration (from 2 × MIC to 4 × MIC) decreased the incidence
of resistant colonies by one to two orders of magnitude. Of the
321 colonies that developed resistance, 303 (94.3%) exhib-
ited a phenotype corresponding to resistance by over expres-
sion of at least one of the three efflux pumps. Though topoiso-
merase mutants confer high-level resistance, these mutations
occur less frequently than efflux mutations after one exposure
to a fluoroquinolone. Over expression of efflux systems can
thus be an important mechanism to initially reduced suscep-
tibility of P. aeruginosa to a fluoroquinolone and allow for
the accumulation of multiple mutations in the gyrase genes
during additional antibiotic exposure.
Few studies have been conducted to differentiate the effect
of ciprofloxacin and newer fluoroquinolones (levofloxacin,
gatifloxacin, and moxifloxacin) on the emergence of ef-
flux mutants in resistant P. aeruginosa. One group com-
pared the selection of efflux mutants by ciprofloxacin and
trovafloxacin using a rat model of acute pneumonia [53].
The frequency of resistant colonies is approximately 10-fold
less with trovafloxacin compared with ciprofloxacin (P <
0.05). Interestingly, trovafloxacin preferentially selected for
MexCD-OprJ over producers while ciprofloxacin selected for
MexEF-OprN over producers.
Since the fluoroquinolones exhibit concentration-
dependent killing activity, effective eradication and
suppression of resistance will depend on meeting the opti-
mal pharmacodynamic targets of AUC/MIC and Cmax /MIC
[54–57]. Currently ciprofloxacin and levofloxacin are the
preferred fluoroquinolones for treating proven or suspected
P. aeruginosa infections. Though ciprofloxacin consistently
demonstrates lower MICs than the respiratory fluoro-
quinolones against P. aeruginosa, levofloxacin achieves
higher peak and AUC values that result in comparable
pharmacokinetic/pharmacodynamic values when treating
these infections [56,57]. However, it must be made clear that
whether a clinician chooses ciprofloxacin or levofloxacin,
combination therapy (preferably using antibiotics with
different mechanisms of action) is recommended for these
patients.
3. Efflux pump inhibitors
With the increased understanding of the significance of
efflux pumps on antibiotic resistance, research is being con-
ducted to discover methods to overcome this mechanism.
One of the more practical strategies involves developing com-
pounds that inhibit the efflux mechanism. A number of re-
ports have been published showing that inhibition of efflux
systems can increase susceptibility while decreasing the fre-
quency of resistance emergence. As mentioned earlier, reser-
pine has been used to inhibit efflux in S. pneumoniae. One
experiment compared the frequency of emergence of resis-
tant isolates when S. pneumoniae was grown in the presence
of ciprofloxacin plus reserpine [58]. Reserpine decreased the
frequency of resistant colonies by 45-fold when grown in the
presence of 3× the MIC of ciprofloxacin. Inhibitors of the
proton motive force, such as CCCP, dinitrophenol (DNP) and
valinomycin, also inhibit efflux pumps in S. pneumoniae, sug-
gesting an energy requirement for drug efflux and possibly
exposing another target for efflux inhibition [59].
Inhibition of efflux pumps in Gram-negative organisms
presents additional challenges. These organisms have multi-
ple efflux systems that must all be inhibited to some degree
since an effect on one system can be compensated by over
expression of another. Large-scale screens of synthetic and
natural compounds have been conducted to identify poten-
tial efflux pump inhibitors (EPIs) that improve the activity
of antibiotics that are susceptible to efflux mechanisms by
P. aeruginosa. One experimental compound (MC-207, 110,
formerly Microcide Technologies, Mountain View, CA) il-
lustrated the potential of EPIs as adjunctive therapy to an-
tibiotics for treating P. aeruginosa infections [43,60,61]. The
EPI reduced the intrinsic resistance of P. aeruginosa to lev-
ofloxacin by eight-fold, while resistance in a strain overex-
 G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535
pressing efflux pumps decreased 32–64-fold, as measured by
levofloxacin MIC values. Importantly, the frequency of the
emergence of resistant isolates decreased from around 10−7
in the presence of levofloxacin alone, to <10−11 when the
EPI was included. When 50 clinical isolates were tested, the
presence of the EPI reduced the MIC90 by about 16-fold.
Research on derivatives of this compound is attempting to
identify more potent inhibitors that may be used in the clini-
cal setting in the future [62–66].
The clinical relevance of using EPIs is clearly evident.
Yet, the main obstacle continues to be the development of
an inhibitor that specifically acts on prokaryotic cells with-
out affecting eukaryotic mechanisms. Currently available in-
hibitors of Gram-positive efflux pumps, such as reserpine
and CCCP, are too toxic to use safely in patients, while a
safe Gram-negative inhibitor is still under development. Fu-
ture development of EPIs may help to increase the potency
of the fluoroquinolone, as well as other classes of agents,
against P. aeruginosa. Some Gram-positive antibiotics, such
as members of the macrolides and ␤-lactams, may be effective
against Gram-negative strains if the efflux mechanisms are in-
hibited. These agents have been shown to be potent against E.
coli and P. aeruginosa mutants lacking efflux pumps [15,67].
However, until an effective and tolerable EPI is developed,
more practical strategies and considerations must be taken
to reduce the emergence of resistance stemming from efflux
mechanisms.
4. Clinical implications
In the absence of a clinically practical efflux pump in-
hibitor, physicians must recognize the role efflux systems
play in the emergence of bacterial resistance. Some of the
clinical consequences of ciprofloxacin use on S. pneumo-
niae resistance development have been discussed previously.
Of the currently available fluoroquinolones, ciprofloxacin is
the most susceptible to efflux mechanisms in S. pneumoniae
and ciprofloxacin exposure may increase the risk of efflux
and/or single-step mutants from developing. Subsequent flu-
oroquinolone exposure may then lead to the development of
high-level fluoroquinolone resistance. Whether this occurs
frequently in the clinical setting remains to be proven. How-
ever, in one study that characterized ciprofloxacin-resistant
clinical isolates of S. pneumoniae, 10 of the 12 isolates ex-
hibiting a reserpine-sensitive phenotype also contained mu-
tations in gyrA and/or parC [21]. Fortunately, the newer flu-
oroquinolones (gatifloxacin, levofloxacin, moxifloxacin, and
gemifloxacin) do not exhibit efflux by S. pneumoniae to as
high a degree as ciprofloxacin. These agents also provide
effective activity to eradicate efflux or single-step mutants,
which may reduce the risk of high-level fluoroquinolone re-
sistant strains from emerging [12,34].
P. aeruginosa shows indiscriminate efflux with the flu-
oroquinolones and probably accounts for its high intrinsic
resistance of these agents. For respiratory infections caused
533
by this pathogen, ciprofloxacin and levofloxacin remain the
most commonly used fluoroquinolones for treatment, though
combination therapy with an antipseudomonal agent is rec-
ommended, especially for severely ill patients, such as those
with nosocomial pneumonia. In vitro studies have demon-
strated that efflux mutants occur more frequently than target
site mutants after an initial exposure to a fluoroquinolone.
Isolates from patients that underwent multiple ciprofloxacin
treatments contained mutations in both efflux genes and tar-
get sites, suggesting a sequence of events of first low level
resistance by P. aeruginosa followed by high-level resistance
by acquisition of target site mutations [49].
Increasing the drug concentration at the site of infection
may decrease the emergence of resistant isolates [52]. Clin-
ically, increasing the drug dosage is not always practical be-
cause of tolerability concerns. For the fluoroquinolones, us-
ing a higher dose of levofloxacin (750 mg QD) approximately
doubles the concentration in plasma and pulmonary epithe-
lial lining fluid when compared with the 500 mg dose [68].
The higher concentrations attained by the 750 mg dose in-
crease the probability of achieving PK/PD targets necessary
for effective eradication and prevention of resistant P. aerugi-
nosa clones during therapy [57]. In one study, a mathematical
model was used to identify rational dosing regimens of fluoro-
quinolones that suppress amplification of resistant P. aerug-
inosa subpopulations at an infection site [56]. The model
predicted that the 750 mg levofloxacin dose (QD) would be
comparable with 400 mg ciprofloxacin TID for P. aeruginosa
infections based on AUC/MIC target attainment data. In the
same study, all resistant isolates resulting from suboptimal
dosing strategies in a mouse thigh-infection model demon-
strated efflux pump over expression while no resistant isolates
were identified that contained a mutation in the QRDR.
The challenge with treating P. aeruginosa infections re-
lates to its ability to acquire adaptive resistance during a
course of therapy, in addition to the high prevalence of resis-
tance. Though fluoroquinolones can provide effective treat-
ment, combination therapy is recommended for infections
proven or suspected of being caused by this organism. Opti-
mally, the adjunctive agent would not be susceptible to efflux
mechanisms, such as an antipseudomonal ␤-lactam. Only
through effective and responsible use of the fluoroquinolones
will this class of agents maintain its clinical value in the years
to come for the treatment of serious respiratory infections.
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