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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 otic in the cell [1,7]. This review will concentrate on the
latter mechanism, particularly for two bacteria, S. pneumo-
Since the introduction of the fluoroquinolones in the niae and P. aeruginosa. These two clinically important respi-
1980s, the use of this class of drugs has rapidly increased to ratory pathogens will provide examples of the differences in
become one of the more common choices of antimicrobials efflux mechanisms associated with Gram-positive and Gram-
for various types of bacterial infection [1]. This widespread negative bacteria. The clinical implications of efflux mecha-
use has had its consequences, as selective pressure has in- nisms will be discussed as well as possible future strategies
creased resistance for some species over the years [1]. Though that may be used to combat efflux-mediated resistance and
resistance to common respiratory pathogens, including Strep- limit resistance emergence.
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, 2. Efflux pumps
has increased significantly [5,6]. Fluoroquinolone resistance
can involve two well-documented and separate mechanisms: Efflux pumps (or drug transporters) are essential mecha-
(1) point mutations in the genes of the quinolone target nisms used by prokaryotes to transport various substances,
enzymes, topoisomerase and gyrase, or (2) expression of including toxic compounds such as antibiotics, out of cells to
drug efflux pumps that reduce the accumulation of antibi- protect themselves from the adverse effects. The importance
of efflux pumps is reflected in the large portion of a bac-
∗ Corresponding author. Tel.: +1 204 787 4902; fax: +1 204 787 4699. terium’s genetic makeup dedicated to this function, where an
E-mail address: ggzhanel@pcs.mb.ca (G.G. Zhanel). 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 strain demonstrating an efflux phenotype resulted in restora-
tetracycline resistance [9,10]. Through the years, the role of tion of drug susceptibility to norfloxacin. With the complete
efflux mechanisms in eliciting resistance to other antimicro- sequence of the S. pneumoniae genome now available, [23]
bial classes was identified and it was soon established that other putative efflux pump genes have been identified. Yet,
efflux was a common mechanism for bacterial resistance. at present no other pumps have been identified that actively
To varying extents, the fluoroquinolones have been shown efflux fluoroquinolones, though alternative pumps are sus-
to be vulnerable to efflux pumps, and may depend on cer- pected [24]. Efflux pump mutants in S. pneumoniae are usu-
tain structural characteristics, such as size and hydrophobic- ally identified by comparing the MIC value of an isolate ei-
ity of the fluoroquinolone molecule [11]. Numerous stud- ther with or without the presence of a known efflux pump in-
ies have assessed the role of efflux in fluoroquinolone resis- hibitor, such as reserpine. Chemically unrelated efflux pump
tance [7,12,13]. One concern of these studies is determining substrates, such as ethidium bromide and acriflavine, are also
if efflux is truly involved in resistance in a particular isolate used as controls to distinguish further the role of efflux pumps
or if an alternate mechanism is involved. Certain applica- in a particular isolate.
tions have been used to inhibit the efflux pump mechanism, It is important to note that all fluoroquinolones do
such as reserpine or the energy inhibitor carbonyl cyanide m- not demonstrate equivalent susceptibility to efflux mecha-
chlorophenylhydrazone (CCCP) for Gram-positive bacteria nisms in S. pneumoniae. Accumulation of fluoroquinolones
[14]. Gram-negative bacteria employ multiple efflux systems, within wild-type strains has been shown to be dependent on
so it is more difficult to identify inhibitors that specifically size and hydrophobicity of the agent [11]. Smaller agents
affect one or all of these systems. Because of this, genetic (ciprofloxacin, norfloxacin, and ofloxacin) and more hy-
techniques are employed to study the role of efflux in these drophobic compounds (ciprofloxacin and norfloxacin) show
organisms. greater accumulation in the cell than other agents. However,
Strains exhibiting efflux-mediated fluoroquinolone resis- in studies with an efflux pump mutant strain, the greatest de-
tance frequently demonstrate cross-resistance to other struc- crease in drug accumulation was observed with ciprofloxacin,
turally unrelated antimicrobials [15,16]. This may be due ofloxacin and gatifloxacin, while there was no significant
to the broad substrate specificity of fluoroquinolone efflux change in the accumulation of levofloxacin, moxifloxacin,
pumps and raises concern over the use of fluoroquinolones and sitafloxacin, among others [11].
and the impending increase in the prevalence of multidrug Fluoroquinolone-resistant strains developed from in vitro
resistant strains. Therefore, a thorough understanding of the selection commonly demonstrate efflux phenotypes along
use of fluoroquinolones and the impact of efflux pumps may with point mutation in the target genes. One study showed
help limit resistance development and preserve the clinical that 23 efflux mutants selected in the laboratory exhib-
value of this class of antibiotics. ited 4–8-fold increases in MIC values for norfloxacin and
ciprofloxacin, without significantly changing the MICs of
2.1. Streptococcus pneumoniae moxifloxacin or sparfloxacin [25]. A larger in vitro study
of 80 fluoroquinolone-resistant strains reported that the pres-
S. pneumoniae is a predominant pathogen in community- ence of reserpine decreased the MIC values 4–32-fold for
acquired respiratory tract infections and its resistance to an- ciprofloxacin in 53 mutants, and for gemifloxacin in 39 mu-
tibiotics has presented an increasing global concern over tants, while decreasing the MIC values for 13 mutants when
the past decade. Surveillance studies in the US have shown tested against gatifloxacin [26]. Reserpine had little effect on
high-level penicillin resistance approaching 20% and resis- the MICs of moxifloxacin and trovafloxacin.
tance to the macrolides at over 25% [2,17]. Fortunately, flu- Several studies have investigated the prevalence of efflux
oroquinolone resistance (to the respiratory fluoroquinolones pump mutants in clinical isolates of S. pneumoniae [27,28].
such as gatifloxacin, levofloxacin and moxifloxacin) has re- Of 1037 clinical isolates collected in the United Kingdom,
mained low throughout the United States and Canada (at or 273 (26.3%) showed reduced susceptibility to norfloxacin
below 1%) but signs of increasing resistance are apparent and ciprofloxacin, while 124 (12.0%) demonstrated an efflux
[18,19]. S. pneumoniae resistance to the fluoroquinolones phenotype [25]. In a Canadian study of fluoroquinolone-
is caused by mutations in the genes of the target enzymes, resistant clinical isolates of S. pneumoniae, 12 of 34 (35.3%)
topoisomerase and gyrase. A single mutation (frequently in clinical isolates demonstrated reserpine-sensitive efflux of
the quinolone resistance determining region—QRDR) usu- ciprofloxacin, decreasing the MIC in the presence of reser-
ally results in low-level resistance, while high-level fluo- pine by 4–8-fold [21]. However, reserpine did not affect the
roquinolone resistance requires at least two mutations in MIC values of any isolate for the newer fluoroquinolones,
these genes [7,20,21]. However, efflux mechanisms have including gatifloxacin, levofloxacin, moxifloxacin, and
been shown to play a role in conferring quinolone resistance, gemifloxacin. Other studies have reported a high prevalence
though at lower levels [13,14]. of reserpine-inhibited efflux of ciprofloxacin in quinolone-
Identification of a putative efflux pump gene, pmrA, re- resistant clinical isolates of S. pneumoniae [7,29] while
sponsible for fluoroquinolone resistance in S. pneumoniae newer fluoroquinolones tend to be less affected by efflux
was first reported in 1999 [22]. Inactivation of this gene in a mechanisms [30].
G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535 531

The high susceptibility of ciprofloxacin to efflux mech- [38]. The size and complexity of the genome may explain its
anisms can have significant implications in the emergence ability to adapt to diverse environments and resist a variety
of fluoroquinolone-resistance. Though efflux mutants only of antimicrobial agents.
moderately increase S. pneumoniae resistance in most cases P. aeruginosa resistance to the fluoroquinolones began in-
(four-fold increase in MIC), this may be adequate to en- creasing with widespread use of ciprofloxacin. One surveil-
sure the survival of a single-step mutant (in topoiso- lance study of US intensive care units reported a decrease
merase or gyrase), and increase the probability of multi- in P. aeruginosa susceptibility to ciprofloxacin from 89% in
step fluoroquinolone-resistant strains. In vitro pharmacody- 1990–1993, to 86% in 1994, to 76% in 2000 [5]. Data from the
namic models show ineffective killing of S. pneumoniae National Nosocomial Infections Surveillance (NNIS) System
by ciprofloxacin at normal pharmacological concentrations reported fluoroquinolone resistance by P. aeruginosa in noso-
[31–33]. Initial killing by ciprofloxacin is usually followed comial infections to be 23% in 1999 [39]. Due to the preva-
by regrowth and the emergence of resistant strains with MIC lence of P. aeruginosa resistance to the fluoroquinolones, an
values 2–8-fold higher than the initial MIC [31,32]. Similar adjunctive agent with antipseudomonal activity is commonly
studies with gatifloxacin, levofloxacin and moxifloxacin re- recommended for infections proven or suspected to be caused
sult in effective killing of these strains and no emergence of by this pathogen.
resistance as long as the pharmacokinetic/pharmacodynamic Unlike efflux pumps in Gram-positive bacteria, the ef-
target of AUC/MIC >30 is met. Newer fluoroquinolones (gat- flux mechanisms in Gram-negative organisms contain many
ifloxacin, levofloxacin, moxifloxacin, and gemifloxacin) have components that coordinate the expulsion of compounds
been reported to effectively kill efflux mutants that exhibited from within the cytosole across the cytoplasmic membrane,
ciprofloxacin resistance [12,34,35]. through the periplasmic space and past the outer mem-
Though ciprofloxacin is not recommended for respiratory brane. P. aeruginosa transporters belong to the resistance-
infections caused by S. pneumoniae, use of ciprofloxacin for nodulation-division (RND) family and are composed of a
other indications, such as urinary tract infections, skin infec- transporter, linker, and outer membrane (OM) pore [15]. This
tions, and nosocomial pneumonia, may potentially impact the system ensures that efflux of molecules through the cyto-
spread of fluoroquinolone resistance by this pathogen. It is plasmic membrane will not accumulate in the periplasmic
presently unclear whether S. pneumoniae (present in the com- space, which may lead to rapid diffusion back into the cy-
mensal flora of the nasopharynx) exposed to subinhibitory toplasm. The various efflux systems of P. aeruginosa have
doses of ciprofloxacin will result in a single-step mutant that been well studied over the past decade. Currently, there are
is now non-susceptible to ciprofloxacin. However, once these five multicomponent efflux pumps (MexAB-OprM, MexCD-
strains are present, subsequent exposure to ciprofloxacin Opr-J, MexEF-OprN, MexXY-OprM, and the recently iden-
or another fluoroquinolone may favour the selection of a tified MexVW-OprM) that have been shown to efflux fluo-
multi-step mutant that exhibits high-level ciprofloxacin re- roquinolones [16,40–42]. Of the known efflux pumps in P.
sistance and cross-resistance to the newer fluoroquinolones. aeruginosa, only MexAB-OprM is expressed constitutively
Use of newer fluoroquinolones that are less vulnerable to at sufficient levels to result in intrinsic fluoroquinolone resis-
efflux mechanisms and are effective against efflux mutants tance in wild-type cells.
or single-step mutants may limit the emergence of fluoro- Genetic approaches are commonly used to decipher the
quinolone resistance, especially when the pharmacodynamic role of efflux pumps and resistance in P. aeruginosa, either
targets are achieved [33,36]. by disrupting or over expressing genes coding for efflux pump
proteins. In one study, deletion of the genes coding for the
2.2. Pseudomonas aeruginosa MexAB-OprM pump resulted in decreased resistance to flu-
oroquinolones (16-fold reduction in the levofloxacin MIC),
Gram-negative bacteria typically present higher intrinsic while over expression of any of the three pumps caused a
resistance than Gram-positive organisms. This is a result of 4–8-fold increase in levofloxacin MIC values [43]. Another
a selectively permeable outer membrane that can act as a study compared the effect of efflux mutants to various flu-
barrier to antibiotics, in addition to the presence of multi- oroquinolones, including ciprofloxacin, gemifloxacin, mox-
ple multidrug efflux systems that continually extrude toxic ifloxacin, and trovafloxacin [44]. Mutant strains that either
compounds out of the cell [15,37]. This is particularly true over express efflux pump genes or contain deletions of these
for P. aeruginosa, which can cause difficult-to-treat respi- genes showed that all fluoroquinolones were substrates for
ratory infections because of its intrinsic resistance to many efflux mechanisms in P. aeruginosa. Overproduction of the
available antibiotics. The outer membrane of P. aeruginosa MexAB-OprM caused the greatest effect, raising the MIC
has very low nonspecific permeability to small hydrophobic values of most fluoroquinolones tested by 16-fold. An in-
molecules, which may contribute to the high intrinsic resis- depth study that compared substrate specificities of three ef-
tance to fluoroquinolones [37]. Sequencing the P. aeruginosa flux pump systems (by systematically over expressing one
genome revealed that this organism contains a high propor- pump system and deleting the other two systems) showed
tion of regulatory genes and a large number of genes involved that the fluoroquinolones (as well as the macrolides, tetra-
in the catabolism, transport and efflux of organic compounds 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 less with trovafloxacin compared with ciprofloxacin (P <
and polymyxin B were unaffected by overproduction of any 0.05). Interestingly, trovafloxacin preferentially selected for
of the efflux pumps. MexCD-OprJ over producers while ciprofloxacin selected for
Clinical isolates provide additional evidence regarding the MexEF-OprN over producers.
importance of efflux mechanisms in emerging resistance of Since the fluoroquinolones exhibit concentration-
P. aeruginosa to the fluoroquinolones. Exposure to the fluo- dependent killing activity, effective eradication and
roquinolones typically produces resistant strains with mu- suppression of resistance will depend on meeting the opti-
tations in the topoisomerase genes and efflux genes [45]. mal pharmacodynamic targets of AUC/MIC and Cmax /MIC
Target site mutations in tandem with mutations in the ef- [54–57]. Currently ciprofloxacin and levofloxacin are the
flux regulatory genes tend to result in magnified levels of preferred fluoroquinolones for treating proven or suspected
fluoroquinolone resistance [46,47]. One case reported the P. aeruginosa infections. Though ciprofloxacin consistently
emergence of a resistant P. aeruginosa strain after 4 days demonstrates lower MICs than the respiratory fluoro-
of ciprofloxacin therapy [48]. The isolate had mutations in quinolones against P. aeruginosa, levofloxacin achieves
the gyrB gene and over expressed the MexAB-OprM sys- higher peak and AUC values that result in comparable
tem, causing a 128-fold increase in MIC to ciprofloxacin. pharmacokinetic/pharmacodynamic values when treating
A larger study of cystic fibrosis patients that underwent re- these infections [56,57]. However, it must be made clear that
peated ciprofloxacin regimens reported an accumulation of whether a clinician chooses ciprofloxacin or levofloxacin,
resistance mutations in gyrA and parC genes, as well as in combination therapy (preferably using antibiotics with
the efflux mechanisms during the 3 years of the study [49]. different mechanisms of action) is recommended for these
Alterations in two efflux systems, MexCD-OprJ and MexEF- patients.
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 3. Efflux pump inhibitors
study period. Overall, 80% (16 of 20) of isolates contained
efflux mutations, suggesting that this mechanism was respon- With the increased understanding of the significance of
sible for at least partial resistance in a large proportion of re- efflux pumps on antibiotic resistance, research is being con-
sistant clinical isolates. Clinical isolates that simultaneously ducted to discover methods to overcome this mechanism.
overproduce multiple efflux pumps have been identified, and One of the more practical strategies involves developing com-
these strains tend to have broader resistance profiles and have pounds that inhibit the efflux mechanism. A number of re-
additive effects on the MICs of effluxed substrates [49–51]. ports have been published showing that inhibition of efflux
All fluoroquinolones seem to be affected by P. aeruginosa systems can increase susceptibility while decreasing the fre-
efflux mechanisms [40,44]. One study compared resistance quency of resistance emergence. As mentioned earlier, reser-
selection of P. aeruginosa by exposing the bacteria to each of pine has been used to inhibit efflux in S. pneumoniae. One
12 quinolones at 1–8 times the MIC value [52]. For a given experiment compared the frequency of emergence of resis-
antibiotic concentration, the frequencies of resistant colonies tant isolates when S. pneumoniae was grown in the presence
were comparable for most of the agents (between 1.2 × 10−6 of ciprofloxacin plus reserpine [58]. Reserpine decreased the
and 1.3 × 10−7 at twice the MIC). Doubling the drug concen- frequency of resistant colonies by 45-fold when grown in the
tration (from 2 × MIC to 4 × MIC) decreased the incidence presence of 3× the MIC of ciprofloxacin. Inhibitors of the
of resistant colonies by one to two orders of magnitude. Of the proton motive force, such as CCCP, dinitrophenol (DNP) and
321 colonies that developed resistance, 303 (94.3%) exhib- valinomycin, also inhibit efflux pumps in S. pneumoniae, sug-
ited a phenotype corresponding to resistance by over expres- gesting an energy requirement for drug efflux and possibly
sion of at least one of the three efflux pumps. Though topoiso- exposing another target for efflux inhibition [59].
merase mutants confer high-level resistance, these mutations Inhibition of efflux pumps in Gram-negative organisms
occur less frequently than efflux mutations after one exposure presents additional challenges. These organisms have multi-
to a fluoroquinolone. Over expression of efflux systems can ple efflux systems that must all be inhibited to some degree
thus be an important mechanism to initially reduced suscep- since an effect on one system can be compensated by over
tibility of P. aeruginosa to a fluoroquinolone and allow for expression of another. Large-scale screens of synthetic and
the accumulation of multiple mutations in the gyrase genes natural compounds have been conducted to identify poten-
during additional antibiotic exposure. tial efflux pump inhibitors (EPIs) that improve the activity
Few studies have been conducted to differentiate the effect of antibiotics that are susceptible to efflux mechanisms by
of ciprofloxacin and newer fluoroquinolones (levofloxacin, P. aeruginosa. One experimental compound (MC-207, 110,
gatifloxacin, and moxifloxacin) on the emergence of ef- formerly Microcide Technologies, Mountain View, CA) il-
flux mutants in resistant P. aeruginosa. One group com- lustrated the potential of EPIs as adjunctive therapy to an-
pared the selection of efflux mutants by ciprofloxacin and tibiotics for treating P. aeruginosa infections [43,60,61]. The
trovafloxacin using a rat model of acute pneumonia [53]. EPI reduced the intrinsic resistance of P. aeruginosa to lev-
The frequency of resistant colonies is approximately 10-fold ofloxacin by eight-fold, while resistance in a strain overex-
G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535 533

pressing efflux pumps decreased 32–64-fold, as measured by by this pathogen, ciprofloxacin and levofloxacin remain the
levofloxacin MIC values. Importantly, the frequency of the most commonly used fluoroquinolones for treatment, though
emergence of resistant isolates decreased from around 10−7 combination therapy with an antipseudomonal agent is rec-
in the presence of levofloxacin alone, to <10−11 when the ommended, especially for severely ill patients, such as those
EPI was included. When 50 clinical isolates were tested, the with nosocomial pneumonia. In vitro studies have demon-
presence of the EPI reduced the MIC90 by about 16-fold. strated that efflux mutants occur more frequently than target
Research on derivatives of this compound is attempting to site mutants after an initial exposure to a fluoroquinolone.
identify more potent inhibitors that may be used in the clini- Isolates from patients that underwent multiple ciprofloxacin
cal setting in the future [62–66]. treatments contained mutations in both efflux genes and tar-
The clinical relevance of using EPIs is clearly evident. get sites, suggesting a sequence of events of first low level
Yet, the main obstacle continues to be the development of resistance by P. aeruginosa followed by high-level resistance
an inhibitor that specifically acts on prokaryotic cells with- by acquisition of target site mutations [49].
out affecting eukaryotic mechanisms. Currently available in- Increasing the drug concentration at the site of infection
hibitors of Gram-positive efflux pumps, such as reserpine may decrease the emergence of resistant isolates [52]. Clin-
and CCCP, are too toxic to use safely in patients, while a ically, increasing the drug dosage is not always practical be-
safe Gram-negative inhibitor is still under development. Fu- cause of tolerability concerns. For the fluoroquinolones, us-
ture development of EPIs may help to increase the potency ing a higher dose of levofloxacin (750 mg QD) approximately
of the fluoroquinolone, as well as other classes of agents, doubles the concentration in plasma and pulmonary epithe-
against P. aeruginosa. Some Gram-positive antibiotics, such lial lining fluid when compared with the 500 mg dose [68].
as members of the macrolides and ␤-lactams, may be effective The higher concentrations attained by the 750 mg dose in-
against Gram-negative strains if the efflux mechanisms are in- crease the probability of achieving PK/PD targets necessary
hibited. These agents have been shown to be potent against E. for effective eradication and prevention of resistant P. aerugi-
coli and P. aeruginosa mutants lacking efflux pumps [15,67]. nosa clones during therapy [57]. In one study, a mathematical
However, until an effective and tolerable EPI is developed, model was used to identify rational dosing regimens of fluoro-
more practical strategies and considerations must be taken quinolones that suppress amplification of resistant P. aerug-
to reduce the emergence of resistance stemming from efflux inosa subpopulations at an infection site [56]. The model
mechanisms. 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
4. Clinical implications same study, all resistant isolates resulting from suboptimal
dosing strategies in a mouse thigh-infection model demon-
In the absence of a clinically practical efflux pump in- strated efflux pump over expression while no resistant isolates
hibitor, physicians must recognize the role efflux systems were identified that contained a mutation in the QRDR.
play in the emergence of bacterial resistance. Some of the The challenge with treating P. aeruginosa infections re-
clinical consequences of ciprofloxacin use on S. pneumo- lates to its ability to acquire adaptive resistance during a
niae resistance development have been discussed previously. course of therapy, in addition to the high prevalence of resis-
Of the currently available fluoroquinolones, ciprofloxacin is tance. Though fluoroquinolones can provide effective treat-
the most susceptible to efflux mechanisms in S. pneumoniae ment, combination therapy is recommended for infections
and ciprofloxacin exposure may increase the risk of efflux proven or suspected of being caused by this organism. Opti-
and/or single-step mutants from developing. Subsequent flu- mally, the adjunctive agent would not be susceptible to efflux
oroquinolone exposure may then lead to the development of mechanisms, such as an antipseudomonal ␤-lactam. Only
high-level fluoroquinolone resistance. Whether this occurs through effective and responsible use of the fluoroquinolones
frequently in the clinical setting remains to be proven. How- will this class of agents maintain its clinical value in the years
ever, in one study that characterized ciprofloxacin-resistant to come for the treatment of serious respiratory infections.
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- References
oroquinolones (gatifloxacin, levofloxacin, moxifloxacin, and
[1] Zhanel GG, Ennis K, Vercaigne L, et al. A critical review of the fluo-
gemifloxacin) do not exhibit efflux by S. pneumoniae to as roquinolones: focus on respiratory infections. Drugs 2002;62:13–59.
high a degree as ciprofloxacin. These agents also provide [2] Karlowsky JA, Thornsberry C, Jones ME, Evangelista AT, Critchley
effective activity to eradicate efflux or single-step mutants, IA, Sahm DF. Factors associated with relative rates of antimicrobial
which may reduce the risk of high-level fluoroquinolone re- resistance among Streptococcus pneumoniae in the United States:
sistant strains from emerging [12,34]. results from the TRUST Surveillance Program (1998–2002). Clin
Infect Dis 2003;36:963–70.
P. aeruginosa shows indiscriminate efflux with the flu- [3] Doern GV, Brown SD. Antimicrobial susceptibility among
oroquinolones and probably accounts for its high intrinsic community-acquired respiratory tract pathogens in the USA: data
resistance of these agents. For respiratory infections caused from PROTEKT US 2000–2001. J Infect 2004;48:56–65.
534 G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535

[4] Jones ME, Blosser-Middleton RS, Thornsberry C, Karlowsky JA, [22] Gill MJ, Brenwald NP, Wise R. Identification of an efflux pump gene,
Sahm DF. The activity of levofloxacin and other antimicro- pmrA, associated with fluoroquinolone resistance in Streptococcus
bials against clinical isolates of Streptococcus pneumoniae col- pneumoniae. Antimicrob Agents Chemother 1999;43:187–9.
lected worldwide during 1999–2002. Diagn Microbiol Infect Dis [23] Tettelin H, Nelson KE, Paulsen IT, et al. Complete genome se-
2003;47:579–86. quence of a virulent isolate of Streptococcus pneumoniae. Science
[5] Neuhauser MM, Weinstein RA, Rydman R, Danziger LH, Karam G, 2001;293(5529):498–506.
Quinn JP. Antibiotic resistance among gram-negative bacilli in US [24] Brenwald NP, Appelbaum P, Davies T, Gill MJ. Evidence for ef-
intensive care units: implications for fluoroquinolone use. JAMA flux pumps, other than PmrA, associated with fluoroquinolone resis-
2003;289:885–8. tance in Streptococcus pneumoniae. Clin Microbiol Infect 2003;9:
[6] NNIS System. National Nosocomial Infections Surveillance (NNIS) 140–3.
System Report, data summary from January 1992 to June 2002. [25] Brenwald NP, Gill MJ, Wise R. Prevalence of a putative efflux mech-
Issued August 2002. Am J Infect Control 2002;30:458–75. anism among fluoroquinolone-resistant clinical isolates of Strepto-
[7] Broskey J, Coleman K, Gwynn MN, et al. Efflux and target mu- coccus pneumoniae. Antimicrob Agents Chemother 1998;42:2032–5.
tations as quinolone resistance mechanisms in clinical isolates of [26] Nagai K, Davies TA, Dewasse BE, Jacobs MR, Appelbaum PC.
Streptococcus pneumoniae. J Antimicrob Chemother 2000;45:95– Single- and multi-step resistance selection study of gemifloxacin
9. compared with trovafloxacin, ciprofloxacin, gatifloxacin and mox-
[8] Webber MA, Piddock LJ. The importance of efflux pumps in ifloxacin in Streptococcus pneumoniae. J Antimicrob Chemother
bacterial antibiotic resistance. J Antimicrob Chemother 2003;51:9– 2001;48:365–74.
11. [27] Johnson AP, Sheppard CL, Harnett SJ, et al. Emergence of a
[9] McMurry L, Petrucci Jr RE, Levy SB. Active efflux of tetracycline fluoroquinolone-resistant strain of Streptococcus pneumoniae in Eng-
encoded by four genetically different tetracycline resistance determi- land. J Antimicrob Chemother 2003;52:953–60.
nants in Escherichia coli. Proc Natl Acad Sci USA 1980;77:3974–7. [28] Piddock LJV, Johnson MM, Simgee S, Pumbwe L. Expression of
[10] Ball PR, Shales SW, Chopra I. Plasmid-mediated tetracycline resis- efflux pump gene pmrA in fluoroquinolone-resistant and -susceptible
tance in Escherichia coli involves increased efflux of the antibiotic. clinical isolates of Streptococcus pneumoniae. Antimicrob Agents
Biochem Biophys Res Commun 1980;93:74–81. Chemother 2002;46:808–12.
[11] Piddock LJ, Johnson MM. Accumulation of 10 fluoroquinolones by [29] Ho PL, Yung RW, Tsang DN, et al. Increasing resistance of Strep-
wild-type or efflux mutant Streptococcus pneumoniae. Antimicrob tococcus pneumoniae to fluoroquinolones: results of a Hong Kong
Agents Chemother 2002;46:813–20. multicentre study in 2001;48:659–65.
[12] Heaton VJ, Goldsmith CE, Ambler JE, Fisher LM. Activ- [30] Piddock LJV, Johnson M, Ricci V, Hill SL. Activities of new fluoro-
ity of gemifloxacin against penicillin- and ciprofloxacin-resistant quinolones against fluoroquinolone-resistant pathogens of the lower
Streptococcus pneumoniae displaying topoisomerase- and efflux- respiratory tract. Antimicrob Agents Chemother 1998;42:2956–
mediated resistance mechanisms. Antimicrob Agents Chemother 60.
1999;43:2998–3000. [31] Lacy MK, Lu W, Xu X, et al. Pharmacodynamic comparisons
[13] Madaras-Kelly KJ, Daniels C, Hegbloom M, Thompson M. Pharma- of levofloxacin, ciprofloxacin, and ampicillin against Streptococcus
codynamic characterization of efflux and topoisomerase IV-mediated pneumoniae in an in vitro model of infection. Antimicrob Agents
fluoroquinolone resistance in Streptococcus pneumoniae. J Antimi- Chemother 1999;43:672–7.
crob Chemother 2002;50:211–8. [32] Zhanel GG, Walters M, Laing N, Hoban DJ. In vitro pharmaco-
[14] Brenwald NP, Gill MJ, Wise R. The effect of reserpine, an in- dynamic modelling simulating free serum concentrations of fluoro-
hibitor of multidrug efflux pumps, on the in-vitro susceptibilities quinolones against multidrug-resistant Streptococcus pneumoniae. J
of fluoroquinolone-resistant strains of Streptococcus pneumoniae to Antimicrob Chemother 2001;47:435–40.
norfloxacin. J Antimicrob Chemother 1997;40:458–60. [33] Coyle EA, Kaatz GW, Rybak MJ. Activities of newer fluoro-
[15] Nikaido H. Antibiotic resistance caused by gram-negative multidrug quinolones against ciprofloxacin-resistant Streptococcus pneumoniae.
efflux pumps. Clin Infect Dis 1998;27:S32–41. Antimicrob Agents Chemother 2001;45:1654–9.
[16] Aeschlimann JR. The role of multidrug efflux pumps in the antibi- [34] Zhanel GG, Roberts D, Waltky A, et al. Pharmacodynamic activ-
otic resistance of Pseudomonas aeruginosa and other Gram-negative ity of fluoroquinolones against ciprofloxacin-resistant Streptococcus
bacteria. Pharmacotherapy 2003;23:916–24. pneumoniae. J Antimicrob Chemother 2002;49:807–12.
[17] Jones ME, Karlowsky JA, Blosser-Middleton R, et al. Longitudinal [35] MacGowan AP, Bowker KE. Mechanism of fluoroquinolone re-
assessment of antipneumococcal susceptibility in the United States. sistance is an important factor in determining the antimicrobial
Antimicrob Agents Chemother 2002;46:2651–5. effect of gemifloxacin against Streptococcus pneumoniae in an
[18] Jones RN, Pfaller MA. Macrolide and fluoroquinolone (levofloxacin) in vitro pharmacokinetic model. Antimicrob Agents Chemother
resistances among Streptococcus pneumoniae strains: significant 2003;47:1096–100.
trends from the SENTRY antimicrobial surveillance program (North [36] Lister PD. Pharmacodynamics of 750 mg and 500 mg doses of
America, 1997–1999). J Clin Microbiol 2000;38:4298–9. levofloxacin against ciprofloxacin-resistant strains of Streptococcus
[19] Zhanel GG, Palatnick L, Nichol KA, Bellyou T, Low DE, Hoban DJ. pneumoniae. Diagn Microbiol Infect Dis 2002;44:43–9.
Antimicrobial resistance in respiratory tract Streptococcus pneumo- [37] Hancock RE. Resistance mechanisms in Pseudomonas aeruginosa
niae isolates: results of the Canadian Respiratory Organism Sus- and other nonfermentative gram-negative bacteria. Clin Infect Dis
ceptibility Study, 1997 to 2002. Antimicrob Agents Chemother 1998;27:S93–9.
2003;47:1867–74. [38] Stover CK, Pham XQ, Erwin AL, et al. Complete genome sequence
[20] Davies TA, Evangelista A, Pfleger S, Bush K, Sahm DF, Gold- of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature
schmidt R. Prevalence of single mutations in topoisomerase type II 2000;406(6799):959–64.
genes among levofloxacin-susceptible clinical strains of Streptococ- [39] NNIS System. National Nosocomial Infections Surveillance (NNIS)
cus pneumoniae isolated in the United States in 1992 to 1996 and System report, data summary from January 1990 to May 1999. Is-
1999 to 2000. Antimicrob Agents Chemother 2002;46:119–24. sued June 1999. Am J Infect Control 1999;27:520–32.
[21] Zhanel GG, Walkty A, Nichol K, Smith H, Noreddin A, Hoban DJ. [40] Masuda N, Sakagawa E, Ohya S, Gotoh N, Tsujimoto H, Nishino T.
Molecular characterization of fluoroquinolone resistant Streptococcus Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-
pneumoniae clinical isolates obtained from across Canada. Diagn oprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents
Microbiol Infect Dis 2003;45:63–7. Chemother 2000;44:3322–7.
G.G. Zhanel et al. / International Journal of Antimicrobial Agents 24 (2004) 529–535 535

[41] Maseda H, Yoneyama H, Nakae T. Assignment of the substrate- [54] MacGowan AP, Wootton M, Holt HA. The antibacterial efficacy of
selective subunits of the MexEF-OprN multidrug efflux pump of levofloxacin and ciprofloxacin against Pseudomonas aeruginosa as-
Pseudomonas aeruginosa. Antimicrob Agents Chemother 2000; sessed by combining antibiotic exposure and bacterial susceptibility.
44:658–64. J Antimicrob Chemother 1999;43:345–9.
[42] Li Y, Mima T, Komori Y, et al. A new member of the tripartitie mul- [55] Madaras-Kelly KJ, Larsson AJ, Rotschafer JC. A pharmacodynamic
tidrug efflux pumps, Mex-VW-OprM, in Pseudomonas aeruginosa. evaluation of ciprofloxacin and ofloxacin against two strains of Pseu-
J Antimicrob Chemother 2003;52:572–5. domonas aeruginosa. J Antimicrob Chemother 1996;37:703–10.
[43] Lomovskaya O, Lee A, Hoshino K, et al. Use of a genetic [56] Jumbe N, Louie A, Leary R, et al. Application of a mathematical
approach to evaluate the consequences of inhibition of efflux model to prevent in vivo amplification of antibiotic-resistant bacterial
pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother populations during therapy. J Clin Invest 2003;112:275–85.
1999;43:1340–6. [57] Drusano GL, Preston SL, Fowler C, Corrado M, Wiesinger B,
[44] Zhang L, Li XZ, Poole K. Fluoroquinolone susceptibilities Kahn J. Relationship between fluoroquinolone area under the
of efflux-mediated multidrug-resistant Pseudomonas aeruginosa, curve:minimum inhibitory concentration ratio and the probability of
Stenotrophomonas maltophilia and Burkholderia cepacia. J Antimi- eradication of the infecting pathogen, in patients with nosocomial
crob Chemother 2001;48:549–52. pneumonia. J Infect Dis 2004;189:1590–7.
[45] Oh H, Stenhoff J, Jalal S, Wretlind B. Role of efflux pumps and [58] Markham PN. Inhibition of the emergence of ciprofloxacin resis-
mutations in genes for topoisomerase II and IV in fluoroquinolone- tance in Streptococcus pneumoniae by the multidrug efflux inhibitor
resistant Pseudomonas aeruginosa strains. Microb Drug Resist reserpine. Antimicrob Agents Chemother 1999;43:988–9.
2003;9:323–8. [59] Zeller V, Janoir C, Kitzis M-D, Gutmann L, Moreau NJ. Active
[46] Nakajima A, Sugimoto Y, Yoneyama H, Nakae T. High-level fluo- efflux as a mechanism of resistance to ciprofloxacin in Streptococcus
roquinolone resistance in Pseudomonas aeruginosa due to interplay pneumoniae. Antimicrob Agents Chemother 1997;41:1973–8.
of the MexAB-OprM efflux pump and the DNA gyrase mutation. [60] Renau TE, Leger R, Flamme EM, et al. Inhibitors of efflux pumps
Microbiol Immunol 2002;46:391–5. in Pseudomonas aeruginosa potentiate the activity of the fluoro-
[47] Higgins PG, Fluit AC, Milatovic D, Verhoef J, Schmitz FJ. Mu- quinolone antibacterial levofloxacin. J Med Chem 1999;42:4928–31.
tations in GyrA, ParC, MexR and NfxB in clinical isolates of [61] Coban AY, Ekinci B, Durupinar B. A multidrug efflux inhibitor re-
Pseudomonas aeruginosa. Int J Antimicrob Agents 2003;21:409– duces fluoroquinolone resistance in Pseudomonas aeruginosa iso-
13. lates. Chemotherapy 2004;50:22–6.
[48] Le Thomas I, Couetdic G, Clermont O, Brahimi N, Plesiat P, [62] Nakayama K, Ishida Y, Ohtsuka M, et al. MexAB-OprM-specific
Bingen E. In vivo selection of a target/efflux double mutant of efflux pump inhibitors in Pseudomonas aeruginosa. Part 1: discovery
Pseudomonas aeruginosa by ciprofloxacin therapy. J Antimicrob and early strategies for lead optimization. Bioorg Med Chem Lett
Chemother 2001;48:553–5. 2003;13:4201–4.
[49] Jalal S, Ciofu O, Hoiby N, Gotoh N, Wretlind B. Molecular mech- [63] Nakayama K, Ishida Y, Ohtsuka M, et al. MexAB-OprM specific
anisms of fluoroquinolone resistance in Pseudomonas aeruginosa efflux pump inhibitors in Pseudomonas aeruginosa. Part 2: achieving
isolates from cystic fibrosis patients. Antimicrob Agents Chemother activity in vivo through the use of alternative scaffolds. Bioorg Med
2000;44:710–2. Chem Lett 2003;13:4205–8.
[50] Llanes C, Hocquet D, Vogne C, Benali-Baitich D, Neuwirth C, Ple- [64] Renau TE, Leger R, Filonova L, et al. Conformationally-restricted
siat P. Clinical strains of Pseudomonas areuginosa overproducing analogues of efflux pump inhibitors that potentiate the activity of
Mex-AB-OprM and MexXY efflux pumps simultaneously. Antimi- levofloxacin in Pseudomonas aeruginosa. Bioorg Med Chem Lett
crob Agents Chemother 2004;48:1797–802. 2003;13:2755–8.
[51] Lee A, Mao W, Warren MS, et al. Interplay between efflux pumps [65] Renau TE, Leger R, Yen R, et al. Peptidomimetics of efflux pump in-
may provide either additive or multiplicative effects on drug resis- hibitors potentiate the activity of levofloxacin in Pseudomonas aerug-
tance. J Bacteriol 2000;182:3142–50. inosa. Bioorg Med Chem Lett 2002;12:763–6.
[52] Kohler T, Michea-Hamzehpour M, Plesiat P, Kahr A-L, Pechere J- [66] Watkins WJ, Landaverry Y, Leger R, et al. The relationship between
C. Differential selection of multidrug efflux systems by quinolones physicochemical properties, in vitro activity and pharmacokinetic
in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1997; profiles of analogues of diamine-containing efflux pump inhibitors.
41:2540–3. Bioorg Med Chem Lett 2003;13:4241–4.
[53] Join-Lambert OF, Michea-Hamzehpour M, Kohler T, et al. Differ- [67] Nikaido H. Multiple antibiotic resistance and efflux. Curr Opin Mi-
ential selection of multidrug efflux mutants by trovafloxacin and crobiol 1998;1:516–23.
ciprofloxacin in an experimental model of Pseudomonas aerug- [68] Gotfried MH, Danziger LH, Rodvold KA. Steady-state plasma and
inosa acute pneumonia in rats. Antimicrob Agents Chemother intrapulmonary concentrations of levofloxacin and ciprofloxacin in
2001;45:571–6. healthy adult subjects. Chest 2001;119:1114–22.