TYPE Mini Review

PUBLISHED 14 August 2023

DOI 10.3389/fbrio.2023.1231657

A brief update on the

OPEN ACCESS controversial and opposing
EDITED BY
Myron Christodoulides,
University of Southampton,
roles of Pseudomonas
United Kingdom

REVIEWED BY
aeruginosa efflux pumps
Ilyas Alav,
University of Birmingham, United Kingdom in virulence regulation
*CORRESPONDENCE
Peter Jorth
[email protected] Sheryl Erica Fernandes 1 and Peter Jorth 1,2,3*
RECEIVED 30 May 2023 1
Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles,
ACCEPTED 27 July 2023 CA, United States, 2 Department of Medicine, Cedars-Sinai Medical Center, Los Angeles,
PUBLISHED 14 August 2023 CA, United States, 3 Department of Biomedical Sciences, Cedars-Sinai Medical Center,
Los Angeles, CA, United States
CITATION
Fernandes SE and Jorth P (2023) A brief
update on the controversial and opposing
roles of Pseudomonas aeruginosa efflux
pumps in virulence regulation. Pseudomonas aeruginosa is highly adaptable and constantly mutates to resist
Front. Bacteriol. 2:1231657.
doi: 10.3389/fbrio.2023.1231657 natural and synthetic antibiotic stresses. Listed as a serious threat by the Centers
COPYRIGHT
for Disease Control, novel antimicrobials are urgently needed for drug resistant P.
© 2023 Fernandes and Jorth. This is an aeruginosa infections. Multidrug efflux pumps which contribute to antibiotic
open-access article distributed under the resistance are genetically encoded, highly conserved, and have evolved long
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or before the rampant clinical use of antibiotics. Hence, efflux pumps may have been
reproduction in other forums is permitted, selected for functions beyond the mere exclusion of antibiotics. In this review, we
provided the original author(s) and the
copyright owner(s) are credited and that
discuss recent updates and controversies surrounding how alternative functions of
the original publication in this journal is multidrug efflux pumps can influence the virulence of P. aeruginosa. We conclude
cited, in accordance with accepted by highlighting unexpected consequences of targeting efflux pumps with therapies,
academic practice. No use, distribution or
reproduction is permitted which does not including potential risks and benefits. Understanding these consequences will be
comply with these terms. critical to the development of successful therapeutic strategies that consider aspects
of both antimicrobial resistance and bacterial pathogenesis.

KEYWORDS

Pseudomonas aeruginosa, efflux pump, virulence, pathogenesis, antibiotic resistance

Introduction
Pseudomonas aeruginosa (Pa) is a Gram-negative bacterium that is ubiquitously
present and has the potential to cause a wide spectrum of opportunistic infections in
vulnerable human hosts. During infection, host antimicrobial responses, nutrient
limitation and antibiotics constitute the major stresses encountered by Pa. These
stressors select for traits that promote bacterial survival and proliferation. Antibiotic
efflux pumps eject antibiotics from the bacterial cell and thus strains overexpressing efflux
pumps are regularly isolated from infections. However, Pa mutants that either lack specific
efflux pumps (Vettoretti et al., 2009) or fail to express efflux pump genes (Jorth et al., 2015;
Marvig et al., 2015; Horna et al., 2018) have also been identified. A growing body of studies
have shown that Pa strains with altered efflux pump expression often exhibit dysregulated

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Fernandes and Jorth 10.3389/fbrio.2023.1231657

virulence gene expression and salient findings from these studies wild type (WT) PAO1 levels. The enhanced efflux of HHQ from the
have been discussed in this review. nfxB mutant resulted in a lower intracellular accumulation of PQS
In Pa, there are six superfamilies of efflux pumps: the ATP- compared to WT PAO1 and a consequential reduction in the
binding cassette (ABC); the major facilitator (MFS); the multidrug expression of PQS-dependent virulence factors (Figure 1A)
and toxic compound extrusion (MATE); the proteobacterial (Alcalde-Rico et al., 2018). Coherent with these findings,
antimicrobial compound efflux (PACE); the small multidrug Martı́n ez-Ramos et al. showed that the same nfxB mutant
resistance (SMR) and the resistance/nodulation/cell division resulted in reduced BALB/c lung infections compared to WT
(RND) (Blanco et al., 2016; Lorusso et al., 2022). However, for PAO1 (Martı́nez-Ramos et al., 2014) and deletion of mexD in the
Pa, the RND efflux pumps have a clinically significant correlation to nfxB mutant resulted in similar lung bacterial burden as WT PAO1
drug resistance and hence are the most widely studied (Zahedi infected mice. In yet another recent study, phenylethylamine was
bialvaei et al., 2021). RND efflux pumps are tripartite consisting of found to induce MexCD-OprJ in PAO1 and its overexpression
one or two periplasmic membrane fusion proteins (MFPs), an inner correlated with reduced pyocyanin, elastase and swarming
membrane associated RND transporter and an outer membrane compared to WT PAO1 (Figure 1A) (Muñoz-Cazalla et al., 2023).
factor (OMF) that forms a continuous channel across the inner and Jeannot et al. also observed reduced rhamnolipid, elastase and
outer bacterial membranes for efficient removal of antibiotics from pyocyanin expression in ciprofloxacin evolved nfxB mutants of
the bacterial cell (Nikaido, 2011). Inactivating any of the PAO1, PA14 and PA19.1 compared to their respective parental
components in the complex can abolish the efflux pump function strains (Jeannot et al., 2008).
(Tikhonova and Zgurskaya, 2004). Twelve efflux pumps belonging Similar to observations with MexCD-OprJ, Kohler et al.
to the RND superfamily have been identified in Pa as multidrug associated MexEF-OprN overexpression in a PAO1 nfxC mutant
efflux pumps: MexAB-OprM, MexCD-OprJ, MexEF-OprN, evolved on ciprofloxacin with reduced levels of intracellular PQS
MexXY-OprM, MexJK-OprM, MexVW-OprM, MexMN-OprM, and C4-Homoserine lactone (HSL), showing exogenous addition of
MexPQ-OpmE, MexGHI-OpmD, MuxABC-OpmB, CzcABC, and PQS and C4-HSL restored rhamnolipid gene expression in the nfxC
TriABC-OpmH (Scoffone et al., 2021; Lorusso et al., 2022). In this mutant to WT PAO1 levels (Köhler et al., 2001). In later studies,
review, we will discuss the expanding and, at times controversial, Lamarche and Dé ziel used PA14 transposon (Tn) mutants of mexS
roles of three RND efflux pumps in Pa virulence: MexCD-OprJ, and Olivares et al. used PAO1 nfxC mutants evolved on
MexEF-OprN, and MexAB-OprM. Since these efflux pumps affect ciprofloxacin to demonstrate that MexEF-OprN overexpression in
both antimicrobial resistance and virulence, they have emerged as PA14 and PAO1 leads to excessive efflux of HHQ and kynurenine,
important targets of newer therapeutic strategies. Recent advances respectively, and correlated with reduced swarming and reduced
in the field of efflux pump directed therapies will also be critically expression of Pa pathogenic factors like elastase and pyocyanin
analyzed in this review as they relate to altered virulence traits. (Figure 1B) (Lamarche and Dé ziel, 2011; Olivares et al., 2012).
Further, deletion of MexEF-OprN in these overexpression strains
reduced HHQ and kynurenine efflux to WT PA14/PAO1 levels.
Role of antibiotic efflux pumps in Olivares et al. also observed an increased survival of C. elegans
regulating Pa virulence infected with the nfxC mutant compared to WT PAO1 indicating
that the overall virulence of the Pa strain was significantly
The roles of MexCD-OprJ, MexEF-OprN and MexAB-OprM in compromised by MexEF-OprN overexpression (Figure 1B). In
altered Pa virulence have been most well-studied. The downstream agreement with these findings, our group recently showed that
effects of overexpression and deletion of these efflux pumps on acute lung infection with PAO1 DmexEF enhanced lethality of
quorum sensing (QS) and subsequent effects on Pa virulence in C57BL/6 mice and this mutant had increased rhamnolipid
different model systems are discussed below. production (Figure 1B) (Vaillancourt et al., 2021).
PQS is required for outer membrane vesicle formation, and
vesicles can help deliver Pa virulence factors and augment biofilm
MexCD-OprJ and MexEF-OprN studies formation (Cooke et al., 2020). Since overexpression of MexCD-
agree on roles in virulence gene regulation OprJ or MexEF-OprN decreases PQS signaling, generation of outer
membrane vesicles and biofilm formation in these overexpressing
Kynurenine and 4-hydroxy-2-heptylquinoline (HHQ) are mutants may also be defective and worth investigating. Besides
precursors of the Pseudomonas Quinolone Signal (PQS) which is regulating Pa virulence via PQS signaling, overexpression of
a QS signal that binds to its response regulator MvfR and induces MexCD-OprJ and MexEF-OprN has been associated with
the expression of Pa virulence factors like elastase, rhamnolipids, reduced expression of the Type 3 secretion system (T3SS) in
and pyocyanin (Olivares et al., 2012; Garcı́a-Reyes et al., 2020; PAO1 (Linares et al., 2005), suggesting an alternative mechanism
Huang et al., 2022). Using Pa PAO1 nfxB mutants that overexpress of virulence regulation by these efflux pumps. Although these
MexCD-OprJ and were evolved on norfloxacin and erythromycin, findings correlated with low levels of the transcription factor exsA
Alcalde-Rico et al. demonstrated that MexCD-OprJ overexpression that activates T3SS expression, the mechanism linking exsA to
in PAO1 leads to excessive extrusion of HHQ (Figure 1A) (Alcalde- MexEF-OprN and MexCD-OprJ still remains elusive.
Rico et al., 2018). Importantly, deletion of mexD in this MexCD- Altogether these studies suggest a common function of both
OprJ overexpression background restored extracellular HHQ to MexCD-OprJ and MexEF-OprN: overexpression of either system

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Fernandes and Jorth 10.3389/fbrio.2023.1231657

A B

FIGURE 1
Overexpression of MexCD-OprJ or MexEF-OprN reduces Pa virulence. (A) Increased efflux of HHQ from PAO1 overexpressing MexCD-OprJ causes
reduced intracellular levels of PQS and reduced expression of PQS-dependent virulence factors-elastase, rhamnolipids, and pyocyanin (Alcalde-Rico
et al., 2018; Muñoz-Cazalla et al., 2023), as well as reduced swarming (Muñoz-Cazalla et al., 2023) and lethality in mice (Martın ́ ez-Ramos et al., 2014;
Muñoz-Cazalla et al., 2023). (B) Increased efflux of kynurenine and HHQ from Pa PA14 and PAO1 overexpressing MexEF-OprN causes reduced
intracellular levels of PQS and reduced expression of PQS-dependent virulence factors: elastase, rhamnolipids, and pyocyanin; (Lamarche and
Dé ziel, 2011; Olivares et al., 2012) reduced swarming (Lamarche and Dé ziel, 2011); as well as reduced lethality in C. elegans (Olivares et al., 2012) and
mice (Vaillancourt et al., 2021).

decreases virulence, while deletion increases virulence. Thus, recent report looked at both intracellular and extracellular levels of
targeting either of these efflux pumps with inhibitors could QS signals in a PAO1 mexR mutant overexpressing MexAB-OprM
unintentionally increase Pa virulence. that was evolved on tetracycline. Their findings showed that
reduced intracellular and extracellular levels of PQS/HHQ in the
MexAB-OprM overexpressing mutant compared to WT PAO1
MexAB-OprM studies disagree on the role resulted in reduced expression of PQS dependent virulence
of the efflux pump in Pa virulence factors and there was no change in intracellular or extracellular 3-
oxo-C12-HSL levels (Figure 2B) (Alcalde-Rico et al., 2020).
Unlike multiple reports for MexEF-OprN and MexCD-OprJ Consistent with these findings that MexAB-OprM overexpression
which agree that overexpression of these efflux pumps decreases Pa reduces Pa virulence factor levels, Hwang and Yoon demonstrated
virulence, the association between MexAB-OprM and Pa virulence lower lung burdens of Pa multidrug resistant (MDR) clinical
remains controversial. First, Evans et al. reported that MexAB isolates overexpressing MexAB-OprM in BALB/c mouse
overexpression in an ofloxacin-cefsulodin evolved nalB mutant infections and observed that fewer mice succumbed to infection
strain of PAO1 was associated with increased efflux of another compared to WT PAO1 infected mice (Figure 2C) (Hwang and
virulence factor enhancing QS signal, 3-oxo-C12-Homoserine Yoon, 2019). However, the studies by Alcalde-Rico et al. and Hwang
lactone (3-oxo-C12-HSL) from PAO1 (Figure 2A) (Evans et al., and Yoon only used evolved antibiotic resistant Pa strains which
1998). Although, in this study, a reduction in pyocyanin, elastase, may have multiple undefined mutations that could potentially affect
and protease was observed and deletion of mexAB-OprM increased other Pa virulence regulating genes. Therefore, deleting mexAB-
their expression and 3-oxo-C12-HSL efflux to WT PAO1 levels, the oprM in these strains would be highly critical to ascertain the
lack of data measuring intracellular levels of the 3-oxo-C12-HSL proposed association between MexAB-OprM overexpression,
makes it difficult to conclude if there is indeed any correlation increased PQS/HHQ extrusion and reduced Pa virulence.
between MexAB-OprM overexpression, 3-oxo-C12-HSL efflux, and Alternatively, reports from our group and several others suggest
thereby reduction in the expression of Pa virulence factors. A more that MexAB-OprM overexpression is associated with increased Pa

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A B C D E

FIGURE 2
The controversial role of MexAB-OprM in Pa virulence. Studies have linked the overexpression of MexAB-OprM to reduced virulence (A-C) and
increased virulence (D) and loss of MexAB-OprM to decreased virulence (E). (A) MexAB-OprM overexpression in PAO1 has been associated with
increased efflux of 3-oxo-C12-HSL and reduced expression of Pa virulence factors-elastase, protease, pyocyanin (Evans et al., 1998). (B) MexAB-
OprM overexpression in PAO1 can potentially decrease intracellular PQS levels, thereby reducing the expression of PQS-dependent virulence genes
(Alcalde-Rico et al., 2020). (C) Pa clinical isolates with undefined mutations overexpressed MexAB-OprM and showed reduced lethality of
intratracheally infected BALB/c mice (Hwang and Yoon, 2019). (D) PAO1 mexR mutants overexpressing MexAB-OprM show increased lethality of
intratracheally infected C57BL/6 mice (Jorth et al., 2017; Vaillancourt et al., 2021). (E) PAO1 mutants defective in MexAB-OprM expression showed
reduced invasion in MDCK cells and reduced lethality in orally infected, germ-free BALB/c mice (Hirakata et al., 2002). PA14 Tn mutants of mexA
also showed reduced virulence in a murine pneumonia model using C3H/HeN mice (Roux et al., 2015) and a burn and wound infection model using
AKR/J mice (Mahajan-Miklos et al., 2000). The molecular mechanisms underlying reduced virulence are unknown. For (A-E), the lower panels
indicate the test and control strains used in each study to determine given phenotypes above.

virulence. Recently, we showed increased lethality of PAO1 mexR expression and no other genetic perturbations. Thus, these studies
mutants overexpressing mexAB-oprM relative to WT PAO1 in an collectively suggest that overexpression of mexAB-oprM increases
acute C57BL/6 lung infection model (Figure 2D) (Jorth et al., 2017; Pa virulence while deletion of mexAB-oprM reduces Pa virulence.
Vaillancourt et al., 2021). In another study, invasion of Madin-
Darby canine kidney (MDCK) cells by MexAB-OprM
overexpressing ofloxacin-cefsulodin evolved PAO1 nalB mutants Inactivation of two other efflux pumps
was significantly higher than WT PAO1 and PAO1 deficient in reduces Pa virulence
MexAB-OprM were avirulent in orally infected germ-free BALB/c
mice compared to WT PAO1 infection in the germ-free mice that Other investigators have explored how inactivating other Pa
led to 100% mortality (Figure 2E) (Hirakata et al., 2002). Likewise, efflux pumps affects virulence; however, these individual studies
Roux et al. found that oprM and mexA Tn mutants of PA14 were have not been followed up by other work to date. One study
less competitive with WT PA14 in a murine gastro-intestinal (GI) demonstrated that insertional inactivation of muxA of the
colonization model and were also less lethal than WT PA14 to C3H/ muxABC-OpmB efflux pump reduces the virulence of PAO1 in
HeN mice in a murine pneumonia model (Roux et al., 2015). These Drosophila melanogaster (Yang et al., 2011) and another showed
data are consistent with another study showing PA14 mexA Tn that insertional inactivation of mexI or opmD of the mexGHI-opmD
mutants were less lethal than WT PA14 to AKR/J mice in a burn efflux pump in PAO1 results in reduced PQS biosynthesis and lung
wound infection model (Mahajan-Miklos et al., 2000). Despite, the infection in a rat infection model (Aendekerk et al., 2005). Besides
controversial evidence for MexAB-OprM and Pa virulence, since this research, the roles of other RND efflux pumps in Pa virulence
the mutant strains used by Roux et al, and Mahajan-Miklos et al. remains to be understood.
had Tn insertions only in mexAB or oprM, the associated decrease In the future, it will also be crucial to test how different Pa strain
in virulence can be specifically attributed to reduced MexAB-OprM backgrounds and secondary mutations identified in Pa clinical

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isolates alter the virulence phenotype associated with MexAB- ceftazidime, tigecycline, chloramphenicol, ciprofloxacin,
OprM, MexCD-OprJ, or MexEF-OprN overexpression or loss of erythromycin, piperacillin and levofloxacin against Pa ATCC BAA-
function through inhibitors or mutations. In Escherichia coli, the 2795 overexpressing the MexAB-OprM efflux pump (Tambat et al.,
deletion of an RND efflux pump resulted in the overexpression of 2022). More importantly, unlike other EPIs which were found to have
other RND efflux pumps (Cudkowicz and Schuldiner, 2019). toxicity issues (Spengler et al., 2017), RP1 was well tolerated in
Hence, studies with functionally inactive efflux pump mutants in murine acute toxicity studies and significantly reduced Pa BAA-2795
which the expression of the efflux pump components is similar to lung burdens in a neutropenic murine lung infection model when
the WT strain will be important to ascertain the effect of efflux subcutaneously administered along with levofloxacin compared to
pumps on Pa virulence. levofloxacin or RP1 treatment alone (Tambat et al., 2022). However,
it is unknown if RP1 can inhibit other RND efflux pumps limiting its
application only to infections caused by Pa overexpressing MexAB-
Efflux pump directed strategies to OprM. Yet, this may be preferable since mutants lacking mexA were
overcome drug resistant Pa infection less virulent in some infection models, so RP1 could have the double
benefit of increasing antibiotic susceptibility while also decreasing
The rapid onset of drug resistance due to overexpression of Pa virulence.
antibiotic efflux pumps is one of the major hurdles to successfully
combatting Pa infections. To counter this, therapeutic strategies
targeting RND efflux pumps are being explored, including Antisense RNA suppression of efflux
inhibitors and phage therapies. pump genes

Another efflux pump targeting strategy that has emerged is the

Efflux pump inhibitors use of phosphorodiamidate morpholino oligomers (PPMOs)
(Sturge et al., 2019). PPMOs are antisense RNA consisting of
EPIs are being explored for their ability to increase antibiotic phosphorodiamidate linkage and morpholino ring that make it
sensitivity in drug resistant Pa mutants. PAbN (MC-207,110) was resistant to cellular DNases and RNases. By binding to
one of the first compounds to be discovered and tested as an EPI for complementary mRNA sequences, PPMOs obstruct ribosome
Pa (Renau et al., 1999; Lomovskaya et al., 2001). Mesaros et al. binding and initiation of protein synthesis. PPMOs targeting
showed that PAbN reduced the minimum inhibitory concentration mexA and mexB were shown to enhance cefotaxime, piperacillin–
(MIC) of canonical antibiotic substrates by at least 10-fold in PAO1 tazobactam, and azithromycin sensitivity of PAO1 and two clinical
and clinical Pa isolates overexpressing MexAB-OprM (carbenicillin), Pa isolates (Sturge et al., 2019). Using a CF human bronchial
MexCD-OprJ (erythromycin), MexEF-OprN (norfloxacin), and epithelial cell infection model, Sturge et al. further demonstrated
MexXY-OprM (gentamicin) and therefore inhibits multiple Pa that PPMO increased piperacillin–tazobactam antimicrobial
RND efflux pumps (Mesaros et al., 2007). PAbN also reduced the activity against PAO1 and another Pa clinical isolate in infected
virulence of cystic fibrosis (CF) isolates in the Galleria mellonella cells. However, the efficacy of PPMO in animal infection models
infection model and inhibited pyocyanin production and swarming remains to be tested.
to varying extents in different urinary tract infection (UTI) or CF Pa
isolates (Rampioni et al., 2017). In another study, PAbN was shown
to reduce the levels of QS signals 3-oxo-C12-HSL and C4-HSL and Phage therapy
QS regulated virulence factors like elastase, pyocyanin and protease in
wound and UTI Pa isolates (El-Shaer et al., 2016). Therefore, the Phage therapy has seen fair clinical success with initial studies
discovery of PAbN has led to a large number of in silico studies indicating phage tolerance and resolution of antibiotic resistant Pa
identifying other structurally related compounds as EPIs with infections (Aslam et al., 2020; Mitropoulou et al., 2022). Since the
potential therapeutic ability (Shriram et al., 2018). Despite the porin components of efflux pumps are surface associated and are
promising results in laboratory conditions, EPIs can be toxic to overexpressed due to antibiotic exposure, selecting lytic phages
eukaryotic cells, limiting progress to human clinical trials (Renau which use efflux pumps as binding targets can be exploited for
et al., 2001; Spengler et al., 2017). Perhaps more worryingly, we also therapy. In a novel strategy, the OMKO1 lytic bacteriophage that
recently showed that EPIs can increase the expression of virulence utilizes OprM as a receptor-binding site was used to eliminate
factors like rhamnolipids in PAO1 (Vaillancourt et al., 2021), raising mutants overexpressing the efflux pump, restoring susceptibility of
concerns that increasing Pa antibiotic sensitivity may come at the cost the Pa population to ceftazidime and ciprofloxacin (Chan et al.,
of increasing its pathogenicity. 2016). This restoration of antibiotic sensitivity was consistent across
Recently, Tambat et al. demonstrated that Ethyl 4-bromopyrrole- PAO1, PA14, 3 clinical, and 3 environmental Pa isolates. Thus, it
2-carboxylate (RP1) produced by the soil bacterium Streptomyces Sp. has been proposed that an alternating regimen of phage and
IMTB 2501 can also inhibit MexAB-OprM, decreasing the MIC of antibiotics could be used to exploit the evolutionary trade-off

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Fernandes and Jorth 10.3389/fbrio.2023.1231657

whereby resistance to phages results in selection of low efflux pump Author contributions
expressing antibiotic sensitive Pa populations.
PJ and SF wrote and edited the manuscript. All authors
contributed to the article and approved the submitted version.
Conclusion and future perspectives
Funding
Chronic exposure of Pa to antibiotics can lead to the selection of
efflux pump overexpressing mutants. Out of the 12 RND efflux This research was funded by grant R01AI14642 from the NIH/
pumps in Pa, the roles of only MexCD-OprJ, MexAB-OprM and National Institute of Allergy and Infectious Diseases.
MexEF-OprN have been considerably studied with respect to Pa
virulence. However, clinical isolates often overexpress other efflux
pumps like MexXY and MuxABC which can result in antimicrobial Acknowledgments
resistance but whether they also affect virulence factors and Pa
pathogenesis remains to be tested in animal models of infection. The figures in the paper were created using BioRender.
Importantly, the search for novel EPIs such as RP1 has been gaining
more attention after the discovery of PAbN which is a non-specific Conflict of interest
inhibitor of multiple Pseudomonas efflux pumps. The possible efficacy
of RP1 in reducing murine lung infections could be related to its The authors declare that the research was conducted in the
ability to inhibit MexAB-OprM which as several infection studies absence of any commercial or financial relationships that could be
cited in this article show can lead to enhanced virulence if construed as a potential conflict of interest.
overexpressed (Mahajan-Miklos et al., 2000; Hirakata et al., 2002; The author PJ declared that they were an editorial board
Roux et al., 2015; Jorth et al., 2017; Vaillancourt et al., 2021). member of Frontiers, at the time of submission. This had no
However, since Pa overexpressing MexCD-OprJ or MexEF-OprN impact on the peer review process and the final decision.
showed reduced virulence (Lamarche and Dé ziel, 2011; Olivares et al.,
2012; Martı́nez-Ramos et al., 2014; Alcalde-Rico et al., 2018;
Vaillancourt et al., 2021; Muñoz-Cazalla et al., 2023), EPIs targeting Publisher’s note
these RND transporters could be expected to result in worse infection
outcomes. For any therapeutic strategy to be successful in the clinic, All claims expressed in this article are solely those of the authors
increasing antibiotic susceptibility may not be best achieved at the and do not necessarily represent those of their affiliated
cost of increasing pathogen virulence. Hence, the consequences of organizations, or those of the publisher, the editors and the
alterations in efflux pump on Pa virulence requires an urgent reviewers. Any product that may be evaluated in this article, or
understanding to inform the development of novel therapeutic claim that may be made by its manufacturer, is not guaranteed or
strategies to combat Pa infections. endorsed by the publisher.

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