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Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens

Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, 27100 Pavia, Italy
Department of Biology, University of Florence, 50019 Sesto Fiorentino, Italy
Authors to whom correspondence should be addressed.
Antibiotics 2021, 10(7), 863;
Submission received: 28 May 2021 / Revised: 1 July 2021 / Accepted: 13 July 2021 / Published: 15 July 2021


Drug resistance represents a great concern among people with cystic fibrosis (CF), due to the recurrent and prolonged antibiotic therapy they should often undergo. Among Multi Drug Resistance (MDR) determinants, Resistance-Nodulation-cell Division (RND) efflux pumps have been reported as the main contributors, due to their ability to extrude a wide variety of molecules out of the bacterial cell. In this review, we summarize the principal RND efflux pump families described in CF pathogens, focusing on the main Gram-negative bacterial species (Pseudomonas aeruginosa, Burkholderia cenocepacia, Achromobacter xylosoxidans, Stenotrophomonas maltophilia) for which a predominant role of RND pumps has been associated to MDR phenotypes.

1. Introduction

According to the Cystic Fibrosis Foundation Patient Registry, worldwide more than 70,000 people suffer from Cystic Fibrosis (CF) [1]. Mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene are responsible for the insurgence of a pathological condition, with different severities based on the type of mutation [2]. The CFTR channel is required for the homeostatic control of chloride and bicarbonate ions in the lung. Its malfunctioning leads to mucin overproduction along airways and disruption of the regular mucociliary activity [3,4]. Together, these defects promote polymicrobial proliferation in the respiratory tract, where bacteria are trapped in the mucus and their clearance becomes harder and harder [4]. Moreover, their presence stimulates an exaggerated inflammatory response, making CF pathology characterized by a progressive loss of lung function.
It is noteworthy that the microbial community in CF lungs changes during the lifetime: in 3–5 year-old children, one or a few CF pathogens are detected [1], while in adolescents and adults a polymicrobial community or the prevalence of one typical CF bacterium (e.g., Pseudomonas, Staphylococcus, Stenotrophomonas, or Burkholderia) has been reported [5]. The introduction of CFTR modulator therapy has greatly improved the general health conditions of CF people; however, the effects of lumacaftor-ivacaftor, tezacaftor-ivacaftor, and elexacaftor-tezacaftor-ivacaftor therapy in patients with diverse genetic backgrounds, as well as their effects on the airway microbiota, need to be addressed [6].
A major concern regards the Multi Drug Resistance (MDR) phenotype of CF lung-associated pathogens. Beside the classical drug resistance mechanisms (i.e., drug modification and inactivation, decreased membrane permeability, modification of antibiotic targets, target protection, drug efflux), during the progression of infection, Pseudomonas aeruginosa may switch to the mucoid phenotype, which is very difficult to eradicate [7]. In addition, the highly resistant small-colony variant phenotype of Staphylococcus aureus and P. aeruginosa may be induced by repetitive antibiotic therapy [8,9]. Also, the proportion of methicillin-resistant S. aureus (MRSA), together with metallo-β-lactamase-producing P. aeruginosa strains is worrisome [10,11]. Moreover, during the COVID-19 pandemic, the increased usage of antibiotics to control secondary bacterial infections may further accelerate the spread of antibiotic resistance among nosocomial pathogens [12].
Indeed, while early infections by CF pathogens can be intermittent and involve different strains with multiple levels of antibiotic resistance (AR) profiles, subsequently, people with CF are chronically colonized with well adapted strains with properties (among which high levels of MDR) that differ significantly from those exhibited by the isolate which gave rise to the infection [13,14].This change is related to the adaptation of bacteria to the fluctuating and heterogeneous conditions of the CF lung environment, which exerts a high selective pressure [15]. CF lung is indeed an ecological niche characterized by several selective elements, including the host immune response, the oxidative stresses especially derived from the liberation of reactive oxygen species (ROS) by neutrophils, the interactions among different microorganisms, the nutrient availability, the modified acidity and salinity of the surrounding environment, and the oxygen deprivation in mucus [14,15,16]. Moreover, a strong selective pressure is exerted by the high levels of antibiotics used to treat the infections caused by CF pathogens (a summary of the antibiotic treatment used for the CF pathogens described in this review is reported in Supplementary Table S1) [14,15,16].
Among the consequences of this high selective pressure, there is the emergence of hypermutable strains, whose presence has been strongly associated with bacteria adaptation to the lung environment [12,13]. Hypermutable strains, together with the characteristic transition from the planktonic to the biofilm lifestyle of CF pathogens during chronic infections, lead to the development of high levels of AR in strains adapted to the CF lung. Together, all these factors increase the rate of AR through horizontal gene transfer [12,13]. Although no single mutations can lead to MDR profiles, the use of all antibiotics is prone to be compromised by the acquisition of mutations that can lead to overexpression of efflux pumps, hyperproduction of antibiotic degrading enzymes, porin loss or altered antibiotic targets [13]. Among efflux pumps, those belonging to the Resistance-Nodulation-cell Division (RND) family are able to translocate different molecules (including drugs) out of the bacterial cell in an aspecific manner, thus increasing the ability of bacteria to resist a wide range of treatments [17] RND efflux systems are tripartite complexes composed of an inner membrane protein, a periplasm associated subunit (membrane fusion protein or MFP), and an outer membrane protein (OMP), that span the inner and outer Gram-negative membranes. These pumps are activated by a proton motive force to export compounds into the extracellular environment. The best-described members of this family are the AcrAB-TolC and the MexAB-OprM of Escherichia coli and P. aeruginosa, respectively [18,19].
In this review, we will describe the principal RND efflux pump families which have been found in CF pathogens, then we will focus on the main Gram-negative bacterial species (P. aeruginosa, Burkholderia cenocepacia, Achromobacter xylosoxidans, Stenotrophomonas maltophilia) for which a predominant role of RND pumps has been associated to MDR phenotypes.

2. RND Efflux Pump Families in CF Pathogens

The RND superfamily is a ubiquitous group of efflux pumps conserved in all domains of life (for a recent review see [17]). This superfamily is divided into nine functionally recognized families, six of which have representatives in Gram-negative bacteria [17,20].
In particular, the SecDF efflux pumps are involved in the general secretion (Sec) pathway and members of this family are present in both Bacteria and Archea [17,20]. However, most of the characterized RND proteins of Gram-negative bacteria belong to the Hydrophobe/Amphiphile Efflux 1 (HAE-1) and Heavy Metal Efflux (HME) families, involved in the export of multiple drugs and heavy metals respectively [17,20]. In addition, three other families with few representatives have been found in Gram-negative bacteria that are less known and characterized: (i) the Nodulation Factor Exporter (NFE) family that was identified as a probable nodulation factor exporter, although recently added members of this family are drug exporters; (ii) the Aryl Polyene Pigment Exporters (APPEs), that have been found in Xanthomonas oryzae where they are involved in exporting a pigment [17,20]; (iii) the Hydrophobe/Amphiphile Efflux 3 (HAE-3) family that included some Archea transporters but also HpnN proteins, a group of Gram-negative pumps apparently involved in the transport of hopanoids to the outer membrane [17,20]. The RND proteins of the HAE-1, HME and NFE families are generally associated with an MFP and an OMP protein to form a complex that allows the extrusion of substrates directly out of the cells. The genes coding for these three proteins are usually associated in an operon [17,20].
Most of the RND systems identified and experimentally characterized in cystic fibrosis pathogens belong to the HAE-1 family and are involved in antibiotic efflux. In P. aeruginosa, twelve different RND operons have been found (mexAB-oprM, mexCD-oprJ, mexEF-oprN, mexXY, mexJK, mexGHI-opmD, mexPQ-opmE, mexMN, muxABC-ompB, mexVW, triABC and czcABC) [21]. The CzcABC system belongs to the HME family, while all the others belong to the HAE-1 family [22]. All the twelve systems have been experimentally characterized and most of them are conserved among different strains (in particular, MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY and MexJK) [22,23,24,25,26].
In the Burkholderia cepacia complex at least 19 different putative HAE-1 RND efflux pumps are present, four of which (operon RND-4 or bpeAB-oprB, operon RND-6 RND-7, operon RND-10 or ceoAB-opcM or bpeEF-oprC and operon RND-13) are being conserved among several different strains [20,27,28]. Most of these proteins belong to the HAE-1 family and for several of them, the role in antibiotic efflux have been experimentally confirmed in several Burkholderia species (RND-1, RND-3 or AmrAB-OprA, RND-4 or BpeAB-OprB, RND-8 and RND-9, RND-10 or CeoAB-OpcM or BpeEF-OprC) [29]. Moreover, for two systems (RND-11 or CusABC and RND-12 or CzcABC) identified as belonging to the HME family [20,30], the role in heavy-metal efflux has been experimentally validated [31]. The genes coding for putative SecDF, HpnN/HAE-3 and APPE proteins have been found but not experimentally confirmed [20]. Finally, in this genus, a group of operons which appear not to belong to any of the recognized RND families have been identified and defined as Uncertain Function (UF) [20,30].
In the genome of the type strain of A. xylosoxidans, ATCC 27061, the genes coding for 9 different RND efflux pumps have been identified [32]. Three of these efflux pumps have been functionally characterized: AxyABM (homolog of MexAB-OprM) [33], AxyXY-OprZ (with homology to MexXY-OprM) [34] and AxyEF-OprN [35]. All these systems are involved in the transport of several different antibiotics and belong to the HAE1 family of RND transporters [33,34,35]. The substrates and the family of the other six pumps have yet to be determined. A comparative genomic analysis showed that the genes coding for most of these nine systems are conserved among different A. xylosoxidans strains [34], with one of them, axyABM, conserved in all the sequenced Achromobacter genomes [36], while axyXY–oprZ has been found also in Achromobacter ruhlandii [37]. Regarding proteins belonging to the HME family, RND transport systems homologous of CzcABC and CusABC are present in the genomes of other Achromobacter strains [38,39].
Finally, in S. maltophilia, the genes coding for fifteen putative HAE-1 RND systems have been found, seven of which (smeVWX, smeYZ, smeGH, smeMN, smeOP, smeDEF, smeIJK) seem to be conserved among different strains [40,41]. Eight out of these fifteen pumps (SmeVWX, SmeYZ, SmeOP, SmeDEF, SmeIJK, SmeABC, SmeGH, SmeMN) have also been experimentally characterized, confirming that they are actually involved in AR [40]. In addition, the genes coding for six others putative HME RND efflux pumps have been found in the genome of the K279a strain [41], but none of them have been experimentally validated.

3. RND in Pseudomonas aeruginosa

3.1. Pseudomonas aeruginosa Infections in CF

P. aeruginosa is a Gram-negative bacterium that belongs to the family of Pseudomonadaceae. Thanks to its metabolic versatility it is able to colonize many different environments and to establish opportunistic infections [42]. The World Health Organization classified as a priority one P. aeruginosa carbapenem resistant [43]. P. aeruginosa is the most common causative agent of Gram-negative nosocomial infections and lung infection in CF patients [44]. MDR P. aeruginosa is responsible for over 72,000 infections and 4800 deaths annually in Europe and the majority of these cases are attributed to carbapenem and colistin-resistant strains [45].
P. aeruginosa has a relatively large genome of 5.5–7 million base pairs, encoding a large number of regulatory enzymes involved in metabolism, transport and efflux [46]. During childhood, CF patients are colonized by both P. aeruginosa and S. aureus, while in adulthood P. aeruginosa is predominant and induces lung function decline [47]. The interaction between P. aeruginosa and its hosts is still poorly understood and its persistence in the airways is due to highly complex and multifactorial reasons [48]. The CF airways environment helps P. aeruginosa colonization over other bacteria (S. aureus) and the consequence of this is the prevalence of P. aeruginosa in adults, ranging from 31 to 47% [49]. One possible reason for this prevalence is that the physiological defects linked to CFTR mutations (such as mucus viscosity, production of reactive oxygen species, impaired autophagy, reduced airway acidity and accumulation of ceramides) induce advantages to P. aeruginosa [50].
During the course of the infection, the genetic and phenotypic traits of P. aeruginosa strains in CF airways are subjected to evolutionary changes in response to the selective pressure of the environment [51]. Chronic P. aeruginosa infections are recalcitrant to antibiotic treatment, which are extremely challenging due to the ability of the bacterium to resist the commonly used compounds thanks to its numerous mechanisms of resistance (efflux pumps, ability to form biofilm, persistence) [52]
P. aeruginosa is resistant to numerous antibiotics belonging to the aminoglycosides, quinolones, and β-lactams families [53]. Mechanisms of AR of P. aeruginosa are classified into intrinsic, acquired, and adaptive. Mechanisms of intrinsic AR are encoded by the core genome of the organism, adaptive resistance is induced by environmental stimuli, while acquired resistance depends on the gain of resistance genes derived from other organisms or those which originated after the selection of mutations [54]. Among intrinsic resistance mechanisms there are: the low outer membrane permeability, the expression of efflux pumps, lipopolysaccharides modification, and the production of enzymes that inactivate antibiotics. The adaptive resistance is related to biofilm formation that limits antibiotic access to bacterial cells, decreases bacterial motility and promotes the formation of persister cells [55]. Acquired resistance is the result of horizontal transfer of resistance-related genes or of mutational changes [56].

3.2. Pseudomonas aeruginosa RND Efflux Systems

Antibiotic extrusion and resistance in P. aeruginosa can be closely related to tripartite RND efflux pumps [57]. Efflux pumps are also involved in cellular stress response. Stress signals such as host factors, detergents and endogenous inducers of bacterial stress could help to select mutants, which over-express efflux systems [58]. The constant inflammation of CF lungs exposes P. aeruginosa to reactive oxygen species (ROS), which might induce the prevalence of strains over-expressing efflux pumps (MexAB-OprM and MexXY-OprM) [59]. Moreover, Fraud and colleagues showed that ROS over-exposure selects resistant mutants expressing the RND MexXY-OprM [60].
Among the 12 RND efflux pumps identified in P. aeruginosa, six contribute to AR [61]. These RNDs are: MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM, MexJK-OprM and MexVW-OprM (Table 1) [52,62]. MexAB-OprM and MexXY-OprM are constitutively expressed at the basal level in wild type strains and are induced by antibiotic substrates, while the other systems are not expressed in wild type strains [52,63]. The genes encoding these tripartite efflux pumps are organized in operons, but in certain cases the operon does not contain the OMF gene, such as in the case of MexXY, MexJK and MexVW.
MexAB-OprM extrudes carbapenems, chloramphenicol, fluoroquinolones, lincomycin, macrolides, novobiocin, tetracyclines, and all β-lactams except imipenem. It is also involved in the efflux of triclosan (antiseptic compound) and of sodium dodecyl sulfate (surfactant). While deletion of mexAB-oprM results in a P. aeruginosa strains sensitive to all the above-mentioned antibiotics, a mutant overexpressing MexAB-OprM is characterized by a significant level of resistance [64,65]. The efflux pump MexAB-OprM is composed of an inner membrane protein MexA, a fusion protein MexB and the outer membrane protein OprM [66]. Genes encoding these proteins constitute an operon which is controlled by the transcriptional regulator MexR [67]. The mexR gene is localized upstream of the mexAB-oprM operon and encodes a transcriptional repressor which binds the intergenic region between mexA and mexR, in proximity to their promoters [68]. When MexR is not functional, there is MexAB-OprM overexpression. P. aeruginosa clinical isolates showed different types of mexR mutations, leading to the production of a protein unable to dimerize, to bind the DNA and to repress mexAB-oprM operon or mutations that result in the complete absence of a functional MexR (such as peptide premature termination) [69,70]. A recent study focused on the evolution of resistance during infections showed that the frequency of mutations (frameshift in either mexA or oprM) in mexAB-oprM rises rapidly during infection, providing evidence that the loss of this pump is adaptive [71]. Mutants have a low meropenem resistance, suggesting that these mutations arise in a sub-population of cells of the ancestral strain that are protected from meropenem by physical barriers, such as biofilm, or by phenotypic resistance (tolerance or persistence) [72,73].
The RND efflux pump MexCD-OprJ is expressed in nfxB P. aeruginosa mutants only. NfxB is the negative regulator of MexCD-OprJ and clinical isolates with diverse mutations in nfxB gene were isolated. These mutants showed different levels of resistance to the antibiotics effluxed by MexCD-OprJ, such as chloramphenicol, erythromycin, fluoroquinolones, and tetracyclines [74].
Another RND efflux pump is MexEF-OprN that, unlike the other efflux systems, is positively regulated by the transcriptional activator MexT [64]. This efflux pump extrudes chloramphenicol, fluoroquinolones, tetracycline, and trimethoprim [75]. In most laboratory strains deriving from reference P. aeruginosa strain PAO1, the mexT gene is frequently unfunctional, causing the suppression of mexEF-oprN operon [76]. On the other hand, when MexT is active, it also works as a repressor of the OprD porin, inducing an increase of resistance to carbapenem [77]. P. aeruginosa mutants in the nfxC gene (norfloxacin resistance gene) are characterized by the over-expression of mexEF-oprN operon and are more resistant to chloramphenicol, fluoroquinolones, tetracycline, trimethoprim, and imipenem [78].
One of the most studied RND of P. aeruginosa is the efflux system MexXY-OprM, which contributes to intrinsic resistance to aminoglycosides, tetracyclines, erythromycin, and cefepime [79]. The MexXY can form functional complexes with two different outer membrane proteins, OprM and OprA, in P. aeruginosa PA7 [80]. Recently, it has been shown that the substrate specificities of MexXY can change depending on which OM protein it complexes with [81]. Both OprM and OprA are involved in aminoglycosides efflux, while carbenicillin and sulbenicillin are substrates only of the MexXY-OprA complex [81]. The regulator of this RND is the repressor MexZ and mutations in its gene, or in the regulatory region, lead to overexpression of MexXY [82,83]. In P. aeruginosa CF clinical isolates, the most common mutations are localized in the mexZ gene, inducing MexXY-OprM overproduction. These mexZ mutations arise during chronic infections in CF patients, contributing to tobramycin resistance, one of the first-line antibiotics used in CF [84]. The expression of mexY and mexZ was found to be higher in adults with chronic infection than in children with new or chronic infections, suggesting that these mutations are subjected to positive selection [85].
Another RND efflux pump, MexJK, was identified using triclosan (biocide) as selective agent in mexL mutants in a ΔmexAB-oprM and ΔmexCD-oprJ strains [86]. Furthermore, MexCD-OprJ expression is selected by triclosan and could be considered an interesting selective tool to study efflux systems [86] MexJK expression is controlled by the product of an upstream regulatory gene, mexL, similar to what has been described in other RND efflux pumps. MexJK lacks its own outer membrane protein and requires OprM for the efflux of antibiotics [86].
Using a P. aeruginosa mutant lacking mexAB, mexCDoprJ, mexEFoprN and mexXY, the RND efflux pump MexVW was characterized [87]. In the proximity of the mexVW genes, no ORFs are present that could encode a regulatory protein; similarly, no genes coding for an outer membrane protein are present in the downstream region. MexVW works as a multidrug efflux pump and uses OprM as OMP. Overexpression of mexVW was demonstrated to confer resistance to norfloxacin, ofloxacin, chloramphenicol, cefpirome, tetracycline, and ethidium bromide [87].

3.3. P. aeruginosa RND Efflux Pumps Inhibitors

Among the P. aeruginosa efflux pump inhibitors, the most studied is Phe-Arg-β- naphthylamide (PAβN), a broad spectrum peptidomimetic compound. PAβN was shown to interfere with the four RND systems of P. aeruginosa: MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM. The association of chloramphenicol, fluoroquinolones, macrolides, ketolides, oxazolidinones, and rifampicin with PAβN increases their effects [88]. PAβN functions as substrate of Mex efflux pumps and competes with antibiotics, preventing their extrusion [89]. Unfortunately, PAβN and its derivatives during phase 1 clinical trials showed adverse toxicity and pharmacokinetic profile [90].
Another efflux pump inhibitor is the pyridopyrimidine derivative D13-9001 [89]. It blocks MexAB-OprM in vivo and in vitro and it showed low toxicity profiles [91]. The mechanism of action of this compound relies on a tight interaction with the hydrophobic trap of the pump, preventing its conformational changes. At the same time, D13-9001 blocks the substrate binding to MexB [92]. The limit of this molecule is its specificity for MexAB-OprM: in fact, efflux pump inhibitors should be broad spectrum compounds in order to be used as adjuvants together with antibiotics that are substrates of several efflux pumps. Moreover, different mechanisms of resistance were identified when the compound was administered with carbenicillin. The resistance occurred due to a mutation in the residue F628 of MexB, a site involved in inhibitor binding [93,94].
A polyamine scaffold was identified as an efflux pump inhibitor by Fleeman and co-workers [95]. Polyamines are essential organic polycations ubiquitous in all forms of life and are composed of an aliphatic carbon chain with numerous amino groups. Five polyamine derivatives were demonstrated to potentiate the effect of aztreonam, chloramphenicol, and tetracycline, inducing an MIC90 decrease of 5- to 8-fold. These compounds have limited toxicity and no inhibitory effects on the eukaryotic Ca2+ channel of human kidney cells [95].
Among the natural products that target MDR efflux pumps, there are EA-371α and EA-371δ, identified by screening a library of 78,000 microbial fermentation extracts [96]. These compounds are the products of a Streptomyces strain and are potent MexAB-OprM inhibitors, with a MPC8 (minimum potentiation concentration decreasing the MIC of 8-fold) values of 4.29 μM (EA-371α) and 2.15 μM (EA-371δ) for levofloxacin against strain PAM103. Unfortunately, EA-371α could not be considered a lead compound because of its moderate cytotoxicity [96].
Another type of RND efflux inhibition relies on the application of phage therapy. While the traditional phage therapy is based on the administration of phages to block bacterial cell growth, another approach used phages to steer AR evolution. An example is the lytic Myoviridae bacteriophage OMKO1 that uses OprM as a receptor binding site. Bacteria resistant to OMKO1, lacking OprM, are more sensitive to ciprofloxacin, tetracycline, ceftazidime, and erythromycin due to the counterselection of MDR P. aeruginosa and, possibly, to a change in the efflux pump mechanism [97].

4. RND in Burkholderia cenocepacia

4.1. Burkholderia cenocepacia Infections in CF

Burkholderia cepacia complex (Bcc) species are abundant in the polymicrobial communities inhabiting the lungs of adult CF patients [98]. Within this group of 24 phenotypically related but genetically distinct bacterial species, Burkholderia cenocepacia and Burkholderia multivorans are responsible for approximately 70–85% of all Bcc infections in this cohort of patients [99,100]. The wide variety of potential virulence factors (e.g., catalases, proteases and siderophores) produced by these bacteria to evade host defenses, their innate resistance to many antibiotics and disinfectants, their ability to adhere and invade epithelial cells and to survive inside macrophages, render B. cenocepacia infections very difficult to treat [101,102,103,104]. Clinical effects vary from transient carriage to chronic lung infection, which can rapidly deteriorate to necrotizing pneumonia and sepsis, the so-called “cepacia syndrome”, resulting in a significant decrease in patients’ survival [105,106]. Moreover, the poor post lung transplant outcomes of individuals affected by B. cenocepacia renders chronic infection as a contraindication for lung transplantation [107]. In this scenario, despite the relatively low and stable prevalence of B. cenocepacia infections, affecting around 3% of CF patients in Europe [108], this opportunistic pathogen represents a serious burden for the management of people affected by CF.
The main challenges in the treatment of B. cenocepacia infections are represented by the intrinsic resistance of this species to clinically relevant antibiotics and by their tolerance to antibiotic exposure, typically associated with a biofilm lifestyle [109,110]. In the absence of evidence-based guidelines for treatment [111], various therapeutic protocols based on the use of single or multiple antibiotics administered by different routes (intravenous, oral, inhaled, or combined) for varying periods of time have been employed in clinics. However, complete eradication of the infection is difficult to achieve [112,113]. Strategies based on compounds that improve the activity of antibiotics (helper compounds) by blocking the main resistance mechanisms or altering the physiological state of antibiotic-tolerant cells are in clinical trials or under study [114,115,116,117]. These molecules generally act by impairing bacterial growth, permeabilizing bacteria through the alteration of the structure of the outer membrane, inhibiting biofilm formation and eradicating established biofilms [114,116]. Alternative approaches based on molecules used for other diseases, natural products, quorum-sensing inhibitors and antimicrobial peptides are under investigation [118,119,120,121]. Finally, interest in the design of B. cenocepacia vaccines has recently risen [122].

4.2. Burkholderia cenocepacia RND Efflux Systems

The ability to produce a variety of efflux pumps significantly contributes to the inherent multidrug resistance of B. cenocepacia [112,123]. After the identification of the gene cluster encoding the conserved salicylate-regulated RND-10 efflux pump responsible for chloramphenicol, trimethoprim, and ciprofloxacin resistance [124], sixteen genes encoding transporters of the RND family, organized in 14 operons, have been identified in the genome of the reference B. cenocepacia strain J2315 (Table 2) [20,125,126]. This CF isolate, belonging to the highly transmissible epidemic ET12 lineage, was used for the preparation of a collection of mutant strains, each carrying a marker-less deletion of a single RND operon, thus allowing the investigation of the role of these systems in B. cenocepacia physiology and antibiotic susceptibility [127,128,129]. While the RND-deleted strains did not show any defect in their growth characteristics, the absence of a few specific RND-systems resulted in increased antibiotic susceptibility and, in some cases, alterations in the production of biofilm matrix compared to their parental strain [129]. In particular, when grown in planktonic cultures, mutants lacking the RND-3 and RND-4 efflux systems displayed a higher susceptibility to both ciprofloxacin and tobramycin and a reduced secretion of quorum-sensing molecules [127,129]. Interestingly, lifestyle specific effects could be observed for the different mutants. While the contribution of the RND-3 system to the intrinsic AR of B. cenocepacia J2315 was exerted both in planktonic and sessile cells, the RND-4 efflux pump played a major role in the efflux of ciprofloxacin, tobramycin, minocycline, and chloramphenicol only in planktonic cells. On the contrary, the RND-8 and RND-9 efflux systems were demonstrated to confer protection against tobramycin only in biofilms, but not in planktonic cultures [129].
The lifestyle-specific activity of these pumps appears as a cellular response to regulatory signals governing the physiology of the cell. In fact, besides contributing to the extrusion of antibiotics and of a variety of compounds toxic for cellular metabolism, RND systems play a role in the control of physiological processes and virulence of B. cenocepacia [128]. Deletion of RND-efflux pumps was reported to affect motility-related phenotypes and biofilm formation, with RND-4 and RND-9 mutant deletion strains showing an enhanced biofilm formation ability and an increased and reduced swimming motility, respectively [128]. As revealed by transcriptomic analysis, while the motility phenotype could be easily correlated to a differential expression of motility genes in the mutant strains compared to wild type, the increased ability to form a biofilm could not be linked to an altered expression of genes involved in biofilm formation, suggesting indirect regulatory mechanisms, possibly activated by altered concentrations of toxic compounds or metabolic signals that accumulate in the cell as a consequence of efflux pump inactivation.
The presence of multiple operons encoding RND-efflux pumps in the B. cenocepacia genome suggests a functional redundancy and synergistic activity, accounting for the lack of alterations in the phenotype and in the antibiotic susceptibility of the majority of single RND deletion mutants [127,129]. Interestingly, the high level of conservation of the RND-4 operon in the genomes of Burkholderia species is consistent with the multiple functions in which this system is involved and with the effects of its inactivation on the increased susceptibility to different antimicrobial compounds, including essential oils and disinfectants [127,130,131,132]. On the other hand, RNDs with a narrow phylogenetic distribution, like RND-9, show a more specific activity, with consequent milder phenotypic changes observed in the corresponding J2315 deletion strain [128,129]. Noteworthy, when the conserved RND-4 efflux pump is missing or inactivated, overexpression of the RND-9 system can compensate for its function. For example, in a B. cenocepacia RND-4 deletion strain, mutations in a gene (bcam1948) encoding a transcriptional repressor of the RND-9 operon were demonstrated to confer resistance to a new antitubercular thiopyridine compound whose antimicrobial activity was previously demonstrated to be impaired by RND-4 mediated extrusion [130,133]. Interestingly, mutations in the same regulator confer resistance to a 2,1,3-benzothiadiazol-5-yl family compound and to multiple antibiotics (chloramphenicol, ciprofloxacin, levofloxacin, norfloxacin, sparfloxacin and nalidixic acid) [134]. It is noteworthy that, despite the important contribution of RND-4 in facilitating multiple AR in the B. cenocepacia J2315 laboratory strain, no significant differences in the expression of the RND-4 gene (bcal2822) was detected in multidrug-resistant clinical isolates which, on the contrary, displayed a high expression level of RND-3 (bcal1674) and RND-9 (bcal1947) [135]. However, the upregulation of the genes encoding RND-6 and RND-4 were found to be involved in conferring resistance to different classes of antimicrobials (aminoglycosides, β-lactams, fluoroquinolones, folate-pathway inhibitors) in a clonal variant of B. cenocepacia isolated during long-term infection in CF lungs [136].
Phylogenetic analysis revealed a high degree of sequence similarity between RND-4 and the functionally distinct RND-2 operon, encoding a system present in only some Bcc species [27]. RND-2 is not expressed in bacteria growing in LB medium and its ability to confer resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin and ethidium bromide could be identified only by overexpression experiments in E. coli [125]. Noteworthy, RND-2 overexpression is able to restore resistance to some antibiotics in an RND-4 deletion mutant, supporting the hypothesis that this operon originated from an RND-4 duplication event that led to the creation of a system maintaining the ancestral substrate specificity but subjected it to different regulatory mechanisms [28].

5. RND in Achromobacter xylosoxidans

5.1. Achromobacter Infections in CF

The Achromobacter genus consists of 19 species [137] of motile, non-lactose fermenting Gram-negative environmental bacilli isolated from soil and water sources. Even though they are not intrinsically pathogenic bacteria, they can represent a threat for critically ill, immunocompromised and CF patients. A. xylosoxidans has been known to cause pulmonary infections in CF patients since the 1980s [138], but only recently has it been recognized as one of the main CF pathogens. There is a high regional variability in its infection rate [139] but different reports highlight a worldwide rise in prevalence [140,141,142]. This increase could be due both to the selective antimicrobial pressure present on the CF lung bacterial community, and to the recent improvement of the detection methods, which allow the unequivocal identification of Achromobacter isolates at the species level [143]. This highlighted the presence in CF of different species aside from A. xylosoxidans, which still remains the most prevalent, such as Achromobacter ruhlandii, Achromobacter dolens, and Achromobacter insuavis [143]. Although the impact of these infections on lung function is not fully understood yet [144,145], it is known that these bacterial species, so closely related to the pathogenic Bordetella genus, have a high host adaptation potential, possessing several virulence-associated genes [146].
The treatment of Achromobacter spp. infections is extremely challenging since they show inherent resistance to most penicillins and cephalosporins, as well as to aztreonam, fluoroquinolones, and aminoglycosides [147]. Besides the intrinsic resistance mechanisms, Achromobacter often exhibits acquired resistances, especially towards β-lactams, but also to aminoglycosides and trimethoprim, achieved by horizontal gene transfer [148]. This array of resistance determinants makes these bacteria potentially resistant to every class of antibiotics, and cases of pan-drug-resistant Achromobacter spp. have been already reported [149]. For this reason, the optimal antibiotic therapy for these infections is patient-specific, even if piperacillin–tazobactam, trimethoprim–sulfamethoxazole, and meropenem are usually the most active agents [147]. Concerning the innate resistance mechanisms, initially some β-lactamases were biochemically characterized [150,151,152,153], but the class D β-lactamases OXA-114, in A. xylosoxidans, and OXA-258, in A. ruhlandii, are nowadays the best characterized enzymes, although their role in the β-lactams resistance profile is likely secondary [37,154]. To better study the resistance potential of A. xylosoxidans, Hu and colleagues performed a genome-wide analysis, predicting the presence of 50 drug resistance genes, 38 of which were efflux pump genes [32].

5.2. Achromobacter spp. RND Efflux Systems

The genome of Achromobacter spp. contains a significantly higher number of efflux pump-related genes compared with other genera [155]. Only three of nine RND efflux systems have been studied so far (Table 3), and a lot of work is still needed to have a comprehensive overview of the intrinsic and acquired antimicrobial resistance patterns in the Achromobacter genus.
The first RND-type multidrug efflux system described in A. xylosoxidans (even though the strain used in this work was later reclassified as A. insuavis) was the AxyABM [33]. This RND system is the ortholog of the MexAB-OprM system of P. aeruginosa (60–72% protein identity) and shares with it the same operon organization. Indeed, the genes composing the multiprotein complex are grouped in a cluster of three open reading frames, axyA (the MFP), axyB (the RND transporter protein), and axyM (oprM; the OMP). Moreover, upstream of the operon a gene coding for a transcriptional regulator, namely axyR, is present, as already seen in P. aeruginosa for mexR, although the two genes do not share any homology [33]. By inactivation of axyB, it was also demonstrated that the spectrum of activity of AxyABM is comparable, even if not identical, to the one of MexAB-OprM, being involved in the innate resistance to a broad spectrum of antibiotics, in particular most cephalosporins and aztreonam, but also nalidixic acid, fluoroquinolones, and chloramphenicol [33]. This RND system is present in all the sequenced Achromobacter genomes [146], but it was better characterized only in A. ruhlandii, where it seems to have a narrower spectrum of activity. Indeed, by cloning the axyABM operon in E. coli, it was demonstrated to be only involved in the extrusion of chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole [37]. Finally, besides the innate antibiotic tolerance, AxyABM is probably involved also in persistence and biofilm metabolism of A. xylosoxidans, since the gene axyA was found to be 21-fold upregulated upon the establishment of chronic infections in CF lungs [156]. Moreover, in the same strain, the expression of axyA increased more than 7-fold in sessile cells, highlighting the importance of this efflux system in biofilm formation [156].
To identify the mechanism(s) responsible for the high-levels of innate resistance of A. xylosoxidans towards aminoglycosides, a genomic comparison with P. aeruginosa was performed. This approach led to the characterization of the AxyXY-OprZ efflux pump, the ortholog of the MexXY-OprM RND system of P. aeruginosa [34]. AxyXY-OprZ is encoded by an operon conserved in many Achromobacter species, predominantly in those often recovered from CF patients, and it is described as the major resistance mechanism to aminoglycosides, since its presence is always associated with a resistant phenotype, whereas its absence leads to a sensitive phenotype [157].
The axyXY-oprZ operon is under the negative control of AxyZ, a TetR-type transcriptional repressor homolog of the P. aeruginosa MexZ and is encoded by the gene axyZ, found upstream of the cluster [158]. Surprisingly, this transcription factor plays a role also in the regulation of a novel carbapenemase, Axc, highly expressed in meropenem-resistant A. xylosoxidans clinical isolates [159]. Indeed, loss of function mutations in the axyZ sequence, and especially the V29G substitution localized in the DNA-binding domain of the protein, lead to the overexpression of AxyXY-OprZ, but also of the Axc carbapenemase, increasing the MICs of antibiotic substrates of these proteins [158,159]. This demonstrates that AxyZ is involved in a wide regulatory pathway controlling the activation of disparate AR mechanisms. The AxyZ mutations can be quite easily selected in vitro by exposure of the bacterium to aminoglycosides [158], a class of antibiotic extensively used for CF infections treatment, and consequently these are reported to be frequently associated with the pathoadaptive process of A. xylosoxidans, A. ruhlandii and A. insuavis in CF lung [160].
The AxyXY-OprZ possesses the ability to extrude a broad spectrum of antibiotics, since its inactivation leads to a drastic decrease in the MICs of aminoglycosides and, to a lesser extent, of carbapenems, cefepime (the only cephalosporin not extruded by AxyABM), ceftazidime, some fluoroquinolones, tetracyclines, and erythromycin. Moreover, this RND pump seems to be partially involved in the Achromobacter spp. acquired resistance to carbapenems, since its impairment leads to a significant decrease of carbapenem MICs in a resistant clinical isolate [34]. However, the MIC value results higher than the carbapenem-sensitive Achromobacter strains, suggesting the presence of additional resistance mechanisms, such as the recently described carbapenemase Axc. Despite the high similarity between AxyXY-OprZ and its P. aeruginosa counterpart, the Achromobacter efflux pump confers up to a 32-fold higher level of resistance to aminoglycosides. It was hypothesized that this difference is probably due to the different Opr protein associated with the RND complex, since OprZ is the homolog of OprA (not OprM), the outer membrane protein coupled with MexXY in some P. aeruginosa genetic lineages [34].
The last RND efflux pump characterized in Achromobacter spp. was the AxyEF-OprN, the ortholog of the P. aeruginosa MexEF-OprN [35]. In contrast to the other two RND systems, this pump has a narrow spectrum of activity and was initially demonstrated to have a role in the Achromobacter innate resistance to few fluoroquinolones, carbapenems, and tetracyclines. Indeed, by analyzing the effect of axyE deletion in the AX08 clinical isolate, Nielsen and collaborators showed a decrease of the MIC of levofloxacin, making this strain susceptible to this antibiotic according to the EUCAST interpretative criterion for Pseudomonas spp. Moreover, a 2-fold decrease in the MIC of ertapenem, ciprofloxacin, and doxycycline was reported [35]. Surprisingly, in the same paper they also described an increase in the MICs of some β-lactams as a consequence of the pump inactivation, but this aspect was not further investigated [35]. AxyEF-OprN was also characterized as the main mechanism responsible for acquired fluoroquinolone resistance in Achromobacter [161]. Indeed, it was demonstrated that, different to many Gram-negative bacilli, the fluoroquinolones-resistant phenotype is not due to amino acid substitutions within the Quinolone Resistance Determining Regions (QRDRs) of the targets (DNA gyrase and topoisomerase IV), but it is mainly due to AxyEF-OprN overexpression. In particular, the overproduction of the efflux pump in Achromobacter-resistant clinical isolates is often caused by gain-of-function mutations of AxyT, the transcriptional activator of the axyEF-oprN operon, although the big difference found in fold change in strains owning the same mutation suggests an interplay between different regulatory pathways [161].

5.3. Achromobacter spp. RND Efflux Pumps Inhibitors

Until now, despite the prominent role of RND efflux pumps in Achromobacter innate and acquired AR, no specific inhibitors have been studied. The only active compound present in the literature is berberine, a benzylisoquinoline alkaloid isolated from many medicinal plants, and its derivatives, characterized as specific inhibitors of the P. aeruginosa MexXY system, but tested also against A. xylosoxidans [162,163]. Indeed, in this bacterium berberine significantly reduced the tolerance to aminoglycosides, decreasing the MICs of amikacin, arbekacin, gentamicin, and tobramycin (the substrates of the AxyXY-OprZ efflux pump) up to 32-fold [162]. Moreover, among eleven berberine derivatives, the 13-(2-methylbenzyl) berberine (13-o-MBB) showed the best activity against P. aeruginosa and thus it was tested against A. xylosoxidans. The presence of 13-o-MBB resulted in a further increased sensitivity to aminoglycosides, and the most impressive result was obtained in combination with gentamicin, reducing its MIC of more than 512-fold [163]. However, even low concentrations (30 μg/mL) of this molecule are cytotoxic to human cells in vitro [163], making the development of less toxic derivatives fundamental for future application in humans.

6. RND in Stenotrophomonas maltophilia

6.1. Stenotrophomonas maltophilia Infections in CF

Stenotrophomonas maltophilia is a Gram-negative, aerobic, non-fermentative bacillus, belonging to the class of gammaproteobacteria. It is an ubiquitous contaminant in soil, water, food, and hospital settings [164]. Its major presence in healthcare centers, after Acinetobacter spp. and Pseudomonas aeruginosa, is linked to opportunistic infections with relevant morbidity among patients with underlying pathologies, such as cystic fibrosis, or immunocompromised subjects, with an incidence in USA intensive care units of 4.3% of all Gram-negative infections [41,165]. Risk factors include malignancy, chronic respiratory diseases, and long-term hospitalization. In CF patients, S. maltophilia isolation in the respiratory tract is linked to intravenous antibiotic use and oral quinolone administration, as for the use of anti-pseudomonal antibiotics; approximately 11% of CF patients are colonized by this bacterium, even if its role in such condition is not clear [164]. S. maltophilia chronic infection is correlated to a lower mean percent predicted Forced Expiratory Volume in the 1st second (FEV1) compared to the uninfected control, with a significantly higher risk of pulmonary exacerbation [164]. Combinatorial treatments are efficient in avoiding clone selection, e.g., with trimethoprim-sulfamethoxazole and ticarcillin-clavulanate, doxycycline and ticarcillin-clavulanate, trimethoprim-sulfamethoxazole and piperacillin-tazobactam, ciprofloxacin and ticarcillin-clavulanate. Nevertheless, MDR strains were isolated from topical antiseptic, hand-washing soap, bottled water, and intravenous cannulae, nebulizers and prosthetic devices, showing how hazardous direct-contact transmission and how tolerant this pathogen can be [164]. Such persistence in the environment is adjuvanted by a broad array of intrinsic AR determinants against β-lactams, macrolides, aminoglycosides, cephalosporins, polymyxins, tetracyclines, chloramphenicol, fluoroquinolones, carbapenems, and trimethoprim-sulfamethoxazole [164]. Such phenotype results from the interaction of different layers, as poor membrane permeability, the presence of chromosomally encoded L1 and L2 β-lactamases [166], AAC(6′)-Iz and APH(3′)-IIc aminoglycoside-modifying enzymes [167], and multidrug resistance efflux pumps [164].

6.2. Stenotrophomonas RND Efflux Systems

In the S. maltophilia K279a strain genome eight pumps have been annotated, while seven (smeABC, smeDEF, smeGH, smeIJK, smeOP, smeU1VWU2X, smeYZ) of them have been characterized as hydrophobic and amphiphilic efflux (HAE)-RND pumps (Table 4) [167,168,169,170,171,172,173].
One of the first identified HAE-RND pumps has been SmeABC, which shows similarities to different efflux pumps, such as MexAB-OprM in P. aeruginosa, TtgABC in P. putida and AcrAB in E. coli [172]. This tripartite efflux pump, whose operon is controlled by the SmeSR sensor proteins, confers resistance to third-generation β-lactams, aminoglycosides, and fluoroquinolones and leads to trimethoprim susceptibility once overexpressed [166,172], while physiologically it does not confer intrinsic resistance due to its low-basal expression level. The determinants involved in MDR are being identified as smeC and smeR, whose deletion leads to the reversal of the resistance phenotype [172].
A similar quiescent behaviour is provided by the smeU1VWU2X operon, whose encoded SmeVWX proteins show 51%, 56%, and 48% amino acid identity with P. aeruginosa MexEF-OprN, respectively. The SmeRv protein, a LysR-type regulator, negatively regulates the operon in the S. maltophilia KJ strain, but it acts as a positive regulator in the S. maltophilia MDR KJ09C strain [170]. No mutations have been identified in smeRv, so the presence of an activator ligand could be able to switch on the expression of the entire operon. Differently from the other RND-efflux pumps, it possesses two additional sensor proteins, SmeU1 and SmeU2, belonging to the Short-chain Dehydrogenase/Reductase (SDR) family. The latter has been shown to mediate alleviation from environmental oxidative stress, which is found to trigger the expression of smeU1VWU2X [170,174]. KJ09C mutant overexpressing this operon shows increased resistance to chloramphenicol, quinolones, and tetracycline, with the MICs of aminoglycosides unexpectedly decreased [170]. Interestingly, the smeX deletion of KJ09C mutant reverts both the resistance and the susceptibility patterns, while smeU2 deletion in the same strain leads only to a slight decrease in the resistance, up to a 2-fold MIC decrease in the case of aminoglycosides, suggesting an additional control exerted by SmeU2 on SmeX overexpression [174].
SmeDEF intrinsically confers a two- to eight-fold increase in the MICs of quinolones, tetracycline, chloramphenicol, and novobiocin [175]. Its components show several homologies with different Gram-negative bacterial efflux pumps: SmeD and SmeE share the highest similarities to E. coli AcrA and AcrE (48%) and AcrB and AcrF (61% and 58%), while SmeF is similar to SmeC (42%) [168]. The smeDEF operon is directly regulated by the SmeT protein, which acts as a negative regulator [176]. Different mutations in smeT have been linked to the acquisition of the resistance phenotype, such as L166Q and T197P, allowing tigecycline, aztreonam, and quinolones tolerance, but also fosfomycin susceptibility [169]. This pattern is reasonable, as overexpression of smeD and ameF has been linked to levofloxacin, moxifloxacin, ceftazidime, and tetracycline resistance and amikacin resistance, respectively; in addition, deletion of the smeF gene in K1385 and K1439 MDR strains leads to the reversion of the MDR phenotype [175]. Indirectly, the expression of this efflux pump is influenced by the SmeRySy two-component regulatory system, the main regulator of the smeYZ operon [176]. The deletion of these particular sensor proteins is linked with smeDEF up-regulation and to subsequent chloramphenicol, ciprofloxacin, tetracycline, and macrolide resistance. Counterintuitively, such deletion also increases also smeT expression: a possible explanation involves the presence of an intermediary modulator, whose expression is altered by smeRySy deletion and which mediates the interaction between SmeT and its operator, resulting in the derepression of both smeDEF and smeT [176]. Interestingly, a biocide called triclosan acts as a SmeT inactivator, consequently leading to smeDEF overexpression and MDR strain selection [177,178].
Two highly expressed efflux pumps, SmeYZ, and SmeIJK, play a major role in the intrinsic resistance to antimicrobials [171,173]. The smeYZ operon, sharing 44% and 59% amino acid identity with Acinetobacter baumanii AdeAB [41], confers resistance to amikacin, gentamicin, kanamycin, and leucomycin. Parallelly, its deletion leads to both aminoglycosides and trimethoprim-sulfamethoxazole susceptibility [173]. As previously stated, the operon is controlled by SmeRySy, with smeRy deletion downregulating smeZ expression and conferring aminoglycoside susceptibility, in addition to the acquired resistances involving smeDEF pump expression [176]. Celastrol, an anti-inflammatory natural terpenoid compound, can down-regulate smeYZ expression, thus proposing a possible candidate to control virulence in S. maltophilia [179].
smeIJK has a particular genetic organization, as it is the only efflux pump in S. maltophilia coding for two inner membrane proteins, SmeJ and SmeK, both showing high similarity (59%) among them [171]. The smeIJK operon shares 41%, 50%, and 44% amino acid identity, respectively, to MtdABC of E. coli [41]. SmeIJK confers intrinsic resistance to tetracycline and, to a lesser extent, to aminoglycosides; overexpression can be found in S. maltophilia KJ and KM5 strains leads to an up to 16-fold increase in aminoglycosides MICs and to an increase in fluoroquinolones and tetracyclines resistance, phenotypes reverted after smeJK deletion [167,171]. In addition, deletion of the entire operon in the KJ mutant is linked to polymyxin E susceptibility, thus suggesting a role for SmeI in membrane integrity and permeability [171].
SmeOP proteins are not conserved in other Gram-negative bacteria, as they share less than 30% of the amino acid identity of other antimicrobial efflux pumps [41]. In the strain KJ, this efflux pump is involved in the extrusion of nalidixic acid, doxycycline, macrolides, and more relevantly aminoglycosides, and in the elimination of some toxic compounds such as carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide (TCS) [180]. The operon is controlled by a TetR-type transcription regulator SmeRo, which represses the expression of the genes [180]. Its deletion only produces a slight increase in the MICs of chloramphenicol, quinolones, and tetracyclines. To properly work, the pump requires the cognate OMP TolCSm: deletion of the corresponding gene has been associated with higher decreases in the MICs than those caused by smeOP inactivation, suggesting the involvement of this OMP in the function of another uncharacterized efflux system [180].
Finally, smeGH is the last operon characterized, whose components share 39% and 49% amino acid identity to Morganella morganii AcrAB [41,181]. It is controlled by a TetR-type regulator, which acts as a repressor. In the S. maltophilia D457 strain, a smeH deletion mutant shows an increased susceptibility to ceftazidime, β-lactams, quinolones, and fluoroquinolones, and polymyxin B, suggesting the role of this pump in intrinsic resistance. In the same mutant, a wide variety of other noxious compounds are identified as substrates, such as menadione, benzalkonium chloride and naringenin [181]. Overexpression of smeH in the S. maltophilia clinical strain L1301 is linked to quinolones, macrolides, chloramphenicol, and tetracycline resistance; a similar effect is observed in another strain, named C2206, except for macrolide MIC, which remains unchanged [182]. Through an approach of laboratory experimental evolution, where the D457 strain was exposed to increased ceftazidime concentrations, two subsequent mutations in smeH were identified as linked to MDR [181]: the first to be acquired was P326Q, which confers a 5-fold and 2-fold increase in the MICs of ceftazidime and cefazolin and for aztreonam, respectively; the second acquired mutation was Q663R, which further increased the resistance to ceftazidime, cefazolin, and aztreonam, and conferred a 2-fold and 3-fold increase in the MICs of cefotaxime and norfloxacin, respectively. Finally, the role of Q663R mutation alone was explored, resulting only in a 4-fold increase in MIC of tetracycline: this suggests how relevant the order of mutation acquisition for the final phenotypic outcome is [181].

7. Conclusions

Multidrug-resistant strains represent a major threat for cystic fibrosis patients, who undergo heavy antibiotic therapies to face the recurrent bacterial infections that damage their lungs especially.
Major contributors to the MDR phenotype are the efflux pumps belonging to the Resistance-Nodulation-cell Division family. These transporters are able to translocate a lot of unrelated compounds out of the bacterial cell, thus impairing the effect of the antibiotic therapy, even when a new molecule is administered for the first time [134]. Although nine families of RND proteins have been described, the Hydrophobe/Amphiphile Efflux 1 (HAE-1) is the most represented among CF bacteria, mainly being involved in the extrusion of drugs.
The contribution of this RND family in MDR has been particularly highlighted in P. aeruginosa, B. cenocepacia, A. xylosoxidans, and S. maltophilia.
In P. aeruginosa, six RND systems have been demonstrated to be related to the insurgence of drug resistance in clinical isolates. These pumps are involved in the extrusion of drugs belonging to different categories and were all used for the treatment of CF infections (beta-lactams, tetracyclines, fluoroquinolones, aminoglycosides, etc., Supplementary Table S1), but also detergents, dyes, and quorum-sensing signal molecules. Whole-genome sequencing of P. aeruginosa clinical isolates derived from CF patients revealed that, among the gene-encoding efflux pumps or their regulators, MexZ presents a high rate of mutation [183]). Indeed, a study by Henrichfreise and collaborators [184] reported that the 82% of multidrug-resistant P. aeruginosa strains overproduced MexXY-OprM. However, another work revealed mutations also in the efflux regulator genes mexR, mexT, and nfxB [185]. Non-synonymous mutations have been reported also in the transcriptional regulator of MexAB-OprM, nalC [186]. The same clinical isolates have mutations which lead to the activation of MexT, the positive regulator of MexEF-OprN [186]. A high mutation rate was identified also in the genes encoding the components of RND efflux pumps, such as mexA, mexY, oprM [187] and mexB [188].
In B. cenocepacia, sixteen genes encoding RND pumps have been identified, although a differential contribution to drug resistance has been reported when bacterial cells grow as planktonic or sessile ones [129]. Also in this case, their major role has been described for unrelated compounds, such as antimicrobial compounds, essential oils, disinfectants, and new molecules [20,130,131,132,189]. A study aimed at dissecting the mechanisms responsible for antibiotic resistance in clinical B. cepacia complex isolates revealed that the majority of them exhibited efflux pump activity, which correlated with resistance to various antimicrobial agents, including those used for the treatment of infections in CF patients (e.g., meropenem, ceftazidime, trimethoprim/sulfamethoxazole, Supplementary Table S1) [135]. In particular, RND-3 and RND-9 overexpression was observed in all clinical isolates, with RND-3 being the most up-regulated among the RND pumps tested [135].
During chronic infections, the long-term colonization of the lungs of CF patients is accompanied by an adaptive remodeling of the B. cenocepacia transcriptome. Adaptive changes include the overexpression of various genes encoding drug efflux pumps, like RND-6 and RND-4. As a consequence, the higher active drug export capacity of clinical isolates from the lungs of CF patients affected by long-term chronic infections is accompanied by an increased resistance to clinically relevant antibiotics with very different biological targets [136].
In A. xylosoxidans, seventeen predicted efflux systems have been reported [32]. Only three of these efflux systems have been fully characterized so far, showing the ability to confer resistance to CF used drugs, such as fluoroquinolones andtrimethoprim/sulfamethoxazole (Supplementary Table S1). As an example, Gabrielaite and collaborators [160], performing a genomic analysis on 101 clinical strains isolated within a time span of 20 years in a Denmark CF center, found that in 38% of the analyzed lineages mutations in the gene axyZ (axyXY-oprZ transcriptional repressor) were present. The presence of axyZ mutations led to an overall increase of tolerance to antibiotics since AxyXY-OprZ has a broad spectrum of activity.
Finally, in the S. maltophilia K279a genome eight pumps belonging to the HAE family have been annotated [168]. Their involvement in the resistance has been assessed in 102 clinical isolates, where 70%, 77%, 59% and 61% overexpressed smeB, smeC, smeD, and smeF, respectively [190]. In particular, as regarding the drugs currently used to treat S. maltophilia infections in CF (Supplementary Table S1), smeD overexpression was responsible for levofloxacin and minocycline resistance, smeC for ceftazidime and ticarcillin-clavulonate-nonsusceptibility, while smeF overexpression was significantly correlated with ceftazidime and levofloxacin resistance [190].
Another interesting point is that all the described pathogens are able to chronically colonize the CF airway. This implies their ability to adapt to the host environment, characterized by peculiar nutrient and oxygen availabilities, to interact with the host immune response and to deal with the presence of drugs administered to try to clear the infections. In order to understand this phenomenon, different papers reported results achieved through transcriptomics, which analyzed differential gene expression of strains isolated from CF patients, or genomic analyses which evaluated the presence of mutations in clinical isolates. Interestingly, efflux pump encoding genes were listed among those in which altered level of expression or mutations were reported as contributors to CF lung adaptation in P. aeruginosa [191], B. cenocepacia [136], Achromobacter sp. [146] and S. maltophilia [16]. This has been mainly ascribed both to their role in biofilm formation and in bacterial virulence [61], highlighting a wider role of efflux systems. Indeed, the role of RND efflux pumps in drug resistance can be demonstrated in vitro, where the amount of antibiotic can be measured, while in the clinical environment it is much more complicated to evaluate the achieved antibiotic concentrations and the consequent contribution of efflux to MDR, which might allow the acquisition of other resistance mechanisms.
Despite the recognized role in drug resistance of RND efflux transporters, more efforts are necessary to find efflux inhibitors to be administered to patients. As some molecules were shown to be effective against P. aeruginosa and A. xylosoxidans, the high degree of similarity found among the RND systems of all the described CF colonizing bacteria could lead to the discovery of new inhibitors effective against a broad range of pathogens. These molecules could be used in combination with antibiotics to avoid extrusion and MDR insurgence. Indeed, given the important contribution of specific efflux systems in the insurgence of MDR, the combined use of antibiotics and specific efflux inhibitors could represent a promising therapeutic strategy for CF patients. Interestingly, phage therapy has been shown to target specific efflux pumps in P. aeruginosa: this also represents a new route in the fight against drug resistance.

Supplementary Materials

The following are available online at, Table S1: Antibitoiotics used for the treatment of P. aeruginosa, B. cenocepacia, A. xylosoxidans ans S. maltophilia infections in CF patients and efflux pumps involved in resistance.


This research was funded by the Italian Ministry of Education, University and Research (MIUR) (Dipartimenti di Eccellenza, Program 2018–2022) to Department of Biology and Biotechnology, “L. Spallanzani”, University of Pavia (to G.B. and S.B.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Cystic Fibrosis Foundation. Cystic Fibrosis Foundation Patient Registry: 2018 Annual Data Report; Cystic Fibrosis Foundation: Bethesda, MD, USA, 2019. [Google Scholar]
  2. Burgener, E.B.; Moss, R.B. Cystic fibrosis transmembrane conductance regulator modulators: Precision medicine in cystic fibrosis. Curr. Opin. Pediatr. 2018, 30, 372–377. [Google Scholar] [CrossRef] [PubMed]
  3. Cribbs, S.K.; Beck, J.M. Microbiome in the pathogenesis of cystic fibrosis and lung transplant-related disease. Transl. Res. 2017, 179, 84–96. [Google Scholar] [CrossRef]
  4. Françoise, A.; Héry-Arnaud, G. The microbiome in cystic fibrosis pulmonary disease. Genes 2020, 11, 536. [Google Scholar] [CrossRef]
  5. Zemanick, E.T.; Wagner, B.D.; Robertson, C.E.; Ahrens, R.C.; Chmiel, J.F.; Clancy, J.P.; Gibson, R.L.; Harris, W.T.; Kurland, G.; Laguna, T.A.; et al. Airway microbiota across age and disease spectrum in cystic fibrosis. Eur. Respir. J. 2017, 50, 1700832. [Google Scholar] [CrossRef]
  6. Yi, B.; Dalpke, A.H.; Boutin, S. Changes in the cystic fibrosis airway microbiome in response to CFTR Modulator therapy. Front. Cell Infect. Microbiol. 2021, 11, 548613. [Google Scholar] [CrossRef]
  7. Li, Z.; Kosorok, M.R.; Farrell, P.M.; Laxova, A.; West, S.E.; Green, C.G.; Collins, J.; Rock, M.J.; Splaingard, M.L. Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in children with cystic fibrosis. JAMA 2005, 293, 581–588. [Google Scholar] [CrossRef] [Green Version]
  8. Besier, S.; Smaczny, C.; von Mallinckrodt, C.; Krahl, A.; Ackermann, H.; Brade, V.; Wichelhaus, T.A. Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease. J. Clin. Microbiol. 2007, 45, 168–172. [Google Scholar] [CrossRef] [Green Version]
  9. Haussler, S.; Ziegler, I.; Lottel, A.; Götz, F.V.; Rohde, M.; Wehmhöhner, D.; Saravanamuthu, S.; Tümmler, B.; Steinmetz, I. Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. J. Med. Microbiol. 2003, 52, 295–301. [Google Scholar] [CrossRef] [PubMed]
  10. Nadesalingam, K.; Conway, S.P.; Denton, M. Risk factors for acquisition of methicillin-resistant Staphylococcus aureus (MRSA) by patients with cystic fibrosis. J. Cyst. Fibros. 2005, 4, 49–52. [Google Scholar] [CrossRef] [Green Version]
  11. Senda, K.; Arakawa, Y.; Ichiyama, S.; Nakashima, K.; Ito, H.; Ohsuka, S.; Shimokata, K.; Kato, N.; Ohta, M. PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams. J. Clin. Microbiol. 1996, 34, 2909–2913. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Langendonk, R.F.; Neill, D.R.; Fothergill, J.L. The Building Blocks of Antimicrobial Resistance in Pseudomonas aeruginosa: Implications for Current Resistance-Breaking Therapies. Front. Cell Infect. Microbiol. 2021, 11, 665759. [Google Scholar] [CrossRef]
  13. López-Causapé, C.; Rojo-Molinero, E.; Macià, M.D.; Oliver, A. The problems of antibiotic resistance in cystic fibrosis and solutions. Expert Rev. Respir. Med. 2015, 9, 73–88. [Google Scholar] [CrossRef] [PubMed]
  14. Coutinho, C.P.; Dos Santos, S.C.; Madeira, A.; Mira, N.P.; Moreira, A.S.; Sá-Correia, I. Long-term colonization of the cystic fibrosis lung by Burkholderia cepacia complex bacteria: Epidemiology, clonal variation, and genome-wide expression alterations. Front. Cell. Infect. Microbiol. 2011, 1, 12. [Google Scholar] [CrossRef] [Green Version]
  15. Hogardt, M.; Heesemann, J. Microevolution of Pseudomonas aeruginosa to a chronic pathogen of the cystic fibrosis lung. Curr. Top. Microbiol. Immunol. 2013, 358, 91–118. [Google Scholar]
  16. Menetrey, Q.; Sorlin, P.; Jumas-Bilak, E.; Chiron, R.; Dupont, C.; Marchandin, H. Achromobacter xylosoxidans and Stenotrophomonas maltophilia: Emerging Pathogens Well-Armed for Life in the Cystic Fibrosis Patients’ Lung. Genes 2021, 12, 610. [Google Scholar] [CrossRef]
  17. Nikaido, H. RND transporters in the living world. Res. Microbiol. 2018, 169, 363–371. [Google Scholar] [CrossRef]
  18. Du, D.; Wang, Z.; James, N.R.; Voss, J.E.; Klimont, E.; Ohene-Agyei, T.; Venter, H.; Chiu, W.; Luisi, B.F. Structure of the AcrAB-TolC multidrug efflux pump. Nature 2014, 509, 512–515. [Google Scholar] [CrossRef] [Green Version]
  19. Glavier, M.; Puvanendran, D.; Salvador, D.; Decossas, M.; Phan, G.; Garnier, C.; Frezza, E.; Cece, Q.; Schoehn, G.; Picard, M.; et al. Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex. Nat. Commun. 2020, 11, 4948. [Google Scholar] [CrossRef] [PubMed]
  20. Perrin, E.; Fondi, M.; Papaleo, M.C.; Maida, I.; Emiliani, G.; Buroni, S.; Pasca, M.R.; Riccardi, G.; Fani, R. A census of RND superfamily proteins in the Burkholderia genus. Future Microbiol. 2013, 8, 923–937. [Google Scholar] [CrossRef] [PubMed]
  21. Schweizer, H.P. Efflux as a mechanism of resistance to antimicrobials in Pseudomonas aeruginosa and related bacteria: Unanswered questions. Genet. Mol. Res. 2003, 2, 48–62. [Google Scholar] [PubMed]
  22. Milton, H.S., Jr.; Vamsee, S.R.; Gabriel, M.H.; Kevin, J.H.; Yichi, Z.; Vasu, I.; Katie, J.K.L.; Nuo, T.; Steven, R.; Jianing, W.; et al. The Transporter Classification Database (TCDB): 2021 update. Nucleic Acids Res. 2021, 49, D461–D467. [Google Scholar]
  23. Teixeira, P.; Tacão, M.; Alves, A.; Henriques, I. Antibiotic and metal resistance in a ST395 Pseudomonas aeruginosa environmental isolate: A genomics approach. Mar. Pollut. Bull. 2016, 110, 75–81. [Google Scholar] [CrossRef] [PubMed]
  24. McFarland, A.G.; Bertucci, H.K.; Littman, E.; Shen, J.; Huttenhower, C.; Hartmann, E.M. Triclosan Tolerance Is Driven by a Conserved Mechanism in Diverse Pseudomonas Species. Appl. Environ. Microbiol. 2021, 87, e02924–e03020. [Google Scholar] [CrossRef]
  25. Sood, U.; Hira, P.; Kumar, R.; Bajaj, A.; Rao, D.L.N.; Lal, R.; Shakarad, M. Comparative Genomic Analyses Reveal Core-Genome-Wide Genes under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa. Front. Microbiol. 2019, 10, 53. [Google Scholar] [CrossRef] [Green Version]
  26. Jeukens, J.; Kukavica-Ibrulj, I.; Emond-Rheault, J.G.; Freschi, L.; Levesque, R.C. Comparative genomics of a drug-resistant Pseudomonas aeruginosa panel and the challenges of antimicrobial resistance prediction from genomes. FEMS Microbiol. Lett. 2017, 364. [Google Scholar] [CrossRef]
  27. Perrin, E.; Fondi, M.; Papaleo, M.C. Exploring the HME and HAE1 efflux systems in the genus Burkholderia. BMC Evol. Biol. 2010, 10, 164. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Perrin, E.; Fondi, M.; Bosi, E.; Mengoni, A.; Buroni, S.; Scoffone, V.C.; Valvano, M.; Fani, R. Subfunctionalization influences the expansion of bacterial multidrug antibiotic resistance. BMC Genom. 2017, 18, 834. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Podnecky, N.L.; Rhodes, K.A.; Schweizer, H.P. Efflux pump-mediated drug resistance in Burkholderia. Front. Microbiol. 2015, 6, 305. [Google Scholar] [CrossRef] [Green Version]
  30. Zhang, J.; Li, Q.; Zeng, Y.; Zhang, J.; Lu, G.; Dang, Z.; Guo, C. Bioaccumulation and distribution of cadmium by Burkholderia cepacia GYP1 under oligotrophic condition and mechanism analysis at proteome level. Ecotoxicol. Environ. Saf. 2019, 176, 162–169. [Google Scholar] [CrossRef] [PubMed]
  31. Wang, X.; Zhang, X.; Liu, X.; Huang, Z.; Niu, S.; Xu, T.; Zeng, J.; Li, H.; Wang, T.; Gao, Y.; et al. Physiological, biochemical and proteomic insight into integrated strategies of an endophytic bacterium Burkholderia cenocepacia strain YG-3 response to cadmium stress. Metallomics 2019, 11, 1252–1264. [Google Scholar] [CrossRef]
  32. Hu, Y.; Zhu, Y.; Ma, Y.; Liu, F.; Lu, N.; Yang, X.; Luan, C.; Yi, Y.; Zhu, B. Genomic insights into intrinsic and acquired drug resistance mechanisms in Achromobacter xylosoxidans. Antimicrob. Agents Chemother. 2015, 59, 1152–1161. [Google Scholar] [CrossRef] [Green Version]
  33. Bador, J.; Amoureux, L.; Duez, J.M.; Drabowicz, A.; Siebor, E.; Llanes, C.; Neuwirth, C. First description of an RND-type multidrug efflux pump in Achromobacter xylosoxidans, AxyABM. Antimicrob. Agents Chemother. 2011, 55, 4912–4914. [Google Scholar] [CrossRef] [Green Version]
  34. Bador, J.; Amoureux, L.; Blanc, E.; Neuwirth, C. Innate aminoglycoside resistance of Achromobacter xylosoxidans is due to AxyXY-OprZ, an RND-type multidrug efflux pump. Antimicrob. Agents Chemother. 2013, 57, 603–605. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Nielsen, S.M.; Penstoft, L.N.; Nørskov-Lauritsen, N. Motility, Biofilm Formation and Antimicrobial Efflux of Sessile and Planktonic Cells of Achromobacter xylosoxidans. Pathogens 2019, 8, 14. [Google Scholar] [CrossRef] [Green Version]
  36. Isler, B.; Kidd, T.J.; Stewart, A.G.; Harris, P.; Paterson, D.L. Achromobacter Infections and Treatment Options. Antimicrob. Agents Chemother. 2020, 64, e01025–e01120. [Google Scholar] [CrossRef] [PubMed]
  37. Papalia, M.; Traglia, G.; Ruggiero, M.; Almuzara, M.; Vay, C.; Gutkind, G.; Ramírez, M.S.; Radice, M. Characterisation of OXA-258 enzymes and AxyABM efflux pump in Achromobacter ruhlandii. J. Glob. Antimicrob. Resist. 2018, 14, 233–237. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Schmidt, T.; Schlegel, H.G. Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A. J. Bacteriol. 1994, 176, 7045–7754. [Google Scholar] [CrossRef] [Green Version]
  39. Hložková, K.; Suman, J.; Strnad, H.; Ruml, T.; Paces, V.; Kotrba, P. Characterization of pbt genes conferring increased Pb2+ and Cd2+ tolerance upon Achromobacter xylosoxidans A8. Res. Microbiol. 2013, 164, 1009–1018. [Google Scholar] [CrossRef] [PubMed]
  40. Youenou, B.; Favre-Bonté, S.; Bodilis, J.; Brothier, E.; Dubost, A.; Muller, D.; Nazaret, S. Comparative Genomics of Environmental and Clinical Stenotrophomonas maltophilia Strains with Different Antibiotic Resistance Profiles. Genome Biol. Evol. 2015, 7, 2484–2505. [Google Scholar] [CrossRef] [Green Version]
  41. Crossman, L.C.; Gould, V.C.; Dow, J.M.; Vernikos, G.S.; Okazaki, A.; Sebaihia, M.; Saunders, D.; Arrowsmith, C.; Carver, T.; Peters, N.; et al. The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants. Genome Biol. 2008, 9, R74. [Google Scholar] [CrossRef] [Green Version]
  42. Mathee, K.; Narasimhan, G.; Valdes, C.; Qiu, X.; Matewish, J.M.; Koehrsen, M.; Rokas, A.; Yandava, C.N.; Engels, R.; Zeng, E.; et al. Dynamics of Pseudomonas aeruginosa genome evolution. Proc. Natl. Acad. Sci. USA 2008, 105, 3100–3105. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. WHO Publishes List of Bacteria for Which New Antibiotics Are Urgently Needed. Available online: (accessed on 27 May 2021).
  44. Pendleton, J.N.; Gorman, S.P.; Gilmore, B.F. Clinical relevance of the ESKAPE pathogens. Expert Rev. Anti-Infect. Ther. 2013, 11, 297–308. [Google Scholar] [CrossRef]
  45. Cassini, A.; Diaz Högberg, L.; Plachouras, D.; Quattrocchi, A.; Hoxha, A.; Skov Simonsen, G.; Colomb-Cotinat, M.; Kretzschmar, M.E.; Devleesschauwer, B.; Cecchini, M.; et al. Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: A population-level modelling analysis. Lancet Infect. Dis. 2019, 19, 56–66. [Google Scholar] [CrossRef] [Green Version]
  46. Klockgether, J.; Cramer, N.; Wiehlmann, L.; Davenport, C.F.; Tummler, B. Pseudomonas aeruginosa genomic structure and diversity. Front. Microbiol. 2011, 2, 150. [Google Scholar] [CrossRef] [Green Version]
  47. Elborn, J.S. Cystic fibrosis. Lancet 2016, 388, 2519–2531. [Google Scholar] [CrossRef]
  48. Maurice, N.M.; Bedi, B.; Sadikot, R.T. Pseudomonas aeruginosa Biofilms: Host Response and Clinical Implications in Lung Infections. Am. J. Respir. Cell Mol. Biol. 2018, 58, 428–439. [Google Scholar] [CrossRef]
  49. Reece, E.; Segurado, R.; Jackson, A.; McClean, S.; Renwick, J.; Greally, P. Co-colonisation with Aspergillus fumigatus and Pseudomonas aeruginosa is associated with poorer health in cystic fibrosis patients: An Irish registry analysis. BMC Pulm. Med. 2017, 17, 70. [Google Scholar] [CrossRef] [Green Version]
  50. Riquelme, S.A.; Ahn, D.; Prince, A. Pseudomonas aeruginosa and Klebsiella pneumoniae Adaptation to Innate Immune Clearance Mechanisms in the Lung. J. Innate Immun. 2018, 10, 442–454. [Google Scholar] [CrossRef] [PubMed]
  51. Winstanley, C.; O’Brien, S.; Brockhurst, M.A. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections. Trends Microbiol. 2016, 24, 327–337. [Google Scholar] [CrossRef] [Green Version]
  52. Lister, P.D.; Wolter, D.J.; Hanson, N.D. Antibacterial-resistant Pseudomonas aeruginosa: Clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin. Microbiol. Rev. 2009, 22, 582–610. [Google Scholar] [CrossRef] [Green Version]
  53. Hancock, R.E.; Speert, D.P. Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and impact on treatment. Drug Resist. Updates 2000, 3, 247–255. [Google Scholar] [CrossRef] [Green Version]
  54. Tenover, F.C. Mechanisms of Antimicrobial Resistance in Bacteria. Am. J. Med. 2006, 119, S3–S10. [Google Scholar] [CrossRef] [PubMed]
  55. Drenkard, E. Antimicrobial resistance of Pseudomonas aeruginosa biofilms. Microbes Infect. 2003, 5, 1213–1219. [Google Scholar] [CrossRef] [PubMed]
  56. Breidenstein, E.B.; de la Fuente-Nunez, C.; Hancock, R.E. Pseudomonas aeruginosa: All roads lead to resistance. Trends Microbiol. 2011, 19, 419–426. [Google Scholar] [CrossRef]
  57. Daury, L.; Orange, F.; Taveau, J.C.; Verchere, A.; Monlezun, L.; Gounou, C.; Marreddy, R.K.; Picard, M.; Broutin, I.; Pos, K.M.; et al. Tripartite assembly of RND multidrug efflux pumps. Nat. Commun. 2016, 7, 10731. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Dreier, J.; Ruggerone, P. Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa. Front. Microbiol. 2015, 6, 660. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Poole, K. Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2005, 49, 479–487. [Google Scholar] [CrossRef] [Green Version]
  60. Fraud, S.; Campigotto, A.J.; Chen, Z.; Poole, K. MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa: Involvement in chlorhexidine resistance and induction by membrane-damaging agents dependent upon the AlgU stress response sigma factor. Antimicrob. Agents Chemother. 2008, 52, 4478–4482. [Google Scholar] [CrossRef] [Green Version]
  61. Alcalde-Rico, M.; Olivares-Pacheco, J.; Alvarez-Ortega, C.; Cámara, M.; Martınez, J.L. Role of the multidrug resistance effluxpump MexCD-OprJ in the Pseudomonas aeruginosa quorum sensing response. Front. Microbiol. 2018, 9, 2752. [Google Scholar] [CrossRef]
  62. Li, X.Z.; Plésiat, P.; Nikaido, H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin. Microbiol. Rev. 2015, 28, 337–418. [Google Scholar] [CrossRef] [Green Version]
  63. Goli, H.R.; Nahaei, M.R.; Rezaee, M.A.; Hasani, A.; Samadi Kafil, H.; Aghazadeh, M.; Sheikhalizadeh, V. Contribution of mexAB-oprM and mexXY (-oprA) efflux operons in antibiotic resistance of clinical Pseudomonas aeruginosa isolates in Tabriz, Iran. Infect. Genet. Evol. 2016, 45, 75–82. [Google Scholar] [CrossRef]
  64. Köhler, T.; Michea-Hamzehpour, M.; Epp, S.F.; Pechere, J.C. Carbapenem activities against Pseudomonas aeruginosa: Respective contributions of OprD and efflux systems. Antimicrob. Agents Chemother. 1999, 43, 424–427. [Google Scholar] [CrossRef] [Green Version]
  65. Chen, W.; Wang, D.; Zhou, W.; Sang, H.; Liu, X.; Ge, Z.; Zhang, J.; Lan, L.; Yang, C.G.; Chen, H. Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa. Mol. Microbiol. 2016, 100, 749–758. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Nehme, D.; Li, X.Z.; Elliot, R.; Poole, K. Assembly of the MexAB- OprM multidrug efflux system of Pseudomonas aeruginosa: Identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB. J. Bacteriol. 2004, 186, 2973–2983. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  67. Andrésen, C.; Jalal, S.; Aili, D.; Wang, Y.; Islam, S.; Jarl, A.; Liedberg, B.; Wretlind, B.; Mårtensson, L.G.; Sunnerhagen, M. Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance. Protein Sci. 2010, 19, 680–692. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Evans, K.; Adewoye, L.; Poole, K. MexR repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa: Identification of MexR binding sites in the mexA-mexR intergenic region. J. Bacteriol. 2001, 183, 807–812. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Choudhury, D.; Ghosh, A.; Dhar Chanda, D.; Das Talukdar, A.; Dutta Choudhury, M.; Paul, D.; Maurya, A.P.; Chakravarty, A.; Bhattacharjee, A. Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India. PLoS ONE 2016, 11, e0149156. [Google Scholar]
  70. Pan, Y.P.; Xu, Y.H.; Wang, Z.X.; Fang, Y.P.; Shen, J.L. Overexpression of MexAB-OprM efflux pump in carbapenem-resistant Pseudomonas aeruginosa. Arch. Microbiol. 2016, 198, 565–571. [Google Scholar] [CrossRef]
  71. Wheatley, R.; Diaz Caballero, J.; Kapel, N.; de Winter, F.H.R.; Jangir, P.; Quinn, A.; Del Barrio-Tofiño, E.; López-Causapé, C.; Hedge, J.; Torrens, G.; et al. Rapid evolution and host immunity drive the rise and fall of carbapenem resistance during an acute Pseudomonas aeruginosa infection. Nat. Commun. 2021, 12, 2460. [Google Scholar] [CrossRef]
  72. Mah, T.F.C.; O’Toole, G.A. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001, 9, 34–39. [Google Scholar] [CrossRef]
  73. Brauner, A.; Fridman, O.; Gefen, O.; Balaban, N.Q. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat. Rev. Microbiol. 2016, 14, 320–330. [Google Scholar] [CrossRef] [PubMed]
  74. Masuda, N.; Gotoh, N.; Ohya, S.; Nishino, T. Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1996, 40, 909–913. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Sobel, M.L.; Neshat, S.; Poole, K. Mutations in PA2491 (mexS) promote MexT-dependent mexEF-oprN expression and multidrug resistance in a clinical strain of Pseudomonas aeruginosa. J. Bacteriol. 2005, 187, 1246–1253. [Google Scholar] [CrossRef] [Green Version]
  76. Maseda, H.; Yoneyama, H.; Nakae, T. Assignment of the substrate- selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2000, 44, 658–664. [Google Scholar] [CrossRef] [Green Version]
  77. Ochs, M.M.; McCusker, M.P.; Bains, M.; Hancock, R.E.W. Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids. Antimicrob. Agents Chemother. 1999, 43, 1085–1090. [Google Scholar] [CrossRef] [Green Version]
  78. Köhler, T.; Michéa-Hamzehpour, M.; Henze, U.; Gotoh, N.; Curty, L.K.; Pechère, J.C. Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol. Microbiol. 1997, 23, 345–354. [Google Scholar] [CrossRef]
  79. Hocquet, D.; Nordmann, P.; El Garch, F.; Cabanne, L.; Plésiat, P. Involvement of the MexXY-OprM efflux system in emergence of cefepime resistance in clinical strains of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2006, 50, 1347–1351. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  80. Morita, Y.; Tomida, J.; Kawamura, Y. Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7. Microbiology 2012, 158, 1071–1083. [Google Scholar] [CrossRef]
  81. Singh, M.; Sykes, E.M.E.; Li, Y.; Kumar, A. MexXY RND pump of Pseudomonas aeruginosa PA7 effluxes bi-anionic β-lactams carbenicillin and sulbenicillin when it partners with the outer membrane factor OprA but not with OprM. Microbiology 2020, 166, 1095–1106. [Google Scholar] [CrossRef] [PubMed]
  82. Llanes, C.; Hocquet, D.; Vogne, C.; Benali-Baitich, D.; Neuwirth, C.; Plésiat, P. Clinical Strains of Pseudomonas aeruginosa Overproducing MexAB-OprM and MexXY Efflux Pumps Simultaneously. Antimicrob. Agents Chemother. 2004, 48, 1797–1802. [Google Scholar] [CrossRef] [Green Version]
  83. Vogne, C.; Aires, J.R.; Bailly, C.; Hocquet, D.; Plésiat, P. Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob. Agents Chemother. 2004, 48, 1676–1680. [Google Scholar] [CrossRef] [Green Version]
  84. Marvig, R.L.; Sommer, L.M.; Molin, S.; Johansen, H.K. Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis. Nat. Genet. 2015, 47, 57–64. [Google Scholar] [CrossRef]
  85. Prickett, M.H.; Hauser, A.R.; McColley, S.A.; Cullina, J.; Potter, E.; Powers, C.; Jain, M. Aminoglycoside resistance of Pseudomonas aeruginosa in cystic fibrosis results from convergent evolution in the mexZ gene. Thorax 2017, 72, 40–47. [Google Scholar] [CrossRef] [Green Version]
  86. Chuanchuen, R.; Narasaki, C.T.; Schweizer, H.P. The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for efflux of triclosan. J. Bacteriol. 2002, 184, 5036–5044. [Google Scholar] [CrossRef] [Green Version]
  87. Li, Y.; Mima, T.; Komori, Y.; Morita, Y.; Kuroda, T.; Mizushima, T.; Tsuchiya, T. A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 2003, 52, 572–575. [Google Scholar] [CrossRef] [Green Version]
  88. Lomovskaya, O.; Bostian, K.A. Practical applications and feasibility of efflux pump inhibitors in the clinic—A vision for applied use. Biochem. Pharmacol. 2006, 71, 910–918. [Google Scholar] [CrossRef]
  89. Mahmood, H.Y.; Jamshidi, S.; Mark Sutton, J.; Rahman, K.M. Current Advances in Developing Inhibitors of Bacterial Multidrug Efflux Pumps. Curr. Med. Chem. 2016, 23, 1062–1081. [Google Scholar] [CrossRef]
  90. Renau, T.E.; Léger, R.; Filonova, L.; Flamme, E.M.; Wang, M.; Yen, R.; Madsen, D.; Griffith, D.; Chamberland, S.; Dudley, M.N.; et al. Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa. Bioorg. Med. Chem. Lett. 2003, 13, 2755–2758. [Google Scholar] [CrossRef]
  91. Yoshida, K.; Nakayama, K.; Ohtsuka, M.; Kuru, N.; Yokomizo, Y.; Sakamoto, A.; Takemura, M.; Hoshino, K.; Kanda, H.; Nitanai, H.; et al. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: Highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate. Bioorg. Med. Chem. 2007, 15, 7087–7097. [Google Scholar] [CrossRef] [PubMed]
  92. Nakashima, R.; Sakurai, K.; Yamasaki, S.; Hayashi, K.; Nagata, C.; Hoshino, K.; Onodera, Y.; Nishino, K.; Yamaguchi, A. Structural basis for the inhibition of bacterial multidrug exporters. Nature 2013, 500, 102–106. [Google Scholar] [CrossRef] [PubMed]
  93. Ranjitkar, S.; Jones, A.K.; Mostafavi, M.; Zwirko, Z.; Iartchouk, O.; Barnes, S.W.; Walker, J.R.; Willis, T.W.; Lee, P.S.; Dean, C.R. Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of b-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression. Antimicrob. Agents Chemother. 2019, 63, e01718-18. [Google Scholar]
  94. Rathi, E.; Kumar, A.; Kini, S.G. Computational approaches in efflux pump inhibitors: Current status and prospects. Drug Discov. Today 2020. [Google Scholar] [CrossRef] [PubMed]
  95. Fleeman, R.M.; Debevec, G.; Antonen, K.; Adams, J.L.; Santos, R.G.; Welmaker, G.S.; Houghten, R.A.; Giulianotti, M.A.; Shaw, L.N. Identification of a Novel Polyamine Scaffold With Potent Efflux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens. Front. Microbiol. 2018, 9, 1301. [Google Scholar] [CrossRef]
  96. Lee, M.D.; Galazzo, J.L.; Staley, A.L.; Lee, J.C.; Warren, M.S.; Fuernkranz, H.; Chamberland, S.; Lomovskaya, O.; Miller, G.H. Microbial fermentation-derived inhibitors of efflux-pump-mediated drug resistance. Farmaco 2001, 56, 81–85. [Google Scholar] [CrossRef]
  97. Chan, B.K.; Sistrom, M.; Wertz, J.E.; Kortright, K.E.; Narayan, D.; Turner, P.E. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa. Sci. Rep. 2016, 6, 1–8. [Google Scholar] [CrossRef]
  98. Einarsson, G.G.; Zhao, J.; LiPuma, J.J.; Downey, D.G.; Tunney, M.M.; Elborn, J.S. Community analysis and co-occurrence patterns in airway microbial communities during health and disease. ERJ Open Res. 2019, 5, 00128–02017. [Google Scholar] [CrossRef]
  99. LiPuma, J.J. The changing microbial epidemiology in cystic fibrosis. Clin. Microbiol. Rev. 2010, 23, 299–323. [Google Scholar] [CrossRef] [Green Version]
  100. Drevinek, P.; Mahenthiralingam, E. Burkholderia cenocepacia in cystic fibrosis: Epidemiology and molecular mechanisms of virulence. Clin. Microbiol. Infect. 2010, 16, 821–830. [Google Scholar] [CrossRef] [Green Version]
  101. Leitão, J.H.; Sousa, S.A.; Ferreira, A.S.; Ramos, C.G.; Silva, I.N.; Moreira, L.M. Pathogenicity, virulence factors, and strategies to fight against Burkholderia cepacia complex pathogens and related species. Appl. Microbiol. Biotechnol. 2010, 87, 31–40. [Google Scholar] [CrossRef]
  102. Valvano, M.A. Intracellular survival of Burkholderia cepacia complex in phagocytic cells. Can. J. Microbiol. 2015, 61, 607–615. [Google Scholar] [CrossRef] [Green Version]
  103. McClean, S.; Healy, M.E.; Collins, C.; Carberry, S.; O’Shaughnessy, L.; Dennehy, R.; Adams, Á.; Kennelly, H.; Corbett, J.M.; Carty, F.; et al. Linocin and OmpW are involved in attachment of the cystic fibrosis-associated pathogen Burkholderia cepacia complex to lung epithelial cells and protect mice against infection. Infect. Immun. 2016, 84, 1424–1437. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  104. Mesureur, J.; Feliciano, J.R.; Wagner, N.; Gomes, M.C.; Zhang, L.; Blanco-Gonzalez, M.; van der Vaart, M.; O’Callaghan, D.; Meijer, A.H.; Vergunst, A.C. Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation. PLoS Pathog. 2017, 13, e1006437. [Google Scholar] [CrossRef] [PubMed]
  105. Isles, A.; Maclusky, I.; Corey, M.; Gold, R.; Prober, C.; Fleming, P.; Levison, H. Pseudomonas cepacia infection in cystic fibrosis: An emerging problem. J. Pediatr. 1984, 104, 206–210. [Google Scholar] [CrossRef]
  106. Gibson, R.L.; Burns, J.L.; Ramsey, B.W. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am. J. Respir. Crit. Care Med. 2003, 168, 918–951. [Google Scholar] [CrossRef] [PubMed]
  107. Snell, G.; Reed, A.; Stern, M.; Hadjiliadis, D. The evolution of lung transplantation for cystic fibrosis: A 2017 update. J. Cyst. Fibros. 2017, 16, 553–564. [Google Scholar] [CrossRef] [Green Version]
  108. Hatziagorou, E.; Orenti, A.; Drevinek, P.; Kashirskaya, N.; Mei-Zahav, M.; De Boeck, K.; Pfleger, A.; Sciensano, M.T.; Lammertyn, E.; Macek, M.; et al. Changing epidemiology of the respiratory bacteriology of patients with cystic fibrosis–data from the european cystic fibrosis society patient registry. J. Cyst. Fibros. 2020, 19, 376–383. [Google Scholar] [CrossRef]
  109. Rose, H.; Baldwin, A.; Dowson, C.G.; Mahenthiralingam, E. Biocide susceptibility of the Burkholderia cepacia complex. J. Antimicrob. Chemother. 2009, 63, 502–510. [Google Scholar] [CrossRef]
  110. Peeters, E.; Nelis, H.J.; Coenye, T. In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria. J. Antimicrob. Chemother. 2009, 64, 801–809. [Google Scholar] [CrossRef] [Green Version]
  111. Lord, R.; Jones, A.M.; Horsley, A. Antibiotic treatment for Burkholderia cepacia complex in people with cystic fibrosis experiencing a pulmonary exacerbation. Cochrane Database Syst. Rev. 2020, 4, CD009529. [Google Scholar] [CrossRef]
  112. Rhodes, K.A.; Schweizer, H.P. Antibiotic resistance in Burkholderia species. Drug Resist. Updates 2016, 28, 82–90. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  113. Sputael, V.; Van Schandevyl, G.; Hanssens, L. A case report of successful eradication of new isolates of Burkholderia cenocepacia in a child with cystic fibrosis. Acta Clin. Belg. 2020, 75, 421–423. [Google Scholar] [CrossRef]
  114. Khan, S.; Tøndervik, A.; Sletta, H.; Klinkenberg, G.; Emanuel, C.; Onsøyen, E.; Myrvold, R.; Howe, R.A.; Walsh, T.R.; Hill, K.E.; et al. Overcoming drug resistance with alginate oligosaccharides able to potentiate the action of selected antibiotics. Antimicrob. Agents Chemother. 2012, 56, 5134–5141. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  115. Van den Driessche, F.; Vanhoutte, B.; Brackman, G.; Crabbé, A.; Rigole, P.; Vercruysse, J.; Verstraete, G.; Cappoen, D.; Vervaet, C.; Cos, P.; et al. Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models. PLoS ONE 2017, 12, e0172723. [Google Scholar] [CrossRef]
  116. Narayanaswamy, V.P.; Duncan, A.P.; LiPuma, J.J.; Wiesmann, W.P.; Baker, S.M.; Townsend, S.M. In vitro activity of a novel glycopolymer against biofilms of Burkholderia cepacia complex cystic fibrosis clinical isolates. Antimicrob. Agents Chemother. 2019, 63, e00498-19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  117. Silva, E.; Monteiro, R.; Grainha, T.; Alves, D.; Pereira, M.O.; Sousa, A.M. Fostering innovation in the treatment of chronic polymicrobial cystic fibrosis-associated infections exploring aspartic acid and succinic acid as ciprofloxacin adjuvants. Front. Cell. Infect. Microbiol. 2020, 10, 441. [Google Scholar] [CrossRef]
  118. de la Fuente-Núñez, C.; Reffuveille, F.; Haney, E.F.; Straus, S.K.; Hancock, R.E.W. Broad-spectrum antibiofilm peptide that targets a cellular stress response. PLoS Pathog. 2014, 10, e1004152. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  119. Vasireddy, L.; Bingle, L.E.H.; Davies, M.S. Antimicrobial activity of essential oils against multidrug-resistant clinical isolates of the Burkholderia cepacia complex. PLoS ONE 2018, 13, e0201835. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  120. Shrestha, C.L.; Zhang, S.; Wisniewski, B.; Häfner, S.; Elie, J.; Meijer, L.; Kopp, B.T. (R)-Roscovitine and CFTR modulators enhance killing of multi-drug resistant Burkholderia cenocepacia by cystic fibrosis macrophages. Sci. Rep. 2020, 10, 21700. [Google Scholar] [CrossRef]
  121. Ganesh, P.S.; Vishnupriya, S.; Vadivelu, J.; Mariappan, V.; Vellasamy, K.M.; Shankar, E.M. Intracellular survival and innate immune evasion of Burkholderia cepacia: Improved understanding of quorum sensing-controlled virulence factors, biofilm, and inhibitors. Microbiol. Immunol. 2020, 64, 87–98. [Google Scholar] [CrossRef]
  122. Scoffone, V.C.; Barbieri, G.; Buroni, S.; Scarselli, M.; Pizza, M.; Rappuoli, R.; Riccardi, G. Vaccines to overcome antibiotic resistance: The challenge of Burkholderia cenocepacia. Trends Microbiol. 2020, 28, 315–326. [Google Scholar] [CrossRef]
  123. Scoffone, V.C.; Chiarelli, L.R.; Trespidi, G.; Mentasti, M.; Riccardi, G.; Buroni, S. Burkholderia cenocepacia infections in cystic fibrosis patients: Drug resistance and therapeutic approaches. Front. Microbiol. 2017, 8, 1592. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  124. Nair, B.M.; Cheung, K.-J.; Griffith, A.; Burns, J.L. Salicylate induces an antibiotic efflux pump in Burkholderia cepacia complex genomovar III (B. cenocepacia). J. Clin. Investig. 2004, 113, 464–473. [Google Scholar] [CrossRef] [Green Version]
  125. Guglierame, P.; Pasca, M.R.; De Rossi, E.; Buroni, S.; Arrigo, P.; Manina, G.; Riccardi, G. Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome. BMC Microbiol. 2006, 6, 66. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  126. Holden, M.T.G.; Seth-Smith, H.M.B.; Crossman, L.C.; Sebaihia, M.; Bentley, S.D.; Cerdeño-Tárraga, A.M.; Thomson, N.R.; Bason, N.; Quail, M.A.; Sharp, S.; et al. The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients. J. Bacteriol. 2009, 191, 261–277. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  127. Buroni, S.; Pasca, M.R.; Flannagan, R.S.; Bazzini, S.; Milano, A.; Bertani, I.; Venturi, V.; Valvano, M.A.; Riccardi, G. Assessment of three resistance-nodulation-cell division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance. BMC Microbiol. 2009, 9, 200. [Google Scholar] [CrossRef] [PubMed]
  128. Bazzini, S.; Udine, C.; Sass, A.; Pasca, M.R.; Longo, F.; Emiliani, G.; Fondi, M.; Perrin, E.; Decorosi, F.; Viti, C.; et al. Deciphering the role of RND efflux transporters in Burkholderia cenocepacia. PLoS ONE 2011, 6, e18902. [Google Scholar] [CrossRef]
  129. Buroni, S.; Matthijs, N.; Spadaro, F.; Van Acker, H.; Scoffone, V.C.; Pasca, M.R.; Riccardi, G.; Coenye, T. Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells. Antimicrob. Agents Chemother. 2014, 58, 7424–7429. [Google Scholar] [CrossRef] [Green Version]
  130. Scoffone, V.C.; Spadaro, F.; Udine, C.; Makarov, V.; Fondi, M.; Fani, R.; De Rossi, E.; Riccardi, G.; Buroni, S. Mechanism of resistance to an antitubercular 2-thiopyridine derivative that is also active against Burkholderia cenocepacia. Antimicrob. Agents Chemother. 2014, 58, 2415–2417. [Google Scholar] [CrossRef] [Green Version]
  131. Coenye, T.; Van Acker, H.; Peeters, E.; Sass, A.; Buroni, S.; Riccardi, G.; Mahenthiralingam, E. molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms. Antimicrob. Agents Chemother. 2011, 55, 1912–1919. [Google Scholar] [CrossRef] [Green Version]
  132. Perrin, E.; Maggini, V.; Maida, I.; Gallo, E.; Lombardo, K.; Madarena, M.P.; Buroni, S.; Scoffone, V.C.; Firenzuoli, F.; Mengoni, A.; et al. Antimicrobial activity of six essential oils against Burkholderia cepacia complex: Insights into mechanism(s) of action. Future Microbiol. 2018, 13, 59–67. [Google Scholar] [CrossRef]
  133. Nunvar, J.; Hogan, A.M.; Buroni, S.; Savina, S.; Makarov, V.; Cardona, S.T.; Drevinek, P. The effect of 2-thiocyanatopyridine derivative 11026103 on Burkholderia cenocepacia: Resistance mechanisms and systemic impact. Antibiotics 2019, 8, 159. [Google Scholar] [CrossRef] [Green Version]
  134. Scoffone, V.C.; Ryabova, O.; Makarov, V.; Iadarola, P.; Fumagalli, M.; Fondi, M.; Fani, R.; De Rossi, E.; Riccardi, G.; Buroni, S. Efflux-mediated resistance to a benzothiadiazol derivative effective against Burkholderia cenocepacia. Front. Microbiol. 2015, 6, 815. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  135. Tseng, S.-P.; Tsai, W.-C.; Liang, C.-Y.; Lin, Y.-S.; Huang, J.-W.; Chang, C.-Y.; Tyan, Y.-C.; Lu, P.-L. The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: An emphasis on efflux pump activity. PLoS ONE 2014, 9, e104986. [Google Scholar] [CrossRef]
  136. Mira, N.P.; Madeira, A.; Moreira, A.S.; Coutinho, C.P.; Sá-Correia, I. Genomic expression analysis reveals strategies of Burkholderia cenocepacia to adapt to cystic fibrosis patients’ airways and antimicrobial therapy. PLoS ONE 2011, 6, e28831. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  137. Dumolin, C.; Peeters, C.; Ehsani, E.; Tahon, G.; De Canck, E.; Cnockaert, M.; Boon, N.; Vandamme, P. Achromobacter veterisilvae sp. nov., from a mixed hydrogen-oxidizing bacteria enrichment reactor for microbial protein production. Int. J. Syst. Evol. Microbiol. 2020, 70, 530–536. [Google Scholar] [CrossRef]
  138. Klinger, J.D.; Thomassen, M.J. Occurrence and antimicrobial susceptibility of gram-negative nonfermentative bacilli in cystic fibrosis patients. Diagn. Microbiol. Infect. Dis. 1985, 3, 149–158. [Google Scholar] [CrossRef]
  139. European Cystic Fibrosis Society Patient Registry. In Annual Data Report 2018; 2020. Available online: (accessed on 27 May 2021).
  140. Amoureux, L.; Bador, J.; Siebor, E.; Taillefumier, N.; Fanton, A.; Neuwirth, C. Epidemiology and resistance of Achromobacter xylosoxidans from cystic fibrosis patients in Dijon, Burgundy: First French data. J. Cyst. Fibros. 2013, 12, 170–176. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  141. Trancassini, M.; Iebba, V.; Citerà, N.; Tuccio, V.; Magni, A.; Varesi, P.; De Biase, R.V.; Totino, V.; Santangelo, F.; Gagliardi, A.; et al. Outbreak of Achromobacter xylosoxidans in an Italian Cystic fibrosis center: Genome variability, biofilm production, antibiotic resistance, and motility in isolated strains. Front. Microbiol. 2014, 5, 138. [Google Scholar] [CrossRef]
  142. Firmida, M.C.; Pereira, R.H.; Silva, E.A.; Marques, E.A.; Lopes, A.J. Clinical impact of Achromobacter xylosoxidans colonization/infection in patients with cystic fibrosis. Braz. J. Med. Biol. Res. 2016, 49, e5097. [Google Scholar] [CrossRef] [Green Version]
  143. Papalia, M.; Steffanowski, C.; Traglia, G.; Almuzara, M.; Martina, P.; Galanternik, L.; Vay, C.; Gutkind, G.; Ramírez, M.S.; Radice, M. Diversity of Achromobacter species recovered from patients with cystic fibrosis, in Argentina. Rev. Argent. Microbiol. 2020, 52, 13–18. [Google Scholar] [CrossRef]
  144. Edwards, B.D.; Greysson-Wong, J.; Somayaji, R.; Waddell, B.; Whelan, F.J.; Storey, D.G.; Rabin, H.R.; Surette, M.G.; Parkins, M.D. Prevalence and Outcomes of Achromobacter Species Infections in Adults with Cystic Fibrosis: A North American Cohort Study. J. Clin. Microbiol. 2017, 55, 2074–2085. [Google Scholar] [CrossRef] [Green Version]
  145. Tetart, M.; Wallet, F.; Kyheng, M.; Leroy, S.; Perez, T.; Le Rouzic, O.; Wallaert, B.; Prevotat, A. Impact of Achromobacter xylosoxidans isolation on the respiratory function of adult patients with cystic fibrosis. ERJ Open Res. 2019, 5, 00051–02019. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  146. Jeukens, J.; Freschi, L.; Vincent, A.T.; Emond-Rheault, J.G.; Kukavica-Ibrulj, I.; Charette, S.J.; Levesque, R.C. A Pan-Genomic Approach to Understand the Basis of Host Adaptation in Achromobacter. Genome Biol. Evol. 2017, 9, 1030–1046. [Google Scholar] [CrossRef]
  147. Abbott, I.J.; Peleg, A.Y. Stenotrophomonas, Achromobacter, and nonmelioid Burkholderia species: Antimicrobial resistance and therapeutic strategies. Semin. Respir. Crit. Care Med. 2015, 36, 99–110. [Google Scholar] [PubMed] [Green Version]
  148. Pongchaikul, P.; Santanirand, P.; Antonyuk, S.; Winstanley, C.; Darby, A.C. AcGI1, a novel genomic island carrying antibiotic resistance integron In687 in multidrug resistant Achromobacter xylosoxidans in a teaching hospital in Thailand. FEMS Microbiol. Lett. 2020, 367, fnaa109. [Google Scholar] [CrossRef]
  149. Gainey, A.B.; Burch, A.K.; Brownstein, M.J.; Brown, D.E.; Fackler, J.; Horne, B.; Biswas, B.; Bivens, B.N.; Malagon, F.; Daniels, R. Combining bacteriophages with cefiderocol and meropenem/vaborbactam to treat a pan-drug resistant Achromobacter species infection in a pediatric cystic fibrosis patient. Pediatr. Pulmonol. 2020, 55, 2990–2994. [Google Scholar] [CrossRef]
  150. Levesque, R.; Letarte, R.; Pechère, J.C. Comparative study of the beta-lactamase activity found in Achromobacter. Can. J. Microbiol. 1983, 29, 819–826. [Google Scholar] [CrossRef] [PubMed]
  151. Fujii, T.; Sato, K.; Inoue, M.; Mitsuhashi, S. Purification and properties of a beta-lactamase from Alcaligenes dentrificans subsp. xylosoxydans. J. Antimicrob. Chemother. 1985, 16, 297–304. [Google Scholar] [CrossRef]
  152. Philippon, A.; Mensah, K.; Fournier, G.; Freney, J. Two resistance phenotypes to beta-lactams of Alcaligenes denitrificans subsp. xylosoxydans in relation to beta-lactamase types. J. Antimicrob. Chemother. 1990, 25, 698–700. [Google Scholar] [CrossRef]
  153. Decré, D.; Arlet, G.; Bergogne-Bérézin, E.; Philippon, A. Identification of a carbenicillin-hydrolyzing beta-lactamase in Alcaligenes denitrificans subsp. xylosoxydans. Antimicrob. Agents Chemother. 1995, 39, 771–774. [Google Scholar] [CrossRef] [Green Version]
  154. Doi, Y.; Poirel, L.; Paterson, D.L.; Nordmann, P. Characterization of a naturally occurring class D beta-lactamase from Achromobacter xylosoxidans. Antimicrob. Agents Chemother. 2008, 52, 1952–1956. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  155. Li, X.; Hu, Y.; Gong, J.; Zhang, L.; Wang, G. Comparative genome characterization of Achromobacter members reveals potential genetic determinants facilitating the adaptation to a pathogenic lifestyle. Appl. Microbiol. Biotechnol. 2013, 97, 6413–6425. [Google Scholar] [CrossRef]
  156. Nielsen, S.M.; Meyer, R.L.; Nørskov-Lauritsen, N. Differences in Gene Expression Profiles between Early and Late Isolates in Monospecies Achromobacter Biofilm. Pathogens 2017, 6, 20. [Google Scholar] [CrossRef] [Green Version]
  157. Bador, J.; Neuwirth, C.; Liszczynski, P.; Mézier, M.C.; Chrétiennot, M.; Grenot, E.; Chapuis, A.; de Curraize, C.; Amoureux, L. Distribution of innate efflux-mediated aminoglycoside resistance among different Achromobacter species. New Microbes New Infect. 2015, 10, 1–5. [Google Scholar] [CrossRef] [Green Version]
  158. Bador, J.; Neuwirth, C.; Grangier, N.; Muniz, M.; Germé, L.; Bonnet, J.; Pillay, V.G.; Llanes, C.; de Curraize, C.; Amoureux, L. Role of AxyZ Transcriptional Regulator in Overproduction of AxyXY-OprZ Multidrug Efflux System in Achromobacter Species Mutants Selected by Tobramycin. Antimicrob. Agents Chemother. 2017, 61, e00290-17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  159. Fleurbaaij, F.; Henneman, A.A.; Corver, J.; Knetsch, C.W.; Smits, W.K.; Nauta, S.T.; Giera, M.; Dragan, I.; Kumar, N.; Lawley, T.D.; et al. Proteomic identification of Axc, a novel beta-lactamase with carbapenemase activity in a meropenem-resistant clinical isolate of Achromobacter xylosoxidans. Sci. Rep. 2018, 8, 8181. [Google Scholar] [CrossRef] [Green Version]
  160. Gabrielaite, M.; Nielsen, F.C.; Johansen, H.K.; Marvig, R.L. Achromobacter genetic adaptation in cystic fibrosis. bioRxiv 2021, 426490. Available online: (accessed on 27 May 2021).
  161. Magallon, A.; Roussel, M.; Neuwirthm, C.; Tetu, J.; Cheiakh, A.C.; Boulet, B.; Varin, V.; Urbain, V.; Bador, J.; Amoureux, L. Fluoroquinolone resistance in Achromobacter spp.: Substitutions in QRDRs of GyrA, GyrB, ParC and ParE and implication of the RND efflux system AxyEF-OprN. J. Antimicrob. Chemother. 2021, 76, 297–304. [Google Scholar] [CrossRef]
  162. Morita, Y.; Nakashima, K.; Nishino, K.; Kotani, K.; Tomida, J.; Inoue, M.; Kawamura, Y. Berberine Is a Novel Type Efflux Inhibitor Which Attenuates the MexXY-Mediated Aminoglycoside Resistance in Pseudomonas aeruginosa. Front. Microbiol. 2016, 7, 1223. [Google Scholar] [CrossRef]
  163. Kotani, K.; Matsumura, M.; Morita, Y.; Tomida, J.; Kutsuna, R.; Nishino, K.; Yasuike, S.; Kawamura, Y. 13-(2-Methylbenzyl) Berberine Is a More Potent Inhibitor of MexXY-Dependent Aminoglycoside Resistance than Berberine. Antibiotics 2019, 8, 212. [Google Scholar] [CrossRef] [Green Version]
  164. Brooke, J.S. Stenotrophomonas maltophilia: An emerging global opportunistic pathogen. Clin. Microbiol. Rev. 2012, 25, 2–41. [Google Scholar] [CrossRef] [Green Version]
  165. Lira, F.; Berg, G.; Martínez, J.L. Double-face meets the bacterial world: The opportunistic pathogen Stenotrophomonas maltophilia. Front. Microbiol. 2017, 8, 1–15. [Google Scholar] [CrossRef]
  166. Biagi, M.; Lamm, D.; Meyer, K.; Vialichka, A.; Jurkovic, M.; Patel, S.; Mendes, R.E.; Bulman, Z.P.; Wenzler, E. Activity of Aztreonam in Combination with Avibactam, Clavulanate, Relebactam, and Vaborbactam against Multidrug-Resistant Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2020, 64, e00297-20. [Google Scholar] [CrossRef] [PubMed]
  167. Gould, V.C.; Okazaki, A.; Avison, M.B. Coordinate hyperproduction of SmeZ and SmeJK efflux pumps extends drug resistance in Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2013, 57, 655–657. [Google Scholar] [CrossRef] [Green Version]
  168. Alonso, A.; Martinez, J.L. Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2000, 44, 3079–3086. [Google Scholar] [CrossRef] [Green Version]
  169. Blanco, P.; Corona, F.; Martinez, J.L. Mechanisms and phenotypic consequences of acquisition of tigecycline resistance by Stenotrophomonas maltophilia. J. Antimicrob. Chemother. 2019, 74, 3221–3230. [Google Scholar] [CrossRef]
  170. Chen, C.H.; Huang, C.C.; Chung, T.C.; Hu, R.M.; Huang, Y.W.; Yang, T.C. Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2011, 55, 5826–5833. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  171. Huang, Y.W.; Liou, R.S.; Lin, Y.T.; Huang, H.H.; Yang, T.C. A linkage between SmeIJK efflux pump, cell envelope integrity, and σe-mediated envelope stress response in Stenotrophomonas maltophilia. PLoS ONE 2014, 9, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  172. Li, X.Z.; Zhang, L.; Poole, K. SmeC, an outer membrane multidrug efflux protein of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2002, 46, 333–343. [Google Scholar] [CrossRef] [Green Version]
  173. Lin, Y.T.; Huang, Y.W.; Chen, S.J.; Chang, C.W.; Yang, T.C. The SmeYZ efflux pump of Stenotrophomonas maltophilia contributes to drug resistance, virulence-related characteristics, and virulence in mice. Antimicrob. Agents Chemother. 2015, 59, 4067–4073. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  174. Wu, C.J.; Chiu, T.T.; Lin, Y.T.; Huang, Y.W.; Li, L.H.; Yang, T.C. Role of smeU1VWU2X operon in alleviation of oxidative stresses and occurrence of sulfamethoxazole-trimethoprim-resistant mutants in Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2018, 62, 1–12. [Google Scholar] [CrossRef] [Green Version]
  175. Zhang, L.; Li, X.Z.; Poole, K. SmeDEF multidrug efflux pump contributes to intrinsic multidrug resistance in Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2001, 45, 3497–3503. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  176. Wu, C.J.; Huang, Y.W.; Lin, Y.T.; Ning, H.C.; Yang, T.C. Inactivation of SmeSyRy two-component regulatory system inversely regulates the expression of SmeYZ and SmeDEF efflux pumps in Stenotrophomonas maltophilia. PLoS ONE 2016, 11, 1–14. [Google Scholar] [CrossRef] [PubMed]
  177. Hernández, A.; Ruiz, F.M.; Romero, A.; Martínez, J.L. The binding of triclosan to SmeT, the repressor of the multidrug efflux pump SmeDEF, induces antibiotic resistance in Stenotrophomonas maltophilia. PLoS Pathog. 2011, 7, 1–12. [Google Scholar] [CrossRef] [Green Version]
  178. Sanchez, P.; Moreno, E.; Martinez, J.L. The biocide triclosan selects Stenotrophomonas maltophilia mutants that overproduce the SmeDEF multidrug efflux pump. Antimicrob. Agents Chemother. 2005, 49, 781–782. [Google Scholar] [CrossRef] [Green Version]
  179. Kim, H.R.; Lee, D.; Eom, Y.B. Anti-biofilm and anti-virulence efficacy of celastrol against Stenotrophomonas maltophilia. Int. J. Res. Med. Sci. 2018, 15, 617–627. [Google Scholar] [CrossRef] [Green Version]
  180. Lin, C.W.; Huang, Y.W.; Hu, R.M.; Yang, T.C. SmeOP-TolCSm efflux pump contributes to the multidrug resistance of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 2014, 58, 2405–2408. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  181. Blanco, P.; Corona, F.; Martínez, J.L. Involvement of the RND efflux pump transporter SmeH in the acquisition of resistance to ceftazidime in Stenotrophomonas maltophilia. Sci. Rep. 2019, 9, 1–14. [Google Scholar] [CrossRef]
  182. Li, L.H.; Zhang, M.S.; Wu, C.J.; Lin, Y.T.; Yang, T.C. Overexpression of SmeGH contributes to the acquired MDR of Stenotrophomonas maltophilia. J. Antimicrob. Chemother. 2019, 74, 2225–2229. [Google Scholar] [CrossRef] [PubMed]
  183. Faure, E.; Kwong, K.; Nguyen, D. Pseudomonas aeruginosa in Chronic Lung Infections: How to Adapt Within the Host? Front. Immunol. 2018, 9, 2416. [Google Scholar] [CrossRef] [Green Version]
  184. Henrichfreise, B.; Wiegand, I.; Pfister, W.; Wiedemann, B. Resistance mechanisms of multiresistant Pseudomonas aeruginosa strains from Germany and correlation with hypermutation. Antimicrob. Agents Chemother. 2007, 51, 4062–4070. [Google Scholar] [CrossRef] [Green Version]
  185. Rees, V.E.; Deveson Lucas, D.S.; López-Causapé, C.; Huang, Y.; Kotsimbos, T.; Bulitta, J.B.; Rees, M.C.; Barugahare, A.; Peleg, A.Y.; Nation, R.L.; et al. Characterization of Hypermutator Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis in Australia. Antimicrob. Agents Chemother. 2019, 63, e02538-18. [Google Scholar] [CrossRef] [Green Version]
  186. Díaz-Ríos, C.; Hernández, M.; Abad, D.; Álvarez-Montes, L.; Varsaki, A.; Iturbe, D.; Calvo, J.; Ocampo-Sosa, A.A. New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient. Antibiotics 2021, 10, 491. [Google Scholar] [CrossRef]
  187. Greipel, L.; Fischer, S.; Klockgether, J.; Dorda, M.; Mielke, S.; Wiehlmann, L.; Cramer, N.; Tümmler, B. Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients. Antimicrob. Agents Chemother. 2016, 60, 6726–6734. [Google Scholar] [CrossRef] [Green Version]
  188. López-Causapé, C.; Sommer, L.M.; Cabot, G.; Rubio, R.; Ocampo-Sosa, A.A.; Johansen, H.K.; Figuerola, J.; Cantón, R.; Kidd, T.J.; Molin, S.; et al. Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone. Sci. Rep. 2017, 7, 5555. [Google Scholar] [CrossRef] [Green Version]
  189. Costabile, G.; Provenzano, R.; Azzalin, A.; Scoffone, V.C.; Chiarelli, L.R.; Rondelli, V.; Grillo, I.; Zinn, T.; Lepioshkin, A.; Savina, S.; et al. PEGylated mucus-penetrating nanocrystals for lung delivery of a new FtsZ inhibitor against Burkholderia cenocepacia infection. Nanomedicine 2020, 23, 102113. [Google Scholar] [CrossRef] [PubMed]
  190. Chong, S.Y.; Lee, K.; Chung, H.S.; Hong, S.G.; Suh, Y.; Chong, Y. Levofloxacin Efflux and smeD in Clinical Isolates of Stenotrophomonas maltophilia. Microb. Drug Resist. 2017, 23, 163–168. [Google Scholar] [CrossRef] [PubMed]
  191. Smith, E.E.; Buckley, D.G.; Wu, Z.; Saenphimmachak, C.; Hoffman, L.R.; D’Argenio, D.A.; Miller, S.I.; Ramsey, B.W.; Speert, D.P.; Moskowitz, S.M. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl. Acad. Sci. USA 2006, 103, 8487–8492. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Table 1. RND efflux pumps in P. aeruginosa.
Table 1. RND efflux pumps in P. aeruginosa.
RND Efflux PumpSystematic IDFamilyIdentified Regulator(s)Substrates
MexAB-OprMPA0425-PA0427HAE-1MexR, repressor (MarR-type regulator)β-Lactams (except imipenem), β-lactam inhibitors, fluoroquinolones, tetracycline, chloramphenicol, novobiocin, macrolides, trimethoprim, triclosan (irgasan), ethidium bromide, SDS, aromatic hydrocarbons, thiolactomycin, cerulenin, acylated homoserine lactones
MexCD-OprJPA4599- PA4597HAE-1NfxB, repressor (TetR/AcrR-type regulator)β-Lactams, fluoroquinolones, chloramphenicol, tetracycline, novobiocin, trimethoprim, macrolides, crystal violet, ethidium bromide, acriflavine, SDS, aromatic hydrocarbons, triclosan
MexEF-OprNPA2493-PA2495HAE-1MexT, activator (LysR-type regulator) Fluoroquinolones, chloramphenicol, trimethoprim, aromatic hydrocarbons, triclosan, Pseudomonas quinolone signal
MexXYPA2019-PA2018HAE-1MexZ, repressor (TetR-type regulatorFluoroquinolones, aminoglycosides, tetracycline, erythromycin
MexJKPA3677-PA3676HAE-1MexL, repressor (TetR/AcrR-type regulator)Tetracycline, erythromycin, triclosan
MexVWPA4374-PA4375HAE-1N.D.Norfloxacin, ofloxacin, chloramphenicol, cefpirome, tetracycline, ethidium bromide and acriflavine
Table 2. RND efflux pumps in B. cenocepacia.
Table 2. RND efflux pumps in B. cenocepacia.
RND-Efflux PumpSystematic IDFamilyIdentified Regulator(s)Antibiotic Substrates
RND-2BCAS0766- BCAS0764HAE-RNDLysR family transcriptional regulator (BCAS0767) AraC family transcriptional regulator (BCAS0768)Fluoroquinolones, tetracycline, rifampicin, novobiocin, EO
RND-3BCAL1674-BCAL1676HAE-RNDTet-R type regulator(BCAL1672)Nalidixic acid, ciprofloxacin, tobramycin, meropenem, chlorhexidine
RND-4BCAL2820-BCAL2822HAE-RNDTet-R type regulator(BCAL2823)Aztreonam, chloramphenicol, fluoroquinolones, tobramycin, tetracycline, rifampicin, novobiocin, essential oils, ethidium bromide, 2-thiocyanatopyridine derivative (11026103)
RND-9BCAM1945-BCAM1947HAE-RNDMer-R type regulator(BCAM1948)Tobramycin, chlorhexidine, EO, 2-thiocyanatopyridine derivative (11026103), 2,1,3-benzothiadiazol-5-yl family compound (10126109)
RND-10BCAM2549-BCAM2551HAE-RNDTet-R type regulator (BCAM2548)Chloramphenicol, fluoroquinolones, Trimethoprim, EO
RND-11BCAM0711-BCAM0713HME-RNDN.A.Divalent cations (Zn2+, Co2+, Cd2+ and Ni2+)
RND-12BCAM0433-BCAM0435HME-RNDN.A.Monovalent cations (Cu+ and Ag+), EO
RND-16BCAL2134-BCAL2136U.F.-RNDN.A.Minocycline, meropenem ciprofloxacin
HAE: Hydrophobe/Amphiphile Efflux-1; HME = Heavy-Metal Efflux; U.F. = Uncertain Function. N.A. Not available; EO: Essential oils.
Table 3. Characterized RND efflux pumps in A. xylosoxidans.
Table 3. Characterized RND efflux pumps in A. xylosoxidans.
RND Efflux-PumpPAO1 Orthologous (% of Identity)Identified Regulator(s)Antibiotic Substrates
AxyABMMexAB-OprM (60-72-60%)AxyR (putative LysR-type regulator)Cephalosporins, aztreonam, nalidixic acid, fluoroquinolones, chloramphenicol, trimethoprim/sulfamethoxazole
AxyXY-OprZMexXY-OprM (62-74-48%)AxyZ (TetR-type regulator)Aminoglycosides, carbapenems, cefepime, ceftazidime, fluoroquinolones, tetracyclines, erythromycin
AxyEF-OprNMexEF-OprN (50-65-31%)AxyT (LysR-type regulator)Fluoroquinolones, carbapenems, tetracyclines
Table 4. RND efflux pumps in S. maltophilia.
Table 4. RND efflux pumps in S. maltophilia.
RND-Efflux PumpSystematic ID FamilyIdentified Regulator(s)Antibiotic Substrates
SmeABCSmlt4474-4476 HAE-RNDTwo-component regulator SmeSRtrimethoprim; third-generation β-lactams; aminoglycosides; fluoroquinolones
SmeDEFSmlt4070-4072 HAE-RNDTet-R type regulator SmeT; Two component regulator SmeRySychloramphenicol; ceftazidime; amikacin; aztreonam; novobiocin; fosfomycin; quinolones
SmeGHSmlt3170-3171 HAE-RNDTet-R type regulatorceftazidime; tetracycline; polymyxin B; β-lactams; quinolones; fluoroquinolones
SmeIJKSmlt4279/4281 HAE-RNDN.D.tetracyclines; fluoroquinolones; aminoglycosides
SmeMNSmlt3788-3787 HAE-RNDN.D.N.D.
SmeOPSmlt3925-3924 HAE-RNDTet-R type regulator SmeRonalidixic acid; doxocycline; aminoglycosides; macrolides
SmeYZSmlt2201-2202 HAE-RNDTwo-component regulator SmeRySytrimethoprim-sulfamethoxazole; leucomycin; aminoglycosides
SmeU1VWU2ZSmlt1829-1833 HAE-RNDLys-R type regulator SmeRvchloramphenicol; tetracycline; quinolones
N.D. Not Determined.
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Scoffone, V.C.; Trespidi, G.; Barbieri, G.; Irudal, S.; Perrin, E.; Buroni, S. Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens. Antibiotics 2021, 10, 863.

AMA Style

Scoffone VC, Trespidi G, Barbieri G, Irudal S, Perrin E, Buroni S. Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens. Antibiotics. 2021; 10(7):863.

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Scoffone, Viola Camilla, Gabriele Trespidi, Giulia Barbieri, Samuele Irudal, Elena Perrin, and Silvia Buroni. 2021. "Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens" Antibiotics 10, no. 7: 863.

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