Search

Pseudomonas aeruginosa, a bacterium characterized for its low antibiotics' susceptibility, is one of the most relevant opportunistic pathogens, causing infections at hospitals and in cystic fibrosis patients. Besides its relevance for human health, P. aeruginosa colonizes environmental ecosystems; therefore the elements driving its infectivity and antibiotic resistance must be analyzed from a One-Health perspective. Although some epidemic clones have been described, there are not specific lineages linked to infections, suggesting that P. aeruginosa virulence and antibiotic resistance determinants evolved in nature to play functions other than infecting the human host and avoiding antimicrobial treatment. Herein, we review current information on the population structure of P. aeruginosa and on the functional role that its resistance and virulence determinants have in non-clinical ecosystems.

Antibiotic pollution of non-clinical environments might have a relevant impact on human health if resistant pathogens are selected. However, this potential risk is often overlooked, since drug concentrations in nature are usually below their minimal inhibitory concentrations (MICs). Albeit, antibiotic resistant bacteria can be selected even at sub-MIC concentrations, in a range known as the sub-MIC selective window. Using short-term evolution experiments, we have determined the sub-MIC selective windows of the opportunistic pathogen Pseudomonas aeruginosa for seven antibiotics of clinical relevance, finding the ones of quinolones to be the widest, and the ones of polymyxin B and imipenem, the narrowest. Clinically relevant multidrug-resistant mutants arose within the sub-MIC selective windows of most antibiotics tested, being some of these phenotypes mediated by efflux pumps' activity. The fact that the concentration of antibiotics reported in aquatic ecosystems - colonizable by P. aeruginosa - are, in occasions, higher than the ones that select multidrug-resistant mutants in our assays, has implications for understanding the role of different ecosystems and conditions in the emergence of antibiotic resistance from a One-Health perspective. Further, it reinforces the importance of procuring accurate information on the sub-MIC selective windows for drugs of clinical value in pathogens with environmental niches.

The rise of antibiotic resistance and the reduced amount of novel antibiotics support the need of developing novel strategies to fight infections, based on improving the use of the antibiotics we already have. Collateral sensitivity is an evolutionary trade-off associated with the acquisition of antibiotic resistance that can be exploited to tackle this relevant health problem. However, different works have shown that patterns of collateral sensitivity are not always conserved, thus precluding the exploitation of this evolutionary trade-off to fight infections. In this work, we identify a robust pattern of collateral sensitivity to fosfomycin in Pseudomonas aeruginosa antibiotic-resistant mutants, selected by antibiotics belonging to different structural families. We characterize the underlying mechanism of the collateral sensitivity observed, which is a reduced expression of the genes encoding the peptidoglycan-recycling pathway, which preserves the peptidoglycan synthesis in situations where its de novo synthesis is blocked, and a reduced expression of fosA, encoding a fosfomycin-inactivating enzyme. We propose that the identification of robust collateral sensitivity patterns, as well as the understanding of the molecular mechanisms behind these phenotypes, would provide valuable information to design evolution-based strategies to treat bacterial infections.

The use and misuse of antibiotics have made antibiotic-resistant bacteria widespread nowadays, constituting one of the most relevant challenges for human health at present. Among these bacteria, opportunistic pathogens with an environmental, non-clinical, primary habitat stand as an increasing matter of concern at hospitals. These organisms usually present low susceptibility to antibiotics currently used for therapy. They are also proficient in acquiring increased resistance levels, a situation that limits the therapeutic options for treating the infections they cause. In this article, we analyse the most predominant opportunistic pathogens with an environmental origin, focusing on the mechanisms of antibiotic resistance they present. Further, we discuss the functions, beyond antibiotic resistance, that these determinants may have in the natural ecosystems that these bacteria usually colonize. Given the capacity of these organisms for colonizing different habitats, from clinical settings to natural environments, and for infecting different hosts, from plants to humans, deciphering their population structure, their mechanisms of resistance and the role that these mechanisms may play in natural ecosystems is of relevance for understanding the dissemination of antibiotic resistance under a One-Health point of view.

Different works have explored independently the evolution toward antibiotic resistance and the role of eco-adaptive mutations in the adaptation to a new habitat (as the infected host) of bacterial pathogens. However, knowledge about the connection between both processes is still limited. We address this issue by comparing the evolutionary trajectories toward antibiotic resistance of a Pseudomonas aeruginosa lasR defective mutant and its parental wild-type strain, when growing in presence of two ribosome-targeting antibiotics. Quorum-sensing lasR defective mutants are selected in P. aeruginosa populations causing chronic infections. Further, we observed they are also selected in vitro as a first adaptation for growing in culture medium. By using experimental evolution and whole-genome sequencing, we found that the evolutionary trajectories of P. aeruginosa in presence of these antibiotics are different in lasR defective and in wild-type backgrounds, both at the phenotypic and the genotypic levels. Recreation of a set of mutants in both genomic backgrounds (either wild type or lasR defective) allowed us to determine the existence of negative epistatic interactions between lasR and antibiotic resistance determinants. These epistatic interactions could lead to mutual contingency in the evolution of antibiotic resistance when P. aeruginosa colonizes a new habitat in presence of antibiotics. If lasR mutants are selected first, this would constraint antibiotic resistance evolution. Conversely, when resistance mutations (at least those studied in the present work) are selected, lasR mutants may not be selected in presence of antibiotics. These results underlie the importance of contingency and epistatic interactions in modulating antibiotic resistance evolution., European Development Regional Fund “A Way to Achieve Europe” by Grant S2017/BMD-3691InGEMICS-CM, funded by Comunidad de Madrid (Spain) and European Structural and Investment Funds and by the Spanish Ministry of Economy and Competitivity (BIO2017-83128-R).

BACKGROUNDAntibiotic pollution of non-clinical environments might have a relevant impact on human health if resistant pathogens are selected. However, this potential risk is often overlooked, since drug concentrations in nature are usually below their minimal inhibitory concentrations (MICs). Albeit, antibiotic resistant bacteria can be selected even at sub-MIC concentrations, in a range that is dubbed the sub-MIC selective window, which depends on both the antibiotic and the pathogen.OBJECTIVESDetermine the sub-MIC selective windows of seven antibiotics of clinical relevance in the opportunistic pathogen Pseudomonas aeruginosa and evaluate the risk for selecting resistant mutants in nature, based on published data about the amount of antimicrobials detected in natural environments.METHODSWe conducted evolution experiments of P. aeruginosa PA14 in presence of sub-MIC concentrations of ceftazidime, amikacin, levofloxacin, ciprofloxacin, tetracycline, polymyxin B or imipenem, and measured drug susceptibility of the evolved populations.RESULTSSub-MIC selective window of quinolones was the largest, and the ones of polymyxin B and imipenem, the narrowest. Clinically relevant multidrug resistant (MDR) mutants (presenting MICs above EUCAST clinical breakpoints) arose within the sub-MIC selective windows of the majority of antibiotics tested, being these phenotypes probably mediated by efflux pumps′ activity.DISCUSSIONOur data show that the concentration of antibiotics reported in aquatic ecosystems -colonizable by P. aeruginosa- are, in occasions, higher than the ones able to select MDR mutants. This finding has implications for understanding the role of different ecosystems and conditions in the emergence of antibiotic resistance from a One-Health point of view. Further, it highlights the importance of delineating the sub-MIC selective windows for drugs of clinical value in pathogens with environmental niches, in order to evaluate the health risks due to antibiotic pollution of natural ecosystems and ultimately tackle antibiotic resistance.

It is generally accepted that antibiotic-resistant mutants are selected in a range of concentrations ranging from the minimum inhibitory concentration (MIC) to the mutant preventive concentration. More recently, it has been found that antibiotic-resistant mutants can also be selected at concentrations below MIC, which expands the conditions where this selection may occur. Using experimental evolution approaches followed by whole-genome sequencing, the current study compares the evolutionary trajectories of Pseudomonas aeruginosa in the presence of tobramycin or tigecycline at lethal and sublethal concentrations. Mutants were selected at sublethal concentrations of tigecycline (1/10 and 1/50 MIC), whereas no mutants were selected in the case of tobramycin, indicating that the width of sub-MIC selective windows is antibiotic-specific. In addition, the patterns of evolution towards tigecycline resistance depend on selection strength. Sublethal concentrations of tigecycline select mutants with lower tigecycline MICs and higher MICs to other antibiotics belonging to different structural families than mutants selected under lethal concentrations. This indicates that the strength of the cross-resistance phenotype associated with tigecycline resistance is decoupled from selection strength. Accurate information on the sublethal selection window for each antibiotic of clinical value, including the phenotypes of cross-resistance of mutants selected at each antibiotic concentration, is needed to understand the role of ecosystems polluted with different antibiotic concentrations in the selection of antibiotic resistance. Integration of this information into clinical and environmental safety controls may help to tackle the problem of antibiotic resistance., Work in the authors’ laboratory is supported by grants from the Instituto de Salud Carlos III (Grant RD16/0016/0011), cofinanced by the European Development Regional Fund ‘A Way to Achieve Europe’; Grant S2017/BMD-3691 InGEMICS-CM; Comunidad de Madrid (Spain) and European Structural and Investment Funds; and the Spanish Ministry of Economy and Competitivity (BIO2017-83128-R). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. FSG is the recipient of an FPU fellowship from MINECO.

Acquisition of antibiotic resistance is a relevant problem for human health. The selection and spread of antibiotic-resistant organisms not only compromise the treatment of infectious diseases, but also the implementation of different therapeutic procedures as organ transplantation, advanced surgery or chemotherapy, all of which require proficient methods for avoiding infections. It has been generally accepted that the acquisition of antibiotic resistance will produce a general metabolic burden: in the absence of selection, the resistant organisms would be outcompeted by the susceptible ones. If that was always true, discontinuation of antibiotic use would render the disappearance of resistant microorganisms. However, several studies have shown that, once resistance emerges, the recovery of a fully susceptible population even in the absence of antibiotics is not easy. In the present study, we review updated information on the effect of the acquisition of antibiotic resistance in bacterial physiology as well as on the mechanisms that allow the compensation of the fitness costs associated with the acquisition of resistance.

Several interconnected human, animal and environmental habitats can contribute to the emergence, evolution and spread of antibiotic resistance, and the health of these contiguous habitats (the focus of the One Health approach) may represent a risk to human health. Additionally, the expansion of resistant clones and antibiotic resistance determinants among human-associated, animal-associated and environmental microbiomes have the potential to alter bacterial population genetics at local and global levels, thereby modifying the structure, and eventually the productivity, of microbiomes where antibiotic-resistant bacteria can expand. Conversely, any change in these habitats (including pollution by antibiotics or by antibiotic-resistant organisms) may influence the structures of their associated bacterial populations, which might affect the spread of antibiotic resistance to, and among, the above-mentioned microbiomes. Besides local transmission among connected habitats—the focus of studies under the One Health concept—the transmission of resistant microorganisms might occur on a broader (even worldwide) scale, requiring coordinated Global Health actions. This Review provides updated information on the elements involved in the evolution and spread of antibiotic resistance at local and global levels, and proposes studies to be performed and strategies to be followed that may help reduce the burden of antibiotic resistance as well as its impact on human and planetary health., J.L.M. is supported by grants from the Instituto de Salud Carlos III (grant no. RD16/0016/0011)—co-financed by the European Development Regional Fund ‘A Way to Achieve Europe’ (grant no. S2017/BMD-3691); InGEMICS-CM, funded by Comunidad de Madrid (Spain) and European Structural and Investment Funds; and by the Spanish Ministry of Economy and Competitivity (grant no. BIO2017-83128-R). T.M.C. and F.B. are supported by the Joint Programming Initiative on Antimicrobial Resistance (grant nos. ST131 JPIAMR2016-AC16/00036 and JPIAMR2016-AC16/00039), the European Development Regional Fund ‘A Way to Achieve Europe’ for co-funding the Spanish R&D National Plan 2012–2019 (grant nos. P15-1581 and PI18-1942), the CIBER (CIBER in Epidemiology and Public Health; grant no. CB06/02/0053), the Regional Government of Madrid (InGeMICS B2017/BMD-3691) and the Fundación Ramón Areces.

High-throughput screening of transposon insertion libraries is a useful strategy for unveiling bacterial genes whose inactivation results in an altered susceptibility to antibiotics. A potential drawback of these studies is they are usually based on just one model antibiotic for each structural family, under the assumption that the results can be extrapolated to all members of said family. To determine if this simplification is appropriate, we have analyzed the susceptibility of mutants of Pseudomonas aeruginosa to four aminoglycosides. Our results indicate that each mutation produces different effects on susceptibility to the tested aminoglycosides, with only two mutants showing similar changes in the susceptibility to all studied aminoglycosides. This indicates that the role of a particular gene in the resistome of a given antibiotic should not be generalized to other members of the same structural family. Five aminoglycoside-hypersusceptible mutants inactivating glnD, hflK, PA2798, PA3016, and hpf were chosen for further analysis in order to elucidate if lower aminoglycoside susceptibility correlates with cross-hypersusceptibility to other antibiotics and with impaired virulence. Our results indicate that glnD inactivation leads to increased cross-susceptibility to different antibiotics. The mutant in this gene is strongly impaired in virulence traits such as pyocyanin production, biofilm formation, elastase activity, and swarming motility and the ability to kill Caenorhabditis elegans Thus, GlnD might be an interesting target for developing antibiotic coadjuvants with antiresistance and antivirulence properties against P. aeruginosa.

The study of the acquisition of antibiotic resistance (AR) has mainly focused on inherited processes, namely, mutations and acquisition of AR genes. However, inducible, noninheritable AR has received less attention, and most information in this field derives from the study of antibiotics as inducers of their associated resistance mechanisms. Less is known about nonantibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tight regulation of their expression. Their contribution to acquired AR relies in their overexpression. Here, we analyzed potential inducers of the expression of the chromosomally encoded Pseudomonas aeruginosa clinically relevant efflux pumps, MexCD-OprJ and MexAB-OprM. For this purpose, we developed a set of luxCDABE-based P. aeruginosa biosensor strains, which allows the high-throughput analysis of compounds able to modify the expression of these efflux pumps. Using these strains, we analyzed a set of 240 compounds present in Biolog phenotype microarrays. Several inducers of the expression of the genes that encode these efflux pumps were found. The study focused in dequalinium chloride, procaine, and atropine, compounds that can be found in clinical settings. Using real-time PCR, we confirmed that these compounds indeed induce the expression of the mexCD-oprJ operon. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine, or atropine are present. This study emphasizes the need to study compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests., This study was supported by grants from the Instituto de Salud Carlos III (Spanish Network for Research on Infectious Diseases [RD16/0016/0011]); the Spanish Ministry of Economy, Industry, and Competitivity (BIO2017-83128-R); JPI Water StARE (JPIW2013-089-C02-01); and the Autonomous Community of Madrid (B2017/BMD-3691).

Multidrug efflux pumps constitute a group of transporters that are ubiquitously found in any organism. In addition to other functions with relevance for the cell physiology, efflux pumps contribute to the resistance to compounds used for treating different diseases, including resistance to anticancer drugs, antibiotics or antifungal compounds. In the case of antimicrobials, efflux pumps are major players in both intrinsic and acquired resistance to drugs currently in use for the treatment of infectious diseases. One important aspect not fully explored of efflux pumps consists on the identification of effectors able to induce their expression. Indeed, whereas the analysis of clinical isolates have shown that mutants overexpressing these resistance elements are frequently found, less is known on the conditions that may trigger expression of efflux pumps, hence leading to transient induction of resistance in vivo , a situation that is barely detectable using classical susceptibility tests. In the current article we review the structure and mechanisms of regulation of the expression of bacterial and fungal efflux pumps, with a particular focus in those for which a role in clinically relevant resistance has been reported.

Most efforts in the development of antimicrobials have focused on the screening of lethal targets. Nevertheless, the constant expansion of antimicrobial resistance makes the antibiotic resistance determinants themselves suitable targets for finding inhibitors to be used in combination with antibiotics. Among them, inhibitors of antibiotic inactivating enzymes and of multidrug efflux pumps are suitable candidates for improving the efficacy of antibiotics. In addition, the application of systems biology tools is helping to understand the changes in bacterial physiology associated to the acquisition of resistance, including the increased susceptibility to other antibiotics displayed by some antibiotic-resistant mutants. This information is useful for implementing novel strategies based in metabolic interventions or combination of antibiotics for improving the efficacy of antibacterial therapy.

The present work examines the evolutionary trajectories of replicate Pseudomonas aeruginosa cultures in presence of the ribosome-targeting antibiotics tobramycin and tigecycline. It is known that large number of mutations across different genes - and therefore a large number of potential pathways - may be involved in resistance to any single antibiotic. Thus, evolution toward resistance might, to a large degree, rely on stochasticity, which might preclude the use of predictive strategies for fighting antibiotic resistance. However, the present results show that P. aeruginosa populations evolving in parallel in the presence of antibiotics (either tobramycin or tigecycline) follow a set of trajectories that present common elements. In addition, the pattern of resistance mutations involved include common elements for these two ribosome-targeting antimicrobials. This indicates that mutational evolution toward resistance (and perhaps other properties) is to a certain degree deterministic and, consequently, predictable. These findings are of interest, not just for P. aeruginosa, but in understanding the general rules involved in the evolution of antibiotic resistance also. In addition, the results indicate that bacteria can evolve toward higher levels of resistance to antibiotics against which they are considered to be intrinsically resistant, as tigecycline in the case of P. aeruginosa and that this may confer cross-resistance to other antibiotics of therapeutic value. Our results are particularly relevant in the case of patients under empiric treatment with tigecycline, which frequently suffer P. aeruginosa superinfections.

Ceftazidime/avibactam is a combination of beta-lactam/beta-lactamases inhibitor, which use is restricted to some clinical cases including cystic fibrosis patients infected with multidrug resistant Pseudomonas aeruginosa, in which mutation is the main driver of resistance. This study aims to predict the mechanisms of mutation-driven resistance that are selected for when P. aeruginosa is challenged with either ceftazidime or ceftazidime/avibactam. For this purpose, P. aeruginosa PA14 was submitted to experimental evolution in the absence of antibiotics and in the presence of increasing concentrations of ceftazidime or ceftazidime/avibactam for 30 consecutive days. Final populations were analysed by whole-genome sequencing. All evolved populations reached similar levels of ceftazidime resistance. Besides, all of them were more susceptible to amikacin and produced pyomelanin. A first event in the evolution was the selection of large chromosomal deletions containing hmgA (involved in pyomelanin production), galU (involved in β-lactams resistance) and mexXY-oprM (involved in aminoglycoside resistance). Besides mutations in mpl and dacB that regulate β-lactamase expression, mutations related to MexAB-OprM overexpression were prevalent. Ceftazidime/avibactam challenge selected mutants in the putative efflux pump PA14_45890-45910 and in a two-component system (PA14_45870-45880), likely regulating its expression. All populations produce pyomelanin and were more susceptible to aminoglycosides likely due to the selection of large chromosomal deletions. Since pyomelanin-producing mutants, presenting similar deletions are regularly isolated from infections, the potential aminoglycosides hyper-susceptiblity and reduced β-lactams susceptibility of pyomelanin-producing P. aeruginosa should be taken into consideration for treating infections by these isolates.

SummaryThe intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens. Yet, this question remains hypothetical because of the difficulty to identify ARDs from intestinal bacteria. Here, we developed and validated a new annotation method (called pairwise comparative modelling, PCM) based on homology modelling in order to characterize the Human resistome. We were able to predict 6,095 ARDs in a 3.9 million protein catalogue from the Human intestinal microbiota. We found that predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino-acid identity 29.8%). Among 3,651 pdARDs that were identified in metagenomic species, 3,489 (95.6%) were assumed to be located on the bacterial chromosome. Furthermore, genes associated with mobility were found in the neighbourhood of only 7.9% (482/6,095) of pdARDs. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n=663) into 6 “resistotypes”. Eventually, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results support that most ARDs in the intestinal microbiota should be considered as intrinsic genes of commensal microbiota with a low risk of transfer to bacterial pathogens.

Quinolones are synthetic antibiotics, and the main cause of resistance to these antimicrobials is mutation of the genes encoding their targets. However, in contrast to the case for other organisms, such mutations have not been found in quinolone-resistant Stenotrophomonas maltophilia isolates, in which overproduction of the SmeDEF efflux pump is a major cause of quinolone resistance. SmeDEF is chromosomally encoded and highly conserved in all studied S. maltophilia strains; it is an ancient element that evolved over millions of years in this species. It thus seems unlikely that its main function would be resistance to quinolones, a family of synthetic antibiotics not present in natural environments until the last few decades. Expression of SmeDEF is tightly controlled by the transcriptional repressor SmeT. Our work shows that plant-produced flavonoids can bind to SmeT, releasing it from smeDEF and smeT operators. Antibiotics extruded by SmeDEF do not impede the binding of SmeT to DNA. The fact that plant-produced flavonoids specifically induce smeDEF expression indicates that they are bona fide effectors regulating expression of this resistance determinant. Expression of efflux pumps is usually downregulated unless their activity is needed. Since smeDEF expression is triggered by plant-produced flavonoids, we reasoned that this efflux pump may have a role in the colonization of plants by S. maltophilia . Our results showed that, indeed, deletion of smeE impairs S. maltophilia colonization of plant roots. Altogether, our results indicate that quinolone resistance is a recent function of SmeDEF and that colonization of plant roots is likely one original function of this efflux pump.

Multidrug efflux pumps can be involved in bacterial resistance to antibiotics at different levels. Some efflux pumps are constitutively expressed at low levels and contribute to intrinsic resistance. In addition, their overexpression may allow higher levels of resistance. This overexpression can be transient, in the presence of an effector (phenotypic resistance), or constitutive when mutants in the regulatory elements of the expression of efflux pumps are selected (acquired resistance). Efflux pumps are present in all cells, from human to bacteria and are highly conserved, which indicates that they are ancient elements in the evolution of different organisms. Consequently, it has been suggested that, besides antibiotic resistance, bacterial multidrug efflux pumps would likely contribute to other relevant processes of the microbial physiology. In the current article, we discuss some specific examples of the role that efflux pumps may have in the bacterial virulence of animals’ and plants’ pathogens, including the processes of intercellular communication. Based in these evidences, we propose that efflux pumps are at the crossroad between resistance and virulence of bacterial pathogens. Consequently, the comprehensive study of multidrug efflux pumps requires addressing these functions, which are of relevance for the bacterial–host interactions during infection., Work in our laboratory is supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2014-54507-R and JPI Water StARE JPIW2013-089-C02-01); from Madrid Autonomous Community [S2010/BMD2414 (PROMPT)]; and from the Instituto de Salud Carlos III [Spanish Network for Research on Infectious Diseases (REIPI RD12/0015)]. MA-R and PB are recipients of FPI fellowships from MINECO.

It is widely accepted that the acquisition of resistance to antimicrobials confers a fitness cost. Different works have shown that the effect of acquiring resistance in bacterial physiology may be more specific than previously thought. Study of these specific changes may help to predict the outcome of resistant organisms in different ecosystems. In addition to changing bacterial physiology, acquisition of resistance either increases or reduces susceptibility to other antimicrobials. In the current article, we review recent information on the effect of acquiring resistance upon bacterial physiology, with a specific focus on studies using phenotype microarray technology.

Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)., Work in our laboratory is supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2014-54507-R and JPI Water StARE JPIW2013-089-C02-01); from Madrid Autonomous Community (S2010/BMD2414 (PROMPT); from the Instituto de Salud Carlos III (Spanish Network for Research on Infectious Diseases (REIPI RD12/0015)); and from the European Union (HEALTH-F3-2011-282004 (EVOTAR)). FC is a recipient of a JAE fellowship financed by European Social Fund. MAR and PB are recipients of FPI fellowships from MINECO.