Evaluation of critical parameters in the hollow-fibre system for tuberculosis: A case study of moxifloxacin.

AIMS: The hollow­fibre system for tuberculosis (HFS­TB) is a preclinical model qualified by the European Medicines Agency to underpin the anti­TB drug development process. It can mimic in vivo pharmacokinetic (PK)­pharmacodynamic (PD) attributes of selected antimicrobials, which could feed into in silico models to inform the design of clinical trials. However, historical data and published protocols are insufficient and omit key information to allow experiments to be reproducible. Therefore, in this work, we aim to optimize and standardize various HFS­TB operational procedures. METHODS: First, we characterized bacterial growth dynamics with different types of hollow­fibre cartridges, Mycobacterium tuberculosis strains and media. Second, we mimicked a moxifloxacin PK profile within hollow­fibre cartridges, in order to check drug­fibres compatibility. Lastly, we mimicked the moxifloxacin total plasma PK profile in human after once daily oral dose of 400 mg to assess PK­PD after different sampling methods, strains, cartridge size and bacterial adaptation periods before drug infusion into the system. RESULTS: We found that final bacterial load inside the HFS­TB was contingent on the studied variables. Besides, we demonstrated that drug­fibres compatibility tests are critical preliminary HFS­TB assays, which need to be properly reported. Lastly, we uncovered that the sampling method and bacterial adaptation period before drug infusion significantly impact actual experimental conclusions. CONCLUSION: Our data contribute to the necessary standardization of HFS­TB experiments, draw attention to multiple aspects of this preclinical model that should be considered when reporting novel results and warn about critical parameters in the HFS­TB currently overlooked.

The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa.

The identification of targets whose inactivation increases the activity of antibiotics helps to fight antibiotic resistance. Previous work showed that a transposon-insertion mutant in the gene PA14_27940 increases Pseudomonas aeruginosa susceptibility to aminoglycosides. Since polar effects may affect the phenotype, in the present work, we generated an in-frame PA14_27940 deletion mutant. A PA14_27940 deletion increased the susceptibility to aminoglycosides, tetracycline, tigecycline, erythromycin and fosfomycin. Excepting fosfomycin, the other antibiotics are inducers of the MexXY efflux pump. MexXY induction is required for P. aeruginosa resistance to these antibiotics, which is post-transcriptionally regulated by the anti-repressor ArmZ. Although mexXY is inducible by tobramycin in ΔPA14_27940, the induction level is lower than in the parental PA14 strain. Additionally, armZ is induced by tobramycin in PA14 and not in ΔPA14_27940, supporting that ΔPA14_27940 presents an ArmZ-mediated defect in mexXY induction. For its part, hypersusceptibility to fosfomycin may be due to a reduced expression of nagZ and agmK, which encode enzymes of the peptidoglycan recycling pathway. ΔPA14_27940 also presents defects in motility, an element with relevance in P. aeruginosa's virulence. Overall, our results support that PA14_27940 is a good target for the search of adjuvants that will increase the activity of antibiotics and reduce the virulence of P. aeruginosa.

Translating eco-evolutionary biology into therapy to tackle antibiotic resistance.

Antibiotic resistance is currently one of the most important public health problems. The golden age of antibiotic discovery ended decades ago, and new approaches are urgently needed. Therefore, preserving the efficacy of the antibiotics currently in use and developing compounds and strategies that specifically target antibiotic-resistant pathogens is critical. The identification of robust trends of antibiotic resistance evolution and of its associated trade-offs, such as collateral sensitivity or fitness costs, is invaluable for the design of rational evolution-based, ecology-based treatment approaches. In this Review, we discuss these evolutionary trade-offs and how such knowledge can aid in informing combination or alternating antibiotic therapies against bacterial infections. In addition, we discuss how targeting bacterial metabolism can enhance drug activity and impair antibiotic resistance evolution. Finally, we explore how an improved understanding of the original physiological function of antibiotic resistance determinants, which have evolved to reach clinical resistance after a process of historical contingency, may help to tackle antibiotic resistance.

Collateral Sensitivity to Fosfomycin of Tobramycin-Resistant Mutants of Pseudomonas aeruginosa Is Contingent on Bacterial Genomic Background.

Understanding the consequences in bacterial physiology of the acquisition of drug resistance is needed to identify and exploit the weaknesses derived from it. One of them is collateral sensitivity, a potentially exploitable phenotype that, unfortunately, is not always conserved among different isolates. The identification of robust, conserved collateral sensitivity patterns is then relevant for the translation of this knowledge into clinical practice. We have previously identified a robust fosfomycin collateral sensitivity pattern of Pseudomonas aeruginosa that emerged in different tobramycin-resistant clones. To go one step further, here, we studied if the acquisition of resistance to tobramycin is associated with robust collateral sensitivity to fosfomycin among P. aeruginosa isolates. To that aim, we analyzed, using adaptive laboratory evolution approaches, 23 different clinical isolates of P. aeruginosa presenting diverse mutational resistomes. Nine of them showed collateral sensitivity to fosfomycin, indicating that this phenotype is contingent on the genetic background. Interestingly, collateral sensitivity to fosfomycin was linked to a larger increase in tobramycin minimal inhibitory concentration. Further, we unveiled that fosA low expression, rendering a higher intracellular accumulation of fosfomycin, and a reduction in the expression of the P. aeruginosa alternative peptidoglycan-recycling pathway enzymes, might be on the basis of the collateral sensitivity phenotype.

Rapid Phenotypic Convergence towards Collateral Sensitivity in Clinical Isolates of Pseudomonas aeruginosa Presenting Different Genomic Backgrounds.

Collateral sensitivity (CS) is an evolutionary trade-off by which acquisition of resistance to an antibiotic leads to increased susceptibility to another. This Achilles' heel of antibiotic resistance could be exploited to design evolution-based strategies for treating bacterial infections. To date, most studies in the field have focused on the identification of CS patterns in model strains. However, one of the main requirements for the clinical application of this trade-off is that it must be robust and has to emerge in different genomic backgrounds, including preexisting drug-resistant isolates, since infections are frequently caused by pathogens already resistant to antibiotics. Here, we report the first analysis of CS robustness in clinical strains of Pseudomonas aeruginosa presenting different ab initio mutational resistomes. We identified a robust CS pattern associated with short-term evolution in the presence of ciprofloxacin of clinical P. aeruginosa isolates, including representatives of high-risk epidemic clones belonging to sequence type (ST) 111, ST175, and ST244. We observed the acquisition of different ciprofloxacin resistance mutations in strains presenting varied STs and different preexisting mutational resistomes. Importantly, despite these genetic differences, the use of ciprofloxacin led to a robust CS to aztreonam and tobramycin. In addition, we describe the possible application of this evolutionary trade-off to drive P. aeruginosa infections to extinction by using the combination of ciprofloxacin-tobramycin or ciprofloxacin-aztreonam. Our results support the notion that the identification of robust patterns of CS may establish the basis for developing evolution-informed treatment strategies to tackle bacterial infections, including those due to antibiotic-resistant pathogens. IMPORTANCE Collateral sensitivity (CS) is a trade-off of antibiotic resistance evolution that could be exploited to design strategies for treating bacterial infections. Clinical application of CS requires it to robustly emerge in different genomic backgrounds. In this study, we performed an analysis to identify robust patterns of CS associated with the use of ciprofloxacin in clinical isolates of P. aeruginosa presenting different mutational resistomes and including high-risk epidemic clones (ST111, ST175, and ST244). We demonstrate the robustness of CS to tobramycin and aztreonam and the potential application of this evolutionary observation to drive P. aeruginosa infections to extinction. Our results support the notion that the identification of robust CS patterns may establish the basis for developing evolutionary strategies to tackle bacterial infections, including those due to antibiotic-resistant pathogens.

Antibiotic Resistance in Pseudomonas.

Pseudomonas is a bacterial genus, with a bona fide environmental habitat that comprises different species, some of them causing diseases in humans and plants, as well as some strains with biotechnological potential. Amongst them, Pseudomonas aeruginosa is currently one of the most important nosocomial pathogens. In addition, this microorganism is a prevalent cause of chronic infections in cystic fibrosis patients and in people suffering from chronic obstructive pulmonary disease. The success of P. aeruginosa in colonising different habitats largely relies on its metabolic versatility and robustness. Besides, this bacterial pathogen harbours in its core genome a large set of virulence determinants that allows it to colonise/infect a variety of hosts, from unicellular organisms to humans. Nevertheless, these are not just the only conditions needed for infecting patients at hospitals. Taking into consideration that infected patients are regularly under antibiotic treatment, the ability to avoid antibiotics' action is also needed. In this sense, P. aeruginosa displays a characteristic low susceptibility to several antibiotics currently used in therapy. This is due to the reduced permeability of its cellular envelopes and the presence in its genome of an arrangement of genes encoding multidrug efflux pumps and antibiotic-inactivating enzymes that contribute to its resilience to antibiotics. Besides intrinsic resistance, P. aeruginosa is able to evolve towards antibiotic resistance through mutations (particularly relevant in the case of chronic infections) and via acquisition of antibiotic resistance genes. It is worth mentioning that acquired resistance is not the only venue that P. aeruginosa has for avoiding the action of antibiotics. Transient resistance can also confer this phenotype. Indeed, the induction of the expression of intrinsic resistance genes by conditions or compounds that P. aeruginosa could face during infection can compromise the effectiveness of antibiotics for treating such infections. In addition, tolerant cells able to survive during the exposure to bactericidal antibiotics without an increase in their antibiotic resistance phenotype are found as well in these patients, and they are the prelude of the evolution towards antibiotic resistance. Finally, P. aeruginosa biofilms, frequently encountered in the lungs of cystic fibrosis patients, in prostheses, or in catheters, present low antibiotic susceptibility and are associated with recalcitrance and disease worsening.

Low Ciprofloxacin Concentrations Select Multidrug-Resistant Mutants Overproducing Efflux Pumps in Clinical Isolates of Pseudomonas aeruginosa.

Low antibiotic concentrations present in natural environments are a severe and often neglected threat to public health. Even if they are present below their MICs, they may select for antibiotic-resistant pathogens. Notably, the minimal subinhibitory concentrations that select resistant bacteria, and define the respective sub-MIC selective windows, differ between antibiotics. The establishment of these selective concentrations is needed for risk-assessment studies regarding the presence of antibiotics in different habitats. Using short-term evolution experiments in a set of 12 Pseudomonas aeruginosa clinical isolates (including high-risk clones with ubiquitous distribution), we have determined that ciprofloxacin sub-MIC selective windows are strain specific and resistome dependent. Nonetheless, in all cases, clinically relevant multidrug-resistant (MDR) mutants emerged upon exposure to low ciprofloxacin concentrations, with these concentrations being below the levels reported in ciprofloxacin-polluted natural habitats where P. aeruginosa can be present. This feature expands the conditions and habitats where clinically relevant quinolone-resistant mutants can emerge. In addition, we established the lowest concentration threshold beyond which P. aeruginosa, regardless of the strain, becomes resistant to ciprofloxacin. Three days of exposure under this sub-MIC "risk concentration" led to the selection of MDR mutants that displayed resistance mechanisms usually ascribed to high selective pressures, i.e., the overproduction of the efflux pumps MexCD-OprJ and MexEF-OprN. From a One-Health viewpoint, these data stress the transcendent role of low drug concentrations, which can be encountered in natural ecosystems, in aggravating the antibiotic resistance problem, especially when it comes to pathogens of environmental origin. IMPORTANCE It has been established that antibiotic concentrations below MICs can select antibiotic-resistant pathogens, a feature of relevance for analyzing the role of nonclinical ecosystems in antibiotic resistance evolution. The range of concentrations where this selection occurs defines the sub-MIC selective window, whose width depends on the antibiotic. Herein, we have determined the ciprofloxacin sub-MIC selective windows of a set of Pseudomonas aeruginosa clinical isolates (including high-risk clones with worldwide distribution) and established the lowest concentration threshold, notably an amount reported to be present in natural ecosystems, beyond which this pathogen acquires resistance. Importantly, our results show that this ciprofloxacin sub-MIC selects for multidrug-resistant mutants overproducing clinically relevant efflux pumps. From a One-Health angle, this information supports that low antimicrobial concentrations, present in natural environments, may have a relevant role in worsening the antibiotic resistance crisis, particularly regarding pathogens with environmental niches, such as P. aeruginosa.

Wildlife and Antibiotic Resistance.

Antibiotic resistance is a major human health problem. While health care facilities are main contributors to the emergence, evolution and spread of antibiotic resistance, other ecosystems are involved in such dissemination. Wastewater, farm animals and pets have been considered important contributors to the development of antibiotic resistance. Herein, we review the impact of wildlife in such problem. Current evidence supports that the presence of antibiotic resistance genes and/or antibiotic resistant bacteria in wild animals is a sign of anthropic pollution more than of selection of resistance. However, once antibiotic resistance is present in the wild, wildlife can contribute to its transmission across different ecosystems. Further, the finding that antibiotic resistance genes, currently causing problems at hospitals, might spread through horizontal gene transfer among the bacteria present in the microbiomes of ubiquitous animals as cockroaches, fleas or rats, supports the possibility that these organisms might be bioreactors for the horizontal transfer of antibiotic resistance genes among human pathogens. The contribution of wildlife in the spread of antibiotic resistance among different hosts and ecosystems occurs at two levels. Firstly, in the case of non-migrating animals, the transfer will take place locally; a One Health problem. Paradigmatic examples are the above mentioned animals that cohabit with humans and can be reservoirs and vehicles for antibiotic resistance dissemination. Secondly, migrating animals, such as gulls, fishes or turtles may participate in the dissemination of antibiotic resistance across different geographic areas, even between different continents, which constitutes a Global Health issue.

Evolution under low antibiotic concentrations: a risk for the selection of Pseudomonas aeruginosa multidrug-resistant mutants in nature.

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.

Pseudomonas aeruginosa: an antibiotic resilient pathogen with environmental origin.

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.

Coming from the Wild: Multidrug Resistant Opportunistic Pathogens Presenting a Primary, Not Human-Linked, Environmental Habitat.

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.

Rapid and robust evolution of collateral sensitivity in Pseudomonas aeruginosa antibiotic-resistant mutants.

The analysis of trade-offs, as collateral sensitivity, associated with the acquisition of antibiotic resistance, is mainly based on the use of model strains. However, the possibility of exploiting these trade-offs for fighting already resistant isolates has not been addressed in depth, despite the fact that bacterial pathogens are frequently antibiotic-resistant, forming either homogeneous or heterogeneous populations. Using a set of Pseudomonas aeruginosa-resistant mutants, we found that ceftazidime selects pyomelanogenic tobramycin-hypersusceptible mutants presenting chromosomal deletions in the analyzed genetic backgrounds. Since pyomelanogenic resistant mutants frequently coexist with other morphotypes in patients with cystic fibrosis, we analyzed the exploitation of this trade-off to drive extinction of heterogeneous resistant populations by using tobramycin/ceftazidime alternation. Our work shows that this approach is feasible because phenotypic trade-offs associated with the use of ceftazidime are robust. The identification of conserved collateral sensitivity networks may guide the rational design of evolution-based antibiotic therapies in P. aeruginosa infections.

Evolutionary landscapes of Pseudomonas aeruginosa towards ribosome-targeting antibiotic resistance depend on selection strength.

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.

Antimicrobial resistance: A multifaceted problem with multipronged solutions.

Infectious diseases still stand as a major cause of morbidity and mortality, and this problem can be worsened with the current antimicrobial resistance crisis. To tackle this crisis more studies analyzing the causes, routes, and reservoirs where antimicrobial resistance can emerge and expand, together with new antimicrobials and strategies for fighting antimicrobial resistance are needed. In the current special issue of MicrobiologyOpen, a set of articles dealing with the multiple faces of antimicrobial resistance are presented. These articles provide new information for understanding and addressing this problem.

Antibiotic Resistance Evolution Is Contingent on the Quorum-Sensing Response in Pseudomonas aeruginosa.

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.

Analysis of the Pseudomonas aeruginosa Aminoglycoside Differential Resistomes Allows Defining Genes Simultaneously Involved in Intrinsic Antibiotic Resistance and Virulence.

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.

Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles.

Aminoglycoside acetyltransferases are important determinants of resistance to aminoglycoside antibiotics in most bacterial genera. In mycobacteria, however, aminoglycoside acetyltransferases contribute only partially to aminoglycoside susceptibility since they are related with low level resistance to these antibiotics (while high level aminoglycoside resistance is due to mutations in the ribosome). Instead, aminoglycoside acetyltransferases contribute to other bacterial functions, and this can explain its widespread presence along species of genus Mycobacterium. This review is focused on two mycobacterial aminoglycoside acetyltransferase enzymes. First, the aminoglycoside 2'-N-acetyltransferase [AAC(2')], which was identified as a determinant of weak aminoglycoside resistance in M. fortuitum, and later found to be widespread in most mycobacterial species; AAC(2') enzymes have been associated with resistance to cell wall degradative enzymes, and bactericidal mode of action of aminoglycosides. Second, the Eis aminoglycoside acetyltransferase, which was identified originally as a virulence determinant in M. tuberculosis (enhanced intracellular survival); Eis protein in fact controls production of pro-inflammatory cytokines and other pathways. The relation of Eis with aminoglycoside susceptibility was found after the years, and reaches clinical significance only in M. tuberculosis isolates resistant to the second-line drug kanamycin. Given the role of AAC(2') and Eis proteins in mycobacterial biology, inhibitory molecules have been identified, more abundantly in case of Eis. In conclusion, AAC(2') and Eis have evolved from a marginal role as potential drug resistance mechanisms into a promising future as drug targets.

Mutational Evolution of Pseudomonas aeruginosa Resistance to Ribosome-Targeting Antibiotics.

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.

The development of efflux pump inhibitors to treat Gram-negative infections.

INTRODUCTION: One of the possibilities for reducing the emergence and spread of antibiotic resistance is the use of anti-resistance compounds capable of resensitizing resistant microorganisms to current antimicrobials. For this purpose, multidrug efflux pumps, whose inhibition may increase bacterial susceptibility to several antibiotics, including macrolides to which Gram-negatives are considered intrinsically resistant, have emerged as suitable targets. Areas covered: In the current review, the authors discuss different mechanisms that can be exploited for inhibiting multidrug efflux pumps and describe the properties and the potential therapeutic value of already studied efflux pumps inhibitors. Although efforts have already been made to develop these inhibitors, there are currently no good candidates for treating infectious diseases. Consequently, the authors also discuss potential approaches for their development. Expert opinion: Classical anti-resistance drugs such as beta-lactamases inhibitors, while useful, are only purposeful for treating infections caused by beta-lactamase producers. However, inhibitors of multidrug efflux pumps, which are present on all organisms, can sensitize both susceptible and resistant bacteria to antibiotics belonging to several different structural families. Since some efflux pumps are involved in bacterial infections, their inhibition may also reduce the infectivity of Gram-negative bacterial pathogens.

Mutation-Driven Evolution of Pseudomonas aeruginosa in the Presence of either Ceftazidime or Ceftazidime-Avibactam.

Ceftazidime-avibactam is a combination of ß-lactam/ß-lactamase inhibitor, the use of which 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 analyzed by whole-genome sequencing. All evolved populations reached similar levels of ceftazidime resistance. In addition, they were more susceptible to amikacin and produced pyomelanin. A first event in this evolution was the selection of large chromosomal deletions containing hmgA (involved in pyomelanin production), galU (involved in ß-lactams resistance), and mexXY-oprM (involved in aminoglycoside resistance). Besides mutations in mpl and dacB that regulate ß-lactamase expression, mutations related to MexAB-OprM overexpression were prevalent. Ceftazidime-avibactam challenge selected mutants in the putative efflux pump PA14_45890 and PA14_45910 and in a two-component system (PA14_45870 and PA14_45880), likely regulating its expression. All populations produced 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 aminoglycoside hypersusceptiblity and reduced ß-lactam susceptibility of pyomelanin-producing P. aeruginosa should be taken into consideration for treating infections caused by these isolates.