In vivo genetic analysis of Pseudomonas aeruginosa carbon catabolic repression through the study of CrcZ pseudo-revertants shows that Crc-mediated metabolic robustness is needed for proficient bacterial virulence and antibiotic resistance.

IIMPORTANCE: Hfq and Crc regulate P. aeruginosa carbon catabolic repression at the post-transcriptional level. In vitro work has shown that Hfq binds the target RNAs and Crc stabilizes the complex. A third element in the regulation is the small RNA CrcZ, which sequesters the Crc-Hfq complex under no catabolic repression conditions, allowing the translation of the target mRNAs. A ΔcrcZ mutant was generated and presented fitness defects and alterations in its virulence potential and antibiotic resistance. Eight pseudo-revertants that present different degrees of fitness compensation were selected. Notably, although Hfq is the RNA binding protein, most mutations occurred in Crc. This indicates that Crc is strictly needed for P. aeruginosa efficient carbon catabolic repression in vivo. The compensatory mutations restore in a different degree the alterations in antibiotic susceptibility and virulence of the ΔcrcZ mutant, supporting that Crc plays a fundamental role linking P. aeruginosa metabolic robustness, virulence, and antibiotic resistance.

Virulence and Metabolism Crosstalk: Impaired Activity of the Type Three Secretion System (T3SS) in a Pseudomonas aeruginosa Crc-Defective Mutant.

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS.

The Inactivation of Enzymes Belonging to the Central Carbon Metabolism Is a Novel Mechanism of Developing Antibiotic Resistance.

Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate, that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from phosphoenolpyruvate to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly being used, mainly for treating infections caused by Gram-negative multidrug-resistant bacteria. The mechanisms of mutational resistance to fosfomycin in Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to commonly used antibiotics, were studied in the current work. None of the mechanisms reported so far for other organisms, which include the production of fosfomycin-inactivating enzymes, target modification, induction of an alternative peptidoglycan biosynthesis pathway, and the impaired entry of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Instead, the unique cause of resistance in the mutants studied is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants, showing that neither inactivation nor transport of the antibiotic is involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes or any related enzyme. Finally, the mutants did not present an increased phosphoenolpyruvate concentration that might compete with fosfomycin for its binding to MurA. On the basis of these results, we describe a completely novel mechanism of antibiotic resistance based on mutations of genes encoding metabolic enzymes.IMPORTANCE Antibiotic resistance has been largely considered a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated on mechanisms that affect the antibiotics (mutations in transporters, efflux pumps, and antibiotic-modifying enzymes, or their regulators) or their targets (i.e., target mutations, protection, or bypass). Usually, antibiotic resistance-associated metabolic changes were considered a consequence (fitness costs) and not a cause of antibiotic resistance. Herein, we show that alterations in the central carbon bacterial metabolism can also be the cause of antibiotic resistance. In the study presented here, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas pathway. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that antibiotic resistance can be under metabolic control, our results provide evidence that antibiotic resistance is intertwined with the bacterial metabolism.

Mechanisms and phenotypic consequences of acquisition of tigecycline resistance by Stenotrophomonas maltophilia.

OBJECTIVES: To elucidate the potential mutation-driven mechanisms involved in the acquisition of tigecycline resistance by the opportunistic pathogen Stenotrophomonas maltophilia. The mutational trajectories and their effects on bacterial fitness, as well as cross-resistance and/or collateral susceptibility to other antibiotics, were also addressed. METHODS: S. maltophilia populations were submitted to experimental evolution in the presence of increasing concentrations of tigecycline for 30 days. The genetic mechanisms involved in the acquisition of tigecycline resistance were determined by WGS. Resistance was evaluated by performing MIC assays. Fitness of the evolved populations and individual clones was assessed by measurement of the maximum growth rates. RESULTS: All the tigecycline-evolved populations attained high-level resistance to tigecycline following different mutational trajectories, yet with some common elements. Among the mechanisms involved in low susceptibility to tigecycline, mutations in the SmeDEF efflux pump negative regulator smeT, changes in proteins involved in the biogenesis of the ribosome and modifications in the LPS biosynthesis pathway seem to play a major role. Besides tigecycline resistance, the evolved populations presented cross-resistance to other antibiotics, such as aztreonam and quinolones, and they were hypersusceptible to fosfomycin, suggesting a possible combination treatment. Further, we found that the selected resistance mechanisms impose a relevant fitness cost when bacteria grow in the absence of antibiotic. CONCLUSIONS: Mutational resistance to tigecycline was easily selected during exposure to this antibiotic. However, the fitness cost may compromise the maintenance of S. maltophilia tigecycline-resistant populations in the absence of antibiotic.

Involvement of the RND efflux pump transporter SmeH in the acquisition of resistance to ceftazidime in Stenotrophomonas maltophilia.

The emergence of antibiotic resistant Gram-negative bacteria has become a serious global health issue. In this study, we have employed the intrinsically resistant opportunistic pathogen Stenotrophomonas maltophilia as a model to study the mechanisms involved in the acquisition of mutation-driven resistance to antibiotics. To this aim, laboratory experimental evolution studies, followed by whole-genome sequencing, were performed in the presence of the third-generation cephalosporin ceftazidime. Using this approach, we determined that exposure to increasing concentrations of ceftazidime selects high-level resistance in S. maltophilia through a novel mechanism: amino acid substitutions in SmeH, the transporter protein of the SmeGH RND efflux pump. The recreation of these mutants in a wild-type background demonstrated that, in addition to ceftazidime, the existence of these substitutions provides bacteria with cross-resistance to other beta-lactam drugs. This acquired resistance does not impose relevant fitness costs when bacteria grow in the absence of antibiotics. Structural prediction of both amino acid residues points that the observed resistance phenotype could be driven by changes in substrate access and recognition.

The global regulator Crc orchestrates the metabolic robustness underlying oxidative stress resistance in Pseudomonas aeruginosa.

The remarkable metabolic versatility of bacteria of the genus Pseudomonas enable their survival across very diverse environmental conditions. P. aeruginosa, one of the most relevant opportunistic pathogens, is a prime example of this adaptability. The interplay between regulatory networks that mediate these metabolic and physiological features is just starting to be explored in detail. Carbon catabolite repression, governed by the Crc protein, controls the availability of several enzymes and transporters involved in the assimilation of secondary carbon sources. Yet, the regulation exerted by Crc on redox metabolism of P. aeruginosa (hence, on the overall physiology) had hitherto been unexplored. In this study, we address the intimate connection between carbon catabolite repression and metabolic robustness of P. aeruginosa PAO1. In particular, we explored the interplay between oxidative stress, metabolic rearrangements in central carbon metabolism and the cellular redox state. By adopting a combination of quantitative physiology experiments, multiomic analyses, transcriptional patterns of key genes, measurement of metabolic activities in vitro and direct quantification of redox balances both in the wild-type strain and in an isogenic Δcrc derivative, we demonstrate that Crc orchestrates the overall response of P. aeruginosa to oxidative stress via reshaping of the core metabolic architecture in this bacterium.

The development of a new parameter for tracking post-transcriptional regulation allows the detailed map of the Pseudomonas aeruginosa Crc regulon.

Bacterial physiology is regulated at different levels, from mRNA synthesis to translational regulation and protein modification. Herein, we propose a parameter, dubbed post-transcriptional variation (PTV), that allows extracting information on post-transcriptional regulation from the combined analysis of transcriptomic and proteomic data. We have applied this parameter for getting a deeper insight in the regulon of the Pseudomonas aeruginosa post-transcriptional regulator Crc. P. aeruginosa is a free-living microorganism, and part of its ecological success relies on its capability of using a large number of carbon sources. The hierarchical assimilation of these sources when present in combination is regulated by Crc that, together with Hfq (the RNA-binding chaperon in the complex), impedes their translation when catabolite repression is triggered. Most studies on Crc regulation are based either in transcriptomics or in proteomics data, which cannot provide information on post-transcriptional regulation when analysed independently. Using the PTV parameter, we present a comprehensive map of the Crc post-transcriptional regulon. In addition of controlling the use of primary and secondary carbon sources, Crc regulates as well cell respiration, c-di-GMP mediated signalling, and iron utilization. Thus, besides controlling the hyerarchical assimilation of carbon sources, Crc is an important element for keeping bacterial homeostasis and, consequently, metabolic robustness.

Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

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.

Multidrug efflux pumps as main players in intrinsic and acquired resistance to antimicrobials.

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.

The analysis of the antibiotic resistome offers new opportunities for therapeutic intervention.

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.

Use of phenotype microarrays to study the effect of acquisition of resistance to antimicrobials in bacterial physiology.

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.

[Scorpionism causing severe acute flaccid paralysis. Case report]. / Alacranismo severo causante de parálisis flácida aguda. Reporte de caso.

BACKGROUND: Scorpionism is a public health problem in various regions of the world, being Mexico the country with the highest number of cases. Clinical manifestations range from local symptoms to severe disease with an impact on cardiovascular, respiratory and neurological level, and even death. There are no reports of acute flaccid paralysis as a manifestation of the clinical picture of the scorpion sting of the Centruroides gender, Family Buthidae, highly toxic, causes high rates of morbidity and mortality in our region. CLINICAL CASE: We documented a case of scorpionism, caused by a scorpion gender Buthidae, Centruroides family, which caused acute flaccid paralysis, after resolution of other severe manifestations. There is only one case report of scorpionism that produces acute flaccid paralysis in the literature, but it is related to the Parabuthus scorpion, endemic of South Africa. CONCLUSIONS: The knowledge of this complication, new for our region, will maximize efforts to diagnose and appropriately manage this symptoms, with the adequate application of the specific fabotherapy and advanced life support for proper survival in the patients with compromise of vital functions and imminent risk of death mainly by respiratory failure.

Introducción: el alacranismo es un problema de salud pública en diversas regiones del mundo, siendo México el país que tiene mayor número de casos. Las manifestaciones clínicas oscilan desde sintomatología local hasta cuadros graves con repercusiones a nivel cardiovascular, respiratorio y neurológico, e incluso la muerte. No existen reportes de parálisis flácida como una manifestación del cuadro clínico por picadura del alacrán del género Centruroides, familia Buthidae, especie altamente tóxica, endémica y causante de altos índices de morbimortalidad en nuestra región. Caso clínico: documentamos un caso de alacranismo grave, provocado por escorpión de la familia Buthidae del género Centruroides, que causó parálisis flácida aguda, posterior a resolución de otras manifestaciones severas. Solo existe un reporte de caso de alacranismo que produce parálisis flácida aguda en la literatura médica, pero relacionado con el escorpión de la familia Parabuthus, endémico de Sudáfrica, el cual no es endémico en México. Conclusiones: conocer esta complicación, nueva para nuestra región, permitirá maximizar esfuerzos para diagnosticar y manejar oportunamente esta entidad con la aplicación temprana de faboterápico específico y soporte vital avanzado.

The inactivation of RNase G reduces the Stenotrophomonas maltophilia susceptibility to quinolones by triggering the heat shock response.

Quinolone resistance is usually due to mutations in the genes encoding bacterial topoisomerases. However, different reports have shown that neither clinical quinolone resistant isolates nor in vitro obtained Stenotrophomonas maltophilia mutants present mutations in such genes. The mechanisms so far described consist on efflux pumps' overexpression. Our objective is to get information on novel mechanisms of S. maltophilia quinolone resistance. For this purpose, a transposon-insertion mutant library was obtained in S. maltophilia D457. One mutant presenting reduced susceptibility to nalidixic acid was selected. Inverse PCR showed that the inactivated gene encodes RNase G. Complementation of the mutant with wild-type RNase G allele restored the susceptibility to quinolones. Transcriptomic and real-time RT-PCR analyses showed that several genes encoding heat-shock response proteins were expressed at higher levels in the RNase defective mutant than in the wild-type strain. In agreement with this situation, heat-shock reduces the S. maltophilia susceptibility to quinolone. We can then conclude that the inactivation of the RNase G reduces the susceptibility of S. maltophilia to quinolones, most likely by regulating the expression of heat-shock response genes. Heat-shock induces a transient phenotype of quinolone resistance in S. maltophilia.

Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa.

Crc is a post-transcriptional regulator in Pseudomonas aeruginosa that modulates its metabolism, but also its susceptibility to antibiotics and virulence. Most of P. aeruginosa virulence factors are secreted or engulfed in vesicles. A Crc deficient mutant was created and the extracellular vesicles associated exoproteome and the vesicle-free secretome was quantified using iTRAQ. Fifty vesicles-associated proteins were more abundant and 14 less abundant in the Crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Different virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the Crc-defective mutant. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain, in agreement with the low secretion of proteins related to virulence. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals.

Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa.

Recent research indicates that the post-transcriptional regulator Crc modulates susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Several P. aeruginosa virulence factors are secreted or engulfed in vesicles. To decipher the Crc modulation of P. aeruginosa virulence, we constructed a crc deficient mutant and measure the proteome associated extracellular vesicles and the vesicle-free secretome using iTRAQ. Fifty vesicle-associated proteins were more abundant and 14 less abundant in the crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Among them, virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the crc-defective mutant. Transcriptomic analysis revealed that some of the observed changes were post-transcriptional and, thus, could be attributed to a direct Crc regulatory role; whereas, for other differentially secreted proteins, the regulatory role was likely indirect. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals. This article is part of a Special Issue entitled: HUPO 2014.

Remisión sintomática y recuperación funcional en pacientes que padecen esquizofrenia / Symptomatic remission and recovery in patients with schizophrenia

A recently proposed definition for remission and recovery in schizophrenia is receiving increased attention by clinicians and researchers. The interest on these issues is based on the recent proposed definition for symptomatic remission, and the development of operational criteria for its assessment, by the Remission in Schizophrenia Working Group (RSWG), in the United States, in 2005. Remission is assessed using eight items of the Positive and Negative Syndrome Scale (PANSS), all of which have to be scored with a symptom severity of <3 points (mild or better), sustained for a minimum duration of six months. In Europe, since 2006, proposed definition criteria about response and remission were introduced. Response can be assessed, with the PANSS, using a cut-off of at least 50% reduction of the baseline score for the acutely ill, and a cut-off of at least of 25% reduction for refractory patients. Remission could be assessed using a formula for calculating percentage PANSS reduction from baseline. Definition criteria have also been introduced to assess functional recovery that includes the combination of clinical and social outcomes for two consecutive years, including dimensions such as psychosocial functioning, cognition, and quality of life. The purpose of this review is to examine existing research on symptomatic remission and functional recovery in schizophrenia. We included clinical and epidemiological studies, reviews and meta-analyses published between January 1970 and July 2013. Sixty two studies on remission and recovery were included, with a total of 94 940 patients, comprising six months to 37 years follow-up. Thirty two studies on functional recovery were included, with a total of 6 483 patients with a range of six months to 42 years follow-up. Research indicates that symptomatic remission can be achieved in 20-97%, and functional recovery in 10-68% of people with schizophrenia. The use of remission and recovery criteria has ...

Recientemente los conceptos de remisión sintomática y recuperación funcional en los pacientes que padecen esquizofrenia han recibido una considerable atención por parte de los clínicos y los investigadores. El interés en estos aspectos tiene que ver con la propuesta realizada en el 2005 por el "Grupo de trabajo para evaluar la remisión en esquizofrenia", con el objetivo de proponer una definición de remisión sintomática, así como el desarrollo de criterios operacionales para su evaluación. La remisión sintomática se evalúa utilizando ocho reactivos de la escala PANSS, los cuales deben puntuar tres o menos, con una duración mínima de seis meses de remisión. En Europa, desde el 2006, también se han propuesto criterios para evaluar la respuesta al tratamiento, así como la remisión sintomática. La remisión se puede evaluar usando una fórmula para calcular el porcentaje de reducción de síntomas desde el inicio del tratamiento de acuerdo al PANSS. También se han desarrollado criterios para evaluar la recuperación funcional que incluyen la combinación de aspectos clínicos y psicosociales, que se deben mantener por lo menos por dos años consecutivos, incluyendo dimensiones como el funcionamiento psicosocial, el funcionamiento cognitivo y la calidad de vida. En el presente artículo se revisa la investigación respecto a los conceptos de remisión sintomática y recuperación funcional en los pacientes afectados por esta patología, incluyendo estudios clínicos, epidemiológicos, estudios de revisión y meta-análisis publicados entre enero de 1970 a julio de 2013. Se incluyeron 62 estudios sobre remisión sintomática/recuperación funcional, con un total de 94 940 pacientes, con un seguimiento de seis meses a 37 años. También se incluyeron 32 estudios de recuperación funcional, con un total de 6 483 pacientes, con un seguimiento de dos a 42 años. Los resultados indican que entre el 20 y el 97% de los pacientes pueden lograr la remisión sintomática, mientras que entre el 10 y el 68% alcanzan la recuperación funcional. Se ha recomendado el uso de estos criterios en la práctica clínica y en la investigación científica.

The intrinsic resistome of bacterial pathogens.

Intrinsically resistant bacteria have emerged as a relevant health problem in the last years. Those bacterial species, several of them with an environmental origin, present naturally low-level susceptibility to several drugs. It has been proposed that intrinsic resistance is mainly the consequence of the impermeability of cellular envelopes, the activity of multidrug efflux pumps or the lack of appropriate targets for a given family of drugs. However, recently published articles indicate that the characteristic phenotype of susceptibility to antibiotics of a given bacterial species depends on the concerted activity of several elements, what has been named as intrinsic resistome. These determinants comprise not just classical resistance genes. Other elements, several of them involved in basic bacterial metabolic processes, are of relevance for the intrinsic resistance of bacterial pathogens. In the present review we analyze recent publications on the intrinsic resistomes of Escherichia coli and Pseudomonas aeruginosa. We present as well information on the role that global regulators of bacterial metabolism, as Crc from P. aeruginosa, may have on modulating bacterial susceptibility to antibiotics. Finally, we discuss the possibility of searching inhibitors of the intrinsic resistome in the aim of improving the activity of drugs currently in use for clinical practice.

Phenotypic Resistance to Antibiotics.

The development of antibiotic resistance is usually associated with genetic changes, either to the acquisition of resistance genes, or to mutations in elements relevant for the activity of the antibiotic. However, in some situations resistance can be achieved without any genetic alteration; this is called phenotypic resistance. Non-inherited resistance is associated to specific processes such as growth in biofilms, a stationary growth phase or persistence. These situations might occur during infection but they are not usually considered in classical susceptibility tests at the clinical microbiology laboratories. Recent work has also shown that the susceptibility to antibiotics is highly dependent on the bacterial metabolism and that global metabolic regulators can modulate this phenotype. This modulation includes situations in which bacteria can be more resistant or more susceptible to antibiotics. Understanding these processes will thus help in establishing novel therapeutic approaches based on the actual susceptibility shown by bacteria during infection, which might differ from that determined in the laboratory. In this review, we discuss different examples of phenotypic resistance and the mechanisms that regulate the crosstalk between bacterial metabolism and the susceptibility to antibiotics. Finally, information on strategies currently under development for diminishing the phenotypic resistance to antibiotics of bacterial pathogens is presented.