Search

La presente investigación tiene por objeto identificar y examinar las series documentales relativas a la función gubernativa en la orden de Alcántara a través de la documentación custodiada en los Archivos Eclesiásticos de Mérida-Badajoz, que a día de hoy son los custodios de la documentación generada por la extinguida orden alcantarina (siglos XV-XIX). Para ello se analizan exhaustivamente por un lado, las series relativas al gobierno interno de la Orden y por otro, las que tienen que ver con su vertiente externa y que pusieron de relación a la orden alcantarina con entidades civiles, militares y eclesiásticas. El resultado de dicho análisis permite conocer de forma exhaustiva los procesos gubernativos emprendidos por dicha Orden en tierras extremeñas y ofrecer a los investigadores posibles líneas de investigación partiendo del análisis de la citada documentación.

Background and Purpose: Cardiovascular side effects from varenicline, and a case report of a hypertensive crisis event in a patient with pheochromocytoma being treated with varenicline, have been reported. The goal of the present study was to determine if such side effects might derive, in part, from increased exocytosis of secretory vesicles and subsequent catecholamine release triggered by varenicline in chromaffin cells of the adrenal gland. Experimental Approach: We performed electrophysiological plasma membrane capacitance (Cm) and carbon fiber amperometry experiments to evaluate the effect of varenicline on exocytosis and catecholamine release, respectively, at concentrations reached during varenicline therapy (100 nM). Experiments were conducted in the absence or presence of nicotine, at plasma concentrations achieved right after smoking (250 nM) or steady-state concentrations (110 nM), in chromaffin cells of the adrenal gland obtained from human organ donors or rats. Key Results: Varenicline increased the exocytosis of secretory vesicles and the release of catecholamines from human chromaffin cells in the presence of nicotine. Comparable results were found using rat chromaffin cells; varenicline alone or in the presence of acute or steady-state concentrations of nicotine found in human plasma increased exocytosis. These effects were not due to an increase of Cm or currents triggered by the nicotinic agonists alone. Conclusion and Implications: Therapeutic concentrations of varenicline in the presence of nicotine increased exocytosis and catecholamine release from human chromaffin cells. These results should be taken into account in nicotine addiction therapies when varenicline is used.

Recombination is an essential process in bacterial drug resistance evolution. Fluoroquinolones, an important class of antibiotics, are known to stimulate recombination in Escherichia coli. On the other hand, when bacteria are exposed to sublethal concentrations of different antibiotic families, including strong inducers of the SOS system, which upregulates recombination pathways, there is no detectable change in the recombination rate. Here we explore why fluoroquinolones, but not any other tested antimicrobials, increase recombination rates. One feature that makes the fluoroquinolones different from other antibiotics is the generation of DNA double-strand breaks (DSBs). We tested whether other antimicrobials known for their ability to cause double-strand breaks, such as mitomycin C and bleomycin, are also affecting bacterial recombination rate. We found that both drugs indeed affected recombination rates in E. coli. Furthermore, there was a positive correlation between the number of DNA double-strand breaks and the recombination frequency. The manipulation of the level of DSBs directly impacted the recombination frequency. These results imply that recombination, at least in Gram-negative bacteria, is conservative, and even if multiple copies of a DNA sequence are available, the basal recombination rate is low. Our results highlight that homologous recombination is intended more for DNA damage repair than generating diversity, or at least that they occur at a very different scale. We also show that only antibiotics that cause DNA double-strand breaks can increase genetic diversity via recombination. The stimulation of recombination by DSBs-causing antimicrobials is an additional factor leading to the risk of antibiotic resistance evolution.

[EN] Listeria monocytogenes is an important cause of meningoencephalitis associated with high mortality. The treatment of choice for listeriosis is ampicillin alone or in combination with gentamicin or penicillin G. However, only low ampicillin concentrations are recorded in the central nervous system (CNS). In this study, we analysed the effect of subinhibitory concentrations of ampicillin on the morphology, growth and survival of L. monocytogenes. The non-inhibitory concentration (NIC), the minimum inhibitory concentration (MIC) and the MIC/NIC ratio were determined. Gram and Live/Dead staining showed aggregates of L. monocytogenes cells when grown at subinhibitory concentrations of ampicillin, with >90% of viable cells. The L. monocytogenes strains tested showed an intermediate heteroresistance to ampicillin, characterised by a MIC/NIC ratio of 4. Our results seem to indicate that both intermediate heteroresistance and the formation of aggregates could play a role in the clinical failure of ampicillin in the treatment of CNS infections caused by L. monocytogenes. However, more studies are necessary to elucidate this question., [ES] Listeria monocytogenes es una importante causa de meningoencefalitis asociada con una elevada mortalidad. El tratamiento de elección para la listeriosis es ampicilina asociada o no a gentamicina o penicilina G. Sin embargo, en el sistema nervioso central (SNC) solo se alcanzan concentraciones bajas de ampicilina. En este estudio, analizamos el efecto de las concentraciones subinhibitorias de ampicilina sobre la morfología, el crecimiento y la supervivencia de L. monocytogenes. Para ello determinamos la concentración no inhibitoria (CNI), la concentración mínima inhibitoria (CMI) y el cociente CMI/CNI. Además, la tinción de Gram y la de vivos y muertos mostraron agregados de L. monocytogenes cuando crece a concentraciones subinhibitorias de ampicilina, con > 90% de las células viables. Las cepas de L. monocytogenes testadas mostraron heterorresistencia intermedia a la ampicilina, caracterizada por un cociente CMI/CNI de 4. Nuestros resultados parecen indicar que tanto la formación de agregados como la heterorresistencia intermedia podrían jugar un papel en el fracaso terapéutico observado en las infecciones del SNC causadas por L. monocytogenes. Sin embargo, se necesitan más estudios para aclarar esta cuestión.

The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3β4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3β4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3β4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3β4 subtype is dependent on Ca2+. In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca2+. In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3β4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.SIGNIFICANCE STATEMENTDesensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3β4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca2+to cooperate in the case of the α3β4 current increase. Because choline is an α3β4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.

Despite good long-term outcomes of kidney transplants from controlled donation after circulatory death (DCD) donors, there are few uncontrolled DCD (uDCD) programs. This longitudinal study compares outcomes for all uDCD (N = 774) and all donation after brain death (DBD) (N = 613) kidney transplants performed from 1996 to 2015 at our center. DBD transplants were divided into those from standard-criteria (SCD) (N = 366) and expanded-criteria (N = 247) brain-dead donors (ECD). One-, 5-, and 10-year graft survival rates were 91.7%, 85.7%, and 80.6% for SCD; 86.0%, 75.8%, and 61.4% for ECD; and 85.1%, 78.1%, and 72.2% for uDCD, respectively. Graft survival was worse in recipients of uDCD kidneys than of SCD (P = .004) but better than in transplants from ECD (P = .021). The main cause of graft loss in the uDCD transplants was primary nonfunction. Through logistic regression, donor death due to pulmonary embolism (OR 4.31, 95% CI 1.65-11.23), extrahospital CPR time ≥75 minutes (OR1.94, 95%CI 1.18-3.22), and in-hospital CPR time ≥50 minutes (OR 1.79, 95% CI 1.09-2.93) emerged as predictive factors of primary nonunction. According to the outcomes of our long-standing kidney transplantation program, uDCD could help expand the kidney donor pool. post-print 1,71 MB

Suppression of the recA SOS response gene and reactive oxygen species (ROS) overproduction have been shown, separately, to enhance fluoroquinolone activity and lethality. Their putative synergistic impact as a strategy to potentiate the efficacy of bactericidal antimicrobial agents such as fluoroquinolones is unknown. We generated Escherichia coli mutants that exhibited a suppressed ΔrecA gene in combination with inactivated ROS detoxification system genes (ΔsodA, ΔsodB, ΔkatG, ΔkatE, and ΔahpC) or inactivated oxidative stress regulator genes (ΔoxyR and ΔrpoS) to evaluate the interplay of both DNA repair and detoxification systems in drug response. Synergistic sensitization effects, ranging from 7.5- to 30-fold relative to the wild type, were observed with ciprofloxacin in double knockouts of recA and inactivated detoxification system genes. Compared to recA knockout, inactivation of an additional detoxification system gene reduced MIC values up to 8-fold. In growth curves, no growth was evident in mutants doubly deficient for recA gene and oxidative detoxification systems at subinhibitory concentrations of ciprofloxacin, in contrast to the recA-deficient strain. There was a marked reduction of viable bacteria in a short period of time when the recA gene and other detoxification system genes (katG, sodA, or ahpC) were inactivated (using absolute ciprofloxacin concentrations). At 4 h, a bactericidal effect of ciprofloxacin was observed for ΔkatG ΔrecA and ΔahpC ΔrecA double mutants compared to the single ΔrecA mutant (Δ3.4 log10 CFU/ml). Synergistic quinolone sensitization, by targeting the recA gene and oxidative detoxification stress systems, reinforces the role of both DNA repair systems and ROS in antibiotic-induced bacterial cell death, opening up a new pathway for antimicrobial sensitization., This work was supported by the Plan Nacional de I+D+i 2013‐2016 and the Instituto de Salud Carlos III (projects PI14/00940 and PI17/01501), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI; RD16/0016/0001 and REIPI RD16/0016/0009) cofinanced by the European Development Regional Fund “A Way to Achieve Europe,” operative program Intelligent Growth 2014‐2020. Sara Diaz-Diaz is supported by a PFIS grant from the Instituto de Salud Carlos III (FI18/00086).

The objectives of this study were to characterize the role of the uhpT, glpT, and fosA genes in fosfomycin resistance in Klebsiella pneumoniae and evaluate the use of sodium phosphonoformate (PPF) in combination with fosfomycin. Seven clinical isolates of K. pneumoniae and the reference strain (ATCC 700721) were used, and their genomes were sequenced. ΔuhpT, ΔglpT, and ΔfosA mutants were constructed from two isolates and K. pneumoniae ATCC 700721. Fosfomycin susceptibility testing was done by the gradient strip method. Synergy between fosfomycin and PPF was studied by checkerboard assay and analyzed using SynergyFinder. Spontaneous fosfomycin mutant frequencies at 64 and 512 mg/liter, in vitro activity using growth curves with fosfomycin gradient concentrations (0 to 256mg/liter), and time-kill assays at 64 and 307 mg/liter were evaluated with and without PPF (0.623 mM). The MICs of fosfomycin against the clinical isolates ranged from 16 to ≥1,024 mg/liter. The addition of 0.623 mM PPF reduced fosfomycin MIC between 2- and 8-fold. Deletion of fosA led to a 32-fold decrease. Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic area at 0.623 mM). The lowest fosfomycin-resistant mutant frequencies were found in ΔfosA mutants, with decreases in frequency from 1.69 × 10−1 to 1.60 × 10−5 for 64 mg/liter of fosfomycin. In the final growth monitoring and time-kill assays, fosfomycin showed a bactericidal effect only with the deletion of fosA and not with the addition of PPF. We conclude that fosA gene inactivation leads to a decrease in fosfomycin resistance in K. pneumoniae. The pharmacological approach using PPF did not achieve enough activity, and the effect decreased with the presence of fosfomycin-resistant mutations., Miriam Ortiz-Padilla is supported by a PFIS fellowship from Instituto de Salud Carlos III. This study was supported by the Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (PI16/01824, REIPI RD12/0015/0010, and REIPI RD16/0016/0001), cofinanced by European Development Regional Fund A Way To Achieve Europe, Operative Program Intelligent Growth 2014‐2020, and the Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI-0044 to 2013). The funders had no role in the design, collection of data, analysis and writing of the manuscript, or the decision to publish.

We evaluated human-like the efficacy of intravenous doses of fosfomycin of 8 g every 8 h (8 g/Q8h) and of amikacin (15 mg/kg/Q24h) in monotherapy and in combination against six fosfomycin-heteroresistant Escherichia coli isolates using a hollow-fiber infection model (HFIM). Six fosfomycin-heteroresistant E. coli isolates (four with strong mutator phenotype) and the control strain E. coli ATCC 25922 were used. Mutant frequencies for rifampin (100 mg/liter), fosfomycin (50 and 200 mg/liter), and amikacin (32 mg/liter) were determined. Fosfomycin and amikacin MICs were assessed by agar dilution (AD), gradient strip assay (GSA), and broth microdilution (BMD). Fosfomycin and amikacin synergies were studied by checkerboard and time-kill assays at different concentrations. The efficacies of fosfomycin (8 g/Q8h) and amikacin (15 mg/kg/Q24h) alone and in combination were assessed using an HFIM. Five isolates were determined to be resistant to fosfomycin by AD and BMD, but all were determined to be susceptible by GSA. All isolates were determined to be susceptible to amikacin. Antibiotic combinations were synergistic in two isolates, and no antagonism was detected. In time-kill assays, all isolates survived under fosfomycin at 64 mg/liter, although at 307 mg/liter only the normomutators and two hypermutators survived. Four isolates survived under 16 mg/liter amikacin, and none survived at 45 mg/liter. No growth was detected under combination conditions. In HFIM, fosfomycin and amikacin monotherapies failed to sterilize bacterial cultures; however, the combination of fosfomycin and amikacin yielded a rapid eradication. There may be a risk of treatment failure of fosfomycin-heteroresistant E. coli isolates using either amikacin or fosfomycin in monotherapy. These results support that the amikacin-fosfomycin combination can rapidly decrease bacterial burden and prevent the emergence of resistant subpopulations against fosfomycin-heteroresistant strains., This study was supported by Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (PI16/01824, REIPI RD12/0015/0010, and REIPI RD16/0016/0001), cofinanced by European Development Regional Fund “A Way to Achieve Europe” (Operative Program Intelligent Growth 2014-2020). The funders had no role in the design, collection of data, analysis, and writing of the manuscript or the decision to publish. J.R.-B. has been a scientific coordinator of a research project, funded by AstraZeneca, unrelated to the present study and a speaker at accredited educational activities funded by Merck through unrestricted grants. J.R.-B. and A.P. received funding for research from COMBACTE-NET (grant 115523), COMBACTE-CARE (grant 115620), and COMBACTE-MAGNET (grant 115737) projects under the Innovative Medicines Initiative, the European Union, and EFPIA companies in kind.

α7 nicotinic receptors have been involved in numerous pathologies. A hallmark of these receptors is their extremely fast desensitization, a process not fully understood yet. Here we show that human native α7 and α3β4 nicotinic receptors physically interact in human chromaffin cells of adrenal glands. The full activation of this α7-α3β4 receptor complex avoids subtypes receptor desensitization, leading to gradual increase of currents with successive acetylcholine pulses. Instead, full and partial activation with choline of α7 and α3β4 subtypes, respectively, of this linked receptor leads to α7 receptor desensitization. Therefore choline, a product of the acetylcholine hydrolysis, acts as a brake by limiting the increase of currents by acetylcholine. Very importantly, the efficiency of the α7-α3β4 interaction diminishes in subjets older than 50 years, accordingly increasing receptor desensitization and decreasing nicotinic currents. These results open a new line of research to achieve improved therapeutic treatments for nicotinic receptors related diseases.

© 2020 by the authors., Mutations that confer low-level fosfomycin resistance (LLFR) but not clinical resistance in Escherichia coli are increasingly reported. LLFR strains can become clinically resistant under urinary tract physiological conditions or may act as gateways for highly resistant subpopulations by the selection of additional LLFR mutations. Nevertheless, most LLFR strains are impossible to detect under routine fosfomycin susceptibility determinations. Here, we have explored the possibility of detecting LLFR variants by reducing glucose-6-phosphate (G6P) concentration in fosfomycin susceptibility testing for E. coli strains. As a proof of concept, fosfomycin minimal inhibitory concentrations (MICs) and disk diffusion susceptibility tests were performed for E. coli strain BW25113 and 10 isogenic derivatives carrying the most prevalent LLFR chromosomal mutations (∆uhpT, ∆glpT, ∆cyaA, and ∆ptsI) and their double combinations. Whereas standard G6P concentrations detected only ∆uhpT single and double variants, assays with reduced G6P detected all LLFR variants. In addition, G6P levels were determined to be ≤5 µg/mL in urine samples from 30 patients with urinary tract infection (UTI) caused by E. coli and 10 healthy volunteers, suggesting that most bacterial cells in uncomplicated UTIs are facing fosfomycin under low G6P concentration. Reducing G6P allows for the detection of LLFR variants, which may suppose a risk for future resistance development, especially in UTIs., This work was supported by Spanish Plan Nacional de I + D + i 2013–2016 and Instituto de Salud Carlos III (ISCIII), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009) co-financed by the European Development Regional Fund “A way to achieve Europe” Operative Programme Intelligent Growth 2014–2020. J.R.B. is a recipient of a Juan de la Cierva-Incorporación Fellowship (IJC2018-035146-I) co-funded by Agencia Estatal de Investigación del Ministerio de Ciencia e Innovación.

This study was presented in part at the Twenty-Ninth European Congress of Clinical Microbiology and Infectious Diseases, Amsterdam, The Netherlands, 2019 (Poster Presentation P1339)., [Background] SOS response suppression (by RecA inactivation) has been postulated as a therapeutic strategy for potentiating antimicrobials against Enterobacterales., [Objectives] To evaluate the impact of RecA inactivation on the reversion and evolution of quinolone resistance using a collection of Escherichia coli clinical isolates., [Methods] Twenty-three E. coli clinical isolates, including isolates belonging to the high-risk clone ST131, were included. SOS response was suppressed by recA inactivation. Susceptibility to fluoroquinolones was determined by broth microdilution, growth curves and killing curves. Evolution of quinolone resistance was evaluated by mutant frequency and mutant prevention concentration (MPC)., [Results] RecA inactivation resulted in 2–16-fold reductions in fluoroquinolone MICs and modified EUCAST clinical category for several isolates, including ST131 clone isolates. Growth curves and time–kill curves showed a clear disadvantage (up to 10 log10 cfu/mL after 24 h) for survival in strains with an inactivated SOS system. For recA-deficient mutants, MPC values decreased 4–8-fold, with values below the maximum serum concentration of ciprofloxacin. RecA inactivation led to a decrease in mutant frequency (≥103-fold) compared with isolates with unmodified SOS responses at ciprofloxacin concentrations of 4×MIC and 1 mg/L. These effects were also observed in ST131 clone isolates., [Conclusions] While RecA inactivation does not reverse existing resistance, it is a promising strategy for increasing the effectiveness of fluoroquinolones against susceptible clinical isolates, including high-risk clone isolates., This study was funded by the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad—co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015 and RD16/0016). Supported by Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de InvestigaciónCooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (PI14/00940, PI17/01501, AC16/00072, RD16/0016/0001 and REIPI RD16/0016/0009) ‐ co-financed by European Development Regional Fund ‘A way to achieve Europe’, Operative Programme Intelligent Growth 2014‐2020.

10 páginas, 3 figuras, 3 tablas. Material suplementario en: https://advances.sciencemag.org/content/suppl/2020/02/10/6.7.eaay4453.DC1, The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution., This work was supported by Plan Nacional de I + D + i 2013–2016 and the Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—cofinanced by the European Development Regional Fund “A Way to Achieve Europe”—by Operative Program Intelligent Growth 2014-2020 and grants FIS PI17/00159 from the Instituto de Salud Carlos III and SAF2015-72793-EXP from the Spanish Ministry of Science and Competitiveness (MINECO)-FEDER. E.C.-S. was the recipient of a PFIS predoctoral research fellowship (FI18/00036) cofinanced by the Instituto de Salud Carlos III and the European Social Fund. I.C., A.C.-O., and M.T.-P. are funded by projects of the 581 European Research Council (ERC) (638553-TB-ACCELERATE) and Ministerio de Economía y Competitividad research grant SAF2016-77346-R.

The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available &beta, lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of &beta, Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine &beta, &minus, Lactamase (SBLs) KPC-2 and class B1 metallo &beta, Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.

[Objectives]: To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae., [Materials and methods]: We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5–512 mg/L) and time–kill assays (64 and 307 mg/L)., [Results]: Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time–kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula., [Conclusions]: The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established., This study was supported by Plan Nacional de I ! D!i 2013–2016 and Instituto de Salud Carlos III, Subdireccio´n General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (PI10/02021, REIPI RD12/0015/0010 and REIPI RD16/0016/0001)—cofinanced by European Development Regional Fund ‘A way to achieve Europe’, Operative Programme Intelligent Growth 2014–2020.

By targeting essential cellular processes, antibiotics provoke metabolic perturbations and induce stress responses and genetic variation in bacteria. Here we review current knowledge of the mechanisms by which these molecules generate genetic instability. They include production of reactive oxygen species, as well as induction of the stress response regulons, which lead to enhancement of mutation and recombination rates and modulation of horizontal gene transfer. All these phenomena influence the evolution and spread of antibiotic resistance. The use of strategies to stop or decrease the generation of resistant variants is also discussed.

Resumen: Los constantes cambios en la sociedad en la que nos vemos inmersos conllevan la necesidad de investigar metodologías y recursos que supongan una mejora en el aprendizaje de los alumnos e incrementar su motivación. Dentro de los diferentes tipos de metodologías, existe la denominada metodología de aprendizaje basado en proyectos (ABP), la cual ofrece multitud de variantes que se pueden adaptar al sistema educativo actual y futuro. Para que la metodología basada en proyectos logre conectar con los estudiantes y consiga mejorar su rendimiento académico y su nivel motivacional, debe ser interesante y atractiva. Es por este hecho por el que se plantea el uso de un nuevo recurso educativo en las aulas utilizando dicha metodología. Ese recurso didáctico es la impresora 3D, debido a su gran variedad de oportunidades que ofrece para trabajar con ella en el aula y su futuro tan prometedor en diferentes campos profesionales. Con el presente trabajo se pretende por un lado, indagar en las bases teóricas que posicionan la impresión 3D junto con el aprendizaje basado en proyectos como una metodología que potencia la motivación del alumno y su aprendizaje significativo, y por el otro, hacer una propuesta metodológica que incluya los aspectos necesarios para poder llevar a cabo un proyecto de estas características en el aula. Abstract: The current society in constant change where we are immersed requires to investigate methodologies and resources that suppose an improvement in the students´ learning process and an increase of their motivation. Within the different types of methodologies, there is one denominated Project-based learning (PBL), which offers a multitude of variants that are perfectly adapted to the current and future educational system. The project-based learning needs to be interesting and engaging to connect with students and improve their academic performance and motivational level. It is for this reason that the use of a new educational resource in the classrooms is proposed meanwhile this methodology is applied. This didactic resource is the 3D printer due to its wide variety of opportunities to work with it in the classroom and its promising future in different professional fields. The present work intends on the one hand to investigate the theoretical bases that position the 3D impression together with the project based learning as a methodology that enhances the motivation of the student and his significant learning and on the other hand to develop a methodological proposal that includes necessary aspects to be able to carry out a project of these characteristics in the classroom. Resumo: A sociedade atual em constante mudança em que estamos imersos precisa investigar metodologias e recursos que supõem uma melhoria na aprendizagem dos alunos e aumentar sua motivação. Entre as várias metodologias encontra-se a denominada aprendizagem baseada em projetos (ABP), a qual oferece um vasto leque de opções que se adaptam perfeitamente ao sistema educativo atual e futuro. A fim de cativar os estudantes e de melhorar o seu rendimento académico e o seu nível motivacional, ametodologia baseada em projetos deverá ser interessante e atrativa. Perante este facto, propõe-se a adoção de um novo recurso educacional nas salas de aula que permitirá aplicar e desenvolver esta mesma metodologia. O dito recurso trata-se da impressora 3D dada a grande variedade de opções que oferece no trabalho em sala de aula e o seu promissor futuro em diferentes campos profissionais. No presente trabalho, pretende-se por um lado aferir as bases teóricas que posicionam a impressão 3D e a aprendizagem baseada em projetos como uma metodologia que potencia a motivação do aluno e a sua aprendizagem significativa, e por outro lado apresentar uma proposta metodológica que inclua os aspetos necessários para poder levar a cabo um projeto com estas características no contexto da aula.

A fluidic permeable and stable in wet media, MBG-NfGel, device consisting of a mesoporous ceramic embodied in a nanofibrillar biodegradable polymer has been processed using appropriate thermally induced phase separation (TIPS) processing variables of 5.4% (wt/v) gelatin in 50/50 water/ethanol (v/v) ratio. The device comprises high surface area mesoporous bioactive glass (MBG) microparticles within a fibrous matrix of 170 nm average diameter nanofibers gelatin, forming a meshwork of 0.2–1.6 μm range voids. Gentamicin sulphate (GS) antibiotic high loading capacity and sustained release ability, as well as in vitro bioactivity and osteoprogenitor cells biocompatibility supports long-term antibacterial and bone growth stimulation properties. Antibiotic local delivery functionality in vitro of this device has been analysed and discussed in relation to other systems previously reported. The presented device properties as well as its industrial scalability potential, in terms of process reliability and absence of toxic chemical agents, low raw material biopolymer cost and immunogenicity, are other important advantages. These advantages rank MBG-NfGel device as a potential candidate to further development for application as local antibiotic device in bone surgery and therapy., The authors gratefully acknowledge the financial support provided by the Andalusian Ministry of Economy, Science and Innovation (Proyecto Excelencia P10-CTS-6681). SBG acknowledges the Andalusian Ministry of Economy, Science and Innovation-Talentia Program and Prof. Dalby at the Centre for Cell Engineering, University of Glasgow (host Laboratory). LBRS acknowledges the CONACYT-Mexico Fellowship PhD Program.

Cardiovascular side effects of varenicline and a case report of a hypertensive crisis in a varenicline-prescribed patient with pheochromocytoma have been reported. The goal of the present study was to determine whether such side effects might derive, in part, from increased exocytosis of secretory vesicles and subsequent catecholamine release triggered by varenicline in human chromaffin cells of the adrenal gland. In this study, we performed electrophysiological plasma membrane capacitance and carbon fiber amperometry experiments to evaluate the effect of varenicline on exocytosis and catecholamine release, respectively, at concentrations reached during varenicline therapy (100 nM). Experiments were conducted in the absence or presence of nicotine, at plasma concentrations achieved right after smoking (250 nM) or steady-state concentrations (110 nM), in chromaffin cells of the adrenal gland obtained from human organ donors. Cells were stimulated with short pulses (10 ms) of acetylcholine (ACh; 300 μM) applied at 0.2 Hz, in order to closer mimic the physiological situation at the splanchnic nerve-chromaffin cell synapse. In addition, rat chromaffin cells were used to compare the effects obtained in cells from a more readily available species. Varenicline increased the exocytosis of secretory vesicles in human and rat chromaffin cells in the presence of nicotine, effects that were not due to an increase of plasma membrane capacitance or currents triggered by the nicotinic agonists alone. These results should be considered in nicotine addiction therapies when varenicline is used, This work was supported by grants from the Spanish Ministry of Science and Innovation [grant number BFU2015-69092 to A.A.] and the U.S. National Institutes of Health [GM136430 and GM103801 to J.M.M]

Bacterial resistance represents a worldwide emergency threatening the efficacy of all available antibiotics. Among the several resistance mechanisms developed by bacteria, β-lactamase enzymes (BLs), which are able to inactivate most β-lactam core antibiotics, represent a key target to block, thus prolonging antibiotics half-life. Several approaches aimed at inhibiting β-lactamases have been so far undertaken, mainly involving β-lactam-like or covalent inhibitors. Applying a structure-based de novo design approach, we recently discovered a novel, non-covalent and competitive inhibitor of AmpC β-lactamase: lead 1. It has a K i of 1 µM, a ligand efficiency of 0.38 kcal mol−1 and lead-like physical properties. Moreover, it reverts resistance to ceftazidime in bacterial pathogens expressing AmpC and does not up-regulate β-lactamases expression in cell culture. Its features make it a good candidate for chemical optimization: starting from lead 1 crystallographic complex with AmpC, 11 analogs were designed to complement additional AmpC sites, then synthesized and tested against clinically resistant pathogens. While the new inhibitors maintain similar in vitro activity as the starting lead, some of them, in biological assays, extert a higher potency showing improved synergic activity with ceftazidime in resistant clinically isolated strains.

The present study was performed to evaluate the Ca v 1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Ca v 1.2 and Ca v 1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Ca v 1.2 and Ca v 1.3 subtypes, but not Ca v 1.1 or Ca v 1.4. Electrophysiological experiments were conducted to investigate the contribution of Ca v 1 channels to the exocytotic process and cell excitability. Ca v 1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3 μM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200 ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (−34 mV), which elicits 10.4 mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Ca v 1 channels in exocytosis and cell excitability.

Varenicline is a nicotinic acetylcholine receptor (nAChR) agonist used to treat nicotine addiction but a live debate persists concerning its mechanism of action in reducing nicotine consumption. Although initially reported as α4β2-selective, varenicline was subsequently shown to activate other nAChR subtypes implicated in nicotine addiction including α3β4. However, it remains unclear whether activation of α3β4 nAChRs by therapeutically relevant concentrations of varenicline is sufficient to affect the behavior of cells that express this subtype. We used patch-clamp electrophysiology to assess the effects of varenicline on native α3β4* nAChRs (asterisk denotes the possible presence of other subunits) expressed in human adrenal chromaffin cells and compared its effects to those of nicotine. Varenicline and nicotine activated α3β4* nAChRs with EC50 values of 1.8 (1.2-2.7) μM and 19.4 (11.1-33.9) μM, respectively. Stimulation of adrenal chromaffin cells with 10 ms pulses of 300 μM acetylcholine (ACh) in current-clamp mode evoked sodium channel-dependent action potentials (APs). Under these conditions, perfusion of 50 nM or 100 nM varenicline showed very little effect on AP firing compared to control conditions (ACh stimulation alone) but at higher concentrations (250 nM) varenicline increased the number of APs fired up to 436 ± 150%. These results demonstrate that therapeutic concentrations of varenicline are unlikely to alter AP firing in chromaffin cells. In contrast, nicotine showed no effect on AP firing at any of the concentrations tested (50, 100, 250, and 500 nM). However, perfusion of 50 nM nicotine simultaneously with 100 nM varenicline increased AP firing by 290 ± 104% indicating that exposure to varenicline and nicotine concurrently may alter cellular behavior including excitability and neurotransmitter release. This article is protected by copyright. All rights reserved.

This article describes an analysis of keywords which was aimed at revealing publication patterns in the field of Food Science (FS) during the last decade, including the temporal evolution of its different research lines. To this end, the records of the specific subject area of FS were 1st retrieved from Scopus, and then their keywords were processed to resolve the obvious problems of synonymy and to limit the study to those most frequently used. These keywords were grouped into thematic clusters based on a scientometric technique known as co-word analysis. The structure of the clusters, their scientific impact, and their temporal evolution were then analyzed. This type of analysis is of great interest for all researchers in FS-for new researchers because they can form an objective vision of the subject based on the data from papers that have been published in the last decade, and for experienced researchers because they can contrast their own vision of the field with this objective overview. The results showed there to has been a clear increase in scientific production related to FS. This production had a structure corresponding to 5 major clusters which were themselves disaggregated into 18 2nd-level clusters. The cluster that had received most attention was that corresponding to antioxidants in food, being the cluster with the greatest scientific impact and the greatest growth in the period.

[Background]: Fluoroquinolones such as ciprofloxacin induce the mutagenic SOS response and increase the levels of intracellular reactive oxygen species (ROS). Both the SOS response and ROS increase bacterial mutagenesis, fuelling the emergence of resistant mutants during antibiotic treatment. Recently, there has been growing interest in developing new drugs able to diminish the mutagenic effect of antibiotics by modulating ROS production and the SOS response., [Objectives]: To test whether physiological concentrations of N-acetylcysteine, a clinically safe antioxidant drug currently used in human therapy, is able to reduce ROS production, SOS induction and mutagenesis in ciprofloxacin-treated bacteria without affecting antibiotic activity., [Methods]: The Escherichia coli strain IBDS1 and its isogenic mutant deprived of SOS mutagenesis (TLS−) were treated with different concentrations of ciprofloxacin, N-acetylcysteine or both drugs in combination. Relevant parameters such as MICs, growth rates, ROS production, SOS induction, filamentation and antibiotic-induced mutation rates were evaluated., [Results]: Treatment with N-acetylcysteine reduced intracellular ROS levels (by ∼40%), as well as SOS induction (by up to 75%) and bacterial filamentation caused by subinhibitory concentrations of ciprofloxacin, without affecting ciprofloxacin antibacterial activity. Remarkably, N-acetylcysteine completely abolished SOS-mediated mutagenesis., [Conclusions]: Collectively, our data strongly support the notion that ROS are a key factor in antibiotic-induced SOS mutagenesis and open the possibility of using N-acetylcysteine in combination with antibiotic therapy to hinder the development of antibiotic resistance., This study was funded by the Spanish Plan Nacional de I!D!i 2013- 2016; grant SAF2015-72793-EXP (AEI/FEDER, UE) and the Instituto de Salud Carlos III (ISCIII), Subdireccio´n General de Redes y Centros de Investigacio´n Cooperativa, Ministerio de Economı´a, Industria y Competitividad; grant FIS PI17/00159 (ISCIII/FEDER, UE) and Spanish Network for Research in Infectious Diseases; grant REIPI RD16/0016/ 0009, cofinanced by the European Development Regional Fund ‘A Way to Achieve Europe’ and by Operative Program IntelligentGrowth 2014- 2020. E. Y. V. was funded by a postdoctoral fellowship from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil (grant 236914/2012–0). R. S. G. was supported by Fundac¸ao de Amparo ~ a` Pesquisa do Estado de Sao Paulo (FAPESP), Brazil (grant 2014/15982–6) ~ and CNPq, Brazil (grant 407259/2013–9). J. R.-B. is a recipient of a Juan de la Cierva Fellowship, Ministerio de Economı´a Industria y Competitividad (FJCI-2016–30019). J. B. was supported by: the Spanish Plan Nacional de I ! D!i 2013–2016 and the Instituto de Salud Carlos III, Subdireccio´n General de Redes y Centros de Investigacio´n Cooperativa, Ministerio de Economı´a, Industria y Competitividad, Spanish Network for Research in Infectious Diseases; grant REIPI RD16/0016/ 0009, cofinanced by the European Development Regional Fund ‘A Way to Achieve Europe’ and by Operative Program IntelligentGrowth 2014– 2020; and grants FIS PI17/00159 (ISCIII/FEDER, UE) and SAF2015- 72793-EXP (AEI/FEDER, UE).

© The Author(s)., [Background]: In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood., [Results]: In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions., [Conclusions]: The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress., This work was supported by Plan Nacional de I+D+i 2013-2016 and the Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by the European Development Regional Fund “A Way to Achieve Europe”—by Operative Program Intelligent Growth 2014-2020. ARR was also supported SFB 973 (Deutsche Forschungsgemeinschaft, project C5). We acknowledge support by the German Research Foundation and the OpenAccess Publication Fund of Freie Universität Berlin.

One of the main objectives in education is to increase the motivation of the students to achieve meaningful learning. The use of technologies in classrooms which students are familiarized with such as the smartphone or the tablet, is a way to achieve this goal. On the other hand, it has been proven that the inclusion of scenarios supported by games and competition enhance the active participation of students. Therefore, in this work we present the results of a study based on of the application Kahoot with students of secondary education, in the subjects of mathematics, biology &amp, geology and physics &amp, chemistry, during the academic year 2017/2018. This tool allows students to answer to on-line questionnaires created by the teacher, through mobile devices, and check their results in a few seconds as well as those of their classmates. The results obtained on the assessment of the tool by students, in terms of the benefits in the learning process, have been very positive and help us to examine the potential of the use of on-line questionnaires in the classrooms.

Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, &beta, lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine &beta, Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect &beta, lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with &beta, lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors.

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type β-lactamases are often reported as resistant to available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non β-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-β-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the β-lactam antibiotic meropenem by four-fold. publishedVersion

[Background] Suppression of the SOS response has been proposed as a therapeutic strategy for potentiating quinolones against susceptible, low-level quinolone-resistant (LLQR) and resistant Enterobacteriaceae., [Objectives] To monitor the functionality of the SOS response in the evolution towards clinical quinolone resistance and study its impact on the evolution of spatiotemporal resistance., [Methods] An isogenic collection of Escherichia coli (derived from the strain ATCC 25922) carrying combinations of chromosomally and plasmid-mediated quinolone resistance mechanisms (including susceptible, LLQR and resistant phenotypes) and exhibiting a spectrum of SOS activity was used. Relevant clinical parameters such as mutation rate, mutant prevention concentration (MPC), bacterial fitness, biofilm formation and post-antibiotic effect (PAE) were evaluated., [Results] Inactivating the SOS response (recA deletion) led to a decrease in mutation rate (∼103 fold) in LLQR compared with WT strains at ciprofloxacin concentrations of 1 mg/L (the EUCAST breakpoint for resistance) and 2.5 mg/L (Cmax), as well as a remarkable delay in the spatiotemporal evolution of quinolone resistance. For all strains, there was an 8-fold decrease in MPC in RecA-deficient strains, with values for LLQR strains decreasing below the Cmax of ciprofloxacin. Inactivation of the SOS response reduced competitive fitness by 33%–50%, biofilm production by 22%–80% and increased the PAE by ∼3–4 h at sub-MIC concentrations of ciprofloxacin., [Conclusions] Our data indicate that suppression of the SOS response affects key bacterial traits and is a promising strategy for reversing and tackling the evolution of antibiotic resistance in E. coli, including low-level and resistant phenotypes at therapeutic quinolone concentrations., This work was supported by the Plan Nacional de I+D+i 2013–2016 and the Instituto de Salud Carlos III (projects PI14/00940 and PI17/01501), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI; RD16/0016/0001 and REIPI RD16/0016/0009), co‐financed by the European Development Regional Fund ‘A way to achieve Europe’, operative program Intelligent Growth 2014–2020. F. D.-P. is supported by a VPPI-US fellowship from the University of Sevilla.

En abril de 2013, la Sociedad Espanola de Farmacia Familiar y Comunitaria (SEFAC) presento su propuesta sobre servicios profesionales farmaceuticos (SPF) cuyo fin es cubrir las necesidades relacionadas tanto con la atencion de los pacientes que utilizan medicamentos, como con la salud publica. Esta propuesta ofrece un planteamiento sobre la implantacion y desarrollo de los SPF con el objetivo de impulsar su prestacion por las farmacias comunitarias en los proximos anos. De acuerdo con dicha propuesta todos los SPF que constituyen el catalogo de servicios contaran con un documento de especificaciones. El objeto de este documento de especificaciones, aprobado por la Junta Directiva de SEFAC el 20 de abril de 2014, es definir y caracterizar el servicio de cesacion tabaquica con una doble finalidad: • Ayudar al farmaceutico comunitario y a sus representantes en el ofrecimiento, prestacion, difusion, financiacion y concertacion de este servicio. • Servir de guia a los farmaceuticos comunitarios que desean implantar un servicio de cesacion tabaquica en la farmacia o elaborar un procedimiento normalizado de trabajo para su realizacion. Este documento se complementa con el programa CESAR de formacion en cesacion tabaquica, y el Documento de intervencion en Cesacion tabaquica en farmacia comunitaria elaborado y avalado por SEFAC y validado por SEFAC, la Sociedad Espanola de Neumologia y Patologia Toracica (SEPAR), la Sociedad Espanola de Especialistas en Tabaquismo (SEDET) y las sociedades medicas de atencion primaria (semFYC, SEMG y SEMERGEN).

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type β-lactamases are often reported as resistant to available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non β-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-β-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the β-lactam antibiotic meropenem by four fold.