Genomewide overexpression screen for fosfomycin resistance in Escherichia coli: Mura confers clinical resistance at low fitness cost

To determine whether the overexpression of chromosomal genes can confer fosfomycin resistance, genomewide screening of a complete set of 5,272 plasmid-expressed open reading frames of Escherichia coli (ASKA collection) was performed. Major results are that (i) no clinical level of resistance is achieved by overexpressing chromosomal genes, except murA; (ii) this level is reached at a low fitness cost; and (iii) this cost is much lower than that imposed by other mutations conferring fosfomycin resistance.Ministerio de Ciencia e Innovación y Instituto de Salud Carlos III PI10/00105Spanish Network for Research on Infectious Diseases REIPI RD06/000

N-acetylcysteine selectively antagonizes the activity of imipenem in Pseudomonas aeruginosa by an OprD-mediated mechanism

The modulating effect of N-acetylcysteine (NAC) on the activity of different antibiotics has been studied in Pseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained with P. aeruginosa clinical isolates. Our results indicate that imipenem-susceptible P. aeruginosa strains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species, Escherichia coli and Acinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.Ministerio de Ciencia e Innovación y Instituto de Salud Carlos III y European Development Regional Fund (ERDF) y Spanish Network for Research in Infectious Diseases REIPI RD12/0015 FIS PI13/0006

Urinary tract physiological conditions promote ciprofloxacin resistance in low-level-quinolone-resistant Escherichia coli

Escherichia coli isolates carrying chromosomally encoded low-level-quinolone-resistant (LLQR) determinants are frequently found in urinary tract infections (UTIs). LLQR mutations are considered the first step in the evolutionary pathway producing high-level fluoroquinolone resistance. Therefore, their evolution and dissemination might influence the outcome of fluoroquinolone treatments of UTI. Previous studies support the notion that low urine pH decreases susceptibility to ciprofloxacin (CIP) in E. coli. However, the effect of the urinary tract physiological parameters on the activity of ciprofloxacin against LLQR E. coli strains has received little attention. We have studied the activity of ciprofloxacin under physiological urinary tract conditions against a set of well-characterized isogenic E. coli derivatives carrying the most prevalent chromosomal mutations (ΔmarR, gyrA-S83L, gyrA-D87N, and parC-S80R and some combinations). The results presented here demonstrate that all the LLQR strains studied became resistant to ciprofloxacin (according to CLSI guidelines) under physiological conditions whereas the control strain lacking LLQR mutations did not. Moreover, the survival of some LLQR E. coli variants increased up to 100-fold after challenge with a high concentration of ciprofloxacin under UTI conditions compared to the results seen with Mueller-Hinton broth. These selective conditions could explain the high prevalence of LLQR mutations in E. coli. Furthermore, our data strongly suggest that recommended methods for MIC determination produce poor estimations of CIP activity against LLQR E. coli in UTIs.Fondo de Investigación Sanitaria PI13/0006

Intrapopulation variability in mutator prevalence among urinary tract infection isolates of Escherichia coli

Bacteria with elevated mutation rates represent a risk factor for treatment failure and are often found with high frequency in clinical isolates from different sources. How this frequency reflects the among-population and within-population proportion of hypermutators is unknown, despite its importance to the choice of antibiotic therapies that minimize the likelihood of resistance development. Here we screened for hypermutators among the urine of 80 patients with urinary tract infections, at an unprecedented resolution of 24 isolates per sample. We found hypermutators in four patients (5%), at frequencies ranging from 4.2% to 62.5%. Molecular characterization revealed alterations in the oxidized guanine (GO) and methly-directed mistmatch repair (MMR) systems as the genetic basis of hypermutability. These observations suggest that mutators may be present in more patients than previously anticipated, at frequencies that are difficult to detect but still sufficient to impact on adaptation to antibiotics or the host environment.This work was supported by grants FIS PI13/00063 and REIPI RD12/0015 to JB from Instituto de Salud Carlos III (www.isciii.es), Plan Nacional de I+D+i, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases and co-financed by European Development Regional Fund A way to achieve Europe ERDF

Effect of N-acetyl-cysteine on antibiotic mediated bacterial mutagenesis

Póster presentado en la XI Reunión del Grupo de Microbiología Molecular de la Sociedad Española de Microbiología (SEM), celebrada en Sevilla del 6 al 8 de septiembre de 2016.Bactericidal antibiotics increase bacterial mutagenesis mediated by induction of the SOS response and production of Reactive Oxygen Species (ROS) promoting the emergence of antibiotic resistance variants. Interestingly, high levels of ROS can additionally induce SOS. Therefore, antibiotics may produce cumulative mutagenic effect (ROS plus SOS), although the relative contribution of each factor has not been yet clarified. Inhibition of SOS and/or ROS could be useful strategies to diminish the mutagenic effect of antibiotics. Among other molecules, antioxidants have been suggested as possible adjuvants in antibiotic therapy. Nonetheless, ROS production has been linked to lethality of bactericidal antibiotics in bacteria. Thus, antioxidants could have an antagonistic effect on this lethality. Our research is aimed at investigating i) whether antioxidants can affect the killing activity of antibiotics, and ii) the relative contribution of SOS and ROS in bacterial mutagenesis. N-Acetyl-Cysteine, a widely used FDA approved antioxidant, was able to decrease the mutagenesis promoted by ciprofloxacin, a bactericidal antibiotic and well-known SOS inducer without affecting viability of Escherichia coli. The relative contribution of ROS and SOS on mutagenesis will also be presented.Peer reviewe

Peptidoglycan recycling contributes to intrinsic resistance to fosfomycin in Acinetobacter baumannii

[Background] Acinetobacter baumannii is intrinsically resistant to fosfomycin; however, the mechanisms underlying this resistance are poorly understood.[Objectives] To identify and characterize genes that contribute to intrinsic fosfomycin resistance in A. baumannii.[Methods] More than 9000 individual transposon mutants of the A. baumannii ATCC 17978 strain (fosfomycin MIC ≥1024 mg/L) were screened to identify mutations conferring increased susceptibility to fosfomycin. In-frame deletion mutants were constructed for the identified genes and their susceptibility to fosfomycin was characterized by MIC determination and growth in the presence of fosfomycin. The effects of these mutations on membrane permeability and peptidoglycan integrity were characterized. Susceptibilities to 21 antibiotics were determined for the mutant strains.[Results] Screening of the transposon library identified mutants in the ampD and anmK genes, both encoding enzymes of the peptidoglycan recycling pathway, that demonstrated increased susceptibility to fosfomycin. MIC values for in-frame deletion mutants were ≥42-fold (ampD) and ≥8-fold (anmK) lower than those for the parental strain, and growth of the mutant strains in the presence of 32 mg/L fosfomycin was significantly reduced. Neither mutation resulted in increased cell permeability; however, the ampD mutant demonstrated decreased peptidoglycan integrity. Susceptibility to 21 antibiotics was minimally affected by mutations in ampD and anmK.[Conclusions] This study demonstrates that AmpD and AnmK of the peptidoglycan recycling pathway contribute to intrinsic fosfomycin resistance in A. baumannii, indicating that inhibitors of these enzymes could be used in combination with fosfomycin as a novel treatment approach for MDR A. baumannii.This work 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 (REIPI RD16/0016/0009), co-financed by European Development Regional Fund ‘A way to achieve Europ’e, Operative program Intelligent Growth 2014‐2020, and a Grant from the European Society of Clinical Microbiology and Infectious Diseases awarded to M. J. M. M. J. M. is supported by the Subprograma Miguel Servet (CPII16/00061), Instituto de Salud Carlos III, Ministerio de Economía, Industria y Competitividad. M. L. G.-M. is supported by the Formación de Profesorado Universitario Program (FPU13/04545), Ministerio de Educación, Cultura y Deporte, Spain.Peer reviewe

Transmigration across a steady-state blood-brain barrie induces activation of circulating dendritic cells partly mediated by actin cytoskeletal reorganization

The central nervous system (CNS) is considered to be an immunologically unique site, in large part given its extensive protection by the blood–brain barrier (BBB). As our knowledge of the complex interaction between the peripheral immune system and the CNS expands, the mechanisms of immune privilege are being refined. Here, we studied the interaction of dendritic cells (DCs) with the BBB in steady–state conditions and observed that transmigrated DCs display an activated phenotype and stronger T cell-stimulatory capacity as compared to non-migrating DCs. Next, we aimed to gain further insights in the processes underlying activation of DCs following transmigration across the BBB. We investigated the interaction of DCs with endothelial cells as well as the involvement of actin cytoskeletal reorganization. Whereas we were not able to demonstrate that DCs engulf membrane fragments from fluorescently labelled endothelial cells during transmigration across the BBB, we found that blocking actin restructuring of DCs by latrunculin-A significantly impaired in vitro migration of DC across the BBB and subsequent T cell-stimulatory capacity, albeit no effect on migration-induced phenotypic activation could be demonstrated. These observations contribute to the current understanding of the interaction between DCs and the BBB, ultimately leading to the design of targeted therapies capable to inhibit autoimmune inflammation of the CNS

N-acetylcysteine blocks SOS induction and mutagenesis produced by fluoroquinolones in Escherichia coli

[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)