Transcriptional responses of Candida glabrata biofilm cells to fluconazole are modulated by the carbon source

The datasets generated in this study are available at public repositories or from the corresponding author upon request. The raw RNA-seq data in fastq format, as well as the processed data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE121074.Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata, with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections.This study was supported by the Portuguese National Funding Agency for Science, Research, and Technology FCT (grant PTDC/BIAMIC/5184/2014). R.A. received FCT Ph.D fellowship (PD/BD/113813/2015). We gratefully acknowledge Edinburgh Genomics for RNA-Seq library preparation and sequencing. The work on CBMA was supported by the strategic programs UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and UID/BIA/04050/2019. The work on CEB was supported by PEst-OE/EQB/LA0023/2013, from FCT, “BioHealth-Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2–O Novo Norte), QREN, FEDER and the project “Consolidating Research Expertize and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, Ref. FCOMP-01-0124-FEDER-027462. The work in Aberdeen was also supported by the European Research Council through the advanced grant “STRIFE” (C-2009-AdG-249793), by the UK Medical Research Council (MR/M026663/1) and by the Medical Research Council Center for Medical Mycology and the University of Aberdeen (MR/N006364/1). The work at KU Leuven was supported by the Federation of European Biochemical Societies (FEBS) through a short-term fellowship awarded to R.A. and by the Fund for Scientific Research Flanders (FWO; WO.009.16N). We thank Beatriz Herrera-Malaver for technical assistance with the GC-MS analysis.info:eu-repo/semantics/publishedVersio

Broth microdilution protocol for determining antimicrobial susceptibility of Legionella pneumophila to clinically relevant antimicrobials

: Currently there is no detailed, internationally agreed protocol defined to evaluate antimicrobial susceptibility testing (AST) for Legionella pneumophila (required to establish epidemiological cut-off value or "ECOFF" boundaries); therefore, antimicrobial resistance in these isolates cannot be defined. AST methods utilising media containing activated charcoal as an ingredient, to enable Legionella growth, are unreliable as noted in an internationally authored opinion paper and a new gold standard is required. Here we define a detailed protocol for broth microdilution (BMD) using defined cell culture collection-deposited control reference strains (Philadelphia-1 and Knoxville-1) as well as two accessible reference strains with moderately (lpeAB-carrying) and markedly (23S rRNA mutation-carrying) elevated azithromycin minimum inhibitory concentration (MIC). The defined protocol enables up to eight L. pneumophila strains to be set up on a single 96-well plate per antimicrobial tested. Initial ranges to routinely capture an MIC for these reference strains using clinically relevant antimicrobials azithromycin (0.01-0.25 mg/L), levofloxacin (0.008-0.03 mg/L), lefamulin (0.01-2 mg/L), rifampicin (0.0002-0.0008 mg/L) and doxycycline (0.25-16 mg/L) following incubation for 48 h at 37 °C in a shaking incubator have been empirically determined. Establishment of this internationally agreed protocol sets the scene for the next step: validation and comparison of antimicrobial ranges between international Legionella reference laboratories to establish putative resistance cut-off thresholds for these clinically relevant antimicrobials

External quality assessment of SARS-CoV-2-sequencing: An ESGMD-SSM pilot trial across 15 European laboratories

Objective: This first pilot on external quality assessment (EQA) of SARS-CoV-2 whole genome sequencing, initiated by the ESCMID Study Group for Genomic and Molecular Diagnostics (ESGMD) and Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing.Methods: Ten samples with varying viral loads were sent out to 15 clinical laboratories who had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centres were compared on were identification of 1) SNPs and indels, 2) Pango lineages, and 3) clusters between samples.Results: The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to varying depth (up to 100-fold difference across centres). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignment. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data.Conclusions: The pilot EQA was an overall success. It was able to show the high quality of participating labs and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.</p

Genotypic resistance determined by whole genome sequencing versus phenotypic resistance in 234 Escherichia coli isolates

Abstract Whole genome sequencing (WGS) enables detailed characterization of bacteria at single nucleotide resolution. It provides data about acquired resistance genes and mutations leading to resistance. Although WGS is becoming an essential tool to predict resistance patterns accurately, comparing genotype to phenotype with WGS is still in its infancy. Additional data and validation are needed. In this retrospective study, we analysed 234 E. coli isolates from positive blood cultures using WGS as well as microdilution for 11 clinically relevant antibiotics, to compare the two techniques. We performed whole genome sequencing analyses on 234 blood culture isolates (genotype) to detect acquired antibiotic resistance. Minimal inhibitory concentrations (MIC) for E. coli were performed for amoxicillin, cefepime, cefotaxime, ceftazidime, meropenem, amoxicillin/clavulanic acid, piperacillin/tazobactam, amikacin, gentamicin, tobramycin, and ciprofloxacin, using the ISO 20776-1 standard broth microdilution method as recommended by EUCAST (phenotype). We then compared the two methods for statistical ‘agreement’. A perfect (100%) categorical agreement between genotype and phenotype was observed for gentamicin and meropenem. However, no resistance to meropenem was observed. A high categorical agreement (> 95%) was observed for amoxicillin, cefepime, cefotaxime, ceftazidime, amikacin, and tobramycin. A categorical agreement lower than 95% was observed for amoxicillin/clavulanic acid, piperacillin/tazobactam, and ciprofloxacin. Most discrepancies occurred in isolates with MICs within ± 1 doubling dilution of the breakpoint and 22.73% of the major errors were samples that tested phenotypically susceptible at higher antibiotic exposure and were therefore considered as ‘not resistant’. This study shows that WGS can be used as a valuable tool to predict phenotypic resistance against most of the clinically relevant antibiotics used for the treatment of E. coli bloodstream infections

Genotypic resistance determined by whole genome sequencing versus phenotypic resistance in 234 Escherichia coli isolates

AbstractWhole genome sequencing (WGS) enables detailed characterization of bacteria at single nucleotide resolution. It provides data about acquired resistance genes and mutations leading to resistance. Although WGS is becoming an essential tool to predict resistance patterns accurately, comparing genotype to phenotype with WGS is still in its infancy. Additional data and validation are needed. In this retrospective study, we analysed 234 E. coli isolates from positive blood cultures using WGS as well as microdilution for 11 clinically relevant antibiotics, to compare the two techniques. We performed whole genome sequencing analyses on 234 blood culture isolates (genotype) to detect acquired antibiotic resistance. Minimal inhibitory concentrations (MIC) for E. coli were performed for amoxicillin, cefepime, cefotaxime, ceftazidime, meropenem, amoxicillin/clavulanic acid, piperacillin/tazobactam, amikacin, gentamicin, tobramycin, and ciprofloxacin, using the ISO 20776-1 standard broth microdilution method as recommended by EUCAST (phenotype). We then compared the two methods for statistical ‘agreement’. A perfect (100%) categorical agreement between genotype and phenotype was observed for gentamicin and meropenem. However, no resistance to meropenem was observed. A high categorical agreement (&gt; 95%) was observed for amoxicillin, cefepime, cefotaxime, ceftazidime, amikacin, and tobramycin. A categorical agreement lower than 95% was observed for amoxicillin/clavulanic acid, piperacillin/tazobactam, and ciprofloxacin. Most discrepancies occurred in isolates with MICs within ± 1 doubling dilution of the breakpoint and 22.73% of the major errors were samples that tested phenotypically susceptible at higher antibiotic exposure and were therefore considered as ‘not resistant’. This study shows that WGS can be used as a valuable tool to predict phenotypic resistance against most of the clinically relevant antibiotics used for the treatment of E. coli bloodstream infections.</jats:p

Backyard running : pushing the boundaries of human performance

Abstract: Ultrarunning is gaining in popularity but no information is available on the physiological and psychological responses during backyard ultrarunning events. The aim of this study was to determine changes in cognitive function, markers of physiological resilience, and running performance during a backyard-running event. Twelve male ultrarunners (38 +/- 8 years old, BMI: 23.5 +/- 1.6 kg/m(2), and VO2max: 60.8 +/- 4.7 mL/min/kg) were monitored before, during, and after the event. Cognitive performance was determined using a cognitive test battery before, during, and after the event. During the event, the rating of perceived exertion (RPE), blood lactate concentration, and heart rate (HR) were assessed. Physical performance was investigated using the total number of completed laps and running speed per lap. Athletes completed 34 +/- 17 laps equaling 227.8 +/- 113.9 km with average speeds starting at 9.0 km/h and slowing down to 7.5 km/h at the end of the event. Physiological resilience (estimated using HR/speed) varied between athletes, with significantly lower values in the more proficient backyard runners at the end of the event (p < 0.05). HR and lactate levels remained constant, whereas a progressive increase in RPE was noticed (p <= 0.001). A significantly worsened reaction time was observed for several cognitive tasks after the event compared to baseline measures (p <= 0.05). These observations show that physiological resilience differs depending on the level of endurance performance of the athletes. Furthermore, the backyard ultrarunning event negatively impacted psychomotor speed. Therefore, the results suggest that implementing strategies that enhance physiological resilience and/or psychomotor speed could potentially have a positive effect on performance in ultraendurance activities

Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed

The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-) ) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges. J. Comp. Neurol., 2015. © 2015 Wiley Periodicals, Inc.status: publishe

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP’s). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP’s is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1’s internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin

Transcriptional responses of Candida glabrata biofilm cells to fluconazole are modulated by the carbon source

AbstractCandida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata, with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections.</jats:p