TY - CONF A1 - Keshmiri, Hamid T1 - Bidiffractive leaky-mode biosensor N2 - This study details a thorough analysis of leaky and waveguide modes in biperiodic diffractive nanostructures. By tuning diffraction orders and subsequently confining local density of optical states at two distinct resonance wavelengths, we present a highly sensitive refractive index biosensing platform that can resolve 35.5 to 41.3 nm/RIU of spectral shift for two separate biological analytes. T2 - EMBL Symposium: Seeing is Believing - Imaging the Molecular Processes of Life CY - Heidelberg, Germany DA - 04.10.2023 KW - Optics PY - 2023 AN - OPUS4-59247 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kanaris, Orestis A1 - Sobisch, Lydia-Yasmin A1 - Gödt, Annett A1 - Schreiber, Frank A1 - Nordholt, Niclas T1 - Consequences of benzalkonium chloride tolerance for selection dynamics and de novo resistance evolution driven by antibiotics N2 - Biocides are used in large amounts in industrial, medical, and domestic settings. Benzalkonium chloride (BAC) is a commonly used biocide, for which previous research revealed that Escherichia coli can rapidly adapt to tolerate BAC-disinfection, with consequences for antibiotic susceptibility. However, the consequences of BAC tolerance for selection dynamics and resistance evolution to antibiotics remain unknown. Here, we investigated the effect of BAC tolerance in E. coli on its response upon challenge with different antibiotics. Competition assays showed that subinhibitory concentrations of ciprofloxacin—but not ampicillin, colistin and gentamicin—select for the BAC-tolerant strain over the BAC-sensitive ancestor at a minimal selective concentration of 0.0013–0.0022 µg/mL. In contrast, the BAC-sensitive ancestor was more likely to evolve resistance to ciprofloxacin, colistin and gentamicin than the BAC-tolerant strain when adapted to higher concentrations of antibiotics in a serial transfer laboratory evolution experiment. The observed difference in the evolvability of resistance to ciprofloxacin was partly explained by an epistatic interaction between the mutations conferring BAC tolerance and a knockout mutation in ompF encoding for the outer membrane porin F. Taken together, these findings suggest that BAC tolerance can be stabilized in environments containing low concentrations of ciprofloxacin, while it also constrains evolutionary pathways towards antibiotic resistance. KW - AMR KW - Resistance evolution KW - Resistance selection PY - 2026 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-653842 DO - https://doi.org/10.1038/s44259-025-00170-8 SN - 2731-8745 VL - 4 IS - 1 SP - 1 EP - 13 PB - Springer Science and Business Media LLC AN - OPUS4-65384 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kanaris, Orestis A1 - Schreiber, Frank T1 - Refuse in order to resist: metabolic bottlenecks reduce antibiotic susceptibility N2 - The growth of pathogenic bacteria in the host is a prerequisite for infectious diseases. Antibiotic drugs are used to impair bacterial growth and thereby treat infections. In turn, growth of bacteria is underpinned by their primary metabolism. Thus,it has long been recognized that the activity of antibiotics is determined by the metabolic state of cells. However, only recently researchers have begun to systematically interrogate the links between metabolism and resistance. In their recent study, Lubrano and colleagues (Lubranoet al, 2025) apply an elegant CRISPR-based approach to the model bacterium Escherichia coli to systematically screen the effect of 15,120 mutations in genes that encode for 346 proteins which are required for growth of E. coli (also referred to as ‘essential proteins’). The authors identified a multitude of mutations that reduce the susceptibility against two antibiotics related to two very distinct chemical classes; the β-lactam antibiotic carbenicillin and the aminoglycoside gentamicin. Strikingly, the majority of the identified mutations are directly linked to primary metabolism. The work highlights the importance of metabolism in order to understand antibiotic resistance mechanisms and the ecology and evolution of antibiotic resistance. In addition, the work provides leads to design metabolism-based intervention strategies to mitigate antibiotic resistance. KW - Metabolism KW - Antibiotic resistance PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-626608 DO - https://doi.org/10.1038/s44320-025-00089-2 SN - 1744-4292 VL - 21 IS - 3 SP - 211 EP - 213 AN - OPUS4-62660 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Metabolic niches and persistence of antibiotic resistant bacteria in the environment N2 - Background and Aim: Wastewater treatment plants are considered as hotspots for the development and spread of antimicrobial resistance. Antimicrobial resistant bacteria (ARB) can persist in the environment for long periods of time, despite metabolic fitness costs that can arise with resistance. We are interested in understanding the mechanisms, which allow ARB to persist in the environment. More specifically, we want to identify metabolic niches that can select for resistant bacteria. Procedure/Method: 62 E. coli strains isolated from different WWTPs in Norway were used. The isolates have different levels of resistance to 14 antibiotics. The susceptibility of the isolates to 3 disinfectants was determined. In addition, genome scale metabolic models (GEMs) were constructed and the growth of the strains was simulated in the presence of 198 different carbon sources. Furthermore, the growth rates of the isolates were measured in the presence of 3 carbon sources. Findings/Results: Many of the isolates have high resistance to multiple antibiotics but only few of the isolates have higher minimum inhibitory concentrations to the disinfectants, compared to an E. coli laboratory strain. With the GEMs, we identified 40 carbon sources that can be utilized for growth only by a portion of all the isolates. The prediction accuracy of the GEMs was 93% in the case of D-Malate. A group of 10 isolates was identified of which 5 isolates are resistant to ciprofloxacin, gentamicin and tetracycline and can grow on Sucrose but not on D-Malate and the other 5 isolates are susceptible to the same 3 antibiotics but can grow on D-Malate and not on Sucrose. Implications/Applications: Our data suggest that changing the available carbon source could shift the selection advantage between resistant and susceptible bacterial strains. If this strategy is confirmed experimentally, it could be applied to reduce the number of ARB in environments like wastewater. T2 - Conference on the Environmental dimentions of antimicrobial resistance 7 CY - Montreal, Canada DA - 26.05.2024 KW - Antimicrobial resistance KW - Wastewater PY - 2024 AN - OPUS4-61410 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Identification of metabolic niches and their association to the persistence of antibiotic resistant bacteria in wastewater N2 - Wastewater treatment plants (WWTP) are hotspots for the development and spread of antimicrobial resistance. Antimicrobial resistant bacteria (ARB) can persist in the environment for long periods of time, despite metabolic fitness costs that often arise with resistance. Recent research efforts are striving to uncover the role of bacterial metabolism for the ecology and evolution of antibiotic resistance. The aim of this study is to understand the ecological mechanisms, which allow ARB to persist in the environment. More specifically, we aim to identify metabolic niches that can select for and against resistant bacteria. 62 E. coli strains isolated from different WWTPs with different levels of resistance to 14 antibiotics and 3 disinfectants were assembled, sequenced, and phenotypically characterized. Next, genome scale metabolic models (GEMs) were constructed, and the growth of the strains was simulated in the presence of 298 different carbon sources. Furthermore, the growth rates of the isolates were measured in the presence of 3 carbon sources to verify the model predictions. Competition experiments with synthetic microbial communities consisting of a selection of 10 WWTP isolates, 5 of which were antibiotic resistant and 5 sensitive, were carried out in minimal medium with different carbon sources. Population dynamics modelling was used to simulate the competition of isolates under different conditions. The isolates have a wide range of susceptibility to the antibiotics, while disinfectants result in a narrower range of susceptibility. GEMs identified 40 carbon sources that can be utilized for growth only by a portion of all the isolates. The prediction accuracy of the GEMs was 93% in the case of D-malate. A range of WWTP isolates were identified which use D-malate as carbon source and are susceptible to specific antibiotics. In contrast, antibiotic-resistant WWTP isolates were identified that did use sucrose as carbon source but not D-malate. Competition experiments demonstrated that changing the carbon source of the medium from sucrose to D-malate resulted in selection against the resistant isolates. Modelling the competition between isolates under different conditions suggests that adding a carbon source to a bacterial community under specific conditions could exclude resistant bacteria from a microbial community. Our data suggest that changing the available carbon source could shift the selection advantage between resistant and susceptible bacterial strains. If this strategy is confirmed experimentally in complex microbial communities, it could be applied to reduce the number of ARB in environments such as wastewater. T2 - FEMS micro Konferenz CY - Milan, Italy DA - 14.07.2025 KW - AMR KW - Wastewater KW - Genome-scale metabolic model PY - 2025 AN - OPUS4-64659 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Metabolic niches and persistence of antibiotic resistant bacteria in the environment N2 - Background and Aim: Wastewater treatment plants are considered as hotspots for the development and spread of antimicrobial resistance. Antimicrobial resistant bacteria (ARB) can persist in the environment for long periods of time, despite metabolic fitness costs that can arise with resistance. We are interested in understanding the mechanisms, which allow ARB to persist in the environment. More specifically, we want to identify metabolic niches that can select for resistant bacteria. Procedure/Method: 62 E. coli strains isolated from different WWTPs in Norway were used. The isolates have different levels of resistance to 14 antibiotics. The susceptibility of the isolates to 3 disinfectants was determined. In addition, genome scale metabolic models (GEMs) were constructed and the growth of the strains was simulated in the presence of 198 different carbon sources. Furthermore, the growth rates of the isolates were measured in the presence of 3 carbon sources. Findings/Results: Many of the isolates have high resistance to multiple antibiotics but only few of the isolates have higher minimum inhibitory concentrations to the disinfectants, compared to an E. coli laboratory strain. With the GEMs, we identified 40 carbon sources that can be utilized for growth only by a portion of all the isolates. The prediction accuracy of the GEMs was 93% in the case of D-Malate. A group of 10 isolates was identified of which 5 isolates are resistant to ciprofloxacin, gentamicin and tetracycline and can grow on Sucrose but not on D-Malate and the other 5 isolates are susceptible to the same 3 antibiotics but can grow on D-Malate and not on Sucrose. Implications/Applications: Our data suggest that changing the available carbon source could shift the selection advantage between resistant and susceptible bacterial strains. If this strategy is confirmed experimentally, it could be applied to reduce the number of ARB in environments such as wastewater. T2 - International Biodeterioration and Biodegradation Symposium 19 CY - Berlin, Germany DA - 09.09.2024 KW - Antimicrobial resistance KW - Wastewater PY - 2024 AN - OPUS4-61412 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Consequences of benzalkonium chloride tolerance on the development of antibiotic resistance in E. coli N2 - Biocides are used in large amounts in industrial, medical, and domestic settings. Benzalkonium chloride (BAC) is a commonly used biocide, for which previous research revealed that Escherichia coli can rapidly adapt to tolerate BAC-disinfection, with consequences for antibiotic susceptibility. However, the consequences of BAC-tolerance for selection dynamics and resistance evolution to antibiotics remain unknown. Here, we investigated the effect of BAC-tolerance in E. coli on its response upon challenge with different antibiotics. Competition assays showed that subinhibitory concentrations of ciprofloxacin - but not ampicillin, colistin and gentamicin - select for the BAC-tolerant strain over the BAC-sensitive ancestor at a minimal selective concentration of 0.0013-0.0022 µg∙mL-1. In contrast, the BAC-sensitive ancestor was more likely to evolve resistance to ciprofloxacin, colistin and gentamicin than the BAC-tolerant strain when adapted to higher concentrations of antibiotics in a serial transfer laboratory evolution experiment. The observed difference in the evolvability of resistance to ciprofloxacin was partly explained by an epistatic interaction between the mutations conferring BAC-tolerance and a knockout mutation in ompF encoding for the outer membrane porin F. Taken together, these findings suggest that BAC-tolerance can be stabilized in environments containing low concentrations of ciprofloxacin, while it also constrains evolutionary pathways towards antibiotic resistance. T2 - µClub Seminar CY - Berlin, Germany DA - 23.05.2025 KW - Biocides KW - AMR KW - Resistance evolution PY - 2025 AN - OPUS4-64658 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Consequences of tolerance to disinfectants on the evolution of antibiotic resistance in E. coli N2 - Biocides are used as disinfectants and preservatives; one important active substance in biocides is benzalkonium chloride (BAC). BAC-tolerant bacterial strains can survive short treatments with high concentrations of BAC. BAC tolerance and resistance have been linked to antibiotic resistance. Here, the selection dynamics between a BAC-tolerant Escherichia coli strain and a sensitive wild type were investigated under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the BAC-tolerant strain was selected over the wild type at all ciprofloxacin concentrations investigated, with a minimum selection concentration (MSC) of 1/10th of the minimum inhibitory concentration (MIC) of the wild type. Furthermore, the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin was assessed by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type. The importance of these results is highlighted by the fact that concentrations of ciprofloxacin well above the calculated MSC can be found in environmental samples such as hospital wastewaters and livestock slurry. In turn, BAC is used as a disinfectant in the same settings. Thus, the selection of BAC-tolerant strains at sub-inhibitory concentrations of ciprofloxacin can contribute to the stabilization and spread of BAC-tolerance in natural populations. The prevalence of such strains can impair the effects of BAC disinfections. T2 - µClub seminar series CY - Berlin, Germany DA - 15.12.2023 KW - Antimicrobial resistance PY - 2023 AN - OPUS4-59222 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Consequences of benzalkonium chloride tolerance in Escherichia coli: Effects on selection and evolution in the presence of ciprofloxacin N2 - We investigated the selection dynamics between a benzalkonium chloride (BAC)-tolerant Escherichia coli strain (S4) and a sensitive wild type under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the opposite was observed at all ciprofloxacin concentrations investigated.Furthermore, we assessed the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type. T2 - 6th international symposium on the environmental dimention of antibiotic resistance-EDAR 6 CY - Gothenburg, Sweden DA - 22.09.2022 KW - Antimicrobial resistance KW - Tolerance KW - Experimental evolution KW - Selection PY - 2022 AN - OPUS4-56808 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie A1 - Schreiber, Frank T1 - Antibiotic tolerance of biofilms emerging fro multicellular effects of antibiotic efflux N2 - The overarching goal of this project is to develop a predictive model for efflux-mediated antimicrobial tolerance in bacterial multicellular assemblies. Our central hypostasis is that efflux pump activity causes emergent antibiotic tolerance of multicellular bacterial populations, through the interplay of efflux mediated spatial interactions and efflux-linked persistence. To test this hypothesis, we will use a combination of microscopy, microbial killing assays, computational modelling, and data analysis, integrating information from 3 types of multicellular assembly: colonies, cell-to-cell interactions in a monolayer microfluidic device, and 3D flow chamber biofilms. Building on our preliminary observations, we will experimentally characterize the link between colony structure and spatial patterns of efflux gene expression in strains that differ in their levels of efflux. We will develop a mathematical model to test whether local growth inhibition of neighbors due to effluxing cells, coupled with local environment-dependent regulation of efflux, can account qualitatively for these results. By including persister cell formation in our model we will predict, and measure, the emergent function of antimicrobial tolerance in our colonies. To fully understand how tolerance emerges from the interplay between efflux-mediated spatial interactions and efflux-linked persister cell formation, we need quantitative measurements at the single cell level. To this end, we will use a microfluidic setup with cells growing in a monolayer to qualify in detail the dependence of efflux expression and persister cell formation on nutrient conditions, the correlation between efflux and persister formation, and the spatial range of efflux-mediated neighbour growth inhibition. To predict and quantitatively understand the emergent multicellular function of tolerance, we will perform individual-based modelling of biofilm growth, using as input the parameters measured on the single-cell level with our microfluidics experiments. Our simulations will predict biofilm spatial structure development, patterns of efflux and persister formation and, ultimately, tolerance to antimicrobial challenge. These predictions will be directly tested in flow-cell biofilm experiments. We are currently generating acrAB-tolC knockout-strain, without efflux activity, and a strain with an inducible acrAB-tolC efflux pump. To distinguish the different strains under the microscope, they were labeled with genes encoding for different fluorescent proteins. All strains are currently characterized in terms of growth, minimum inhibitory concentration of different antimicrobial substances, colony morphology, and biofilm formation ability. On the theoretical side, we are currently working on modeling the system at various scales and degree of detail, ranging from coarse-grained continuum models to stochastic, individual-based models. Some exploratory work was doe to test existing software for individual-based modelling that may be adapted for our purpose. Furthermore, we are in the process of developing more coarse-grained models. This work involves some physiological modelling and literature search, focusing on working mechanisms of efflux pumps and kinetic models for import and export of antibiotics. T2 - SPP Meeting CY - Jena, Germany DA - 04.10.2023 KW - Antibiotic KW - Bioilm KW - Tolerance KW - Efflux PY - 2023 AN - OPUS4-59245 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie T1 - Antibiotic tolerance of biofilms emerging from multicellular effects of antibiotic efflux N2 - Biofilms are multicellular assemblies of bacteria living in a self-produced extracellular matrix. One characteristic of biofilms is that they are difficult to kill. Different mechanisms, like the development of persister cells or efflux pumps which pump some antimicrobials out of the cell, make them tolerant. Our central hypothesis is that efflux pump activity causes emergent antimicrobial tolerance of multicellular bacterial populations, through the interplay of efflux-mediated spatial interactions and efflux-linked persistence. To verify the hypothesis, we combine computational modelling with information gained from 3 types of multicellular assemblies. We are currently generating strains that differ in their levels of efflux activity, mixes are then cultivated together in the 3 model systems. In colonies the link between structure and spatial patterns of gene expression will be characterized. Using a microfluidic device, the interactions range of efflux as a response to different antimicrobials will be determined. In a flow chamber a 3D biofilm will be generated, to investigate the biofilm development over time and persister cell formation. All results will be compared with model predictions. T2 - EuroBioFilms2024 CY - Copenhagen, Denmark DA - 25.06.2024 KW - Antibiotic KW - Biofilm KW - Tolerance KW - Efflux PY - 2024 AN - OPUS4-61277 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie T1 - Antibiotic tolerance of biofilms emerging from multicellular effects of antibiotic efflux N2 - Efflux pumps play an important role in the context of antimicrobial resistance, which is the ability to grow in the presence of antimicrobials. Many of these transporters can be categorized into multidrug efflux pumps, extruding various antimicrobials out of the cells, and thereby leading to antimicrobial resistance. It has been shown that efflux pumps can be linked by global regulators that regulate efflux pump expression affecting cell-to-cell-interactions, membrane integrity and biofilm formation. The aim of this study is to investigate the survival of cells in biofilms upon exposure to antimicrobials through the interplay of efflux-mediated spatial interactions . To this end, we generated fluorescently labeled E. coli strains that differ in their levels of AcrAB-TolC efflux pump activity and an acrB knockout-strain. The strains were characterized in terms of their antimicrobial susceptibility of three antibiotics, tetracycline, kanamycin, ampicillin, and the biocide benzalkonium chloride. The knockout strain shows higher susceptibility than the wildtype strain, with highest difference observed upon exposure to benzalkonium chloride. The results were confirmed with an efflux activity assay, which showed decreased efflux for the knockout strain as compared to the wildtype. Interestingly, adding the efflux inhibitor PAβN at intermediate concentrations induced bimodality in efflux activity in the wildtype. To investigate the link between colony structure and spatial patterns of efflux pump gene expression, strains with different fluorescent labels and efflux activity were mixed in a 1:1 ratio and grown on agar supplemented with antimicrobials at sub-inhibitory concentrations. Analysis of the colonies with fluorescence microcopy shows that the absence of the AcrAB efflux pump affects the structure of sector formation and morphology within the colony. We observed relatively large sectors with similar surface area for high efflux and low efflux cells in the absence of antimicrobials and at low concentrations. In contrast, sectors are disappearing due a strong intermixing of high and low efflux strains with increasing antibiotic concentration, specifically upon exposure to tetracycline and kanamycin. As next steps, we will perform quantitative analysis of colony images, to better interpret the results, develop a mathematical model of interacting cell types and investigate efflux-based interactions at the single-cell level. Taken together, out data suggest that efflux shapes cell-to-cell interactions and these interactions affect the spatial arrangement and the morphology of biofilms. Understanding the dynamics can provide insights into the emergence of resistance of bacterial communities to antimicrobial environments, the complex interplay of resistance, efflux, and biofilm formation, which potentially gives information to combat biofilm resistance. T2 - Multi-Drug Efflux Systems Gordon Research Conference CY - Lucca, Italy DA - 26.04.2025 KW - Antimicrobial KW - Resistance KW - Tolerance KW - Efflux KW - E. coli PY - 2025 AN - OPUS4-64561 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie T1 - Antibiotic tolerance of biofilms emerging from multicellular effects of antibiotic efflux N2 - Biofilms are multicellular assemblages of bacteria living in a self-produced extracellular matrix. Different mechanisms, like the development of highly tolerant persister cells or increased expression of efflux pumps make them tolerant. Here we want to investigate the emergency of antimicrobial tolerance of multicellular bacterial populations, through the interplay of efflux-mediated spatial interactions and efflux-linked persistence. To this end, we are combining computational modelling with experimentally observations gained from three types of multicellular assemblages, i.e. colonies on agar, multicellular populations grown in a monolayer microfluidic device, and 3D biofilms grown in flow chambers. We generated fluorescently labeled E. coli strains that differ in their levels of AcrAB-TolC efflux pump activity, an acrB knockout-strain and a strain with inducible expression of acrAB. All strains were characterized in terms of their antimicrobial susceptibility of three antibiotics, tetracycline, kanamycin, ampicillin, and the biocide benzalkonium chloride. The knockout strain shows higher susceptibility than the wild type strain, with highest difference when using benzalkonium chlorid e. To investigate the link between colony structure and spatial patterns of gene expression, the strains were mixed equimolar and grown on agar supplemented with antimicrobials. First results show, antimicrobials affect the structure of sector formation and morphology. Cells grown on tetracycline agar show a more finer sector formation. While kanamycin changes the overall colony structure . We will develop a mathematical model and additional experiments with multicellular assemblages to explain the observed interactions and extrapolate the results to more realistic biofilm models. T2 - SPP Conference CY - Berlin, Germany DA - 06.01.2025 KW - Antimicrobial KW - Resistance KW - Tolerance KW - Efflux KW - E. coli PY - 2025 AN - OPUS4-64563 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie T1 - Antibiotic tolerance of biofilms emerging from multicellular effects of antibiotic efflux N2 - Biofilms are multicellular assemblies of bacteria living in a self-produced extracellular matrix. One characteristic of biofilms is that they are difficult to kill. Different mechanisms, like the development of persister cells or efflux pumps which pump some antimicrobials out of the cell, make them tolerant. Our central hypothesis is that efflux pump activity causes emergent antimicrobial tolerance of multicellular bacterial populations, through the interplay of efflux-mediated spatial interactions and efflux-linked persistence. To verify the hypothesis, we combine computational modelling with information gained from 3 types of multicellular assemblies. We are currently generating strains that differ in their levels of efflux activity, mixes are then cultivated together in the 3 model systems. In colonies the link between structure and spatial patterns of gene expression will be characterized. Using a microfluidic device, the interactions range of efflux as a response to different antimicrobials will be determined. In a flow chamber a 3D biofilm will be generated, to investigate the biofilm development over time and persister cell formation. All results will be compared with model predictions. T2 - UNA Workshop CY - Berlin, Germany DA - 29.01.2024 KW - Antibiotic KW - Biofilm KW - Tolerance KW - Efflux PY - 2024 AN - OPUS4-61280 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Huang, Yanliang A1 - Liu, Xiangju A1 - Zhang, Qichao A1 - Xu, Yong A1 - Kunte, Hans-Jörg A1 - De Marco, Roland T1 - Hydrogen release from carbon steel in chloride solution under anodic polarization N2 - The hydrogen permeation current increase was noticed for carbon steel in 0.5 mol/L NaCl solution under strong anodic potentials, which is contrary to the common understanding. Hydrogen permeation under cathodic potentials has been widely studied because of possible hydrogen embrittlement failures of high strength steels in seawater, but investigations of anodic polarization on hydrogen permeation are fairly rare, as the hydrogen evolution reaction shall be retarded. To corroborate the observed phenomenon, experiments were conducted using both as-received and vacuum-annealed sheet specimens. It was verified that the observed phenomena originated from the released hydrogen in traps by metal dissolution under anodic polarization. KW - Energy Engineering and Power Technology KW - Condensed Matter Physics KW - Fuel Technology KW - Renewable Energy, Sustainability and the Environment PY - 2020 DO - https://doi.org/10.1016/j.ijhydene.2019.11.218 SN - 1879-3487 VL - 45 IS - 4 SP - 3307 EP - 3315 PB - Elsevier BV CY - New York, NY AN - OPUS4-59322 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hobmeier, K. A1 - Oppermann, M. A1 - Stasinski, N. A1 - Kremling, A. A1 - Pflüger-Grau, K. A1 - Kunte, Hans-Jörg A1 - Marin Sanguino, A. T1 - Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches N2 - The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13. KW - Osmoadaptation KW - Metabolic engineering PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-555644 DO - https://doi.org/10.3389/fmicb.2022.968983 SN - 1664-302X VL - 13 SP - 1 EP - 13 PB - Frontiers Media CY - Lausanne AN - OPUS4-55564 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hobmeier, K. A1 - Goëss, M. C. A1 - Sehr, C. A1 - Schwaminger, S. A1 - Berensmeier, S. A1 - Kremling, A. A1 - Kunte, Hans-Jörg A1 - Pflüger-Grau, K. A1 - Marin-Sanguino, A. T1 - Anaplerotic Pathways in Halomonas elongata: The Role of the Sodium Gradient N2 - Salt tolerance in the γ-proteobacterium Halomonas elongata is linked to its ability to produce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. Ectoine belongs to the biosynthetic family of aspartate-derived amino-acids. Aspartate is formed from oxaloacetate, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the tricarboxylic acid cycle (TCA cycle). This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work, we combine modeling and experiments to analyze how these different needs shape the anaplerotic reactions in H. elongata. First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic enzymes: phosphoenolpyruvate carboxylase (Ppc) and oxaloacetate decarboxylase (Oad) are experimentally characterized. The results show that the anaplerotic reactions in H. elongata are indeed subject to evolutionary pressures that differ from those faced by other gram-negative bacteria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in H. elongata is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role in vivo. Ppc is necessary for H. elongata to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of oxaloacetate for biosynthetic reactions. KW - Metabolic flux analysis KW - Halophilic bacteria KW - Halomonas elongata KW - Metabolic modeling PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-513124 DO - https://doi.org/10.3389/fmicb.2020.561800 VL - 11 SP - 561800 AN - OPUS4-51312 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hobmeier, K. A1 - Cantone, M. A1 - Nguyen, Q. A. A1 - Pflüger-Grau, K. A1 - Kremling, A. A1 - Kunte, Hans-Jörg A1 - Pfeiffer, F. A1 - Marin-Sanguino, A. T1 - Adaptation to varying salinity in Halomonas elongata: Much more than ectoine accumulation N2 - The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed. KW - Ectoine KW - Osmoadaptation PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545608 DO - https://doi.org/10.3389/fmicb.2022.846677 SN - 1664-302X VL - 13 SP - 1 EP - 19 PB - Frontiers Media CY - Lausanne AN - OPUS4-54560 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - He, Zhiming A1 - Dechesne, Arnaud A1 - Schreiber, Frank A1 - Zhu, Yong-Guan A1 - Larsson, Joakim A1 - Smets, Barth T1 - Understanding Stimulation of Conjugal Gene Transfer by Nonantibiotic Compounds: How Far Are We? N2 - A myriad of nonantibiotic compounds is released into the environment, some of which may contribute to the dissemination of antimicrobial resistance by stimulating conjugation. Here, we analyzed a collection of studies to (i) identify patterns of transfer stimulation across groups and concentrations of chemicals, (ii) evaluate the strength of evidence for the proposed mechanisms behind conjugal stimulation, and (iii) examine the plausibility of alternative mechanisms. We show that stimulatory nonantibiotic compounds act at concentrations from 1/1000 to 1/10 of the minimal inhibitory concentration for the donor strain but that stimulation is always modest (less than 8-fold). The main proposed mechanisms for stimulation via the reactive oxygen species/SOS cascade and/or an increase in cell membrane permeability are not unequivocally supported by the literature. However, we identify the reactive oxygen species/SOS cascade as the most likely mechanism. This remains to be confirmed by firm molecular evidence. Such evidence and more standardized and high-throughput conjugation assays are needed to create technologies and solutions to limit the stimulation of conjugal gene transfer and contribute to mitigating global antibiotic resistance. KW - Antibiotic resistance KW - Horizontal gene transfer KW - Conjugation KW - Chemicals PY - 2024 DO - https://doi.org/10.1021/acs.est.3c06060 SP - 1 EP - 14 PB - ACS Publications AN - OPUS4-60105 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Franziska, Pietsch A1 - Nordholt, Niclas A1 - Heidrich, Gabriele A1 - Schreiber, Frank T1 - Prevalent Synergy and Antagonism Among Antibiotics and Biocides in Pseudomonas aeruginosa N2 - Antimicrobials can exert specific physiological effects when used in combination that are different from those when applied alone. While combination effects have been extensively mapped for antibiotic-antibiotic combinations, the combination effects of antibiotics with antimicrobials used as biocides or antiseptics have not been systematically investigated. Here, we investigated the effects of combinations of antibiotics (meropenem, gentamicin, and ciprofloxacin) and substances used as biocides or antiseptics [octenidine, benzalkonium chloride, cetrimonium bromide, chlorhexidine, Povidone-iodine, silver nitrate (AgNO3), and Ag-nanoparticles] on the planktonic growth rate of Pseudomonas aeruginosa. Combination effects were investigated in growth experiments in microtiter plates at different concentrations and the Bliss interaction scores were calculated. Among the 21 screened combinations, we find prevalent combination effects with synergy occurring six times and antagonism occurring 10 times. The effects are specific to the antibiotic-biocide combination with meropenem showing a tendency for antagonism with biocides (6 of 7), while gentamicin has a tendency for synergy (5 of 7). In conclusion, antibiotics and biocides or antiseptics exert physiological combination effects on the pathogen P. aeruginosa. These effects have consequences for the efficacy of both types of substances and potentially for the selection of antimicrobial resistant strains in clinical applications with combined exposure (e.g., wound care and coated biomaterials). KW - Synergy KW - Antagonism KW - Suppression KW - Biocides KW - Antibiotics KW - Pseudomonas aeruginosa PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-520845 DO - https://doi.org/10.3389/fmicb.2020.615618 VL - 11 SP - Article 615618 PB - Frontiers CY - Lausanne AN - OPUS4-52084 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -