TY - CONF A1 - Schulz, Anne-Christine T1 - Assessing co-selection of biocide and antibiotic resistance in wastewater microbial communities N2 - Biocides are used for a wide range of purposes, including disinfectants or preservatives. Biocides play a major role in the prevention of microbial infections in healthcare and animal husbandry. The use of biocides often leads to the discharge of active biocidal substances into wastewater streams, causing the exposure of wastewater microbial communities to subinhibitory concentrations. In turn, it is known that wastewater treatment plants (WWTP) are hotspots for antibiotic resistant bacteria. Since similar mechanisms confer resistance to biocides and antibiotics, exposure to biocides can result in co-selection of antibiotic resistant bacteria in WWTP due to biocides. Here, we investigate the magnitude and the drivers of co-selection of antibiotic resistance in natural wastewater microbial communities upon biocide exposure. Microbial communities will be sampled at the WWTP Ruhleben in Berlin and characterized regarding their biocide and antibiotic resistance. Changes in the resistance level after exposure to different biocides will be determined by enumerating resistant and non-resistant E. coli and heterotrophic bacteria on selective plates with and without several biocides and antibiotics. Moreover, we are establishing a synthetic community comprising about 100 environmental E. coli isolates each with different antimicrobial resistance traits. Each isolate will be tagged with a unique DNA-barcode. All isolates will be pooled and exposed to different biocides at various concentrations. The barcode labeling enables us to determine the abundance of each isolate at the beginning and end of the experiment by transposon-tag sequencing. The project results will inform risk assessment of the effects of biocidal residues on antimicrobial resistance selection in WWTP. The project is part of the BIOCIDE consortium funded within the call on Aquatic pollutants by JPI-AMR, JPI-OCEANS and JPI-WATER. T2 - 6th International Symposium onn the Environmental Dimension of Antibiotic Resistance - EDAR 6 CY - Gothenburg, Sweden DA - 22.09.2022 KW - Antibiotic resisitance KW - Biocide KW - Wastewater KW - Risk assesment PY - 2022 AN - OPUS4-56796 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Evolution and mechanistic basis of disinfectant tolerance in E. coli N2 - Disinfectants are important to provide hygiene in sensitive areas, to prevent the spread of infections and to preserve materials from biodeterioration. Bacteria can survive disinfection through phenotypic and genotypic adaptation. Phenotypic heterogeneity may be linked to the ability to evolve disinfectant tolerance. The genetic factors which determine the survival of disinfection remain largely unknown. Here, we investigate the effects of phenotypic heterogeneity on the evolvability of disinfectant tolerance. Furthermore, using a whole-genome CRISPRi-library, we uncover genetic determinants that are important for the survival of disifenction. T2 - µClub Seminar Berlin CY - Berlin, Germany DA - 23.05.2025 KW - Biocides KW - Heterogeneity KW - Biocide resistance KW - Evolution KW - Disinfectants PY - 2025 AN - OPUS4-63833 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 - 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 - Schmidt, Selina T1 - Biocides Used as Material Preservatives Modify Rates of de novo Mutation and Horizontal Gene Transfer in Bacteria N2 - Biocides used as material preservatives are in contact with various environments during direct application or passive leaching from protected materials. Antimicrobial resistance (AMR) is a global health problem and the environment is an important compartment for the evolution and transmission of AMR. Soil is an environment with a large reservoir of natural microbial communities and antimicrobial resistance (AMR) genes. Those natural microbial communities are frequently exposed to biocides used as material preservatives. Previous studies have shown that antibiotics, metals and pesticides affect the underlying processes of resistance evolution and spread; namely de novo mutagenesis and horizontal gene transfer by conjugation and transformation in microbial communities. However, it is unknown if active substances used in material preservatives are involved in these processes. We show that biocides used as material preservatives affect rates of mutation and conjugation in microorganism in a species- and substance-dependent manner, while rates of transformation are not directly affected. Our data highlights the importance of assessing the contribution of material preservatives on AMR evolution and spread in the environment. T2 - Microbiome Network Meeting CY - Berlin, Germany DA - 20.07.2022 KW - Biocides KW - Horizontal gene transfer KW - Mutation rate PY - 2022 AN - OPUS4-55263 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Heterogeneous tolerance to biocides and its consequences for the evolution of antimicrobial resistance N2 - The exposure to antimicrobial substances drives the evolution of antimicrobial resistance (AMR). Biocides are antimicrobials used as disinfectants, antiseptics and preservatives. They find application on a large scale in the industrial and medical sector, but also in private households. In terms of mass, the worldwide use of biocides exceeds that of antibiotics. Thus, despite their important role in preventing the spread of pathogens, due to their ubiquity, biocides are suspected to be drivers of the antimicrobial resistance crisis. In our work we try to understand how biocides contribute to the emergence of AMR, what the underlying adaptation principles and mechanisms are and how they compare to those found for antibiotics. Here, I present results which demonstrate that phenotypic heterogeneity in E. coli, namely the formation of tolerant persister cells, can enable the survival of disinfection and consequentially facilitate the evolution of AMR. Using experimental evolution that selects for tolerance, we find that mutations in lipid A biosynthesis arise following periodic treatment with benzalkonium chloride (BAC), a widely used disinfectant. BAC tolerance is associated with additional, diverse mutations as well as changes in the susceptibility to antibiotics from different classes and changes in motility and biofilm formation, suggesting extensive regulatory re-wiring in the evolved clones. Remarkably, we find that fitness costs (growth rate reduction) incurred by BAC tolerance are alleviated in the presence of antibiotics. These findings demonstrate the complexity underlying the adaptation to antimicrobials and highlight the links between persistence to disinfectants and resistance evolution to antimicrobials. T2 - 4th VAAM discussion meeting 'Microbial Cell Biology' CY - Berlin, Germany DA - 09.10.2022 KW - Persistence KW - Biocides KW - Evolution KW - Disinfection KW - Biocide tolerance KW - Heterogeneity PY - 2022 AN - OPUS4-55958 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 - Schmidt, Selina T1 - Bacterial resistance evolution towards disinfectants and antimicrobial surfaces and development of a standardized test N2 - Question Disinfectants and antimicrobial surfaces (AMCs) are important tools to prevent the spread of pathogens and antimicrobial resistant bacteria. However, concerns have been raised about the possibility for the evolution and selection of resistance against disinfectants and AMCs. In turn, resistance against disinfectants and AMCs can be associated to antibiotic resistance due to cross-resistance - a single mechanism conferring resistance to a disinfectant and an antibiotic- and co-resistance - two distinct mechanisms physically linked on e.g. a plasmid. The risk for resistance and cross-resistance during use of biocides (including disinfectants and AMCs) must be evaluated during authorization according to the EU biocidal product regulation. However, to date there is a lack of standardized methods that support risk assessment during the authorization process. Methods We used adaptive laboratory evolution (ALE) experiments which are based on repeated exposure of bacteria to disinfectants or AMCs. The experiments are followed by phenotypic (antimicrobial susceptibility testing) and genotypic (whole genome sequencing) characterization of the evolved strains. The basic idea of these experiments is to expose bacteria to lethal conditions and select for mutants with increased survival. This approach is fundamentally different to other ALE experiments, which commonly select for increased growth at subinhibitory concentrations. However, selection for increased survival represents a selective pressure that more realistically reflects selection under in-use conditions of disinfectants and AMCs. Results First, we studied adaptation of E. coli during repeated disinfection with benzalkonium chloride in a suspension assay. The experiments showed a 2000-fold increase in survival within 5 exposure cycles. The adaptive changes are linked to highly parallel mutations in genes related to lipid A biosynthesis, less negative cell surface charge, reduced growth rate and increased competitive ability in the presence of certain antibiotics. We use the same approach to develop standardizable ALE experiments based upon accepted standards that are used to determine the efficacy of disinfectants (EN 13697) and antimicrobial surfaces (ISO 22196). The results highlight pronounced adaptation of different test strains towards surface disinfection (benzalkonium chloride and isopropanol) and AMCs (copper). Conclusion Bacteria can adapt with increased survival towards lethal stress imposed by disinfectants and AMCs. The adaptive ability of bacteria to disinfectants and AMCs can be determined in a standardized manner. T2 - 74. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie [DGHM] e. V. CY - Berlin, Germany DA - 05.09.2022 KW - Antimicrobial surfaces KW - ISO22196 KW - Antimicrobial resistance KW - Round robin test PY - 2022 AN - OPUS4-56432 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Phenotypic and evolutionary responses of bacteria to disinfection N2 - Disinfectants are important to prevent the transmission of pathogens, especially in the face of the current antibiotic resistance crisis. The crisis is further exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfectant application, persistence to disinfectants and its role for the evolution of tolerance and cross-resistance to antibiotics has not been studied. Our work shows that E. coli displays persistence against several widely used disinfectants, including benzalkonium chloride (BAC), didecyldimethylammoniumchlorid (DDAC) and isopropanol. The molecular mechanism of BAC persistence is triggered in stationary phase and affected by several antibiotic persister genes (hipA, tisB, tolC, relA, spoT). Experimental evolution and population dynamic modeling show that repeated failure of disinfection due to persisters rapidly selects for BAC tolerance underpinned by reduced cell surface charge due to mutations in genes related to lipid A acylation (lpxML). Furthermore, evolved BAC tolerance affects the susceptibility to antibiotics, leading to positive selection of disinfectant tolerant strains at environmentally relevant antibiotic concentrations and variations in evolvability of antibiotic resistance due to epistatic effects. These results highlight the need for faithful application of disinfectants to steward their efficacy and the efficacy of antibiotics. A better understanding of the bacterial response to disinfectants is crucial to understand and avert the ongoing antimicrobial resistance crisis. T2 - Gordon Research Conference - Molecular mechanisms of evolution CY - Easton, MA, United States DA - 25.06.2023 KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2023 AN - OPUS4-57861 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - The disinfectant glutaraldehyde induces antibiotic tolerance N2 - Glutaraldehyde is widely used as a disinfectant and preservative, but little is known about its effects on bacterial susceptibility to antibiotics and the selection of tolerant phenotypes. We found that short-term exposure to sub-inhibitory levels of glutaraldehyde makes E. coli resistant to high doses of bactericidal antibiotics from different classes. This tolerance is associated with delayed, heterogeneous regrowth dynamics and global transcriptome remodeling. We identified over 1200 differentially expressed genes, including those related to antibiotic efflux, metabolic processes, and the cell envelope. The cells entered a disrupted state likely due to the unspecific mode-of-action of glutaraldehyde. Despite this unregulated response, we identified several differentially expressed genes not previously associated with antibiotic tolerance or persistence that induce antibiotic tolerance when overexpressed alone. These findings highlight how the unspecific mode-of-action of disinfectants can make bacteria temporarily resistant to antibiotics. They have implications for settings where disinfectants and antibiotics are used in close proximity, such as hospitals and animal husbandry, and for the selection dynamics of tolerant pheno- and genotypes in fluctuating environments where microorganisms are exposed to these substances, such as sewage systems. A trade-off arises from overcoming the disrupted state as quickly as possible and maintaining antibiotic tolerance. T2 - Gordon Research Conference - Molecular Mechanisms in Evolution CY - Easton, Massachusetts, United States DA - 25.06.2023 KW - Glutaraldehyde KW - Biocides KW - Tolerance KW - Bacteria KW - Disinfection KW - Heterogeneity KW - Antibiotics PY - 2023 AN - OPUS4-58033 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -