TY - CONF A1 - Schreiber, Frank A1 - Nordholt, Niclas A1 - Kanaris, Orestis A1 - Schmidt, Selina A1 - Lewerenz, Dominique 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 - JOUR A1 - Nordholt, Nicolas A1 - Kanaris, Orestis A1 - Schmidt, Selina A1 - Schreiber, Frank T1 - Persistence against benzalkonium chloride promotes rapid evolution of tolerance during periodic disinfection N2 - Biocides used as disinfectants are important to prevent the transmission of pathogens, especially during the current antibiotic resistance crisis. This crisis is exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic Treatment and facilitate resistance evolution. Here, we show that E. coli displays persistence against a widely used disinfectant, benzalkonium chloride (BAC). Periodic, persister-mediated failure of disinfection rapidly selects for BAC tolerance, which is associated with reduced cell Surface charge and mutations in the lpxM locus, encoding an enzyme for lipid A biosynthesis. Moreover, the fitness cost incurred by BAC tolerance turns into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings highlight the links between persistence to disinfectants and resistance evolution to antimicrobials. KW - Persistence KW - Biocides KW - Evolution KW - Cross-resistance KW - Biocide tolerance KW - Disinfection PY - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-538532 SN - 2041-1723 VL - 12 IS - 1 SP - 6792 PB - Springer AN - OPUS4-53853 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nordholt, Niclas A1 - O'Hara, Kate A1 - Resch-Genger, Ute A1 - Blaskovich, M. A1 - Rühle, Bastian A1 - Schreiber, Frank T1 - A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria N2 - Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action. KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-563811 SN - 1664-302X IS - 13 SP - 1 EP - 13 PB - Frontiers Media CY - Lausanne AN - OPUS4-56381 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas A1 - Schreiber, Frank A1 - Kanaris, Orestis T1 - Consequences of BAC tolerance for selection and evolution in the presence of antibiotics N2 - The exposure to antimicrobial substances drives the evolution of antimicrobial resistance. 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 at BAM 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. Within our group, I mainly focus on the following two questions: How does phenotypic heterogeneity in bacteria affect the ability to survive treatment with biocides? And what are the consequences of phenotypic heterogeneity for the evolution of resistance to biocides and antibiotics? I will share published and unpublished results which demonstrate that phenotypic heterogeneity can enable the survival of biocide treatment and, through this, facilitate the evolution of AMR. On the other hand, we find that adaptation to a biocide can unexpectedly impair the ability to evolve resistance against an antibiotic. T2 - FEMS summer school for postdocs 2022: Microbial Evolvability Mechanisms: Resistance, Biology, and Strategies to Defeat and Detect CY - Split, Croatia DA - 27.04.2022 KW - Disinfection KW - Biocides KW - Evolution KW - Resistance KW - Biocide tolerance PY - 2022 AN - OPUS4-54846 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas A1 - Kanaris, Orestis A1 - Schmidt, Selina A1 - Schreiber, Frank T1 - Persistence against benzalkonium chloride promotes rapid evolution of tolerance under periodic disinfection N2 - A presentation given at the VAAM conference 2022, summarizing our findings published in the research paper "Persistence against benzalkonium chloride promotes rapid evolution of tolerance under periodic disinfection" T2 - Annual conference of the association for general and applied microbiology (VAAM) 2022 CY - Düsseldorf, Germany DA - 21.02.2022 KW - Persistence KW - Biocides KW - Evolution KW - Cross-resistance KW - Biocide tolerance KW - Disinfection PY - 2022 AN - OPUS4-54437 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas A1 - Schreiber, Frank T1 - Phenotypic heterogeneity in disinfection: sources and consequences for antimicrobial resistance N2 - A summary of projects here at BAM which investigate the influence of phenotypic heterogeneity on the outcome of disinfection and the influence on antimicrobial resistance. This presentation was given in the Theory Seminar of the Quantitative and Theoretical Biology group of Prof. Oliver Ebenhöh at HHU Düsseldorf T2 - Theory Seminar in the Quantitative and Theoretical Biology group at HHU Düsseldorf CY - Düsseldorf, Germany DA - 24.02.2022 KW - Disinfection KW - Biocides KW - Heterogeneity KW - Resistance PY - 2022 AN - OPUS4-54442 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas A1 - Schreiber, Frank 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 - Nordholt, Niclas T1 - The disinfectant glutaraldehyde induces antibiotic tolerance underpinned by phenotypic heterogeneity and transcriptome remodeling 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 - µClub Seminar Series CY - Berlin, Germany DA - 26.05.2023 KW - Glutaraldehyde KW - Biocides KW - Tolerance KW - Bacteria KW - Disinfection KW - Heterogeneity PY - 2023 AN - OPUS4-58031 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - The disinfectant glutaraldehyde induces antibiotic tolerance underpinned by a Disrupted Cellular State and Heterogenous Regrowth Dynamics 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 - Molecular Mechanisms in Evolution (GRS) Gordon Research Seminar CY - Easton, Massachusetts, USA DA - 24.06.2023 KW - Glutaraldehyde KW - Biocides KW - Tolerance KW - Bacteria KW - Disinfection KW - Heterogeneity KW - Antibiotics KW - AMR PY - 2023 AN - OPUS4-58032 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas A1 - Gustiherdini, Cahyaruri A1 - Lewerenz, Dominique A1 - Kanaris, Orestis A1 - Schreiber, Frank 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 - TY - JOUR A1 - Chacón, Luz A1 - Kuropka, B. A1 - González-Tortuero, E. A1 - Schreiber, Frank A1 - Rojas-Jiménez, K. A1 - Rodríguez-Rojas, A. T1 - Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila N2 - Excessive discharge of quaternary ammoniumdisinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, froma wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three di􀀀erent concentrations of BAC and characterizedmechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 di􀀀erent sequenced A. hydrophila strains, consists of approximately 4.6Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations.Other genes related tomembrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand howbacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments. KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-578555 SN - 1664-302X VL - 14 SP - 1 EP - 18 PB - Frontiers SA CY - Lausanne AN - OPUS4-57855 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -