TY - CONF A1 - Mezera, Marek T1 - Influence of the pulse repetition rate on the chemical and morphological properties of laser generated surface structures N2 - Inter-pulse accumulation of heat could affect the chemical and morphological properties of the laser processed material surface. Hence, the laser pulse repetition rate may restrict the processing parameters for specific laser-induced surface structures. In this study, the evolution of various types of laser-induced micro- and nanostructures at various laser fluence levels, effective number of pulses and at different pulse repetition rates (1 – 400 kHz) are studied for common metals/alloys (e.g. steel or titanium alloy) irradiated by near-infrared ultrashort laser pulses (925 fs, 1030 nm) in air environment. The processed surfaces were characterized by optical and scanning electron microscopy (OM, SEM), energy dispersive X-ray spectroscopy (EDX) as well as time of flight secondary ion mass spectrometry (TOF-SIMS). The results show that not only the surface morphology could change at different laser pulse repetition rates and comparable laser fluence levels and effective number of pulses, but also the surface chemistry is altered. Consequences for medical applications are outlined. T2 - European Materials Research Society Spring 2021 Meeting CY - Online meeting DA - 31.05.2021 KW - Laser-induced pariodic surface structures KW - LIPSS PY - 2021 AN - OPUS4-52778 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Mirabella, Francesca T1 - ToF-SIMS as a new tool for nano-scale investigation of ps-laser-generated surface structures on titanium substrates N2 - In recent years, the fabrication of laser-generated surface structures on metals such as titanium surfaces have gained remarkable interests, being technologically relevant for applications in optics, medicine, fluid transport, tribology, and wetting of surfaces. The morphology of these structures, and so their chemistry, is influenced by the different laser processing parameters such as the laser fluence, wavelength, pulse repetition rate, laser light polarization type and direction, angle of incidence, and the effective number of laser pulses per beam spot area. However, the characterization of the different surface structures can be difficult because of constraints regarding the analytical information from both depth and the topographic artifacts which may limit the lateral and depth resolution of elemental distributions as well as their proper quantification. A promising technique to investigate these structures even at the nano-scale is Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), a very surface sensitive technique that at the same time allows to perform depth-profiling, imaging and 3D-reconstruction of selected ion-sputter fragment distributions on the surface. In this study we combine chemical analyses such as Energy Dispersive X-ray spectroscopy (EDX) and high-resolution scanning electron microscopy (SEM) analyses with ToF-SIMS to fully characterize the evolution of various types of laser-generated micro- and nanostructures formed on Ti and Ti alloys at different laser fluence levels, effective number of pulses and at different pulse repetition rates (1 – 400 kHz), following irradiation by near-infrared ultrashort laser pulses (925 fs, 1030 nm) in air environment or under argon gas flow. We show how this combined surface analytical approach allows to evaluate alteration in the surface chemistry of the laser-generated surface structures depending on the laser processing parameters and the ambient environment. T2 - European Materials Research Society (EMRS) Fall Meeting 2021 CY - Online meeting DA - 20.09.2021 KW - ToF SIMS KW - Nano characterization PY - 2021 AN - OPUS4-53366 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Bonse, Jörn T1 - Bacterial adhesion on ultrashort pulse laser processed surfaces ― more than size matters! N2 - Bacterial biofilms are aggregates of bacterial cells, often attached to a surface and enclosed by a self-produced extracellular matrix which confers increased stress tolerance and resistance to cleaning. Biofilm formation leads to biofouling which gives rise to high costs in numerous technical settings due to biocorrosion and biodegradation. However, biofilms can also be attractive for industrial settings such as wastewater treatment systems or for soil bioremediation processes. Hence, the control of bacterial adhesion to a surface is of major concern. Surface topography strongly influences bacterial adhesion. Therefore, one promising way to achieve bacteria-guiding surfaces lies in the contactless and aseptic large-area laser processing of technical surfaces. We used short and ultrashort pulsed laser systems to generate different surface textures, mainly high-spatial-frequency and low-spatial-frequency laser-induced periodic surface structures, LIPSS (HFSL and LFSL), on Ti, Ti-alloy, steel, and polymers (PET and PE). Pristine (polished) and laser processed samples were subjected to bacterial adhesion experiments with two different Escherichia coli strains and Staphylococcus aureus as test organisms. The bacterial strains differed in their cell wall structure (grampositive vs. gramnegative strains), in size, shape, the occurrence of cell appendages, and in their biofilm forming capabilities. Adhesion patterns were analyzed microscopically and compared regarding the respective test strain and surface topography. Our results revealed that adhesion behavior strongly depends not only on the material’s topography and chemistry, but also on the specific bacterial strain, the presence of cell appendages, and ambient growth conditions. T2 - 13th International Conference on Photoexcited Processes and Applications, ICPEPA-13 CY - Lecce, Italy DA - 14.09.2025 KW - Biofilm KW - Bacterial adhesion KW - Laser-induced periodic surface structures (LIPSS) KW - Ultrashort laser pulses PY - 2025 AN - OPUS4-64166 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Bonse, Jörn T1 - Bacterial adhesion on ultrashort pulse laser processed surfaces ― more than size matters! N2 - Bacterial biofilms are aggregates of bacterial cells, often attached to a surface, and enclosed by a self-produced extracellular matrix which confers increased stress tolerance and resistance to cleaning. Biofilm formation leads to biofouling which gives rise to high costs in numerous technical settings due to biocorrosion and biodegradation. However, biofilms can also be attractive for industrial settings such as wastewater treatment systems or for soil bioremediation processes. Hence, the control of bacterial adhesion to a surface is of major concern. Surface topography strongly influences bacterial adhesion. Therefore, one promising way to achieve bacteria-guiding surfaces lies in the contactless and aseptic large-area laser processing of technical surfaces. We used short and ultrashort pulsed laser systems to generate different surface textures, mainly high-spatial-frequency and low-spatial-frequency laser-induced periodic surface structures, LIPSS (HFSL and LFSL), on Ti, Ti-alloy, steel, and polymers (PET and PE). Pristine (polished) and laser processed samples were subjected to bacterial adhesion experiments with two different Escherichia coli strains and Staphylococcus aureus as test organisms. The bacterial strains differed in their cell wall structure (grampositive vs. gramnegative strains), in size, shape, the occurrence of cell appendages, and in their biofilm forming capabilities. Adhesion patterns were analyzed microscopically and compared regarding the respective test strain and surface topography. Our results revealed that adhesion behavior strongly depends not only on the material’s topography and chemistry, but also on the specific bacterial strain, the presence of cell appendages, and ambient growth conditions. T2 - 13th International LIPSS Workshop CY - Enschede, Netherlands DA - 29.10.2025 KW - Bacterial adhesion KW - Biofilm KW - Laser-induced periodic surface structures (LIPSS) KW - Ultrashort laser pulses PY - 2025 AN - OPUS4-64632 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 - Varadarajan, A. A1 - Allan, R. A1 - Valentin, J. A1 - Castañeda Ocampo, O. A1 - Somerville, V. A1 - Buhmann, M. A1 - West, J. A1 - Skipp, Paul A1 - van der Mei, H. A1 - Ren, Q. A1 - Schreiber, Frank A1 - Webb, J. A1 - Pietsch, Franziska A1 - Ahrens, C. T1 - An integrated model system to gain mechanistic insights into biofilm-associated antimicrobial resistance in Pseudomonas aeruginosa MPAO1 N2 - Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential Protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms. KW - Biofilms PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-515108 DO - https://doi.org/10.1038/s41522-020-00154-8 VL - 6 IS - 1 SP - Article number: 46 PB - Springer Nature CY - Singapore AN - OPUS4-51510 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - 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 - Schreiber, Frank T1 - Hydro-Touch test - Evaluation of Transfer of Microorganisms to Non-porous Surfaces and their antimicrobial Activity N2 - This presentation describes a new test procedure that allows to determine the transfer of microorganisms to surfaces and to measure the antimicrobial efficacy of those surfaces. The main improvement of the method is that is assesses the effect of the surfaces under semi-dry, realistic conditions by transferring microorganisms via a gelatin pad linked to a stamp of defined weight. Comparing the performance of the established antimicrobial materials with currently used wet test methods and the newly developed method shows reduced antimicrobial activity of those materials under semi-dry conditions. T2 - 64. Sitzung des DIN NA 176-03-06 AA „Chemische Desinfektionsmittel und Antiseptika in der Humanmedizin“ CY - Online meeting DA - 29.09.2025 KW - Antimicrobial surfaces KW - Standardization PY - 2025 AN - OPUS4-64870 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kupke, Johannes A1 - Brombach, Julian A1 - Fang, Yuwen A1 - Wolf, Silver A. A1 - Thrukonda, Lakshmipriya A1 - Ghazisaeedi, Fereshteh A1 - Kuropka, Benno A1 - Hanke, Dennis A1 - Semmler, Torsten A1 - Nordholt, Niclas A1 - Schreiber, Frank A1 - Tedin, Karsten A1 - Lübke-Becker, Antina A1 - Steiner, Ulrich K. A1 - Fulde, Marcus T1 - Heteroresistance in Enterobacter cloacae complex caused by variation in transient gene amplification events N2 - Heteroresistance (HR) in bacteria describes a subpopulational phenomenon of antibiotic resistant cells of a generally susceptible population. Here, we investigated the molecular mechanisms and phenotypic characteristics underlying HR to ceftazidime (CAZ) in a clinical Enterobacter cloacae complex strain (ECC). We identified a plasmid-borne gene duplication-amplification (GDA) event of a region harbouring an ampC gene encoding a β-lactamase bla DHA-1 as the key determinant of HR. Individual colonies exhibited variations in the copy number of the genes resulting in resistance level variation which correlated with growth onset (lag times) and growth rates in the presence of CAZ. GDA copy number heterogeneity occurred within single resistant colonies, demonstrating heterogeneity of GDA on the single-cell level. The interdependence between GDA, lag time and antibiotic treatment and the strong plasticity underlying HR underlines the high risk for misdetection of antimicrobial HR and subsequent treatment failure. KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-627057 DO - https://doi.org/10.1038/s44259-025-00082-7 VL - 3 IS - 1 SP - 1 EP - 14 PB - Springer AN - OPUS4-62705 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Srivastava, Priyanka A1 - Tavernaro, Isabella A1 - Scholtz, Lena A1 - Genger, C. A1 - Welker, P. A1 - Schreiber, Frank A1 - Meyer, Klas A1 - Resch-Genger, Ute T1 - Dual color pH probes made from silica and polystyrene nanoparticles and their performance in cell studies N2 - Ratiometric green–red fluorescent nanosensors for fluorometrically monitoring pH in the acidic range were designed from 80 nm-sized polystyrene (PS) and silica (SiO2) nanoparticles (NPs), red emissive reference dyes, and a green emissive naphthalimide pH probe, analytically and spectroscopically characterized, and compared regarding their sensing performance in aqueous dispersion and in cellular uptake studies. Preparation of these optical probes, which are excitable by 405 nm laser or LED light sources, involved the encapsulation of the pH-inert red-fuorescent dye Nile Red (NR) in the core of self-made carboxylated PSNPs by a simple swelling procedure and the fabrication of rhodamine B (RhB)-stained SiO2-NPs from a silane derivative of pH-insensitive RhB. Subsequently, the custom-made naphthalimide pH probe, that utilizes a protonation-controlled photoinduced electron transfer process, was covalently attached to the carboxylic acid groups at the surface of both types of NPs. Fluorescence microscopy studies with the molecular and nanoscale optical probes and A549 lung cancer cells confirmed the cellular uptake of all probes and their penetration into acidic cell compartments, i.e., the lysosomes, indicated by the switching ON of the green naphthalimide fluorescence. This underlines their suitability for intracellular pH sensing, with the SiO2-based nanosensor revealing the best performance regarding uptake speed and stability. KW - Sensors KW - Silica and polystyrene nanoparticles KW - pH probe KW - Fluorescence spectroscopy KW - Cell studies KW - Dye KW - Particle synthesis PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-569198 DO - https://doi.org/10.1038/s41598-023-28203-0 SN - 2045-2322 VL - 13 IS - 1 SP - 1321 EP - 1336 PB - Nature CY - London AN - OPUS4-56919 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Keshmiri, Hamid A1 - Armin, F. A1 - Elsayad, K. A1 - Schreiber, Frank A1 - Moreno, M. T1 - Leaky and waveguide modes in biperiodic holograms N2 - This study details a theoretical analysis of leaky and waveguide modes in biperiodic all-dielectric holograms. By tuning diffraction orders and subsequently confining local density of optical states at two distinct resonance wavelengths, we present a new class of highly sensitive refractive index biosensing platforms that are capable of resolving 35.5 to 41.3 nm/RIU of spectral shift for two separate biological analytes. KW - Antimicrobial resistance KW - Bacteria KW - Photonics KW - Diffractive gratings PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-527632 DO - https://doi.org/10.1038/s41598-021-89971-1 SN - 2045-2322 (online) VL - 11 IS - 1 SP - 10991 PB - Springer Nature AN - OPUS4-52763 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nordholt, Niclas A1 - Sobisch, Lydia-Yasmin A1 - Gödt, Annett A1 - Lewerenz, Dominique A1 - Schreiber, Frank T1 - Heterogeneous survival upon disinfection underlies evolution of increased tolerance N2 - Disinfection is important to limit the spread of infections, but failure of disinfection may foster the evolution of antimicrobial resistance in bacteria. Persisters are phenotypically tolerant subpopulations that survive toxic stress longer than susceptible cells, leading to failure in treatments with antimicrobials and facilitating resistance evolution. To date, little is known about persistence in the context of disinfectants. The aim of this study was to investigate the influence of persisters on disinfection and to determine the consequences of disinfectant persistence for the evolution of increased tolerance to disinfectants. Disinfection kinetics with high temporal resolution were recorded for Escherichia coli exposed to the following six disinfectants: hydrogen peroxide (H2O2), glutaraldehyde (GTA), chlorhexidine (CHX), benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDAC), and isopropanol (ISO). A mathematical model was used to infer the presence of persisters from the time–kill data. Time–kill kinetics for BAC, DDAC, and ISO were indicative of persisters, whereas no or weak evidence was found for H2O2, GTA, and CHX. When subjected to comparative experimental evolution under recurring disinfection, E. coli evolved increased tolerance to substances for which persisters were predicted (BAC and ISO), whereas adaptation failed for substances in which no persisters were predicted (GTA and CHX), causing extinction of exposed populations. Our findings have implications for the risk of disinfection failure, highlighting a potential link between persistence to disinfectants and the ability to evolve disinfectant survival mechanisms. IMPORTANCE: Disinfection is key to control the spread of infections. But the application of disinfectants bears the risk to promote the evolution of reduced susceptibility to antimicrobials if bacteria survive the treatment. The ability of individual bacteria to survive disinfection can display considerable heterogeneity within isogenic populations and may be facilitated by tolerant persister subpopulations. Using time–kill kinetics and interpreting the data within a mathematical framework, we quantify heterogeneity and persistence in Escherichia coli when exposed to six different disinfectants. We find that the level of persistence, and with this the risk for disinfection failure, depends on the disinfectant. Importantly, evolution experiments under recurrent disinfection provide evidence that links the presence of persisters to the ability to evolve reduced susceptibility to disinfectants. This study emphasizes the impact of heterogeneity within bacterial populations on disinfection outcomes and the potential consequences for the evolution of antimicrobial resistances. KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-615566 DO - https://doi.org/10.1128/spectrum.03276-22 SN - 2165-0497 VL - 12 IS - 12 SP - 1 EP - 11 PB - American Society for Microbiology CY - Birmingham, Ala. AN - OPUS4-61556 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Pietsch, Franziska A1 - O'Neill, A. J. A1 - Ivask, A. A1 - Jenssen, H. A1 - Inkinen, J. A1 - Kahru, A. A1 - Ahonen, M. A1 - Schreiber, Frank T1 - Selection of resistance by antimicrobial coatings in the healthcare setting N2 - Antimicrobial touch surfaces have been introduced in healthcare settings with the aim of supporting existing hygiene procedures, and to help combat the increasing threat of antimicrobial resistance. However, concerns have been raised over the potential selection pressure exerted by such surfaces, which may drive the evolution and spread of antimicrobial resistance. This review highlights studies that indicate risks associated with resistance on antimicrobial surfaces by different processes, including evolution by de-novo mutation and horizontal gene transfer, and species sorting of inherently resistant bacteria dispersed on to antimicrobial surfaces. The review focuses on antimicrobial surfaces made of copper, silver and antimicrobial peptides because of the practical application of copper and silver, and the promising characteristics of antimicrobial peptides. The available data point to a potential for resistance selection and a subsequent increase in resistant strains via cross-resistance and co-resistance conferred by metal and antibiotic resistance traits. However, translational studies describing the development of resistance to antimicrobial touch surfaces in healthcare-related environments are rare, and will be needed to assess whether and how antimicrobial surfaces lead to resistance selection in These settings. Such studies will need to consider numerous variables, including the antimicrobial concentrations present in coatings, the occurrence of biofilms on surfaces, and the humidity relevant to dry-surface environments. On-site tests on the efficacy of antimicrobial Coatings should routinely evaluate the risk of selection associated with their use. KW - Antimicrobial resistance KW - Antimicrobial coating KW - Touch surfaces KW - Healthcare KW - Infections KW - COST action CA15114 AMICI PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-510926 DO - https://doi.org/10.1016/j.jhin.2020.06.006 SN - 0195-6701 VL - 106 IS - 1 SP - 115 EP - 125 PB - Elsevier Ltd AN - OPUS4-51092 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nordholt, Niclas 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 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-538532 DO - https://doi.org/10.1038/s41467-021-27019-8 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 - Vareschi, Silvia A1 - Jaut, Valerie A1 - Vijay, Srinivasan A1 - Allen, Rosalind J. A1 - Schreiber, Frank T1 - Antimicrobial efflux and biofilms: an interplay leading to emergent resistance evolution N2 - The biofilm mode of growth and drug efflux are both important factors that impede the treatment of bacterial infections with antimicrobials. Decades of work have uncovered the mechanisms involved in both efflux and biofilm-mediated antimicrobial tolerance, but links between these phenomena have only recently been discovered. Novel findings show how efflux impacts global cellular physiology and antibiotic tolerance, underpinned by phenotypic heterogeneity. In addition efflux can mediate cell-to-cell interactions, relevant in biofilms, via mechanisms including efflux of signaling molecules and metabolites, signaling using pump components and the establishment of local antibiotic gradients via pumping. These recent findings suggest that biofilm antibiotic tolerance and efflux are closely coupled, with synergistic effects leading to the evolution of antimicrobial resistance in the biofilm environment. KW - Evolution KW - Efflux KW - Antibiotics KW - Biofilms KW - Antimicrobial resistance KW - Phenotypic heterogeneity PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-632799 DO - https://doi.org/10.1016/j.tim.2025.04.012 SN - 0966-842X SP - 1 EP - 15 PB - Elsevier Ltd. AN - OPUS4-63279 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Schreiber, Frank A1 - Nordholt, Niclas A1 - Lewerenz, Dominique T1 - Time-kill kinetics reveal heterogeneous tolerance to disinfectants N2 - Disinfection is an important strategy to limit the spread of infections. Failure of disinfection may facilitate evolution of resistance against disinfectants and antibiotics through the processes of cross-resistance and co-resistance. The best possible outcome of disinfection minimizes the number of surviving bacteria and the chance for resistance evolution. Resistance describes the ability to grow in previously inhibitory concentrations of an antimicrobial, whereas tolerance is associated with enhanced survival of lethal doses. Individual bacteria from the same population can display considerable heterogeneity in their ability to survive treatment (i.e. tolerance) with antimicrobials, which can result in unexpected treatment failure. Here, we investigated how phenotypic heterogeneity affects the ability of E. coli to survive treatment with six different substances commonly used as active substances in disinfectants, preservatives and antiseptics. A mathematical model which assumes that phenotypic heterogeneity underlies the observed disinfection kinetics was used to infer whether time-kill kinetics were caused by a tolerant subpopulation. The analysis identified bimodal kill kinetics for benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDAC), and isopropanol (Iso). In contrast, kill kinetics by chlorhexidine (CHX), glutaraldehyde (GTA), and hydrogen peroxide (H2O2) were best explained by unimodal kill kinetics underpinned by a broad distribution of tolerance times for CHX as opposed to a narrow distribution of tolerance times for GTA and H2O2. These findings have implications for the risk of disinfection failure, with potential consequences for the evolution of antimicrobial resistance and tolerance. KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-615572 DO - https://doi.org/https://doi.org/10.1101/2022.06.22.497202 SN - 2692-8205 SP - 1 EP - 20 PB - Cold Spring Harbor Laboratory CY - Cold Spring Harbor, NY AN - OPUS4-61557 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Lee, Mihyun A1 - Wiesli, Luzia A1 - Schreiber, Frank A1 - Ivask, Angela Ivask A1 - Ren, Qun T1 - Quantitative Assessment of Microbial Transmission onto Environmental Surfaces Using Thermoresponsive Gelatin Hydrogels as a Finger Mimetic under In Situ-Mimicking Conditions N2 - Surface-mediated transmission of pathogens plays a key role in healthcare-associated infections. However, proper techniques for its quantitative analysis are lacking, making it challenging to develop novel antimicrobial and anti-fouling surfaces to reduce pathogen spread via environmental surfaces. This study demonstrates a gelatin hydrogel-based touch transfer test, the HydroTouch test, to evaluate pathogen transmission on high-touch surfaces under semi-dry conditions. The HydroTouch test employs gelatin as a finger mimetic, facilitating testing with pathogenic bacteria under controlled conditions. The thermoresponsive sol–gel transition of gelatin allows easy recovery and quantification of bacteria before and after testing. The HydroTouch test demonstrates that methicillin-resistant Staphylococcus aureus has a high transmission efficiency of ≈16% onto stainless steel, compared to <3% for Escherichia coli or Pseudomonas aeruginosa. Polyurethane surfaces exhibit strong resistance to bacterial contamination with a transmission efficiency of ≈0.6%, while polytetrafluoroethylene shows a transmission efficiency approximately four times higher than polyurethane. Additionally, quaternary ammonium-based antimicrobial coatings reduce the transmission efficiency of live bacteria on stainless steel to ≈4% of the original level. The HydroTouch test provides a reliable method for assessing pathogen transmission on various surfaces under semi-dry settings, supporting the development of effective antimicrobial, anti-transmission coatings to reduce healthcare-associated infections. KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-624566 DO - https://doi.org/10.1002/adhm.202403790 SN - 2192-2659 SP - 1 EP - 10 PB - Wiley VHC-Verlag AN - OPUS4-62456 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 - Nordholt, Niclas T1 - Phenotypic heterogeneity in bacterial lag times and antibiotic tolerance induced by the disinfectant glutaraldehydePhenotypic heterogeneity in bacterial lag times and antibiotic tolerance induced by the disinfectant glutaraldehyde N2 - Phenotypic heterogeneity in clonal bacterial populations can be considered a preliminary stage of functional differentiation, which may increase population fitness in fluctuating environments. Here, we investigated how transient exposure of clonal bacterial populations to residual amounts of a commonly used disinfectant, glutaraldehyde (GTA), induces phenotypic heterogeneity, ensuring survival of the population upon sudden challenge with high doses of antibiotics. Using the ScanLag system, we found that exposure to GTA resulted in wide lag-time distributions across different bacterial isolates of E. coli, S. aureus, and P. aeruginosa. Importantly, this was associated with elevated levels of survival (i.e. tolerance) towards lethal doses of antibiotics. As revealed by RNAseq in E. coli, GTA exposure caused global transcriptome remodeling, with more than 1200 differentially expressed genes of diverse biological functions. Several of these genes that were not previously associated with antibiotic tolerance or persistence induced, when overexpressed alone, antibiotic tolerance without showing a lag phenotype. This suggests that exposure to GTA induces unspecific, lag-dependent and specific, lag-independent tolerance to antibiotics in clonal bacterial populations. These findings have implications for 1.) settings where disinfectants and antibiotics are used in close proximity, such as hospitals and animal husbandry, and 2.) for the selection dynamics of tolerant pheno- and genotypes in fluctuating environments because of the trade-off that arises from exiting lag and resuming growth as fast as possible and maintaining antibiotic tolerance. This trade-off may be weakened by phenotypically heterogeneous clonal populations as induced by GTA. T2 - FAST REAL Project Meeting Tartu CY - Tartu, Estonia DA - 16.06.2025 KW - Biocides KW - Biocide resistance KW - Phenotypic heterogeneity KW - Glutaraldehyde KW - Disinfectants PY - 2025 AN - OPUS4-63834 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 - THES A1 - Schmidt, Selina T1 - Effects of biocides on processes underlying resistance evolution N2 - Antimicrobial resistance (AMR) is a global health problem. It is well known that antibiotics can drive evolutionary processes that underlie antimicrobial resistance (AMR) evolution and spread in clinical and environmental settings. In contrast, less is known about the effects of antimicrobial substances that are used as biocides (i.e. disinfectants and preservatives) on AMR evolution and spread. Biocides are present in various settings, interacting with diverse microbial communities. Therefore, it is crucial to evaluate their role in the evolution and dissemination of antimicrobial resistance. Biocides occur in a wide range of concentrations in various environmental settings. By examining how the various concentrations affect selection mechanisms, we gain insights into potential developments related to antimicrobial resistance. The aim of this PhD thesis is to investigate the effects of biocides on processes underlying resistance evolution. Specifically, the work focused on key mechanisms for resistance spread, resistance evolution, and the effect of selection pressures on evolved resistance mechanisms. The thesis is structured around three major objectives: (i) to determine the effect of biocides on the evolution of resistance by affecting the rate of occurrence of de novo mutations, (ii) to determine the effect of biocides on the spread of resistance genes by modifying the rate of horizontal gene transfer (HGT) processes, and (iii) to investigate the selective drivers of the emergence of antimicrobial resistance in adaptive laboratory evolution (ALE) experiments. De-novo mutations are spontaneous mutations that occur at a certain rate in microorganisms. The effect of biocides at subinhibitory environmentally relevant concentrations on the mutation rate in Acinetobacer baylyi, Bacillus subtilis and Escherichia coli was assessed with the fluctuation assay. The results showed that biocides affected mutation rates in a species and substance dependent matter. The bisbiguanide chlorhexidine digluconate, the quaternary ammonium compound didecyldimethylammonium chloride, the metal copper, the pyrethroid-insecticide permethrin, and the azole-fungicide propiconazole increase mutation rates in E. coli, whereas no increases were identified for B. subtilis and A. baylyi. Horizontal gene transfer refers to diverse mechanisms that mediate the transfer of mobile genetic elements between microorganisms. This work focused on conjugation and transformation. Conjugation is a process whereby a conjugative plasmid is transferred from a donor cell to a recipient cell. Transformation is a process whereby exogenous donor DNA is taken up into a recipient cell and integrated into the recipient’s’ genome. The effects of subinhibitory environmentally relevant biocide concentrations on the conjugation rate of E. coli and the transformation rate of the naturally competent organisms A. baylyi in were assessed. The results showed that benzalkonium chloride (BAC), chlorhexidine and permethrin increased conjugation in E. coli, while none of the biocides increased transformation rates in A. baylyi. To further understand the molecular mechanisms underlying the effects on mutation and conjugation rates, I investigated the induction of the RpoS-mediated general stress and the RecA-linked SOS response upon biocide exposure. The results show a link between the general stress and the SOS response with increased rates of mutation and conjugation, but not for all biocides. One major approach to study the evolutionary response of bacteria to antimicrobials are ALE experiments with growth at subinhibitory concentrations linked to serial subculturing over many generations. Such experiments have been used to study resistance evolution to antibiotics and biocides. However, previous work showed that adaptation to biocide stress may be mediated by different evolutionary drivers. Here, I investigated the contributions of evolution for increased survival as opposed to improved growth in ALE experiments with E. coli exposed to subinhibitory BAC concentrations. Two distinct evolutionary treatments selecting for survival only or survival and growth led to specific evolutionary adaptations apparent in the phenotypes and genotypes of the evolved populations. Populations growing in the presence of BAC evolved increased fitness in the presence of BAC associated with higher resistance to BAC and cross-resistance to antibiotics, while this was not the case for populations evolving for increased survival only. Genotypic characterization by whole genome sequencing of the evolved populations revealed parallelism in mutated genes among replicate populations and distinct differences across treatments. Treatments selecting for survival and growth showed mutations in stress response related genes (hslO and tufA), while selection for survival led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ). In summary, this thesis shows that biocides affect AMR evolution and emphasizes the importance of understanding of how biocides impact the molecular and evolutionary process that underlie AMR evolution. KW - Biocides KW - Antimicrobial resistances KW - Microbial survival mechanisms PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:188-refubium-43383-9 SP - 1 EP - 101 PB - Freie Universität CY - Berlin AN - OPUS4-60678 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Braetz, S. A1 - Nordholt, Niclas A1 - Nerlich, A. A1 - Schreiber, Frank A1 - Tedin, K. A1 - Fulde, M. T1 - TisB enables antibiotic tolerance in Salmonella by preventing prophage induction through ATP depletion N2 - Antibiotic persistence comprises drug-tolerant bacteria that can survive treatment with antibacterial agents, despite lacking classical genetic resistance mechanisms. Therefore, persisters are clinically relevant because they can lead to treatment failures and chronic infections. Additionally, antibiotic persistence facilitates the evolution of resistance through genetic mutations. Persisters are triggered by a lack of nutrients, bacterial toxins, low ATP levels, or other stress responses that shut down bacterial metabolism. However, the involvement of prophages, viruses that integrate into bacterial chromosomes, is less well understood. In this study, we tested a tisAB deletion in Salmonella Typhimurium and examined persister cell formation following treatment with the DNA-damaging drug ciprofloxacin. TisB is a bacterial toxin that increases the influx of protons across the inner bacterial membrane into the cytosol, causing ATP depletion. We demonstrate that the deletion of tisAB increases prophage induction and bacterial killing, leading to a reduced persister cell fraction. The tisAB mutant is unable to down regulate its ATP concentration after exposure to ciprofloxacin, which in turn allows for stronger binding of RecA to single-stranded DNA, the activator of both the SOS response and prophage induction. KW - Antimicrobial resistance KW - Bacterial survival mechanisms KW - Escherichia coli KW - Salmonella typhimurium PY - 2025 DO - https://doi.org/10.1371/journal.ppat.1013498 IS - 9 SP - 1 EP - 23 AN - OPUS4-64642 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 - Marano, Roberto T1 - A Customizable Procedure for Predicting Antibiotic Resistance Selection on Novel Antimicrobial Coatings N2 - The global burden of antimicrobial resistance (AR) has manifested in an increasing number of deaths attributable to antibiotic-resistant bacteria (ARB) in recent years, with projections indicating a continued rise. Combating AR requires a multifaceted approach, one aspect of which involves preventing the spread of ARB in hospital environments via high-touch surfaces, which are known contributors to nosocomial infections. In conjunction with routine disinfection protocols and infection prevention measures, antimicrobial surfaces or antimicrobial coatings (AC) are increasingly being investigated and implemented to reduce microbial transmission via high-touch surfaces, thereby mitigating their spread in healthcare settings. However, similar to antibiotics, prolonged use of such surfaces may lead to the direct or indirect selection of ARB. To prevent this, targeted tests must be developed to predict potential AR selection before AC implementation. Materials and Methods As part of the Horizon-Europe-funded project "STOP" (Grant Agreement ID: 101057961), a novel antimicrobial coating is being developed and tested to reduce pathogen transfer on surfaces without selecting for ARB. To this end, two bacterial libraries (80 strains each) were assembled from the two species most associated with AR-attributable mortality worldwide, Escherichia coli and Staphylococcus aureus, as identified in the most recent comprehensive study [1]. Each library maintains a 50/50% ratio of strains resistant or sensitive to third-generation cephalosporins (E. coli) and methicillin (S. aureus), respectively—representing two of the most widespread resistance profiles. An adapted ISO 22196 method was developed using a reference benchmark antimicrobial surface (i.e., copper), with stainless steel serving as a control. The goal was to infer potential advantages of copper-unrelated antibiotic resistance phenotypes on the tested surface. Results Metadata, genomic data, and antibiotic susceptibility testing (AST) data were collected for all selected strains in each library, ensuring diversity in sequence types (ST) of clinical relevance, antibiotic resistance gene profiles, and geographical origins. The method was calibrated on copper surfaces using a reference E. coli strain, establishing the initial parameters required for investigators to customize the test for a given AC. Procedural reproducibility was assessed by comparing results from independent operators. Discussion The two libraries are currently being screened against the selected copper surfaces to identify potential associations between the resistance phenotypes of the tested species and their observed survival rates post-exposure. Three mutually exclusive outcomes are anticipated: (i) a statistically significant survival advantage of resistant strains compared to sensitive strains, (ii) an inverse scenario where sensitive strains exhibit higher survival, or (iii) no significant difference between the two groups. Furthermore, post hoc principal component analysis utilizing metadata and AST data may help elucidate genetic traits that confer a survival advantage on the tested AC. Conclusions This test aims to assist developers of antimicrobial coatings and materials in assessing potential selective pressures toward ARB before these products are implemented and evaluated under real-life conditions. T2 - ESB Conference CY - Torino, Italy DA - 07.09.2025 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64569 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 - Marano, Roberto T1 - Antimicrobial coatings: modes of action, microbiological efficacy testing, and resistance evolution N2 - Within the Horizon-Europe funded project “STOP: Surface Transfer Of Pathogens”, Grant agreement 101057961, Working Package 6 is currently working on developing tests aimed at predicting possible selection and/or evolution of antibiotic resistance onto antimicrobial coatings meant for high-touch surfaces. This presentation describes the devised protocols and the preliminary results of their application on copper as a benchmark antimicrobial material. T2 - STOP-project Meeting CY - Sofia, Bulgaria DA - 27.02.2025 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64567 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Mechanisms and evolution of resistance to antimicrobial biocides N2 - Antimicrobial resistance (AMR) is a global health problem with the environment being an important compartment for the evolution, selection and transmission of AMR. These processes are impacted by pollution with antibiotics. However, antimicrobial biocides used as disinfectants and material preservatives are major pollutants exceeding the antibiotic market in terms of chemical diversity and mass. The aim of our work is to understand the mechanisms and risks of biocides for resistance and antibiotic cross-resistance evolution in bacteria to optimize their application and safeguard their efficacy. Our work shows that biocides have the potential to affect evolutionary processes towards AMR by increasing the rates of de-novo mutation and conjugation. Importantly, widely used compounds such as chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations. Furthermore, we show that single-cell phenotypic heterogeneity regarding tolerance (persistence) determines survival against specific biocides including QACs and isopropanol. Mechanistic investigations reveal that known antibiotic persister mechanisms contribute to persister formation to biocides. The evolution of high-level tolerance to different biocides is linked to the initial persister level and the evolution of specific genetically encoded mechanisms related to properties of the cell envelope. Biocide-tolerant strains have a selective advantage in the presence of environmentally-relevant concentrations of antibiotics, which could lead to the stabilization of biocide tolerance in environments where biocides and antibiotics co-occur (e.g. wastewater, animal stables). Taken together, our work shows the importance of assessing the contribution of biocides on evolution and selection of AMR in the environment. T2 - EMBO Symposium on Mechanisms of drug resistance and tolerance in bacteria, fungi, and cancer CY - Heidelberg, Germany DA - 18.03.2025 KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance PY - 2025 AN - OPUS4-64866 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Marano, Roberto T1 - Antimicrobial coatings: modes of action, microbiological efficacy testing, and resistance evolution N2 - Antimicrobial coatings and materials on high touch surfaces can provide additional level of protection to periodic disinfection cycles by reducing bacterial loads. However, different coatings and materials assert their antimicrobial activity in different ways, and it is not clear whether they could pose detrimental selective pressure towards antibiotic-resistant bacteria. This talk elucidates modes of actions and current knowledge gaps in testing antimicrobial resistance on antimicrobial surfaces.” T2 - STOP Antimicrobial Coating Conference CY - Mons, Belgium DA - 05.12.2024 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64566 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Marano, Roberto T1 - A Customizable Procedure for Predicting Antibiotic Resistance Selection on Novel Antimicrobial Coatings N2 - This presentation introduceda new methodological approach developed to test antimocrobials implemented on high touch surfaces, inlcuding preliminary results on its use on reference antimicrobial materials. The seminar was organized by the 'Fast-real' project, funded by the Horizon Europe (project ID: 101159721). T2 - Novel strategies and considerations in fighting pathogens CY - Tartu, Estonia DA - 16.06.2025 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64568 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 - 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 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-563811 DO - https://doi.org/10.3389/fmicb.2022.1023326 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 - JOUR A1 - Valentin, J. A1 - Straub, H. A1 - Pietsch, Franziska A1 - Lemare, M. A1 - Ahrens, C. A1 - Schreiber, Frank A1 - Webb, J. A1 - van der Mei, H. A1 - Ren, Q. T1 - Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms N2 - Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance. KW - Antimicrobial resistance KW - Bacteria KW - Biofilms KW - Biocides PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-541113 DO - https://doi.org/10.1038/s41396-021-01157-9 SN - 1751-7370 VL - 16 IS - 4 SP - 1176 EP - 1186 PB - Springer Nature AN - OPUS4-54111 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 - TY - JOUR A1 - Valentin, Jules D. P. A1 - Altenried, Stefanie A1 - Varadarajan, Adithi R. A1 - Ahrens, Christian H. A1 - Schreiber, Frank A1 - Webb, Jeremy S. A1 - van der Mei, Henny C. A1 - Ren, Qun T1 - Identification of Potential Antimicrobial Targets of Pseudomonas aeruginosa Biofilms through a Novel Screening Approach N2 - Pseudomonas aeruginosa is an opportunistic pathogen of considerable medical importance, owing to its pronounced antibiotic tolerance and association with cystic fibrosis and other life-threatening diseases. The aim of this study was to highlight the genes responsible for P. aeruginosa biofilm tolerance to antibiotics and thereby identify potential new targets for the development of drugs against biofilm-related infections. By developing a novel screening approach and utilizing a public P. aeruginosa transposon insertion library, several biofilm-relevant genes were identified. The Pf phage gene (PA0720) and flagellin gene (fliC) conferred biofilm-specific tolerance to gentamicin. Compared with the reference biofilms, the biofilms formed by PA0720 and fliC mutants were completely eliminated with a 4-fold-lower gentamicin concentration. Furthermore, the mreC, pprB, coxC, and PA3785 genes were demonstrated to play major roles in enhancing biofilm tolerance to gentamicin. The analysis of biofilm-relevant genes performed in this study provides important novel insights into the understanding of P. aeruginosa antibiotic tolerance, which will facilitate the detection of antibiotic resistance and the development of antibiofilm strategies against P. aeruginosa. KW - Antimicrobial resistance KW - Bacteria KW - Biofilms KW - Pseudomonas aeruginosa PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-570205 DO - https://doi.org/10.1128/spectrum.03099-22 SP - 1 EP - 5 PB - ASM Journals AN - OPUS4-57020 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Resistance evolution towards biocides and antimicrobial surfaces N2 - Biocides, including disinfectants and antimicrobial surfaces (AMCs), are important to prevent the spread of pathogens and antimicrobial resistant bacteria via surfaces. However, concerns have been raised about 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 and co-resistance. We need to understand the mechanisms and risks of disinfectants and AMCs for resistance and cross-resistance evolution to optimize their application and safeguard their long-term efficacy. We used adaptive laboratory evolution (ALE) experiments based on repeated exposure of bacteria to disinfectants. Our results show that repeated disinfection of E. coli with benzalkonium chloride in suspension results in a 2000-fold increase in survival within 5 exposure cycles. Adaption is linked to the initial presence of persister cells highly tolerant to benzalkonium chloride. We used the same approach to develop standardizable ALE experiments to determine resistance evolution to AMCs. The results highlight rapid adaptation of E. coli and P. aeruginosa towards copper surfaces. Moreover, there are multiple situations in the clinic or in the environment in which biocides and antibiotics co-occur and in which combination effects can shape their antimicrobial activity or their selective effects. Our work with P. aeruginosa shows prevalent combination effects of biocides and antibiotics, ranging from synergy to antagonism and resulting in the selection for or against antibiotic resistant strains. The combination effects are dependent on the biofilm mode-of-growth, manifesting in apparent differences in the structural arrangement of antibiotic sensitive and resistant strains in biofilms exposed to combinations. Furthermore, biocides affect rates of mutation and horizontal gene transfer, thereby having a potential facilitating effect on resistance evolution. Taken together, our work shows that the role of biocides as potential drivers of resistance evolution and selection deserves further study and regulative action. T2 - Eurobiofilms 2022 CY - Palma, Spain DA - 31.08.2022 KW - Antimicrobial resistance KW - Bacteria KW - Biofilms KW - Biocides KW - Antimicrobial surfaces PY - 2022 AN - OPUS4-55608 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Biocides as drivers for the evolution of antimicrobial resistance N2 - This presentation provides an overview about how biocides drive the evolution of antimicrobial resistance during application and as pollutants in the environment. The presentation shows that biocides can lead to heterogeneous killing, facilitating tolerance evolution. This evolution is related to decreased susceptibility to antibiotics and has potential for co-selection. In contrast, evolved tolerance can limit antibiotic evolvability via epistatic interactions. Moreover, biocides can co-select for antibiotic resistance in wastewater and affect rates of mutation and horizontal gene transfer. Biocides and antibiotics show strong combination effects with consequences for selection of antibiotic resistance. T2 - Novel strategies and considerations in fighting pathogens CY - Tartu, Estonia DA - 16.06.2025 KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 AN - OPUS4-64867 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Biocides as drivers for the Selection and evolution of antimicrobial resistance N2 - Antimicrobial resistance (AMR) is a global health problem with the environment being an important compartment for the evolution, selection and transmission of AMR. These processes are impacted by pollution with antibiotics. However, antimicrobial biocides used as disinfectants and material preservatives are major pollutants exceeding the antibiotic market in terms of chemical diversity and mass. The aim of our work is to understand the mechanisms and risks of biocides for resistance and antibiotic cross-resistance evolution in bacteria to optimize their application and safeguard their efficacy. We use adaptive laboratory evolution experiments, phenotypic characterization, single-cell analysis, whole genome sequencing, and competition experiments to investigate AMR evolution and selection of the model organism E. coli in the presence of biocides. Our work shows that biocides have the potential to affect evolutionary processes towards AMR by increasing the rates of de-novo mutation and conjugation. Importantly, widely used compounds such as chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations. Furthermore, we show that single-cell phenotypic heterogeneity regarding tolerance (persistence) determines survival against specific biocides including QACs and isopropanol. Mechanistic investigations reveal that known antibiotic persister mechanisms contribute to persister formation to biocides. The evolution of high-level tolerance to different biocides is linked to the initial persister level and the evolution of specific genetically encoded mechanisms related to properties of the cell envelope. Biocide-tolerant strains have a selective advantage in the presence of environmentally-relevant concentrations of antibiotics, which could lead to the stabilization of biocide tolerance in environments where biocides and antibiotics co-occur (e.g. wastewater, animal stables). Taken together, our work shows the importance of assessing the contribution of biocides on evolution and selection of AMR in the environment. T2 - 10th Symposium on Antimicrobial Resistance in Animals and the Environment (ARAE) CY - Berlin, Germany DA - 30.06.2025 KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 AN - OPUS4-64869 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Biocides as drivers for the evolution of antimicrobial resistance N2 - This presentation provides an overview about how biocides drive the evolution of antimicrobial resistance during application and as pollutants in the environment. The presentation shows that biocides can lead to heterogeneous killing, facilitating tolerance evolution. This evolution is related to decreased susceptibility to antibiotics and has potential for co-selection. In contrast, evolved tolerance can limit antibiotic evolvability via epistatic interactions. Moreover, biocides can co-select for antibiotic resistance in wastewater and affect rates of mutation and horizontal gene transfer. Biocides and antibiotics show strong combination effects with consequences for selection of antibiotic resistance. T2 - Vorstellungsvortrag zur Habilitation FU Berlin CY - Berlin, Germany DA - 15.05.2025 KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 AN - OPUS4-64873 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Resistance evolution to antimicrobial biocides N2 - This talk discusses resistance evolution to antimicrobial biocides. It shows (i) that biocides show heterogeneous killing facilitating tolerance evolution, (ii) that serial transfer at subinhibitory concentrations is not appropriate to model evolutionary adaptation to disinfection, (iii) that biocides affect rates of mutation and horizontal gene transfer Biocides, and (iv) that antibiotics show strong combination effects. T2 - Evolutionary Biology meets the Antibiotic Crisis vol. 2 CY - Plön, Germany DA - 24.09.2024 KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2024 AN - OPUS4-61545 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Dr. Frank Schreiber and Work on Biocide Resistance at BAM N2 - This talk details the career path of Dr. Frank Schreiber and his work on biocide resistance at BAM. Biocides are antimicrobial products for defined applications (disinfectants, preservatives, pest control). Biocides show heterogeneous killing facilitating resistance/tolerance evolution. Adaptation to biocides has effects on growth and selection. T2 - Vortrag an der Berliner Hochschule für Technik CY - Berlin, Gemany DA - 07.06.2024 KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2024 AN - OPUS4-61543 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Biocides as drivers for the evolution and selection of antimicrobial resistance N2 - This presentation provides an overview about how biocides drive the evolution of antimicrobial resistance during application and as pollutants in the environment. The presentation shows that biocides can lead to heterogeneous killing, facilitating tolerance evolution. This evolution is related to decreased susceptibility to antibiotics and has potential for co-selection. In contrast, evolved tolerance can limit antibiotic evolvability via epistatic interactions. Moreover, biocides can co-select for antibiotic resistance in wastewater and affect rates of mutation and horizontal gene transfer. Biocides and antibiotics show strong combination effects with consequences for selection of antibiotic resistance. T2 - 12. Dresdner Wasserseminar ' Wasser und Verunreinigung' CY - Dresden, Germany DA - 26.06.2025 KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 AN - OPUS4-64868 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Beyond Antibiotics – Biocides as Drivers of Environmental AMR N2 - This presentation provides an overview about how biocides drive the evolution of antimicrobial resistance during application and as pollutants in the environment. It also discusses the contribution of biocides for the environmental transmission of AMR. T2 - OneBridge: Making environmental AMR Surveillance Fit for Purpose: Data Integration and the Ecology of Resistance CY - Dresden, Germany DA - 06.10.205 KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 AN - OPUS4-64872 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 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-578555 DO - https://doi.org/10.3389/fmicb.2023.1180128 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 - TY - CONF A1 - Schreiber, Frank T1 - Resistance evolution towards biocides and antimicrobial surfaces N2 - This presentation describes our work at BAM on resistance evolution towards biocides and antimicrobial surfaces. T2 - Break biofilms workshop CY - Vienna, Austria DA - 16.01.2023 KW - Antimicrobial resistance KW - Bacteria KW - Biofilms KW - Biocides PY - 2023 AN - OPUS4-57857 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schmidt, Selina T1 - Antimicrobial resistance in soil microbes mediated by resistance evolution and horizontal gene transfer (HGT) N2 - Biocides are in contact with soils through direct application and passive leaching from protected materials. Consequently, soil microorganisms are exposed to biocides even though they are not their primary targets. Soil is a large reservoir of microbial diversity and has been hypothesized to be a crucial factor for the evolution and spread of antimicrobial resistance. Currently, there is little knowledge on how biocides used to protect materials affect the evolution and spread of resistance. Thus, our aim is to investigate the risk for the evolution of biocide resistance and cross-resistance to antibiotics. In addition, we aim to elucidate the affect of biocides on the spread of resistance via horizontal gene transfer (HGT). In adaptive laboratory evolution experiments we cultured selected model soil microorganism with representative biocides under selection regimes with increasing and stable biocide concentrations followed by antibiotic and biocide cross-resistance determination. Moreover, we investigate if the selected biocides affect the rates of de novo mutations and HGT of plasmids that carry resistance genes among soil microorganism. Our results show only small increases of biocide resistance during serial transfers under increasing biocide concentrations. One reason for this might be the narrow selective window for biocide resistance due to steep dose-response relationships. Furthermore, our results indicate that a stable low-level biocide regime did not select for high level cross-resistance to antibiotics and other biocides. Moreover, material preservatives affected the rates of HGT via conjugation and the mutation rates at sub-inhibitory concentrations. The results will enable future risk assessment regarding resistance evolution for biocides used as material preservatives. T2 - 4th Evo Eco PhD Meeting CY - Lutherstadt Wittenberg DA - 04.03.2020 KW - Microbiology KW - Biocides KW - Horizontal gene transfer HGT KW - Resistance evolution KW - Antimicrobial resistance PY - 2020 AN - OPUS4-51313 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 - Schreiber, Frank T1 - Biocide resistance risk assessment N2 - This presentation details the current status of biocide resistance risk assessment. T2 - Risk assessment of biocide and antibiotic resistance CY - Online meeting DA - 09.03.2022 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Standardization KW - Biocides KW - Risk assessment PY - 2022 UR - https://www.gu.se/en/biocide/risk-assessment-of-biocide-and-antibiotic-resistance AN - OPUS4-56235 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schmidt, Selina B. I. A1 - Täschner, Tom A1 - Nordholt, Niclas A1 - Schreiber, Frank T1 - Differential Selection for Survival and for Growth in Adaptive Laboratory Evolution Experiments With Benzalkonium Chloride N2 - Biocides are used to control microorganisms across different applications, but emerging resistance may pose risks for those applications. Resistance to biocides has commonly been studied using adaptive laboratory evolution (ALE) experiments with growth at subinhibitory concentrations linked to serial subculturing. It has been shown recently that Escherichia coli adapts to repeated lethal stress imposed by the biocide benzalkonium chloride (BAC) by increased survival (i.e., tolerance) and not by evolving the ability to grow at increased concentrations (i.e., resistance). Here, we investigate the contributions of evolution for tolerance as opposed to resistance for the outcome of ALE experiments with E. coli exposed to BAC. We find that BAC concentrations close to the half maximal effective concentration (EC50, 4.36 μg mL−1) show initial killing (~40%) before the population resumes growth. This indicates that cells face a two‐fold selection pressure: for increased survival and for increased growth. To disentangle the effects of both selection pressures, we conducted two ALE experiments: (i) one with initial killing and continued stress close to the EC50 during growth and (ii) another with initial killing and no stress during growth. Phenotypic characterization of adapted populations showed that growth at higher BAC concentrations was only selected for when BAC was present during growth. Whole genome sequencing revealed distinct differences in mutated genes across treatments. Treatments selecting for survival‐only led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ), while treatments selecting for growth and survival led to mutations in genes related to stress response (hslO and tufA). Our results demonstrate that serial subculture ALE experiments with an antimicrobial at subinhibitory concentrations can select for increased growth and survival. This finding has implications for the design of ALE experiments to assess resistance risks of antimicrobials in different scenarios such as disinfection, preservation, and environmental pollution. KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-615496 DO - https://doi.org/10.1111/eva.70017 VL - 17 IS - 10 SP - 1 EP - 11 PB - Wiley AN - OPUS4-61549 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 -