TY - CONF A1 - Schulz, Anne-Christine T1 - Co-selection for biocide and antibiotic resistance in microbial wastewater communities N2 - Biocides are used for a wide range of purposes, including disinfectants or preservatives. They 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 Here, we want to investigate co-selection processes of antibiotic resistance in natural WWTP microbial communities upon biocide exposure. Microbial communities were sampled at the WWTP Ruhleben in Berlin and characterized regarding their susceptibility against different clinically relevant antibiotics. To investigate the link between biocide exposure and antibiotic resistance, changes in the susceptibility level after exposure to environmentally relevant concentrations of the commonly used biocide didecyldimethylammonium chloride (DDAC) will be determined by enumerating resistant and non-resistant E. coli on selective plates with and without antibiotics and DDAC. In case of antibiotics, clinical breakpoint concentrations according to EUCAST will be used to discriminate between susceptible and resistant strains. In case of DDAC (and biocides in general), clinical breakpoints do not exist. Therefore, we determined a cut-off concentration at which the majority of naturally-occurring E. coli strains cannot grow anymore based on (I) the MIC (minimal inhibitory concentration) distribution, and (II) by plating wastewater communities onto selective indicator agar plates loaded with increasing DDAC concentration. Additionally, antibiotic cross-resistance will be determined by spotting single colonies, isolated from DDAC-selective plates onto antibiotic plates. The results of our experiments will help to determine selective concentrations and to estimate the risk of antibiotic co-selection and cross-resistance in microbial WWTP communities upon biocide exposure. T2 - Annual Conference 2023 of the Association for General and Applied Microbiology CY - Göttingen, Germany DA - 10.09.2023 KW - Antibiotic resisitance KW - Biocide KW - Wastewater KW - Risk assesment PY - 2023 AN - OPUS4-58510 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie A1 - Schreiber, Frank T1 - Antibiotic tolerance of biofilms emerging fro multicellular effects of antibiotic efflux N2 - The overarching goal of this project is to develop a predictive model for efflux-mediated antimicrobial tolerance in bacterial multicellular assemblies. Our central hypostasis is that efflux pump activity causes emergent antibiotic tolerance of multicellular bacterial populations, through the interplay of efflux mediated spatial interactions and efflux-linked persistence. To test this hypothesis, we will use a combination of microscopy, microbial killing assays, computational modelling, and data analysis, integrating information from 3 types of multicellular assembly: colonies, cell-to-cell interactions in a monolayer microfluidic device, and 3D flow chamber biofilms. Building on our preliminary observations, we will experimentally characterize the link between colony structure and spatial patterns of efflux gene expression in strains that differ in their levels of efflux. We will develop a mathematical model to test whether local growth inhibition of neighbors due to effluxing cells, coupled with local environment-dependent regulation of efflux, can account qualitatively for these results. By including persister cell formation in our model we will predict, and measure, the emergent function of antimicrobial tolerance in our colonies. To fully understand how tolerance emerges from the interplay between efflux-mediated spatial interactions and efflux-linked persister cell formation, we need quantitative measurements at the single cell level. To this end, we will use a microfluidic setup with cells growing in a monolayer to qualify in detail the dependence of efflux expression and persister cell formation on nutrient conditions, the correlation between efflux and persister formation, and the spatial range of efflux-mediated neighbour growth inhibition. To predict and quantitatively understand the emergent multicellular function of tolerance, we will perform individual-based modelling of biofilm growth, using as input the parameters measured on the single-cell level with our microfluidics experiments. Our simulations will predict biofilm spatial structure development, patterns of efflux and persister formation and, ultimately, tolerance to antimicrobial challenge. These predictions will be directly tested in flow-cell biofilm experiments. We are currently generating acrAB-tolC knockout-strain, without efflux activity, and a strain with an inducible acrAB-tolC efflux pump. To distinguish the different strains under the microscope, they were labeled with genes encoding for different fluorescent proteins. All strains are currently characterized in terms of growth, minimum inhibitory concentration of different antimicrobial substances, colony morphology, and biofilm formation ability. On the theoretical side, we are currently working on modeling the system at various scales and degree of detail, ranging from coarse-grained continuum models to stochastic, individual-based models. Some exploratory work was doe to test existing software for individual-based modelling that may be adapted for our purpose. Furthermore, we are in the process of developing more coarse-grained models. This work involves some physiological modelling and literature search, focusing on working mechanisms of efflux pumps and kinetic models for import and export of antibiotics. T2 - SPP Meeting CY - Jena, Germany DA - 04.10.2023 KW - Antibiotic KW - Bioilm KW - Tolerance KW - Efflux PY - 2023 AN - OPUS4-59245 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Die Entstehung von antimikrobiellen Resistenzen durch die Verwendung von Bioziden N2 - Dieser Vortrag gibt einen Überblick über die BAM und die Aktivitäten im Bereich Biozidresistenz. T2 - Berliner Hochschule für Technik Studiengang Biotechnologie CY - Berlin, Germany DA - 05.05.2023 KW - Antimikrobielle Resistenz KW - Antmikrobielle Oberflächen KW - Standardisierung PY - 2023 AN - OPUS4-57859 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Keshmiri, Hamid T1 - Plasmon-enhanced diffractive supercells N2 - Multiple diffractive surface modulations can concurrently couple the light to several electromagnetic surface waves. We present a multi-resonant plasmonic supercell structure with a broad range of applicability in harvesting the light over an extensive wavelength range and angles of incidence. T2 - Molecular Plasmonics 2023 CY - Jena, Germany DA - 11.05.2023 KW - Optics PY - 2023 AN - OPUS4-59248 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - How to regulate and assess resistance risks of co-selecting agents during application and in the environment N2 - This talk deals with the question on How to regulate and assess resistance risks of co-selecting agents during application and in the environment. It shows that there the overview of worldwide activities to regulate co-selecting agents is missing. Regulations for product authorization usually consider resistance in target organisms, but there is a risk of emergence of resistance from non-target organisms as well. Moreover, pollution effects on resistance development in the environment are not explicitly covered during product authorization and few risk assessment schemes and methods available. T2 - EDAR7 - Environmental Dimension of Antimicrobial Resistance Conference 2024 CY - Montreal, Canada DA - 26.05.2024 KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2024 AN - OPUS4-61542 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Metabolic niches and persistence of antibiotic resistant bacteria in the environment N2 - Background and Aim: Wastewater treatment plants are considered as hotspots for the development and spread of antimicrobial resistance. Antimicrobial resistant bacteria (ARB) can persist in the environment for long periods of time, despite metabolic fitness costs that can arise with resistance. We are interested in understanding the mechanisms, which allow ARB to persist in the environment. More specifically, we want to identify metabolic niches that can select for resistant bacteria. Procedure/Method: 62 E. coli strains isolated from different WWTPs in Norway were used. The isolates have different levels of resistance to 14 antibiotics. The susceptibility of the isolates to 3 disinfectants was determined. In addition, genome scale metabolic models (GEMs) were constructed and the growth of the strains was simulated in the presence of 198 different carbon sources. Furthermore, the growth rates of the isolates were measured in the presence of 3 carbon sources. Findings/Results: Many of the isolates have high resistance to multiple antibiotics but only few of the isolates have higher minimum inhibitory concentrations to the disinfectants, compared to an E. coli laboratory strain. With the GEMs, we identified 40 carbon sources that can be utilized for growth only by a portion of all the isolates. The prediction accuracy of the GEMs was 93% in the case of D-Malate. A group of 10 isolates was identified of which 5 isolates are resistant to ciprofloxacin, gentamicin and tetracycline and can grow on Sucrose but not on D-Malate and the other 5 isolates are susceptible to the same 3 antibiotics but can grow on D-Malate and not on Sucrose. Implications/Applications: Our data suggest that changing the available carbon source could shift the selection advantage between resistant and susceptible bacterial strains. If this strategy is confirmed experimentally, it could be applied to reduce the number of ARB in environments like wastewater. T2 - Conference on the Environmental dimentions of antimicrobial resistance 7 CY - Montreal, Canada DA - 26.05.2024 KW - Antimicrobial resistance KW - Wastewater PY - 2024 AN - OPUS4-61410 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dinter, Adelina-Elisa T1 - Examination of biological samples by means of single-cell ICP-ToF-MS N2 - Up to now, different analytical methods for single cell analysis exist focusing on key features such as size, shape, morphology and elemental composition. The combination of the latest ICP-MS techniques - ICP-ToF-MS - together with the latest developments in the field of sample delivery - micro droplet generator (MDG) – will allow a Deep insight into the composition and size of cells. Microbiologically influenced corrosion (MIC) is an oxidation of metals affected by the presence or activity (or both) of microorganisms e.g. Shewanella Putrefaciens in biofilms on the surface of the corroding material. As this can happen for example in the soil on iron pipes of water pipes, in oil tanks or on steel sheet piling, there is great interest in MIC research, not only from various industrial sectors, but also from the environmental aspect. T2 - SALSA - Make & Measure CY - Online Meeting DA - 15.10.2020 KW - SC-ICP-ToF-MS KW - Single cell analysis KW - MDG ICP-ToF-MS KW - Microdroplet generator PY - 2020 AN - OPUS4-52441 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kalbe, Ute T1 - Identifikation und Einordnung von historischen Lehmbauteilen für die Wiederverwendung hinsichtlich Anreicherung von Schadstoffen und bauschädlichen Salzen N2 - Lehmbaustoffe, einschließlich Lehm aus dem historischen Baubestand, bieten ein hohes Potenzial für ihre Wiederverwendbarkeit und die Schonung natürlicher Ressourcen. Zur Bewertung des Wiederverwendungspotenzials gibt es nur wenige Studien hinsichtlich der Schadstoffbelastung und der Anreicherung von Salzen in Gebäuden aus dem historischen Bestand. Tonminerale, die in Lehmbaustoffen als Bindemittel fungieren, können Schadstoffe binden. Die Region Mitteldeutschland ist geprägt durch jahrzehntelange Einwirkung erheblicher Schadstoffemissionen aus der Chemie- und Braunkohleindustrie. Der Eintrag von Schadstoffen in Lehmbaustoffe ist außerdem aus geogenen Hintergrundbelastungen denkbar, z. B. in Auenböden beeinflusst durch Einträge aus dem Erzbergbau. Salzbildende Anionen wie Chloride oder Nitrate können aufgrund ihrer hohen Löslichkeit mittels kapillarer Kräfte durch die Porenstruktur transportiert werden. In Lehmwänden können sie vor allem in oberflächennahen Lagen, in denen höhere Verdunstungsraten vorherrschen, durch Bindung mit in Lösung vorliegenden oder baustoffeigenen Kationen auskristallisieren und bauschädliche Salze ausbilden. Auch durch spezifische Nutzungen akkumulierte Schadstoffe, z. B. durch die Einwirkung von Tabakrauch, könnten theoretisch einer Wiederverwendung von Lehm entgegenstehen. In Anbetracht der Herausforderungen hinsichtlich der klimatischen und gesellschaftlichen Veränderungen, sind wissenschaftliche Erkenntnisse im Lehmbau erforderlich, die eine Integration in eine nachhaltige Kreislaufwirtschaft ermöglichen. Bei bisherigen Gesetzgebungsverfahren zu rezyklierten Baustoffen wurden Lehmbaustoffe noch nicht berücksichtigt. Ziel einer Vorstudie im Rahmen der WIR!-Initiative GOLEHM ist die Überprüfung der Wiederverwendbarkeit Wiederverwendbarkeit von Lehmbaustoffen aus historischen Bauten an Stichproben hinsichtlich der Umwelt- und Nutzerverträglichkeit. Für den Raum Mitteldeutschland können damit Anhaltspunkte darüber gegeben werden, welche Schadstoffe und bauschädlichen Salze in welchem Maße in Lehmbaustoffen aus dem Gebäudebestand erwartet werden können. Daraus können Hinweise zum Umgang mit Lehmbaustoffen aus historischen Gebäuden abgeleitet werden. Darüber hinaus wurden exemplarisch an zwei Gebäuden mikrobiologische Untersuchungen zur Ermittlung einer möglichen Belastung mit Schimmelpilzen vorgenommen. T2 - Lehm 2024 Fachtagung CY - Weimar, Germany DA - 27.09.2024 KW - Lehm KW - Schadstoffe KW - Wiederverwendung KW - Historischer Baubestand PY - 2024 AN - OPUS4-61881 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kalbe, Ute A1 - Schröder, Simon Alexander A1 - Richter, Matthias A1 - Horn, Wolfgang A1 - Stephan, Ina A1 - Hohmann, Gloria A1 - Ziegert, Christof T1 - Identifikation und Einordnung von historischen Lehmbauteilen für die Wiederverwendung hinsichtlich Anreicherung von Schadstoffen und bauschädlichen Salzen T1 - Identification and classification of historical earth construction elements for recycling in respect of accumulation of pollutants and structurally damaging salts N2 - Lehmbaustoffe, einschließlich Lehm aus dem historischen Baubestand, bieten ein hohes Potenzial für ihre Wiederverwendbarkeit und die Schonung natürlicher Ressourcen. Zur Bewertung des Wiederverwendungspotenzials gibt es nur wenige Studien hinsichtlich der Schadstoffbelastung und der Anreicherung von Salzen in Gebäuden aus dem historischen Bestand. Tonminerale, die in Lehmbaustoffen als Bindemittel fungieren, können Schadstoffe binden. Die Region Mitteldeutschland ist geprägt durch jahrzehntelange Einwirkung erheblicher Schadstoffemissionen aus der Chemie- und Braunkohleindustrie. Der Eintrag von Schadstoffen in Lehmbaustoffe ist außerdem aus geogenen Hintergrundbelastungen denkbar, z. B. in Auenböden beeinflusst durch Einträge aus dem Erzbergbau. Salzbildende Anionen wie Chloride oder Nitrate können aufgrund ihrer hohen Löslichkeit mittels kapillarer Kräfte durch die Porenstruktur transportiert werden. In Lehmwänden können sie vor allem in oberflächennahen Lagen, in denen höhere Verdunstungsraten vorherrschen, durch Bindung mit in Lösung vorliegenden oder baustoffeigenen Kationen auskristallisieren und bauschädliche Salze ausbilden. Auch durch spezifische Nutzungen akkumulierte Schadstoffe, z. B. durch die Einwirkung von Tabakrauch, könnten theoretisch einer Wiederverwendung von Lehm entgegenstehen. In Anbetracht der Herausforderungen hinsichtlich der klimatischen und gesellschaftlichen Veränderungen, sind wissenschaftliche Erkenntnisse im Lehmbau erforderlich, die eine Integration in eine nachhaltige Kreislaufwirtschaft ermöglichen. Bei bisherigen Gesetzgebungsverfahren zu rezyklierten Baustoffen wurden Lehmbaustoffe noch nicht berücksichtigt. Ziel einer Vorstudie im Rahmen der WIR!-Initiative GOLEHM ist die Überprüfung der Wiederverwendbarkeit Wiederverwendbarkeit von Lehmbaustoffen aus historischen Bauten an Stichproben hinsichtlich der Umwelt- und Nutzerverträglichkeit. Für den Raum Mitteldeutschland können damit Anhaltspunkte darüber gegeben werden, welche Schadstoffe und bauschädlichen Salze in welchem Maße in Lehmbaustoffen aus dem Gebäudebestand erwartet werden können. Daraus können Hinweise zum Umgang mit Lehmbaustoffen aus historischen Gebäuden abgeleitet werden. Darüber hinaus wurden exemplarisch an zwei Gebäuden mikrobiologische Untersuchungen zur Ermittlung einer möglichen Belastung mit Schimmelpilzen vorgenommen. N2 - Earth construction materials, including earth from historical building stock, offer high potential in terms of their recyclability and sparing use of natural resources. Yet, only a few studies have been conducted on the levels of pollutants and the accumulation of salts in buildings from the historical building stock to enable an assessment of the potential for recycling. The aim of a preliminary study as part of the GOLEHM Initiative from WIR! is to use spot checks to review the recyclability of earth construction materials from historical buildings in terms of environmental impact and tolerance by users. This could provide indicators for the Central Germany region on which pollutants and structurally damaging salts can be expected in earth construction materials from the building stock and to what extent, allowing conclusions to be drawn on how to manage earth construction materials from historical buildings. Additionally, microbiological inspections of two sample buildings were performed to assess a potential contamination with mould. T2 - Lehm 2024 Fachtagung CY - Weimar, Germany DA - 27.09.2024 KW - Lehm KW - Wiederverwendung KW - Kreislaufwirtschaft KW - Schadstoffe KW - Bauschädliche Salze PY - 2024 SP - 1 EP - 8 CY - Weimar AN - OPUS4-61883 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Wen, Keqing A1 - Gorbushina, Anna A1 - Schwibbert, Karin A1 - Bell, Jérémy T1 - A microfluidic platform for monitoring biofilm formation in flow under defined hydrodynamic conditions N2 - Bacterial adhesion on surfaces of medical, water and food applications may lead to infections, water or food spoilage and human illness. In comparison to traditional static and macro flow chamber assays for biofilm formation studies, microfluidic chips allow in situ monitoring of biofilm formation under various flow regimes, have better environment control and smaller sample requirements. In this work, a novel microfluidic platform is developed to investigate biofilm adhesion under precisely controlled bacteria concentration, temperature, and flow conditions. This platform central unit is a single-inlet microfluidic flow cell with a 5 mm wide chamber designed and tested to achieve ultra-homogenous flow in the central area of chamber. Within this area, defined microstructures are integrated that will disturb the homogeneity of the flow, thus changing bacterial adhesion pattern. Here we present the monitoring of bacterial biofilm formation in a microfluidic chip equipped with a microstructure known as micro-trap. This feature is based on a 3D bacteria trap designed by Di Giacomo et al. and successfully used to sequester motile bacteria. At first, fluorescent particles similar in size to Escherichia coli (E. coli) are used to simulate bacteria flow inside the flow cell and at the micro-trap. The turbulences induced by the trap are analyzed by imaging and particle tracking velocimetry (PTV). Secondly, the model strain E. coli TG1, ideal and well described for biofilm studies, is used to analyze biofilm formation in the micro-trap. Therefore, a stable fluorescent strain E. coli TG1-MRE-Tn7-141 is constructed by using Tn7 transposon mutagenesis according to the method described by Schlechter et al. Sequestering of E. coli cells within the micro-trap was followed using epifluorescence microscopy. The novel microfluidic platform shows great potential for assessment of bacterial adhesion under various flow regimes. The performance of structural feature with respect to the generation of turbulences that promote or reduce bacterial adhesion can be systematically examined. The combination of flow analysis and fluorescent strain injection into the microfluidic chip shows that the micro-trap is useful for capturing bacteria at defined positions and to study how flow conditions, especially micro-turbulences, can affect biofilm formation. It represents a powerful and versatile tool for studying the relation between topography and bacteria adhesion. T2 - International Conference on Miniaturized Systems for Chemistry and Life Sciences CY - Katowice, Poland DA - 15.10.2023 KW - Biofilm KW - E. coli KW - Microfluidics KW - Velocimetry KW - Fluorescence PY - 2023 AN - OPUS4-59593 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - ALEE-AMC: Bacterial resistance evolution towards antimicrobial surfaces and development of a standardized test N2 - Background: Antimicrobial surfaces and coatings (AMCs) are important to protect man-made structures from biodeterioration and biodegradation. Advances in nano-structuring methods hold the promise of a new generation of AMCs. However, the evolution and selection of bacterial resistance to AMCs may threaten their efficacy in the long term. Therefore, according to the EU Biocidal Products Regulation, the risk of resistance development upon exposure to AMCs must be evaluated during product authorization. The same applies to the development of cross-resistances to other substances, for instance biocides and antibiotics. However, no standardized method exists to assess the risk of resistance and cross-resistance development upon exposure to AMCs during the authorization process. Objectives: • To develop a standardizable adaptive laboratory evolution experiment to be performed on AMCs (ALEE-AMC) • To assess performance and robustness of ALEE-AMC in a round robin test, using a copper AMC as reference • To uncover the mechanisms underlying evolution of resistance to copper AMC Materials & Methods: ALEE-AMC was developed based on an approved standard to determine the efficacy of antimicrobial surfaces (ISO 22196). ALEE-MC was performed on an antimicrobial copper surface as reference material and Escherichia coli as model organism. A round robin test was conducted with six participants to evaluate the reproducibility and applicability of ALEE-AMC. Evolved E. coli populations from the round robin partners were collected and subjected to phenotypic (antimicrobial susceptibility testing, ISO 22196) and genotypic (whole genome sequencing) characterization at BAM. Results: The results of the ALEE-AMC round robin test indicate that repeated exposure to copper can select for reduced copper susceptibility. However, failure of individual E. coli lineages to adapt to the copper surfaces was also observed. Evolved E. coli exhibited increased survival upon exposure to copper surfaces. Adaptation to copper did not induce cross-resistance to antibiotics. Whole genome sequencing of the evolved E. coli revealed high diversity of mutations among individual evolved strains, indicating the existence of multiple, underexplored evolutionary pathways towards increased survival of antimicrobial copper surfaces. Conclusion & Significance: ALEE-AMC offers a standardizable platform to assess the risk of resistance development towards novel and existing AMCs. Specifically, using ALEE-AMC in a round robin test, insights into evolvable survival mechanisms to copper AMCs have been gained. These mechanistic insights may be exploited to prevent the evolution against copper AMCs. In future steps, criteria need to be defined to provide guidelines for the authorization of AMCs based on the outcomes of ALEE-AMC T2 - International Biodeterioration and Biodegradation Symposium, Berlin, Germany CY - Berlin, Germany DA - 09.09.2024 KW - Biocides KW - Antimicrobial surfaces KW - Resistance KW - Evolution KW - Standardized test PY - 2024 AN - OPUS4-61176 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - RPRT A1 - Stephan, Ina A1 - Boenke, Viola A1 - Drecker, Sigrid T1 - Weiterentwicklung und Verbesserung des Konzeptes zur Begrenzung von Konservierungsmitteln für Produkte mit dem Blauen Engel N2 - Im Rahmen des Forschungsvorhabens wurde die biologische Prüfmethode der Checkliste zur stoffli-chen Bewertung im Rahmen des Aufnahmeverfahrens für weitere Topfkonservierungsmittel in den Anhang 1 zur Vergabegrundlage RAL-UZ102 „Emissionsarme Innenwandfarben“ überarbeitet. Der Test für den Einsatz von Konservierungsmitteln in seiner jetzigen Fassung besteht seit nahezu 20 Jah-ren und erfolgt ausschließlich an weißer Dispersionsfarbe für den Innenraum. Die hierbei ermittelten Höchstmengen für den Einsatz von Topfkonservierern werden z.Zt. auch auf andere Innenraumbau-produkte übertragen. Ziel war es zu klären, ob die Anforderungen an die Topfkonservierung bei Far-ben tatsächlich für andere Innenraumbauprodukte übernommen werden können. Die im Vorhaben durchgeführten Versuche deuten darauf hin, dass dies nicht ohne weiteres möglich ist. Nach Rückspra-che mit Vertretern aus der Industrie wurde das Keimspektrum für die Prüfung zudem um Hefe- und Schimmelpilze erweitert. Des Weiteren wurde eine künstliche Alterung eingeführt und überprüft, ob dies einen Einfluss auf den Gehalt an Isothiazolinonen hat. Die Konzentration von Isothialzolinonen wurde mittels Ultra-Hochleistungsflüssigkeitschromatographie kontrolliert und hinsichtlich einer Kor-relation mit dem Wachstum von Mikroorganismen ausgewertet. Da die in den Innenraumbauproduk-ten zugesetzten Isothiazolinone als Kontaktallergene bekannt sind, ist es ein Ziel des „Blauen Engel“-Umweltzeichens, die geringste erforderliche Menge an Konservierungsmittel in den Produkten zu ver-wenden, ohne die positive Auswirkung der Topfkonservierung zu gefährden. Daher sind die Optimie-rung der Leistungsstärke von Konservierungsmitteln und die Kontrolle der Hygiene im Herstellungs-prozess von zunehmender Bedeutung. Um Einblicke in den Herstellungsprozess von Farben zu gewin-nen, wurden im Rahmen des Vorhabens vier Farbwerke besichtigt, in denen weiße Innenraumfarben produziert werden, die mit dem Umweltzeichen „Blauer Engel“ ausgezeichnet sind. Gemeinsam mit den Fachleuten vor Ort wurde diskutiert, welche Maßnahmen zur Verbesserung der Werkshygiene er-griffen werden können, um den Einsatz von Topfkonservierern möglichst gering zu halten. Des Weite-ren wurde in dem Forschungsvorhaben untersucht, ob eine schnellere Bestimmung der Keimzahl in den Rohstoffen mittels quantitativer Polymerasekettenreaktion bereits im Farbwerk realisierbar ist. KW - Methode KW - Isothiazolinone KW - Gebindekonservierung KW - Bakterien KW - Schimmel KW - Wirksamkeit KW - Biozide PY - 2020 SN - 1862-4804 VL - 54 SP - 4 EP - 149 PB - Umweltbundesamt CY - Dessau AN - OPUS4-50972 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Bacterial resistance evolution on antimicrobial surfaces: Mechanistic insights from a standardizable method N2 - Introduction: Antimicrobial surfaces and coatings (AMCs) are important to prevent the spread of pathogens, especially in hygiene-sensitive areas. However, the evolution and selection of bacterial resistance to AMCs may threaten their efficacy in the long term. In addition, resistance evolution to AMCs may pose the risk for the development of cross-resistance to antibiotics. The assessment of unacceptable resistance risks during the authorization of AMCs is hampered by the lack of standardized test methods that quantify the adaptability of exposed bacteria to AMCs. Objectives: • To develop a standardizable method to determine resistance evolution of bacteria on AMCs (ALEE-AMC) • To assess performance and robustness of ALEE-AMC in a ring trial • To uncover the mechanisms underlying evolution of resistance to a metallic copper AMC • To use ALEE-AMC to assess the evolution of resistance on a novel, nano-particle-based AMC Methods: ALEE-AMC was developed based on an international standard to determine the efficacy of antimicrobial surfaces (ISO 22196). In the ALEE-AMC test, adaptive laboratory evolution is conducted by repeated cycles of AMC exposure and re-growth of surviving cells, selecting for increased survival, followed by isolation of evolved clones. Metallic copper was used as a reference AMC and Escherichia coli as a model microorganism in the ring trial. Evolved E. coli populations from the ring trial partners were subjected to phenotypic (antimicrobial susceptibility testing, ISO 22196) and genotypic (whole genome sequencing) characterization. ALEE-AMC will be used to assess the evolution of resistance on a novel, nano-particle-based AMC currently under development. Findings: The results of the ALEE-AMC ring trial show that repeated exposure to a metallic copper AMC can reproducibly select for reduced copper susceptibility in individual evolutionary lineages across ring trial participants. However, failure to adapt in individual lineages was also observed in all trials. Isolated evolved E. coli clones exhibited increased survival upon exposure to copper surfaces. Adaptation to copper did not induce cross-resistance to antibiotics because the antibiotic susceptibility of copper-adapted clones did not increase above the clinical breakpoint. Whole genome sequencing of the evolved E. coli revealed a high diversity of mutations, including mutations in genes involved in survival to antibiotics. These results indicate the existence of multiple, underexplored evolutionary pathways towards increased survival of antimicrobial copper surfaces. Conclusion: ALEE-AMC offers a standardizable platform to assess the risk of resistance development towards novel and existing AMCs, including nano-particle-based and metallic copper AMCs. Specifically, using ALEE-AMC provided insights into evolvable survival mechanisms to copper AMCs and its consequences for antimicrobial resistance. T2 - FEMS MICRO 2025 CY - Mailand, Italy DA - 14.07.2025 KW - Biocides KW - Antimicrobial surfaces KW - Biocide resistance KW - Standardization KW - ISO 22196 KW - Evolution PY - 2025 AN - OPUS4-63837 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Development of a laboratory method to assess resistance development of microorganisms to biocides – An update N2 - This presentation describes the development of a laboratory method to assess resistance development of microorganisms to biocides and antimicrobial surfaces. T2 - The International Biodeterioration Research Group (IBRG) autumn meeting 2022 CY - Online meeting DA - 11.10.2022 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Standardization KW - Biocides KW - Risk assessment PY - 2022 AN - OPUS4-56264 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Barkeshli, M. A1 - Stephan, Ina ED - Barkeshli, M. ED - Arya, A. ED - Nath, V. ED - Abduraheem, K. T1 - Sizing materials used in Persian manuscripts and their behaviour against the mould fungus Aspergillus flavus N2 - Our earlier study based on historical and scientific analysis showed that Iranians used a considerable range of materials in the sizing process. Unlike many nations who used limited sizing materials to improve the mechanical strength and to smoothen the paper surface, Iranians have used various materials for sizing process from Taimurid (15th century) to Safawid (16th century) and Qajar (19th century) periods. Further to our earlier study on the Sizing Materials used in Persian manuscripts and miniature paintings, scientific analysis was carried out to investigate their behaviour against the mould fungus Aspergillus flavus. In the first stage fourteen different sizing materials that was identified from Persian historical recipes were reconstructed. In the second stage fungicidal property of each sizing samples against Aspergillus flavus fungus were identified that will be presented in this paper. KW - Sizing KW - Materials KW - Persian manuscripts KW - Deterioration KW - Fungus KW - Aspergillus flavus PY - 2023 SN - 978-93-5461-714-0 SP - 3 EP - 20 PB - Daya Publishing House CY - New Delhi AN - OPUS4-58102 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Development of a laboratory method to assess resistance development of microorganisms to biocides – An update N2 - This presentation describes our efforts at BAM towards the development of a laboratory method to assess resistance development of microorganisms to biocides. T2 - The International Biodeterioration Research Group (IBRG) spring meeting 2023 CY - Online meeting DA - 05.03.2023 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Standardization KW - Biocides PY - 2023 AN - OPUS4-57858 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Development of a laboratory method to assess resistance development of microorganisms to biocides N2 - Bacteria are exposed to biocides through surface disinfection or by antimicrobial surfaces. These stressful environments provide a strong selective pressure for bacteria to adapt. Here, we describe the development of a laboratory method to assess adaption of bacteria by resistance development in response to surface disinfection and antimicrobial surfaces. T2 - OECD, 5th Meeting of the Working Party on Biocides CY - Online meeting DA - 26.05.2021 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Standardization PY - 2021 AN - OPUS4-53163 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Development of a laboratory method to assess resistance development of microorganisms to biocides N2 - Bacteria are exposed to biocides through surface disinfection or by antimicrobial surfaces. These stressful environments provide a strong selective pressure for bacteria to adapt. Here, we describe the development of a laboratory method to assess adaption of bacteria by resistance development in response to surface disinfection and antimicrobial surfaces. T2 - The International Biodeterioration Research Group (IBRG) Spring Meeting 2021 CY - Online meeting DA - 28.06.2021 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Standardization PY - 2021 AN - OPUS4-53164 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kalbe, Ute T1 - HistRCL- Vorstudie zur Identifikation und Einordnung von historischen Lehmbauteilen für die Wiederverwendung hinsichtlich der Anreicherung von Schadstoffen und bauschädlichen Salzen N2 - Lehm aus dem historischen Baubestand bietet ein hohes Potenzial für eine Wiederverwendung und die Schonung natürlicher Ressourcen. Anthropogene Einflüsse aus der Industrie der Region Mitteldeutschland und auch die Akkumulation von Schadstoffen durch spezifische Nutzungen sowie durch Feuchteeinfluss angereicherte bauschädliche Salze können einer Wiederverwendung entgegenstehen. Ziel des Projektes war daher die Überprüfung der Rezyklierbarkeit von Lehmbaustoffen aus historischen Bauten hinsichtlich der Umwelt- und Nutzerverträglichkeit. Dafür wurden exemplarisch Wellerlehmgebäude aus dem historischen Bestand mit einer potenziellen Belastung beprobt und untersucht. Die Ergebnisse liefern einen Beitrag, Unsicherheiten bei im Lehmbau tätigen Unternehmen hinsichtlich des sicheren Umganges mit möglichen stofflichen Belastungen abzubauen und diese bei der Einhaltung umweltrechtlicher und kreislaufgerechter Rahmenbedingungen bei der Wiederverwendung von Lehm aus dem historischen Bestand zu unterstützen. T2 - GOLEHM Innovationsforum CY - Halle (Saale), Germany DA - 26.07.2024 KW - Lehm KW - Wiederverwendung KW - Schadstoffe KW - Bauschädliche Salze PY - 2024 AN - OPUS4-61878 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dinter, Adelina-Elisa T1 - Deciphering corrosion processes of MIC organisms - single cell-ICP-ToF-MS analysis of archaea on solid steels N2 - ICP-ToF (time of flight) MS enables the analysis of the multi-element fingerprint of single cells. The single cell ICP-ToF-MS is used in the presented poster for the analysis of archaea involved in microbiologically influenced corrosion (MIC) of steel. By means of sc-ICP-ToF-MS, the possible uptake of individual elements from the respective steel is investigated - the information obtained will be used in the future to elucidate underlying mechanisms and develop possible material protection concepts. The work combines modern methods of analytical sciences with materials. T2 - SALSA - Make & Measure 2021 CY - Online meeting DA - 16.09.2021 KW - Sc-ICP-ToF-MS KW - Single cell analysis KW - Microbiologically influenced corrosion KW - Archaea KW - Poster presentation PY - 2021 AN - OPUS4-53337 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Ulbricht, Alexander T1 - Do Microbes like Additively Manufactured Aluminium? N2 - The use of laser-additively manufactured metallic components in the industrial sector is gaining traction, with the process now entering its application phase. Consequently, the materials are exposed to corrosive microbes, which this study examines in order to determine the implications of this exposure. Specifically, the study examines the exposure of laser powder bed fused AlSi10Mg (PBF-LB/AlSi10Mg) components to sulphate-reducing bacteria in an anaerobic environment. Is the specific PBF-LB microstructure a potential factor in determining or aiding biocorrosion? A prismatic specimen was exposed to sulphate-reducing bacteria (SRB) for two weeks. The sample was scanned by X-ray computed tomography (XCT) before and after SRB exposure. The two data sets were registered to each other (using the internal void distribution as markers, due to the corrosion of the specimen’s surface) to enable the evaluation of the corrosion damage. Virtual cuts of the 3D XCT reconstructions show a loss of volume and localised damage. The damage distribution aligns with the hatching pattern of laser exposure from laser powder bed fusion. T2 - Konferenz iCT 2026 CY - Linz, Austria DA - 10.02.2026 KW - Computed tomography KW - Biokorrosion KW - Additive manufacturing KW - PBF-LB KW - Laser powder bed fusion KW - Sulphate-reducing bacteria PY - 2026 AN - OPUS4-65570 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - THES A1 - Usmani, Shirin T1 - Wood treated with nano metal fluorides - relations between composition, size, and durability N2 - In this study, nanoscopic particles of magnesium Fluoride (MgF2) and calcium fluoride (CaF2) also known as nano metal fluorides (NMFs), were evaluated for their potential to improve wood durability. Even though these fluorides are sparingly soluble, their synthesis in the form of nano-sized particles turns them into promising candidates for wood preservation. Their distinct property of low-water solubility is proposed to maintain long-lasting protection of treated wood by reducing the leaching of fluoride. Analytical methods were used to characterize the synthesized NMFs and their distribution in treated wood specimens. Transmission electron microscopy images showed that these fluoride particles are smaller than 10 nm. In nano metal fluoride (NMF) treated specimens, aggregates of these particles are uniformly distributed in the wood matrix as confirmed with scanning electron microscopy images and their corresponding energy-dispersive X-ray spectroscopy maps. The fluoride aggregates form a protective layer around the tracheid walls and block the bordered pits, thus reducing the possible flow path for water absorption into wood. This is reflected in the reduced swelling and increased hydrophobicity of wood treated with NMFs. The biocidal efficacy of NMFs was tested against brown-rot fungi (Coniophora puteanaand Rhodonia placenta), white-rot fungus (Trametes versicolor), and termites (Coptotermes formosanus). The fungal and termite tests were performed in accordance with the EN 113 (1996) and EN 117 (2012) standards, respectively. Prior to fungal tests, the NMF treated wood specimens were leached according to the EN 84 (1997)standard. Compared to untreated specimens, the NMF treated wood specimens have a higher resistance to decay caused by brown-rot fungi, white-rot fungus, and termites. Although all NMF treatments in wood reduce the mass loss caused by fungal decay, only the combined treatment of MgF2 and CaF2 has efficacy against both brown-rot fungi and white-rot fungus. Similarly, wood treated with the combined NMF formulation is the least susceptible to attack by C. formosanus.It is proposed that combining MgF2 and CaF2changes their overall solubility to promote the release of fluoride ions at the optimal concentration needed for biocidal efficacy against fungi and termites. In this thesis, it was proven that even after leaching, sufficient fluoride was present to protect NMF treated wood from fungal decay. This shows that NMFs are robust enough for above ground contact outdoor applications of wood, where permanent wetness cannot be avoided according to Use Class 3.2, as per the EN 335 (2013) standard. Also, they pose a low risk to human health and the environment because they are sparingly soluble. Since NMFs significantly reduce the decay of wood, the CO2 fixed in it will be retained for longer than in unpreserved wood. Overall, the novel results of this study show the potential of NMFs to increase the service life of building materials made from non-durable wood. KW - Termites KW - Nano metal fluorides KW - Solubility KW - Wood protection KW - Fungi PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-525108 DO - https://doi.org/10.18452/22553 SP - i EP - 118 PB - Humboldt-Universität zu Berlin CY - Berlin AN - OPUS4-52510 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - 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 - 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 - 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 - 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 - 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 - 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 - 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 - CONF A1 - Schmidt, Selina T1 - Biocides Used as Material Preservatives Modify Rates of de novo Mutation and Horizontal Gene Transfer in Bacteria N2 - Biocides used as material preservatives are in contact with various environments during direct application or passive leaching from protected materials. Antimicrobial resistance (AMR) is a global health problem and the environment is an important compartment for the evolution and transmission of AMR. Soil is an environment with a large reservoir of natural microbial communities and antimicrobial resistance (AMR) genes. Those natural microbial communities are frequently exposed to biocides used as material preservatives. Previous studies have shown that antibiotics, metals and pesticides affect the underlying processes of resistance evolution and spread; namely de novo mutagenesis and horizontal gene transfer by conjugation and transformation in microbial communities. However, it is unknown if active substances used in material preservatives are involved in these processes. We show that biocides used as material preservatives affect rates of mutation and conjugation in microorganism in a species- and substance-dependent manner, while rates of transformation are not directly affected. Our data highlights the importance of assessing the contribution of material preservatives on AMR evolution and spread in the environment. T2 - Microbiome Network Meeting CY - Berlin, Germany DA - 20.07.2022 KW - Biocides KW - Horizontal gene transfer KW - Mutation rate PY - 2022 AN - OPUS4-55263 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Heterogeneous tolerance to biocides and its consequences for the evolution of antimicrobial resistance N2 - The exposure to antimicrobial substances drives the evolution of antimicrobial resistance (AMR). Biocides are antimicrobials used as disinfectants, antiseptics and preservatives. They find application on a large scale in the industrial and medical sector, but also in private households. In terms of mass, the worldwide use of biocides exceeds that of antibiotics. Thus, despite their important role in preventing the spread of pathogens, due to their ubiquity, biocides are suspected to be drivers of the antimicrobial resistance crisis. In our work we try to understand how biocides contribute to the emergence of AMR, what the underlying adaptation principles and mechanisms are and how they compare to those found for antibiotics. Here, I present results which demonstrate that phenotypic heterogeneity in E. coli, namely the formation of tolerant persister cells, can enable the survival of disinfection and consequentially facilitate the evolution of AMR. Using experimental evolution that selects for tolerance, we find that mutations in lipid A biosynthesis arise following periodic treatment with benzalkonium chloride (BAC), a widely used disinfectant. BAC tolerance is associated with additional, diverse mutations as well as changes in the susceptibility to antibiotics from different classes and changes in motility and biofilm formation, suggesting extensive regulatory re-wiring in the evolved clones. Remarkably, we find that fitness costs (growth rate reduction) incurred by BAC tolerance are alleviated in the presence of antibiotics. These findings demonstrate the complexity underlying the adaptation to antimicrobials and highlight the links between persistence to disinfectants and resistance evolution to antimicrobials. T2 - 4th VAAM discussion meeting 'Microbial Cell Biology' CY - Berlin, Germany DA - 09.10.2022 KW - Persistence KW - Biocides KW - Evolution KW - Disinfection KW - Biocide tolerance KW - Heterogeneity PY - 2022 AN - OPUS4-55958 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie T1 - Antibiotic tolerance of biofilms emerging from multicellular effects of antibiotic efflux N2 - Biofilms are multicellular assemblages of bacteria living in a self-produced extracellular matrix. Different mechanisms, like the development of highly tolerant persister cells or increased expression of efflux pumps make them tolerant. Here we want to investigate the emergency of antimicrobial tolerance of multicellular bacterial populations, through the interplay of efflux-mediated spatial interactions and efflux-linked persistence. To this end, we are combining computational modelling with experimentally observations gained from three types of multicellular assemblages, i.e. colonies on agar, multicellular populations grown in a monolayer microfluidic device, and 3D biofilms grown in flow chambers. We generated fluorescently labeled E. coli strains that differ in their levels of AcrAB-TolC efflux pump activity, an acrB knockout-strain and a strain with inducible expression of acrAB. All strains were characterized in terms of their antimicrobial susceptibility of three antibiotics, tetracycline, kanamycin, ampicillin, and the biocide benzalkonium chloride. The knockout strain shows higher susceptibility than the wild type strain, with highest difference when using benzalkonium chlorid e. To investigate the link between colony structure and spatial patterns of gene expression, the strains were mixed equimolar and grown on agar supplemented with antimicrobials. First results show, antimicrobials affect the structure of sector formation and morphology. Cells grown on tetracycline agar show a more finer sector formation. While kanamycin changes the overall colony structure . We will develop a mathematical model and additional experiments with multicellular assemblages to explain the observed interactions and extrapolate the results to more realistic biofilm models. T2 - SPP Conference CY - Berlin, Germany DA - 06.01.2025 KW - Antimicrobial KW - Resistance KW - Tolerance KW - Efflux KW - E. coli PY - 2025 AN - OPUS4-64563 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Persistence against benzalkonium chloride promotes rapid evolution of tolerance under periodic disinfection N2 - A presentation given at the VAAM conference 2022, summarizing our findings published in the research paper "Persistence against benzalkonium chloride promotes rapid evolution of tolerance under periodic disinfection" T2 - Annual conference of the association for general and applied microbiology (VAAM) 2022 CY - Düsseldorf, Germany DA - 21.02.2022 KW - Persistence KW - Biocides KW - Evolution KW - Cross-resistance KW - Biocide tolerance KW - Disinfection PY - 2022 AN - OPUS4-54437 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Phenotypic 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 - 1st International Conference on Emergent Functions of Bacterial Multicellularity CY - Berlin, Germany DA - 06.01.2025 KW - Disinfectants KW - Biocides KW - Phenotypic heterogeneity PY - 2025 AN - OPUS4-62462 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schmidt, Selina T1 - Bacterial resistance evolution towards disinfectants and antimicrobial surfaces and development of a standardized test N2 - Question Disinfectants and antimicrobial surfaces (AMCs) are important tools to prevent the spread of pathogens and antimicrobial resistant bacteria. However, concerns have been raised about the possibility for the evolution and selection of resistance against disinfectants and AMCs. In turn, resistance against disinfectants and AMCs can be associated to antibiotic resistance due to cross-resistance - a single mechanism conferring resistance to a disinfectant and an antibiotic- and co-resistance - two distinct mechanisms physically linked on e.g. a plasmid. The risk for resistance and cross-resistance during use of biocides (including disinfectants and AMCs) must be evaluated during authorization according to the EU biocidal product regulation. However, to date there is a lack of standardized methods that support risk assessment during the authorization process. Methods We used adaptive laboratory evolution (ALE) experiments which are based on repeated exposure of bacteria to disinfectants or AMCs. The experiments are followed by phenotypic (antimicrobial susceptibility testing) and genotypic (whole genome sequencing) characterization of the evolved strains. The basic idea of these experiments is to expose bacteria to lethal conditions and select for mutants with increased survival. This approach is fundamentally different to other ALE experiments, which commonly select for increased growth at subinhibitory concentrations. However, selection for increased survival represents a selective pressure that more realistically reflects selection under in-use conditions of disinfectants and AMCs. Results First, we studied adaptation of E. coli during repeated disinfection with benzalkonium chloride in a suspension assay. The experiments showed a 2000-fold increase in survival within 5 exposure cycles. The adaptive changes are linked to highly parallel mutations in genes related to lipid A biosynthesis, less negative cell surface charge, reduced growth rate and increased competitive ability in the presence of certain antibiotics. We use the same approach to develop standardizable ALE experiments based upon accepted standards that are used to determine the efficacy of disinfectants (EN 13697) and antimicrobial surfaces (ISO 22196). The results highlight pronounced adaptation of different test strains towards surface disinfection (benzalkonium chloride and isopropanol) and AMCs (copper). Conclusion Bacteria can adapt with increased survival towards lethal stress imposed by disinfectants and AMCs. The adaptive ability of bacteria to disinfectants and AMCs can be determined in a standardized manner. T2 - 74. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie [DGHM] e. V. CY - Berlin, Germany DA - 05.09.2022 KW - Antimicrobial surfaces KW - ISO22196 KW - Antimicrobial resistance KW - Round robin test PY - 2022 AN - OPUS4-56432 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Phenotypic and evolutionary responses of bacteria to disinfection N2 - Disinfectants are important to prevent the transmission of pathogens, especially in the face of the current antibiotic resistance crisis. The crisis is further exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfectant application, persistence to disinfectants and its role for the evolution of tolerance and cross-resistance to antibiotics has not been studied. Our work shows that E. coli displays persistence against several widely used disinfectants, including benzalkonium chloride (BAC), didecyldimethylammoniumchlorid (DDAC) and isopropanol. The molecular mechanism of BAC persistence is triggered in stationary phase and affected by several antibiotic persister genes (hipA, tisB, tolC, relA, spoT). Experimental evolution and population dynamic modeling show that repeated failure of disinfection due to persisters rapidly selects for BAC tolerance underpinned by reduced cell surface charge due to mutations in genes related to lipid A acylation (lpxML). Furthermore, evolved BAC tolerance affects the susceptibility to antibiotics, leading to positive selection of disinfectant tolerant strains at environmentally relevant antibiotic concentrations and variations in evolvability of antibiotic resistance due to epistatic effects. These results highlight the need for faithful application of disinfectants to steward their efficacy and the efficacy of antibiotics. A better understanding of the bacterial response to disinfectants is crucial to understand and avert the ongoing antimicrobial resistance crisis. T2 - Gordon Research Conference - Molecular mechanisms of evolution CY - Easton, MA, United States DA - 25.06.2023 KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2023 AN - OPUS4-57861 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - The disinfectant glutaraldehyde induces antibiotic tolerance N2 - Glutaraldehyde is widely used as a disinfectant and preservative, but little is known about its effects on bacterial susceptibility to antibiotics and the selection of tolerant phenotypes. We found that short-term exposure to sub-inhibitory levels of glutaraldehyde makes E. coli resistant to high doses of bactericidal antibiotics from different classes. This tolerance is associated with delayed, heterogeneous regrowth dynamics and global transcriptome remodeling. We identified over 1200 differentially expressed genes, including those related to antibiotic efflux, metabolic processes, and the cell envelope. The cells entered a disrupted state likely due to the unspecific mode-of-action of glutaraldehyde. Despite this unregulated response, we identified several differentially expressed genes not previously associated with antibiotic tolerance or persistence that induce antibiotic tolerance when overexpressed alone. These findings highlight how the unspecific mode-of-action of disinfectants can make bacteria temporarily resistant to antibiotics. They have implications for settings where disinfectants and antibiotics are used in close proximity, such as hospitals and animal husbandry, and for the selection dynamics of tolerant pheno- and genotypes in fluctuating environments where microorganisms are exposed to these substances, such as sewage systems. A trade-off arises from overcoming the disrupted state as quickly as possible and maintaining antibiotic tolerance. T2 - Gordon Research Conference - Molecular Mechanisms in Evolution CY - Easton, Massachusetts, United States DA - 25.06.2023 KW - Glutaraldehyde KW - Biocides KW - Tolerance KW - Bacteria KW - Disinfection KW - Heterogeneity KW - Antibiotics PY - 2023 AN - OPUS4-58033 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas A1 - Schreiber, Frank T1 - Persistence against benzalkonium chloride promotes rapid evolution of tolerance under 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 which can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfection, knowledge concerning persistence to disinfectants and its link to resistance evolution is currently lacking. 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. BAC tolerance is associated with reduced cell surface charge and mutations in the novel tolerance locus lpxM. Moreover, the fitness cost incurred by BAC tolerance turned into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings provide a mechanistic underpinning for the faithful application of disinfectants to prevent multi-drug-resistance evolution and to steward the efficacy of biocides and antibiotics. T2 - World Microbe Forum (ASM, FEMS) CY - Online meeting DA - 20.06.2021 KW - Persistence KW - Biocides KW - Evolution KW - Cross-resistance KW - Biocide tolerance PY - 2021 AN - OPUS4-53167 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Keshmiri, Hamid T1 - Bidiffractive leaky-mode biosensor N2 - This study details a thorough analysis of leaky and waveguide modes in biperiodic diffractive nanostructures. By tuning diffraction orders and subsequently confining local density of optical states at two distinct resonance wavelengths, we present a highly sensitive refractive index biosensing platform that can resolve 35.5 to 41.3 nm/RIU of spectral shift for two separate biological analytes. T2 - EMBL Symposium: Seeing is Believing - Imaging the Molecular Processes of Life CY - Heidelberg, Germany DA - 04.10.2023 KW - Optics PY - 2023 AN - OPUS4-59247 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - BIOCIDE N2 - This presentation gives an overview about the BIOCIDE project performed with the Aquatic Pollutants joint call. T2 - Aquatic Pollutants TransNet workshop CY - Online meeting DA - 09.11.2022 KW - Antimicrobial resistance KW - Bacteria KW - Biofilms KW - Biocides KW - Risk assessment KW - Wastewater PY - 2022 AN - OPUS4-56265 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Koerdt, Andrea T1 - Hydrogen storage & Microbiologically influenced corrosion - Improved test system for Stability and Durability of the Materials & Gas N2 - Underground hydrogen storage (UHS) is a strategic step towards implementing the hydrogen economy. Achieving the required infrastructure by 2050 necessitates advancements in hydrogen-dedicated assets and the evaluation of existing infrastructure. The unique conditions in UHS require an experimental set-up to simulate UHS operating conditions, which allows to assess the readiness of current storage and transmission for hydrogen, and develop new technologies for material-resistance, operational-simulations, and risk-assessments. In addition to the physical/chemical conditions in UHS (e.g., salinity, hydrogen concentration, operating temperature/-pressure, water content), biological threats must also be considered. Therefore, we present here a high-pressure-set-up, developed for research/-industrial testing purposes. Currently, UHS-experiments for microbiologically-influenced-corrosion (MIC) are performed in standard autoclaves with relatively high volumes/pressures; they were primarily designed for material-specific investigations. While these methods provided some useful information for biological questions, they had significant limitations. Besides, the rapid depressurization that occurs with standard autoclaves can greatly affect materials, especially amorphous materials like polymers, causing damage that isn't due to the actual hydrogen storage. This presents a challenge, as the test results may not accurately reflect real-world conditions. To address these issues, specialized autoclaves have been developed to allow for slower depressurization while also enabling continuous monitoring of gases and liquids during the experiment. Such modifications could help obtain more accurate and reliable data. The novel UHS-simulation-set-up presented here is designed with a controlled independently temperature and pressure. Field samples can be used to mimic geology, water chemistry, construction materials, and microbiological conditions. Most significant advantages of the set-up are: 1. It allows for liquid addition during the test, enabling the study of biocides or the evaluation of operating setups. 2. It permits liquid/-gas sampling during the test, allowing for more efficient monitoring of testing conditions and a better understanding of the process over time. Additionally, a low-release function is added, which is particularly important for studying MIC to avoid negative side effects, on the material (e.g. polymers/corrosion product-layer/cells itself) which might occur due to the fast pressure release. T2 - MATHEA | MATerials in Hydrogen related Energy Applications 2025 CY - Hamburg, Germany DA - 24.06.2025 KW - MIC KW - MISTRAL KW - Underground storage KW - Hydrogen KW - Polymer KW - Metal PY - 2025 AN - OPUS4-63567 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Consequences of tolerance to disinfectants on the evolution of antibiotic resistance in E. coli N2 - Biocides are used as disinfectants and preservatives; one important active substance in biocides is benzalkonium chloride (BAC). BAC-tolerant bacterial strains can survive short treatments with high concentrations of BAC. BAC tolerance and resistance have been linked to antibiotic resistance. Here, the selection dynamics between a BAC-tolerant Escherichia coli strain and a sensitive wild type were investigated under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the BAC-tolerant strain was selected over the wild type at all ciprofloxacin concentrations investigated, with a minimum selection concentration (MSC) of 1/10th of the minimum inhibitory concentration (MIC) of the wild type. Furthermore, the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin was assessed by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type. The importance of these results is highlighted by the fact that concentrations of ciprofloxacin well above the calculated MSC can be found in environmental samples such as hospital wastewaters and livestock slurry. In turn, BAC is used as a disinfectant in the same settings. Thus, the selection of BAC-tolerant strains at sub-inhibitory concentrations of ciprofloxacin can contribute to the stabilization and spread of BAC-tolerance in natural populations. The prevalence of such strains can impair the effects of BAC disinfections. T2 - µClub seminar series CY - Berlin, Germany DA - 15.12.2023 KW - Antimicrobial resistance PY - 2023 AN - OPUS4-59222 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Resistenzen von Bakterien gegen Biozide – Evolution, Mechanismen und Methoden N2 - Diese Präsentation gibt einen Überblick über die Aktivitäten zum Thema Biozidresistenz an der BAM. T2 - Life Science Nord - Online-Update Hygiene und Infektionsprävention CY - Online meeting DA - 14.06.2022 KW - Antimikrobielle Resistenz KW - Antmikrobielle Oberflächen KW - Standardisierung KW - Biozide KW - Risikobewertung PY - 2022 UR - https://vimeo.com/722198443 AN - OPUS4-56236 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - The disinfectant glutaraldehyde induces antibiotic tolerance underpinned by Phenotypic Heterogeneity and transcriptome remodeling N2 - Glutaraldehyde (GTA) is commonly used to disinfect medical equipment, in animal husbandry and in hydraulic fracturing. Its wide use bears the risk that microorganisms in different environments are exposed to potentially non-lethal doses of glutaraldehyde. To date, little is known about the effects of glutaraldehyde on the susceptibility of bacteria to antibiotics and its role in the selection of tolerant phenotypes. Objectives • To determine the effect of glutaraldehyde exposure on the survival of E. coli, S. aureus and P. aeruginosa to antibiotics • To find the mechanistic basis for antibiotic tolerance upon glutaraldehyde exposure Materials & Methods Four bacterial isolates were exposed to sub-inhibitory glutaraldehyde. Antibiotic tolerance was determined by time-kill assays. Regrowth dynamics (lag times) were determined with ScanLag. E. coli was further investigated, using RNAseq to identify genes and processes involved in antibiotic tolerance. Mutants of candidate genes were screened for their antibiotic tolerance and heterogeneous target gene expression under stressed and unstressed conditions. Results Short-term exposure to sub-inhibitory levels of glutaraldehyde induced tolerance to high doses of bactericidal antibiotics. Tolerance to antibiotics was associated with highly heterogeneous regrowth dynamics and global transcriptome remodeling. Differentially expressed genes represented diverse biological functions and cellular components, including antibiotic efflux, metabolic processes, and the cell envelope. The heterogeneous regrowth dynamics and the diversity of the differentially expressed genes are likely related to the unspecific mode-of-action of glutaraldehyde. Among the many differentially expressed genes, several genes were identified that were not previously associated with antibiotic tolerance or persistence, which, when overexpressed alone, increased antibiotic tolerance. Conclusion Our results highlight how the big advantage of a disinfectant, its unspecific mode-of-action, can induce transient tolerance to antibiotics in bacteria. These findings have implications for 1.) settings where disinfectants and antibiotics are used in proximity, such as hospitals and animal husbandry, and 2.) for the selection dynamics of tolerant bacteria in fluctuating environments because of the trade-off that arises from overcoming the lag phase as fast as possible and maintaining antibiotic tolerance. T2 - VAAM Jahrestagung 2023 CY - Göttingen, Germany DA - 10.09.2023 KW - Glutaraldehyde KW - Disinfectants KW - Biocides KW - Antibiotics KW - Bacteria PY - 2023 AN - OPUS4-58439 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 talk discusses resistance evolution towards biocides and antimicrobial surfaces. It shows (i) that biocides affect rates of mutation and horizontal gene transfer, (ii) that biocides show heterogeneous killing facilitating tolerance evolution, and (iii) that biocides and antibiotics show strong combination effect on growth and selection. T2 - STOP project internal seminar CY - Online meeting DA - 24.04.2024 KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides KW - Antimicrobial surfaces PY - 2024 AN - OPUS4-61546 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Physiological and evolutionary consequences of exposing Pseudomonas aeruginosa to biocide-antibiotic combinations N2 - Antimicrobials can exert specific physiological effects when used in combination that are different from those when applied alone. These effects include physiological effects (i.e. synergy, antagonism and suppression) as well as evolutionary effects on the selection of resistant strains (i.e. cross-resistance and collateral sensitivity). 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 physiological and evolutionary consequences of combinations of antibiotics (meropenem, gentamicin and ciprofloxacin) and substances used as biocides or antiseptics (octenidine, benzalkonium chloride, cetrimonium bromide, chlorhexidine, povidone-iodine, silver) on growth and selection of Pseudomonas aeruginosa. We find prevalent physiological combination effects with synergy occurring 6 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). A particular strong antagonism is apparent for the meropenem-chlorhexidine combination, for which we conducted an in-depth study on the underlying molecular mechanism using RNASeq. Moreover, we find widespread effects of the biocide-antibiotic combinations on selection of P. aeruginosa strains resistant to the antibiotics, including cross-resistance and collateral sensitivity. In conclusion, antibiotics and biocides or antiseptics exert physiological and evolutionary combination effects on the pathogen P. aeruginosa. These effects have consequences for the efficacy of both types of substances and for the selection of antimicrobial resistant strains in clinical applications with combined exposure (e.g. wound care, coated biomaterials). T2 - Antimicrobial Resistance in Biofilms and on Biomaterials CY - Online meeting DA - 10.06.2021 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Biofilms PY - 2021 AN - OPUS4-53162 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Physiological and evolutionary consequences of exposing Pseudomonas aeruginosa to biocide-antibiotic combinations N2 - Antimicrobials can exert specific physiological effects when used in combination that are different from those when applied alone. These effects include physiological effects (i.e. synergy, antagonism and suppression) as well as evolutionary effects on the selection of resistant strains (i.e. cross-resistance and collateral sensitivity). 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 physiological and evolutionary consequences of combinations of antibiotics (meropenem, gentamicin and ciprofloxacin) and substances used as biocides or antiseptics (octenidine, benzalkonium chloride, cetrimonium bromide, chlorhexidine, povidone-iodine, silver) on growth and selection of Pseudomonas aeruginosa. We find prevalent physiological combination effects with synergy occurring 6 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). A particular strong antagonism is apparent for the meropenem-chlorhexidine combination, for which we conducted an in-depth study on the underlying molecular mechanism using RNASeq. Moreover, we find widespread effects of the biocide-antibiotic combinations on selection of P. aeruginosa strains resistant to the antibiotics, including cross-resistance and collateral sensitivity. In conclusion, antibiotics and biocides or antiseptics exert physiological and evolutionary combination effects on the pathogen P. aeruginosa. These effects have consequences for the efficacy of both types of substances and for the selection of antimicrobial resistant strains in clinical applications with combined exposure (e.g. wound care, coated biomaterials). T2 - ASM-FEMS World Microbe Forum CY - Online meeting DA - 20.06.2021 KW - Antimicrobial resistance KW - Antagonism KW - Biofilms PY - 2021 AN - OPUS4-53165 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - NBD-DDA: a tool to study biocide & disinfectant resistance mechanisms 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 that can make bacteria less susceptible to QACs, including phenotypic heterogeneity. Goals To develop a fluorescent quaternary ammonium compound and to use it to investigate the mechanisms underlying QAC susceptibility in bacteria. Material & Methods A fluorescent quaternary ammonium compound, N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl] azanium-iodide (NBD-DDA), was synthesized. Chemical structure and purity of NBD-DDA was confirmed by mass spectrometry and NMR spectroscopy. Antimicrobial activity against S. aureus, P. aeruginosa and E. coli was assessed by determination of minimum inhibitory concentrations and time-kill assays. Uptake, heterogeneity and efflux of NBD-DDA in wild-type and efflux-deficient mutants (ΔtolC) were determined, using flow cytometry and fluorometry. Sub-cellular localization of NBD-DDA was determined using confocal laser scanning microscopy. Results NBD-DDA was readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings. NBD-DDA retained antimicrobial activity comparable to the structurally similar, widely used QAC benzalkonium chloride (BAC). 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. NBD-DDA was preferentially localized to the cell envelope, which is a primary target of BAC and other QACs. Leveraging NBD-DDA‘s fluorescent properties, it was demonstrated that reduced cellular accumulation underlies BAC tolerance in a BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Summary Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study mechanisms of QAC susceptibility and mode-of-action in bacteria, both on the level of populations and single-cells. T2 - VAAM - Jahrestagung 2024 CY - Würzburg, Germany DA - 02.06.2024 KW - Biocides KW - Phenotypic heterogeneity PY - 2024 AN - OPUS4-61177 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Consequences of BAC tolerance for selection and evolution in the presence of antibiotics N2 - The exposure to antimicrobial substances drives the evolution of antimicrobial resistance. Biocides are antimicrobials used as disinfectants, antiseptics and preservatives. They find application on a large scale in the industrial and medical sector, but also in private households. In terms of mass, the worldwide use of biocides exceeds that of antibiotics. Thus, despite their important role in preventing the spread of pathogens, due to their ubiquity, biocides are suspected to be drivers of the antimicrobial resistance crisis. In our work at BAM we try to understand how biocides contribute to the emergence of AMR, what the underlying adaptation principles and mechanisms are and how they compare to those found for antibiotics. Within our group, I mainly focus on the following two questions: How does phenotypic heterogeneity in bacteria affect the ability to survive treatment with biocides? And what are the consequences of phenotypic heterogeneity for the evolution of resistance to biocides and antibiotics? I will share published and unpublished results which demonstrate that phenotypic heterogeneity can enable the survival of biocide treatment and, through this, facilitate the evolution of AMR. On the other hand, we find that adaptation to a biocide can unexpectedly impair the ability to evolve resistance against an antibiotic. T2 - FEMS summer school for postdocs 2022: Microbial Evolvability Mechanisms: Resistance, Biology, and Strategies to Defeat and Detect CY - Split, Croatia DA - 27.04.2022 KW - Disinfection KW - Biocides KW - Evolution KW - Resistance KW - Biocide tolerance PY - 2022 AN - OPUS4-54846 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Sobisch, Lydia-Yasmin T1 - Time-kill kinetics reveal heterogeneous tolerance to disinfectants N2 - Background. Disinfection is an important mitigation strategy to control and prevent the spread of infections. Incomplete or incorrect usage of disinfection may promote evolution of resistance against disinfectants and antibiotics. Ideally, disinfection reduces 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. Objective. In this study, we investigated six active substances of disinfectants, preservatives, and antiseptics against a population of E. coli to identify the presence of a tolerant subpopulation. Methods. We performed time-kill experiments and analyzed the data with a mathematical model to statistically infer whether the data is best explained by the presence of a tolerant subpopulation. Results. The analysis identified bimodal kill kinetics for benzalkonium chloride, didecyldimethylammonium chloride, and isopropanol. In contrast, kill kinetics by chlorhexidine, glutaraldehyde, and hydrogen peroxide were best explained by unimodal kill kinetics. These findings have implications for the risk of disinfection failure. In addition, we are currently performing adaptive laboratory evolution (ALE) experiments with the different disinfectants to investigate the potential consequences of tolerant sub-populations for the evolution of antimicrobial resistance and tolerance. T2 - FEMS Conference CY - Hamburg, Germany DA - 10.07.2023 KW - Biocide KW - Desinfectant KW - E.coli PY - 2023 AN - OPUS4-58471 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Risk Assessment of Biocide Resistance N2 - This presentation details approaches for the risk assessment of biocide resistance. Different methods are presented to acquire the necessary data for such risk assessments. T2 - OECD, 7th Meeting of the Working Party on Biocides CY - Leiden, Netherlands DA - 18.09.2023 KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2023 AN - OPUS4-59062 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Persistence against benzalkonium chloride promotes rapid evolution of tolerance under periodic disinfection 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 - Bacterial Networks (BacNet22) CY - Sant Feliu de Guixols, Spanien DA - 04.09.2022 KW - Persistence KW - Biocides KW - evolution KW - disinfection KW - biocide tolerance PY - 2022 AN - OPUS4-55713 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas A1 - Kanaris, Orestis A1 - Lewerenz, Dominique A1 - Gödt, Annett A1 - Schreiber, Frank T1 - Evolutionary implications of heterogeneous disinfectant tolerance N2 - Introduction: Effective disinfection is crucial to maintain hygiene and to prevent the spread of infections. Phenotypic heterogeneity in disinfection survival (i.e. tolerance) may result in failure of disinfection, which in turn may foster the evolution of resistance to both disinfectants and antibiotics. However, the consequences of phenotypic heterogeneity for disinfection outcome and resistance evolution are not well understood. Goal: This study investigates the impact of phenotypic heterogeneity on the survival and evolution of Escherichia coli during disinfection with six commonly used substances. Furthermore, the consequences of evolved disinfectant tolerance for antibiotic resistance evolution are studied. Materials & Methods: The extent of population heterogeneity during disinfection is derived by determining time-kill kinetics and analysis with mathematical modelling. The link between population heterogeneity and evolvability of disinfectant tolerance was assessed by laboratory evolution experiments under periodic disinfection. The ability of disinfectant tolerant strains to evolve antibiotic resistance is assessed by serial transfer experiments with increasing concentrations of different antibiotics and by whole genome sequencing. Results: Multi-modal time-kill kinetics in three of the six disinfectants suggest the presence of disinfectant-tolerant subpopulations (i.e. persister cells). Importantly, the ability and extent to evolve population-wide tolerance under periodic disinfection is related with the presence of persister cells and the level of phenotypic heterogeneity during disinfection. Interestingly, the probability of high-level resistance evolution to certain antibiotics is attenuated in disinfectant tolerant strains as compared to the sensitive ancestor. Whole-genome sequencing reveals epistatic interactions between disinfectant tolerance and antibiotic resistance mutations, preventing access to canonical evolutionary paths to resistance. Summary: Our findings suggest that phenotypic heterogeneity can facilitate disinfection survival and the evolution of population wide tolerance, which can impact future antibiotic resistance evolution. T2 - Vereinigung Allgemeiner und Angewandter Mikrobiobiologie Jahreskongress 2023 CY - Würzburg, Germany DA - 02.06.2024 KW - Biocide KW - Resistance KW - Persistence KW - Evolution KW - Herteogeneous phenotypes PY - 2024 AN - OPUS4-60244 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 - Antimicrobial resistance (AMR) is an important global health problem. The environment has been regocnized as an improtant compartment for the occurance, evolution and transmission of AMR. Biocides used as material preservatives are in contact with the environment and natural microbial communities through direct application and passive leaching from protected materials. It has been shown that environmental contaminants, such as antibiotics, metals and pesticides, can affect resistance evolution and spread by modifying the underlying processes of de novo mutagenesis, horizontal gene transfer and selection. However, it is unknown if material preservatives are involved in these processes and thereby can also drive AMR in the environment. Here, we investigate the effect of material preservatives on rates of de novo mutation and horizontal gene transfer (HGT) in Escherichia coli and the model soil microorganisms Acinetobacter baylyi and Bacillus subtilis. To this end, we determined the effects of material preservatives on the mutation rates and HGT with the Luria-Delbrück fluctuation assay and a conjugation assay with the broad host-range plasmid pKJK5 and a transformation assay. Our data shows that the quaternary ammonium compound DDAC, copper, the pyrethroid insecticide permethrin and the azole fungicide propiconazole significantly increase mutation rates in E. coli, whereas A. baylyi and B. subtilis are not significantly affected. Moreover, we show that the carbamate IPBC and the insecticide permethrin affect HGT in a concentration dependent manner. Investigations with reporter strains for bacterial stress response pathways show that induction of the general stress response (rpoS) and components of the SOS response (recA) underlie the effects of most biocides on mutation rates and HGT. Taken together, our data is important for assessing the contribution of biocides on AMR evolution and spread in the environment. T2 - World Microbe Forum (ASM, FEMS) CY - Online meeting DA - 20.06.2021 KW - Biocides KW - Horizontal gene transfer KW - Mutation rate PY - 2021 UR - https://www.abstractsonline.com/pp8/#!/9286/presentation/11692 AN - OPUS4-54211 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN 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 E. 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 solely for survival 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 sub-inhibitory 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://www.ncbi.nlm.nih.gov/bioproject/PRJNA1074740 PB - National Library of Biotechnology Information CY - Bethesda AN - OPUS4-61559 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank A1 - Nordholt, Niclas T1 - Persistence against benzalkonium chloride promotes rapid evolution of tolerance under 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 which can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfection, knowledge concerning persistence to disinfectants and its link to resistance evolution is currently lacking. 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. BAC tolerance is associated with reduced cell surface charge and mutations in the novel tolerance locus lpxM. Moreover, the fitness cost incurred by BAC tolerance turned into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings provide a mechanistic underpinning for the faithful application of disinfectants to prevent multi-drug-resistance evolution and to steward the efficacy of biocides and antibiotics. T2 - New Approaches and Concepts in Microbiology CY - Online meeting DA - 07.07.2021 KW - Persistence KW - Biocides KW - Evolution KW - Cross-resistance KW - Biocide tolerance PY - 2021 AN - OPUS4-53168 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Progress on STOP-project WP5 and WP6 at BAM N2 - An overview of the progress that has been made on testing of antimicrobial efficacy and method development in the STOP project at BAM. T2 - STOP project meeting 09/2024 CY - Bukarest, Romania DA - 18.09.2024 KW - Antimicrobial surfaces KW - ISO22196 PY - 2024 AN - OPUS4-61178 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Schreiber, Frank A1 - Kanaris, Orestis A1 - Nordholt, Niclas T1 - Consequences of benzalkonium chloride tolerance for selection dynamics and de novode 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−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. KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Antibiotics PY - 2025 UR - https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1282584 PB - National Library of Medicine CY - Bethesda AN - OPUS4-65221 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schmidt, Selina T1 - Effect of biocides on the evolution and spread of resistance 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 - Antimicrobial resistance KW - Horizontal gene transfer KW - Mutation rate PY - 2022 AN - OPUS4-56426 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Sobisch, Lydia-Yasmin T1 - Biocide resistance evolution of corrosion causing sulfate reducing bacteria N2 - SRB are environmentally and industrially important microorganisms. The disadvantage of their metabolic activity (e.g. sulfate reduction) results in the formation of toxic sulfide that leads to microbial influenced corrosion. SRB have been responsible for biocorrosion of ferrous metal. One of mitigation strategy is the use of biocides. However, it has been shown that various bacteria develop antimicrobial resistance due to excessive use of biocides. Thus, a deeper understanding of the evolution of biocide resistance of SRB is necessary. Three commonly used biocides, THPS, BAC, and GLUT were applied to investigate the susceptibility of Desulfovibrio alaskensis G20.The minimum inhibitory and bactericidal concentration and the killing kinetics of the three biocides was determined. These results will be used to conduct evolution experiments to determine the evolution of resistance towards biocides of SRBs. The outcome of this work can be helpful to improve the management of MIC treatments. T2 - Panel, Pitch & Popcorn by EUROMIC CY - Online meeting DA - 21.06.2021 KW - Biocide KW - Evolution KW - Mircobially influcenced corrosion KW - Sulfate reducing bacteria PY - 2021 AN - OPUS4-56940 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Bakterielle Resistenzen und Toleranzen gegen Desinfektionsmittel und antimikrobielle Oberflächen N2 - Dieser Vortrag beschreibt die Entstehung von bakteriellen Resistenzen und Toleranzen gegen Desinfektionsmittel und antimikrobielle Oberflächen. T2 - Fachtagung für Krankenhaushygiene der Deutschen Gesellschaft für Krankenhaushygiene CY - Essen, Germany DA - 12.05.2023 KW - Antimikrobielle Resistenz KW - Antmikrobielle Oberflächen KW - Standardisierung KW - Biozide PY - 2023 AN - OPUS4-57860 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Sobisch, Lydia-Yasmin T1 - Time-kill kinetics reveal heterogeneous tolerance to disinfectants N2 - Background. Disinfection is an important mitigation strategy to control and prevent the spread of infections. Incomplete or incorrect usage of disinfection may promote evolution of resistance against disinfectants and antibiotics. Ideally, disinfection reduces 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. Objective. In this study, we investigated six active substances of disinfectants, preservatives, and antiseptics against a population of E. coli to identify the presence of a tolerant subpopulation. Methods. We performed time-kill experiments and analyzed the data with a mathematical model to statistically infer whether the data is best explained by the presence of a tolerant subpopulation. Results. The analysis identified bimodal kill kinetics for benzalkonium chloride, didecyldimethylammonium chloride, and isopropanol. In contrast, kill kinetics by chlorhexidine, glutaraldehyde, and hydrogen peroxide were best explained by unimodal kill kinetics. These findings have implications for the risk of disinfection failure. In addition, we are currently performing adaptive laboratory evolution (ALE) experiments with the different disinfectants to investigate the potential consequences of tolerant sub-populations for the evolution of antimicrobial resistance and tolerance. T2 - Vereinigung Allgemeiner und Angewandter Mikrobiobiologie Jahreskongress 2023 CY - Göttingen, Germany DA - 10.09.2023 KW - Biocide KW - Desinfectant KW - E.coli PY - 2023 AN - OPUS4-58470 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Jaut, Valerie T1 - Antibiotic tolerance of biofilms emerging from multicellular effects of antibiotic efflux N2 - Biofilms are multicellular assemblies of bacteria living in a self-produced extracellular matrix. One characteristic of biofilms is that they are difficult to kill. Different mechanisms, like the development of persister cells or efflux pumps which pump some antimicrobials out of the cell, make them tolerant. Our central hypothesis is that efflux pump activity causes emergent antimicrobial tolerance of multicellular bacterial populations, through the interplay of efflux-mediated spatial interactions and efflux-linked persistence. To verify the hypothesis, we combine computational modelling with information gained from 3 types of multicellular assemblies. We are currently generating strains that differ in their levels of efflux activity, mixes are then cultivated together in the 3 model systems. In colonies the link between structure and spatial patterns of gene expression will be characterized. Using a microfluidic device, the interactions range of efflux as a response to different antimicrobials will be determined. In a flow chamber a 3D biofilm will be generated, to investigate the biofilm development over time and persister cell formation. All results will be compared with model predictions. T2 - UNA Workshop CY - Berlin, Germany DA - 29.01.2024 KW - Antibiotic KW - Biofilm KW - Tolerance KW - Efflux PY - 2024 AN - OPUS4-61280 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - RPRT A1 - European Food Safety Authority (EFSA), A1 - European Centre for Disease Prevention and Control (ECDC), A1 - European Chemicals Agency (ECHA), A1 - European Environment Agency (EEA), A1 - European Medicines Agency (EMA), A1 - European Commission's Joint Research Centre (JRC), T1 - Annex to: Scientific report 'Impact of the use of azole fungicides, other than as human medicines, on the development of azole-resistant Aspergillus spp.' doi:10.2903/j.efsa.2025.9200 - Annex E - Detailed answer to Term of Reference 5 'Environmental hotspots' and Term of Reference 6 'Prevention and control options' N2 - The widespread use of azole compounds in various sectors has led to the emergence of azole-resistant Aspergillus fumigatus (ARAf), which poses a significant challenge for treating fungal infections, especially in immunocompromised patients. Certain environmental conditions and practices, particularly in agricultural settings and the use of azoles as biocides, have been identified as hotspots for the selection and dispersal of azole-resistant strains of Aspergillus spp. Factors contributing to the selection of resistance include the use of azoles in crop protection, wood preservation and, to a much lesser extent, veterinary medicine. For plant protection products (PPPs), a number of scenarios (green waste of indoor-grown vegetables, uses with the production of wet pomace used as fertiliser, maize or sugar beet silage, and field heaps including flower bulbs) are deemed high risk for hotspot development. Based on EU authorised use patterns, these scenarios are characterised by the hazard characteristics of the azole fungicides in terms of activity against the wild-type Aspergillus spp. compared to resistant strains, substrate characteristics and residue levels, and environmental conditions that promote the growth of the fungus. For biocidal azole applications, products (biocidal product [BP]) for temporary preservation of freshly cut wood have been identified to have the potential for hotspot formation because freshly cut wood allows the growth of Aspergillus spp., and azole concentrations in treated wood are above the predicted no effect concentration (PNEC) for resistance selection (PNECres) and below the minimum inhibitory concentration (MIC) of ARAf for most analysed products on the EU market. Following identification of environmental hotspots, the report recommends measures to prevent the selection of azole-resistant strains in the environment, including controlled storage of organic waste, proper waste management, and responsible use and disposal of azole-treated products. Azole use in veterinary medicinal products (VMPs) represents a very small percentage of total azole use and is unlikely to be a significant source of selection of resistance in the environment. As such, the focus for mitigating resistance should be on other uses of azoles. The report stresses the importance of ongoing surveillance to monitor the presence of ARAf in the environment and to inform risk assessments and management strategies. As industrial chemicals, the azole substances are mostly used as intermediates (precursors) to manufacture yet a different substance, are formulated into a mixture or are reported to be manufactured as active substances in PPP, BP or VMP (therefore already covered above). There are only a few industrial azole substances with widespread use, and as for the moment, there is no evidence from the literature that industrial azoles would be a source of a possible hotspot; thus, the industrial chemicals were not further investigated. There are several areas where further research is needed, including understanding the environmental conditions that support the growth of Aspergillus spp. in different agricultural matrices or on wood, assessing human exposure to resistant strains, regional waste practices and the impact of active substance combinations for azole resistance selection. There is also a need for more comprehensive data on the use and quantities of azole-containing products. Furthermore, industrial substances with widespread use and having antifungal effects, e.g. an antidandruff substance in cosmetics, could be further investigated. Measures were identified that could be implemented with respect to the use of azole fungicides in PPPs as well as in BPs and with respect to the storage, processing and disposal of crop (waste) materials containing azole residues to prevent or minimise the selection of environmental resistance or to minimise the spread of resistant Aspergillus spp. to patients. Any measures that slow down or prevent growth in the presence of azoles, sporulation and dispersal of Aspergillus spp. should be encouraged. A coordinated effort among various stakeholders, including farmers, manufacturers, industrial users, waste managers, regulatory bodies and scientists, is essential to effectively address the challenge of azole resistance in A. fumigatus. KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Azoles KW - Fungi KW - Wood preservatives PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-652187 UR - https://doi.org/10.5281/zenodo.14223436 DO - https://doi.org/10.5281/zenodo.14223435 SP - 1 EP - 76 PB - Zenodo CY - Geneva AN - OPUS4-65218 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Biocides As Drivers For Antimicrobial Resistance Evolution In The Environment 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, biocides used as disinfectants and material preservatives are major pollutants by far excceding the market for antibiotics in terms of mass. Our work shows that biocides have the potential to affect evolutionary processes towards AMR by increasing the rates of de-novo mutation and conjugation. These effects depend on the species and biocidal substance. Importantly, chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations in E. coli. Moreover, our results show a connection between the RpoS-mediated general stress and the RecA-linked SOS response with increased rates of mutation and conjugation, but not for all biocides. Furthermore, our work highlights the potential of biocides to contribute to selection and transmission of AMR. We show that the application of biocides, especially QAC disinfectants, leads to the rapid evolution of tolerance (i.e. increased survival) in adaptive laboratory evolution (ALE) experiments. The evolved 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). ALE experiments with biocide tolerant strains indicate a decreased evolvability of resistance to antibiotics. Taken together, our work shows the importance of assessing the contribution of biocides on evolution, selection and transmission of AMR in the environment. T2 - 6th Environmental Dimension of Antibiotic Resistance (EDAR6) CY - Gothenburg, Sweden DA - 22.09.2022 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Biofilms KW - Biocides KW - Risk assessment PY - 2022 AN - OPUS4-56262 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 talk discusses the work at BAM concerning Resistance evolution towards biocides and antimicrobial surfaces. It shows (i) that biocides affect rates of mutation and horizontal gene transfer, (ii) that biocides show heterogeneous killing facilitating tolerance evolution, and (iii) that biocides and antibiotics show strong combination effect on growth and selection. T2 - Exchange seminar with Nottingham Trent University CY - Berlin, Germany DA - 03.09.2024 KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides KW - Antimicrobial surfaces PY - 2024 AN - OPUS4-61548 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN 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 E. 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 solely for survival 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 sub-inhibitory 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 DO - https://doi.org/10.5061/dryad.2jm63xszx PB - Dryad AN - OPUS4-61558 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nordholt, Niclas T1 - Phenotypic heterogeneity in disinfection: sources and consequences for antimicrobial resistance N2 - A summary of projects here at BAM which investigate the influence of phenotypic heterogeneity on the outcome of disinfection and the influence on antimicrobial resistance. This presentation was given in the Theory Seminar of the Quantitative and Theoretical Biology group of Prof. Oliver Ebenhöh at HHU Düsseldorf T2 - Theory Seminar in the Quantitative and Theoretical Biology group at HHU Düsseldorf CY - Düsseldorf, Germany DA - 24.02.2022 KW - Disinfection KW - Biocides KW - Heterogeneity KW - Resistance PY - 2022 AN - OPUS4-54442 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Deland, Eric T1 - Environmental conditions affect the corrosion product composition of Methanogen induced microbiologically influenced corrosion (Mi-MIC) N2 - Corrosion is a very expensive and serious problem in the different industry sectors, eg. Petroleum-, On- and off-shore, infrastructure. It is estimated that 20% of all corrosion damage is caused by microorganisms or microbiologically influenced corrosion (MIC). Several microorganisms are known to cause corrosion, including sulfate-reducing bacteria, nitrate-reducing bacteria, methanogens etc. For several years, methanogens were regarded as a mild corroder (~0.065 mm/yr), largely due to a lack of detailed investigation on the corrosion mechanism under real-environment simulated conditions. Resulting in the common belief that siderite, a non-conductive compound, is the sole corrosion product (CP) of methanogen-induced MIC (Mi-MIC). To simulate natural environmental conditions, we developed and introduced the multiport flow column system (MFC), a multi-sectional corrosion flow-cell. Using the MFC, we obtained ten times higher corrosion rates than previously reported. With a combination of several analytical techniques, such as ToF-SIMS, SEM-EDS and FIB-SEM, we found strong indication that siderite is not the sole corrosion product of Mi-MIC. The corrosion layers contained phosphorus, oxygen, magnesium, calcium and iron. The differences in the CP between static and dynamic environments demonstrated the impact of testing procedures on the corrosive potential of methanogens. To further verify and deepen our understanding of Mi-MIC, we are currently studying the influence of additional environmental parameters (e.g. pH, salinity, flow rate) on Mi-MIC. Overall, results of this study will expand the current understanding of MIC from both analytical and mechanistic points of view, thus aiding the development of different mitigation strategies for various industry sectors. T2 - ISMOS-8 CY - Online meeting DA - 08.06.2021 KW - Methanogen-induced microbiologically influenced corrosion KW - Carbon steel KW - ToF-SIMS KW - Modelling studies PY - 2021 AN - OPUS4-54041 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Deland, Eric T1 - Environmental conditions impact the corrosion layer composition of Methanogen induced microbiologically influenced corrosion (Mi-MIC) N2 - Microbiologically influenced corrosion (MIC) is a now well-known challenge and affects industry, society and infrastructure. For a long time, the impact of methanogen-induced MIC (Mi-MIC) was underestimated. This was mainly due to the rather low published corrosion rates and the presumed corrosion product siderite, which is not electrically conductive. In our laboratory, we were able to show that this trivialization or underestimation of Mi-MIC was due to the nature of the testing. The static systems used so far do not provide environmentally relevant information about the corrosion rate and the corrosion product, especially for methanogens To further illustrate the importance of the environment on the MIC process, we established a multiport flow column (MFC), to simulate the natural environment. With this method, and in contrast to published results using the static conditions, we obtained ten times higher corrosion rates. We analyzed the corrosion products with a combination of techniques, like ToF-SIMS, SEM-EDS and FIB-SEM, and found strong evidence that siderite is not the sole corrosion product of Mi-MIC. The corrosion layers contain phosphorus, oxygen, magnesium, calcium and iron and lacked on carbon-related species. It is hypothesized that methanogens may have influenced the nucleation process of siderite, converting bicarbonate into carbon dioxide for methanogenesis. This results in increased localized corrosion and reduced siderite formation. To verify and deepen our understanding of Mi-MIC, we are currently studying the influence of additional environmental parameters (e.g. pH, salinity, flow rate etc.) on Mi-MIC and the subsequent impacts on corrosion rates and the corrosion products. Overall, results of this study will expand the current understanding of MIC from both analytical and mechanistic points of view, thus aiding the development of different mitigation strategies for various industry sectors. T2 - Eurocorr 2021 CY - Online meeting DA - 20.09.2021 KW - Methanogen-induced microbiologically influenced corrosion KW - Carbon steel KW - SEM-EDS KW - Modelling studies PY - 2021 AN - OPUS4-54043 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schmidt, Selina T1 - Effect of biocides on the evolution and spread of resistance N2 - Biocides used as material preservatives are in contact with various environments during direct application or passive leaching from protected materials [1]. Antimicrobial resistance (AMR) is a global health problem and the environment is an important compartment for the evolution and transmission of AMR [2,3]. 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 - RokoCon2022 CY - Berlin, Germany DA - 29.09.2022 KW - Biocide KW - Antimicrobial resistance KW - Tolerance KW - Horizontal gene transfer KW - Mutation rate PY - 2022 AN - OPUS4-56431 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Koerdt, Andrea T1 - The impact and potential of halophilic microorganisms on alternative fuels N2 - As more industrial interests focusing on using salt caverns and repurposed gas or petroleum reservoirs for alternative fuel storage, i.e. CO2/H2, the question raises whether microorganisms may impact the infrastructure, gas purity and storage condition over time. Environments with high salinity (> 1.5 Meq of NaCl) are resided by halophiles (salt-loving microorganisms). To compensate for the intensive osmotic stress, they have resorted to two main adaptation strategies: 1) production of compatible solutes and 2) accumulation of intracellular KCl. Microbial community analysis of several high salinity environments revealed a number of recurring genera, including Halomonas and Halanaerobium. However, the impact of halophiles on the overall integrity and stability of the storage facilities remain largely unknown. To evaluate the suitability and stability of saline storage facilities, several model halophilic microorganisms, such as members of Halomonas, will be selected as testing subjects. First, the impact of halophiles on the infrastructure will be determined using an integrative approach by combining a number of techniques, including electrochemistry, TOF-SIMS, SEM/FIB/EDS and FIB-TEM. Second, the abilities of halophiles to alter the fuel composition (i.e. increase/decrease the fractions of H2) will be monitored using gas chromatography by growing them under high pressure. As a result of climate change and the accompanying mandatory shift to renewable energy resources, microorganisms will continue to play an important role in the energy sector, both to their benefit and detriment. Thus, it is important to achieve a certain level of understanding regarding the activities and mechanisms of halophiles prior to large-scaled excursions. T2 - ISMOS-8 CY - Online meeting DA - 07.06.2021 KW - Microbiologically influenced corrosion KW - Hydrogen KW - Gas storage KW - Contamination PY - 2021 AN - OPUS4-52891 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Selection of resistance by antimicrobials used in coatings N2 - Antimicrobial surfaces have broad use in multiple settings including touch surfaces in hospitals, implanted devices, or consumer products. Their aim is to support 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 might drive the evolution and spread of antimicrobial resistance. In my presentation, I will highlight the risks and knowledge gaps associated with resistance on antimicrobial surfaces by different processes including evolution by de novo mutations and horizontal gene transfer, and species sorting of inherently resistant bacteria dispersed onto antimicrobial surfaces. The latter process has the potential to select for antibiotic resistance via cross-resistance between traits that confer resistance to both the antimicrobial surface coating and antibiotics. Conditions in which antibiotics and antimicrobial coatings are present simultaneously (e.g. implants) will lead to more complex interactions that can either result in the selection for or against antibiotic resistance. We mapped these interactions between several antimicrobials and antibiotics on growth and selection of Pseudomonas aeruginosa. We find prevalent physiological (i.e. synergy and antagonism) and evolutionary (i.e. cross-resistance and collateral sensitivity) combination effects. Understanding these interactions opens the door to tailor therapeutic interventions to select against resistance. In additions, we need new methods and translational studies that investigate resistance development to antimicrobial surfaces under realistic conditions. Therefore, I will present recent developments in our lab on the development of such a method based on existing efficacy standards. T2 - 2021 Fall Meeting of the European Materials Research Society (E-MRS) CY - Online meeting DA - 20.09.2021 KW - Antimicrobial resistance KW - Antimicrobial coating KW - Biofilms KW - Biocides PY - 2021 AN - OPUS4-53645 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Deland, Eric A1 - Kleinbub, Sherin T1 - Is Methanogen-induced microbiologically influenced corrosion (Mi-MIC) underestimated? N2 - The role of methanogens in microbiologically influenced corrosion (Mi-MIC) is often neglected, due to 1) low reported corrosion rates and 2) the suspected corrosion product siderite, which is electrically non-conductive. Typically, MIC corrosion studies are carried out using batch cultures, which did not represent the dynamic conditions, i.e. pipeline and provide insufficient information on the overall corrosion potential. We established a unique approach, a multiport flow column (MFC), to simulate pipeline conditions and obtained 10-times higher corrosion rates than previously published. Our result showed that testing procedures have a large impact on the corrosive potential of methanogens. We found strong indications with a combination of ToF-SIMS, SEM-EDS and FIB-SEM analyses that siderite is not the sole corrosion product. The corrosion layers contain phosphorus, oxygen, magnesium, calcium and iron. To verify and deepen our understanding of Mi-MIC, we are currently studying the influence of other environmental parameters (e.g. pH) on Mi-MIC. T2 - VAAM 2021 CY - Online meeting DA - 18.03.2021 KW - Methanogen-induced microbiologically influenced corrosion KW - Carbon steel KW - MIC Island PY - 2021 AN - OPUS4-54042 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Koerdt, Andrea T1 - Exploring Microbial Impacts on Hydrogen Storage: - A Novel System for Corrosion Testing of Gas- and Material Degradation N2 - Underground hydrogen storage (UHS) is a strategic step towards implementing the hydrogen economy. Achieving the required infrastructure by 2050 necessitates advancements in hydrogen-dedicated assets and the evaluation of existing infrastructure. The unique conditions in UHS require an experimental set-up to simulate UHS operating conditions, which allows to assess the readiness of current storage and transmission for hydrogen, and develop new technologies for material-resistance, operational-simulations, and risk-assessments. In addition to the physical/chemical conditions in UHS (e.g., salinity, hydrogen concentration, operating temperature/-pressure, water content), biological threats must also be considered. Therefore, we present here a high-pressure-set-up, developed for research/-industrial testing purposes. Currently, UHS-experiments for microbiologically-influenced-corrosion (MIC) are performed in standard autoclaves with relatively high volumes/pressures; they were primarily designed for material-specific investigations. While these methods provided some useful information for biological questions, they had significant limitations. The novel UHS-simulation-set-up presented here is designed with a controlled independently temperature and pressure. Field samples can be used to mimic geology, water chemistry, construction materials, and microbiological conditions. Most significant advantages of the set-up are: 1. It allows for liquid addition during the test, enabling the study of biocides or the evaluation of operating setups. 2. It permits liquid/-gas sampling during the test, allowing for more efficient monitoring of testing conditions and a better understanding of the process over time. Additionally, a low-release function is added, which is particularly important for studying MIC to avoid negative side effects, on the material (e.g. polymers/corrosion product-layer/cells itself) which might occur due to the fast pressure release T2 - ISMOS10 CY - Nashville, TN, USA DA - 11.08.2025 KW - MIC KW - MISTRAL KW - Corrosion KW - Environmental simulation KW - Metall KW - Polymer PY - 2025 AN - OPUS4-64297 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Consequences of benzalkonium chloride tolerance in Escherichia coli: Effects on selection and evolution in the presence of ciprofloxacin N2 - We investigated the selection dynamics between a benzalkonium chloride (BAC)-tolerant Escherichia coli strain (S4) and a sensitive wild type under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the opposite was observed at all ciprofloxacin concentrations investigated.Furthermore, we assessed the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type. T2 - 6th international symposium on the environmental dimention of antibiotic resistance-EDAR 6 CY - Gothenburg, Sweden DA - 22.09.2022 KW - Antimicrobial resistance KW - Tolerance KW - Experimental evolution KW - Selection PY - 2022 AN - OPUS4-56808 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Misra, Archismita T1 - Polyoxometalate Ionic Liquids (POM-ILs) as Protective Coatings for CulturalHeritage against Acid Corrosion and Biodeterioration N2 - Corrosion of stone by acid rain anddeterioration from microbial biofilmsare challenges pertinent worldwide forindustrial or residential buildings as wellas cultural heritage artefacts, like statuesor historic buildings. One mitigationoption might be the use of thintransparent films of polyoxometalate-based ionic liquids (POM-ILs). In thisregard, different limestone samples werecoated with hydrophobic, acid resistantPOM-ILs which also have biocidalproperties.1 Exposure of the samples tosimulated acid rain showed negligiblecorrosion compared to the significantdeterioration of unprotected samples(Fig 1. Left). In addition, the biocidalproperties of the POM-ILs suppress theformation of biofilms on coated stoneslabs. The coating is mechanically stableand is not removed even by harshmechanical and chemical treatment.Following studies successfully exploredthe effectiveness of the coating againstlampenflora growing in the PommeryChampagne cellar 2 (Fig 1. Right); andthe long-term performance of POM-ILsunder outdoor environmental conditions3. So, POM-ILs are already proven topossess remarkable anticorrosion andantimicrobial properties against aerobicmicroorganisms and being water-insoluble, they don’t get leached intoaquatic ecosystem, which is extremelybeneficial from an environmentalsustainability and toxicological point ofview. The current project aims tocontinue the journey on protecting thecultural heritage, shifting focus fromstones to metals and employ functionalPOM-IL nanocoatings to prevent MIC(Microbiologically Influenced Corrosion)of cultural heritage artefacts made ofmetal or metal alloy like carbon steel,brass, cast iron or bronze. Performanceof both the coating materials and coatingtechniques via optimization of theadhesion of the nanocoating on themetallic surface on the corrosion rateand corrosion products in the MICcaused by anaerobic microorganismslike methanogenic archaea or SulphateReducing Bacteria (SRB) would betested. The objective is to establishPOM-ILs as efficient environmentallysustainable nanocoating materialsagainst biocorrosion citing the already published success stories; and sketch theongoing endeavours and prospects ofthese very efficient candidates in thecontext of MIC mitigation. T2 - Mitigation of Microbiologically InfluencedCorrosion: Towards Scientific &Industrial Standardization (MIC-STAND) CY - Lisbon, Portugal DA - 24.07.2024 KW - Microbiologically influenced corrosion (MIC) KW - Polyoxometalate Ionic Liquid KW - Nanocoating KW - Cultural heritage PY - 2024 AN - OPUS4-64575 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -