TY - JOUR A1 - He, Zhiming A1 - Dechesne, Arnaud A1 - Schreiber, Frank A1 - Zhu, Yong-Guan A1 - Larsson, Joakim A1 - Smets, Barth T1 - Understanding Stimulation of Conjugal Gene Transfer by Nonantibiotic Compounds: How Far Are We? N2 - A myriad of nonantibiotic compounds is released into the environment, some of which may contribute to the dissemination of antimicrobial resistance by stimulating conjugation. Here, we analyzed a collection of studies to (i) identify patterns of transfer stimulation across groups and concentrations of chemicals, (ii) evaluate the strength of evidence for the proposed mechanisms behind conjugal stimulation, and (iii) examine the plausibility of alternative mechanisms. We show that stimulatory nonantibiotic compounds act at concentrations from 1/1000 to 1/10 of the minimal inhibitory concentration for the donor strain but that stimulation is always modest (less than 8-fold). The main proposed mechanisms for stimulation via the reactive oxygen species/SOS cascade and/or an increase in cell membrane permeability are not unequivocally supported by the literature. However, we identify the reactive oxygen species/SOS cascade as the most likely mechanism. This remains to be confirmed by firm molecular evidence. Such evidence and more standardized and high-throughput conjugation assays are needed to create technologies and solutions to limit the stimulation of conjugal gene transfer and contribute to mitigating global antibiotic resistance. KW - Antibiotic resistance KW - Horizontal gene transfer KW - Conjugation KW - Chemicals PY - 2024 U6 - https://doi.org/10.1021/acs.est.3c06060 SP - 1 EP - 14 PB - ACS Publications AN - OPUS4-60105 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Valentin, Jules D. P. A1 - Altenried, Stefanie A1 - Varadarajan, Adithi R. A1 - Ahrens, Christian H. A1 - Schreiber, Frank A1 - Webb, Jeremy S. A1 - van der Mei, Henny C. A1 - Ren, Qun T1 - Identification of Potential Antimicrobial Targets of Pseudomonas aeruginosa Biofilms through a Novel Screening Approach N2 - Pseudomonas aeruginosa is an opportunistic pathogen of considerable medical importance, owing to its pronounced antibiotic tolerance and association with cystic fibrosis and other life-threatening diseases. The aim of this study was to highlight the genes responsible for P. aeruginosa biofilm tolerance to antibiotics and thereby identify potential new targets for the development of drugs against biofilm-related infections. By developing a novel screening approach and utilizing a public P. aeruginosa transposon insertion library, several biofilm-relevant genes were identified. The Pf phage gene (PA0720) and flagellin gene (fliC) conferred biofilm-specific tolerance to gentamicin. Compared with the reference biofilms, the biofilms formed by PA0720 and fliC mutants were completely eliminated with a 4-fold-lower gentamicin concentration. Furthermore, the mreC, pprB, coxC, and PA3785 genes were demonstrated to play major roles in enhancing biofilm tolerance to gentamicin. The analysis of biofilm-relevant genes performed in this study provides important novel insights into the understanding of P. aeruginosa antibiotic tolerance, which will facilitate the detection of antibiotic resistance and the development of antibiofilm strategies against P. aeruginosa. KW - Antimicrobial resistance KW - Bacteria KW - Biofilms KW - Pseudomonas aeruginosa PY - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-570205 SP - 1 EP - 5 PB - ASM Journals AN - OPUS4-57020 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Chacón, Luz A1 - Kuropka, B. A1 - González-Tortuero, E. A1 - Schreiber, Frank A1 - Rojas-Jiménez, K. A1 - Rodríguez-Rojas, A. T1 - Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila N2 - Excessive discharge of quaternary ammoniumdisinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, froma wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three di􀀀erent concentrations of BAC and characterizedmechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 di􀀀erent sequenced A. hydrophila strains, consists of approximately 4.6Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations.Other genes related tomembrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand howbacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments. KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-578555 SN - 1664-302X VL - 14 SP - 1 EP - 18 PB - Frontiers SA CY - Lausanne AN - OPUS4-57855 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - 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 - 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 - 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 - Schreiber, Frank A1 - Nordholt, Niclas A1 - Kanaris, Orestis A1 - Schmidt, Selina A1 - Lewerenz, Dominique T1 - Phenotypic and evolutionary responses of bacteria to disinfection N2 - Disinfectants are important to prevent the transmission of pathogens, especially in the face of the current antibiotic resistance crisis. The crisis is further exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfectant application, persistence to disinfectants and its role for the evolution of tolerance and cross-resistance to antibiotics has not been studied. Our work shows that E. coli displays persistence against several widely used disinfectants, including benzalkonium chloride (BAC), didecyldimethylammoniumchlorid (DDAC) and isopropanol. The molecular mechanism of BAC persistence is triggered in stationary phase and affected by several antibiotic persister genes (hipA, tisB, tolC, relA, spoT). Experimental evolution and population dynamic modeling show that repeated failure of disinfection due to persisters rapidly selects for BAC tolerance underpinned by reduced cell surface charge due to mutations in genes related to lipid A acylation (lpxML). Furthermore, evolved BAC tolerance affects the susceptibility to antibiotics, leading to positive selection of disinfectant tolerant strains at environmentally relevant antibiotic concentrations and variations in evolvability of antibiotic resistance due to epistatic effects. These results highlight the need for faithful application of disinfectants to steward their efficacy and the efficacy of antibiotics. A better understanding of the bacterial response to disinfectants is crucial to understand and avert the ongoing antimicrobial resistance crisis. T2 - Gordon Research Conference - Molecular mechanisms of evolution CY - Easton, MA, United States DA - 25.06.2023 KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2023 AN - OPUS4-57861 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Srivastava, Priyanka A1 - Tavernaro, Isabella A1 - Scholtz, Lena A1 - Genger, C. A1 - Welker, P. A1 - Schreiber, Frank A1 - Meyer, Klas A1 - Resch-Genger, Ute T1 - Dual color pH probes made from silica and polystyrene nanoparticles and their performance in cell studies N2 - Ratiometric green–red fluorescent nanosensors for fluorometrically monitoring pH in the acidic range were designed from 80 nm-sized polystyrene (PS) and silica (SiO2) nanoparticles (NPs), red emissive reference dyes, and a green emissive naphthalimide pH probe, analytically and spectroscopically characterized, and compared regarding their sensing performance in aqueous dispersion and in cellular uptake studies. Preparation of these optical probes, which are excitable by 405 nm laser or LED light sources, involved the encapsulation of the pH-inert red-fuorescent dye Nile Red (NR) in the core of self-made carboxylated PSNPs by a simple swelling procedure and the fabrication of rhodamine B (RhB)-stained SiO2-NPs from a silane derivative of pH-insensitive RhB. Subsequently, the custom-made naphthalimide pH probe, that utilizes a protonation-controlled photoinduced electron transfer process, was covalently attached to the carboxylic acid groups at the surface of both types of NPs. Fluorescence microscopy studies with the molecular and nanoscale optical probes and A549 lung cancer cells confirmed the cellular uptake of all probes and their penetration into acidic cell compartments, i.e., the lysosomes, indicated by the switching ON of the green naphthalimide fluorescence. This underlines their suitability for intracellular pH sensing, with the SiO2-based nanosensor revealing the best performance regarding uptake speed and stability. KW - Sensors KW - Silica and polystyrene nanoparticles KW - pH probe KW - Fluorescence spectroscopy KW - Cell studies KW - Dye KW - Particle synthesis PY - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-569198 SN - 2045-2322 VL - 13 IS - 1 SP - 1321 EP - 1336 PB - Nature CY - London AN - OPUS4-56919 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - 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 - Jaut, Valerie A1 - Schreiber, Frank A1 - Vareschi, Silvia 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 -