TY - JOUR A1 - Kupke, Johannes A1 - Brombach, Julian A1 - Fang, Yuwen A1 - Wolf, Silver A. A1 - Thrukonda, Lakshmipriya A1 - Ghazisaeedi, Fereshteh A1 - Kuropka, Benno A1 - Hanke, Dennis A1 - Semmler, Torsten A1 - Nordholt, Niclas A1 - Schreiber, Frank A1 - Tedin, Karsten A1 - Lübke-Becker, Antina A1 - Steiner, Ulrich K. A1 - Fulde, Marcus T1 - Heteroresistance in Enterobacter cloacae complex caused by variation in transient gene amplification events N2 - Heteroresistance (HR) in bacteria describes a subpopulational phenomenon of antibiotic resistant cells of a generally susceptible population. Here, we investigated the molecular mechanisms and phenotypic characteristics underlying HR to ceftazidime (CAZ) in a clinical Enterobacter cloacae complex strain (ECC). We identified a plasmid-borne gene duplication-amplification (GDA) event of a region harbouring an ampC gene encoding a β-lactamase bla DHA-1 as the key determinant of HR. Individual colonies exhibited variations in the copy number of the genes resulting in resistance level variation which correlated with growth onset (lag times) and growth rates in the presence of CAZ. GDA copy number heterogeneity occurred within single resistant colonies, demonstrating heterogeneity of GDA on the single-cell level. The interdependence between GDA, lag time and antibiotic treatment and the strong plasticity underlying HR underlines the high risk for misdetection of antimicrobial HR and subsequent treatment failure. KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-627057 DO - https://doi.org/10.1038/s44259-025-00082-7 VL - 3 IS - 1 SP - 1 EP - 14 PB - Springer AN - OPUS4-62705 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nordholt, Niclas A1 - Sobisch, Lydia-Yasmin A1 - Gödt, Annett A1 - Lewerenz, Dominique A1 - Schreiber, Frank T1 - Heterogeneous survival upon disinfection underlies evolution of increased tolerance N2 - Disinfection is important to limit the spread of infections, but failure of disinfection may foster the evolution of antimicrobial resistance in bacteria. Persisters are phenotypically tolerant subpopulations that survive toxic stress longer than susceptible cells, leading to failure in treatments with antimicrobials and facilitating resistance evolution. To date, little is known about persistence in the context of disinfectants. The aim of this study was to investigate the influence of persisters on disinfection and to determine the consequences of disinfectant persistence for the evolution of increased tolerance to disinfectants. Disinfection kinetics with high temporal resolution were recorded for Escherichia coli exposed to the following six disinfectants: hydrogen peroxide (H2O2), glutaraldehyde (GTA), chlorhexidine (CHX), benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDAC), and isopropanol (ISO). A mathematical model was used to infer the presence of persisters from the time–kill data. Time–kill kinetics for BAC, DDAC, and ISO were indicative of persisters, whereas no or weak evidence was found for H2O2, GTA, and CHX. When subjected to comparative experimental evolution under recurring disinfection, E. coli evolved increased tolerance to substances for which persisters were predicted (BAC and ISO), whereas adaptation failed for substances in which no persisters were predicted (GTA and CHX), causing extinction of exposed populations. Our findings have implications for the risk of disinfection failure, highlighting a potential link between persistence to disinfectants and the ability to evolve disinfectant survival mechanisms. IMPORTANCE: Disinfection is key to control the spread of infections. But the application of disinfectants bears the risk to promote the evolution of reduced susceptibility to antimicrobials if bacteria survive the treatment. The ability of individual bacteria to survive disinfection can display considerable heterogeneity within isogenic populations and may be facilitated by tolerant persister subpopulations. Using time–kill kinetics and interpreting the data within a mathematical framework, we quantify heterogeneity and persistence in Escherichia coli when exposed to six different disinfectants. We find that the level of persistence, and with this the risk for disinfection failure, depends on the disinfectant. Importantly, evolution experiments under recurrent disinfection provide evidence that links the presence of persisters to the ability to evolve reduced susceptibility to disinfectants. This study emphasizes the impact of heterogeneity within bacterial populations on disinfection outcomes and the potential consequences for the evolution of antimicrobial resistances. KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-615566 DO - https://doi.org/10.1128/spectrum.03276-22 SN - 2165-0497 VL - 12 IS - 12 SP - 1 EP - 11 PB - American Society for Microbiology CY - Birmingham, Ala. AN - OPUS4-61556 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nordholt, Niclas A1 - Kanaris, Orestis A1 - Schmidt, Selina A1 - Schreiber, Frank T1 - Persistence against benzalkonium chloride promotes rapid evolution of tolerance during periodic disinfection N2 - Biocides used as disinfectants are important to prevent the transmission of pathogens, especially during the current antibiotic resistance crisis. This crisis is exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic Treatment and facilitate resistance evolution. Here, we show that E. coli displays persistence against a widely used disinfectant, benzalkonium chloride (BAC). Periodic, persister-mediated failure of disinfection rapidly selects for BAC tolerance, which is associated with reduced cell Surface charge and mutations in the lpxM locus, encoding an enzyme for lipid A biosynthesis. Moreover, the fitness cost incurred by BAC tolerance turns into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings highlight the links between persistence to disinfectants and resistance evolution to antimicrobials. KW - Persistence KW - Biocides KW - Evolution KW - Cross-resistance KW - Biocide tolerance KW - Disinfection PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-538532 DO - https://doi.org/10.1038/s41467-021-27019-8 SN - 2041-1723 VL - 12 IS - 1 SP - 6792 PB - Springer AN - OPUS4-53853 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Lee, Mihyun A1 - Wiesli, Luzia A1 - Schreiber, Frank A1 - Ivask, Angela Ivask A1 - Ren, Qun T1 - Quantitative Assessment of Microbial Transmission onto Environmental Surfaces Using Thermoresponsive Gelatin Hydrogels as a Finger Mimetic under In Situ-Mimicking Conditions N2 - Surface-mediated transmission of pathogens plays a key role in healthcare-associated infections. However, proper techniques for its quantitative analysis are lacking, making it challenging to develop novel antimicrobial and anti-fouling surfaces to reduce pathogen spread via environmental surfaces. This study demonstrates a gelatin hydrogel-based touch transfer test, the HydroTouch test, to evaluate pathogen transmission on high-touch surfaces under semi-dry conditions. The HydroTouch test employs gelatin as a finger mimetic, facilitating testing with pathogenic bacteria under controlled conditions. The thermoresponsive sol–gel transition of gelatin allows easy recovery and quantification of bacteria before and after testing. The HydroTouch test demonstrates that methicillin-resistant Staphylococcus aureus has a high transmission efficiency of ≈16% onto stainless steel, compared to <3% for Escherichia coli or Pseudomonas aeruginosa. Polyurethane surfaces exhibit strong resistance to bacterial contamination with a transmission efficiency of ≈0.6%, while polytetrafluoroethylene shows a transmission efficiency approximately four times higher than polyurethane. Additionally, quaternary ammonium-based antimicrobial coatings reduce the transmission efficiency of live bacteria on stainless steel to ≈4% of the original level. The HydroTouch test provides a reliable method for assessing pathogen transmission on various surfaces under semi-dry settings, supporting the development of effective antimicrobial, anti-transmission coatings to reduce healthcare-associated infections. KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance KW - Standardization PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-624566 DO - https://doi.org/10.1002/adhm.202403790 SN - 2192-2659 SP - 1 EP - 10 PB - Wiley VHC-Verlag AN - OPUS4-62456 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nordholt, Niclas A1 - O'Hara, Kate A1 - Resch-Genger, Ute A1 - Blaskovich, M. A1 - Rühle, Bastian A1 - Schreiber, Frank T1 - A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria N2 - Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action. KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-563811 DO - https://doi.org/10.3389/fmicb.2022.1023326 SN - 1664-302X IS - 13 SP - 1 EP - 13 PB - Frontiers Media CY - Lausanne AN - OPUS4-56381 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Valentin, J. A1 - Straub, H. A1 - Pietsch, Franziska A1 - Lemare, M. A1 - Ahrens, C. A1 - Schreiber, Frank A1 - Webb, J. A1 - van der Mei, H. A1 - Ren, Q. T1 - Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms N2 - Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance. KW - Antimicrobial resistance KW - Bacteria KW - Biofilms KW - Biocides PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-541113 DO - https://doi.org/10.1038/s41396-021-01157-9 SN - 1751-7370 VL - 16 IS - 4 SP - 1176 EP - 1186 PB - Springer Nature AN - OPUS4-54111 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Franziska, Pietsch A1 - Nordholt, Niclas A1 - Heidrich, Gabriele A1 - Schreiber, Frank T1 - Prevalent Synergy and Antagonism Among Antibiotics and Biocides in Pseudomonas aeruginosa N2 - Antimicrobials can exert specific physiological effects when used in combination that are different from those when applied alone. While combination effects have been extensively mapped for antibiotic-antibiotic combinations, the combination effects of antibiotics with antimicrobials used as biocides or antiseptics have not been systematically investigated. Here, we investigated the effects of combinations of antibiotics (meropenem, gentamicin, and ciprofloxacin) and substances used as biocides or antiseptics [octenidine, benzalkonium chloride, cetrimonium bromide, chlorhexidine, Povidone-iodine, silver nitrate (AgNO3), and Ag-nanoparticles] on the planktonic growth rate of Pseudomonas aeruginosa. Combination effects were investigated in growth experiments in microtiter plates at different concentrations and the Bliss interaction scores were calculated. Among the 21 screened combinations, we find prevalent combination effects with synergy occurring six times and antagonism occurring 10 times. The effects are specific to the antibiotic-biocide combination with meropenem showing a tendency for antagonism with biocides (6 of 7), while gentamicin has a tendency for synergy (5 of 7). In conclusion, antibiotics and biocides or antiseptics exert physiological combination effects on the pathogen P. aeruginosa. These effects have consequences for the efficacy of both types of substances and potentially for the selection of antimicrobial resistant strains in clinical applications with combined exposure (e.g., wound care and coated biomaterials). KW - Synergy KW - Antagonism KW - Suppression KW - Biocides KW - Antibiotics KW - Pseudomonas aeruginosa PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-520845 DO - https://doi.org/10.3389/fmicb.2020.615618 VL - 11 SP - Article 615618 PB - Frontiers CY - Lausanne AN - OPUS4-52084 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Chacón, Luz A1 - Kuropka, B. A1 - González-Tortuero, E. A1 - Schreiber, Frank A1 - Rojas-Jiménez, K. A1 - Rodríguez-Rojas, A. T1 - Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila N2 - Excessive discharge of quaternary ammoniumdisinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, froma wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three di􀀀erent concentrations of BAC and characterizedmechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 di􀀀erent sequenced A. hydrophila strains, consists of approximately 4.6Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations.Other genes related tomembrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand howbacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments. KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-578555 DO - https://doi.org/10.3389/fmicb.2023.1180128 SN - 1664-302X VL - 14 SP - 1 EP - 18 PB - Frontiers SA CY - Lausanne AN - OPUS4-57855 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schmidt, Selina B. I. A1 - Täschner, Tom A1 - Nordholt, Niclas A1 - Schreiber, Frank T1 - Differential Selection for Survival and for Growth in Adaptive Laboratory Evolution Experiments With Benzalkonium Chloride N2 - Biocides are used to control microorganisms across different applications, but emerging resistance may pose risks for those applications. Resistance to biocides has commonly been studied using adaptive laboratory evolution (ALE) experiments with growth at subinhibitory concentrations linked to serial subculturing. It has been shown recently that Escherichia coli adapts to repeated lethal stress imposed by the biocide benzalkonium chloride (BAC) by increased survival (i.e., tolerance) and not by evolving the ability to grow at increased concentrations (i.e., resistance). Here, we investigate the contributions of evolution for tolerance as opposed to resistance for the outcome of ALE experiments with E. coli exposed to BAC. We find that BAC concentrations close to the half maximal effective concentration (EC50, 4.36 μg mL−1) show initial killing (~40%) before the population resumes growth. This indicates that cells face a two‐fold selection pressure: for increased survival and for increased growth. To disentangle the effects of both selection pressures, we conducted two ALE experiments: (i) one with initial killing and continued stress close to the EC50 during growth and (ii) another with initial killing and no stress during growth. Phenotypic characterization of adapted populations showed that growth at higher BAC concentrations was only selected for when BAC was present during growth. Whole genome sequencing revealed distinct differences in mutated genes across treatments. Treatments selecting for survival‐only led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ), while treatments selecting for growth and survival led to mutations in genes related to stress response (hslO and tufA). Our results demonstrate that serial subculture ALE experiments with an antimicrobial at subinhibitory concentrations can select for increased growth and survival. This finding has implications for the design of ALE experiments to assess resistance risks of antimicrobials in different scenarios such as disinfection, preservation, and environmental pollution. KW - Antimicrobial resistance KW - Bacteria KW - Standardization KW - Biocides PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-615496 DO - https://doi.org/10.1111/eva.70017 VL - 17 IS - 10 SP - 1 EP - 11 PB - Wiley AN - OPUS4-61549 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR 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 - Scientific report - Impact of the use of azole fungicides, other than as human medicines, on the development of azole‐resistant Aspergillus spp. N2 - The use of azoles in the European Union and European Economic Area (EU/EEA) other than as human medicines has raised concerns about emergence and spread of azole‐resistant Aspergillus species. EU agencies, with the support of JRC, reviewed the evidence and provided conclusions and recommendations on this topic. Although incomplete, data from 2010 to 2021 showed that around 120,000 tonnes of azoles were sold in EU/EEA for uses other than as human medicines. The majority are used as plant protection products (119,000 tonnes), with a stable temporal trend. Evidence supported a link between environmental azole exposure and cross‐resistance selection to medical azoles in Aspergillus species (primarily shown for A. fumigatus). Prevalence of azole‐resistant A. fumigatus in human A. fumigatus infections ranges from 0.7% to 63.6% among different disease presentations and geographic regions; mortality rates range from 36% to 100% for invasive aspergillosis (IA). It was concluded that azole usage outside the human domain is likely or very likely to contribute to selection of azole‐resistant A. fumigatus isolates that could cause severe disease like IA. Environmental hotspots for resistance selection were identified, including stockpiling of agricultural waste and their possible use as soil amendment/fertiliser for certain agricultural crops (for plant protection products) and freshly cut wood (for biocides). Recommendations were formulated on measures to prevent and control selection of azole resistance in A. fumigatus, including implementation of good agricultural/horticultural practices, proper agricultural and wood waste storage and management, and on approval of new azole fungicides or renewal of existing fungicides. Recommendations on topics to be covered by studies provided when submitting applications for the approval of azole fungicides were listed. For the evaluation of such studies within the approval procedure, a preliminary framework for risk assessment was developed and should be further refined. Data gaps and uncertainties were identified, alongside with respective recommendations to address them. 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:b43-652175 DO - https://doi.org/10.2903/j.efsa.2025.9200 SN - 1831-4732 VL - 23 IS - 1 SP - 1 EP - 35 PB - Wiley CY - Hoboken, NJ AN - OPUS4-65217 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -