Open Access

Managing AMR from a OneHealth perspective requires understanding the ecology of resistant bacteria. Despite the fitness cost of resistance, many resistant strains are not outcompeted in the environment. The aim of this study is to understand how metabolic niches allow resistant bacteria to persist in the environment. We combined antimicrobial susceptibility testing, whole genome sequencing, genome-scale metabolic modeling (GEMs), population dynamics modelling, laboratory competition experiments with synthetic microbial communities and bioinformatic analysis of 20368 publicly available E. coli genomes. 62 E. coli strains isolated from wastewater treatment plants were sequenced and phenotypically characterized. Using GEMs, the growth of the strains was simulated with 298 substrates, identifying 40 substrates that allow growth for a portion of the isolates. The prediction accuracy of the GEMs was phenotypically evaluated to be 93% in the case of D-malate. Strains were identified that use D-malate as C-source and are susceptible to specific antibiotics as opposed to resistant strains that cannot use D-malate. Population dynamics modelling and laboratory competition experiments between these strains in synthetic communities showed that adding a C-source selects for or against resistant bacteria. Bioinformatic analysis of 20368 E. coli genomes for resistance and metabolic genes revealed that bacteria of the pathogenic and multidrug-resistant sequence type 131 cannot utilize D-malate. Our work shows that metabolic niches control the ecological selection of resistant bacteria. Adding a C-source to a bacterial community can select against resistance. This strategy could be used to reduce resistant bacteria in the gut of host organisms or in technical systems.

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 this study 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 combined susceptibility testing, time-kill experiments, experimental evolution, whole genome sequencing, molecular mechanistic methods, and competition experiments to study the link between biocide resistance and antibiotic resistance. We find that widely used compounds such as chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations. Furthermore, 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). However, such biocide-tolerant strains have reduced evolvability to high concentrations of antibiotics due to epistatic interactions of mutations conferring biocide tolerance and antibiotic resistance. Taken together, our work shows the importance of assessing the contribution of biocides on evolution and selection of AMR in the environment.

Evolutionary adaptation of bacteria to antimicrobial materials and coatings (AMCs) threatens their efficacy and may lead to cross-resistance to antibiotics. We develop an adaptive laboratory evolution experiment to be performed on AMCs (ALEE-AMC) based on the efficacy standard ISO 22196:2011, using metallic copper as a reference AMC and Escherichia coli as a model microorganism. The method consists of three steps: (i) determination of a timepoint at which viable cells are reduced upon AMC exposure by ~1000-fold, (ii) repeated cycles of exposure to AMCs and re-growth of surviving cells, and (iii) isolation of clones from the evolved populations and phenotypic testing. Repeated exposure to copper consistently selects for increased survival of evolved populations across participants of a ring trial. We find that laboratory identity did neither have a statistically significant effect on the average adapted antibacterial activity nor on the number of replicate populations that increased their survival to >100-fold. Antimicrobial susceptibility testing of isolated clones and whole genome sequencing showed that ALEE-AMC can detect changes in susceptibility including those above the clinical breakpoint related to mutations in known resistance determinants; specifically cross-resistance to fosfomycin was observed in several copper-evolved clones. ALEE-AMC offers a standardizable platform to assess the risk of bacterial adaptation to AMCs.

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 this study 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 find that widely used compounds such as chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations. Furthermore, 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 rapid 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). However, such biocide-tolerant strains have reduced evolvability to high concentrations of antibiotics due to epistatic interactions of mutations conferring biocide tolerance and antibiotic resistance. Taken together, our work shows the importance of assessing the contribution of biocides for the evolution and selection of AMR in the environment.

This research addresses the growing concern of water pollution with per- and polyfluoroalkyl substances (PFAS). These substances pose a severe global issue because they are highly persistent and are accumulating in water sources, soil, wildlife and humans, potentially causing significant health risks such as cancer, immune system disorders and hormonal disruption. This research is based on a collaborative effort between the Freie Universität Berlin, the Fraunhofer Institute for Applied Polymer Research (IAP) and the Federal Institute for Materials Research and Testing (BAM) with the aim of developing a sustainable adsorber material that can remove PFAS-based surfactants from water. The material is based on Kraft lignin, a renewable resource that is currently generated as an industrial waste stream from pulp and paper mills. During the project, lignin is functionalized as well as crosslinked to produce adsorber beads that are optimized for PFAS adsorption, and which can be used in water treatment. The beads are designed to be reused by removing the adsorbed PFAS in a regeneration process. In this way, the PFAS free beads may be released into the environment at their end of life, where they may serve as water and nutrient retention in soil. Thereby, a truly circular approach is envisioned. While the overall research project is presented, this work focuses particularly on viable ways for lignin functionalization and the formation of cross-linked adsorber beads. To this end, mechanochemical reductive amination is explored as a strategy for lignin functionalization while crosslinking with polyglycerol is explored as a strategy for bead formation.