Final report JRP15-AMR2.1-FED-AMR

FED-AMR project aimed to understand the role of free extracellular DNA (exDNA) in dissemination of antimicrobial resistance (AMR) over ecosystem boundaries along the food/feed chain in one-year crop growing season. As transformation is an important driver for genetic plasticity of bacterial genes and genomes and natural transformation does not require physical contact between donor and recipient bacteria we hypothesized that this may facilitate antibiotic resistance genes (ARG) crossing ecosystem barriers and invading new habitats compared to HGT by conjugation. The WP1 aims were met and were mainly based on project coordination, including scientific and administrative management, organization of regular activities, facilitation of cooperation between the WPs and integration of their outputs and results. WP2 aimed to quantify and assess microbial and AMR diversity with next generation sequencing (metagenomics) in different compartments of food production chain, during one-year-crop growing season. In particular we was interested in studying the role of extracellular DNA in the dissemination of AMR genes. We had 476 samples from six European countries (Austria, Czech Republic, Estonia, Ireland, Portugal, United Kingdom), distributed in four EU regions from up to eleven compartments along the food/feed chain. Both extracellular free DNA (exDNA) and total DNA were subjected to 16S rDNA sequence-based microbial profiling to measure the bacterial diversity. The Phyla in exDNA are dominated by Proteobacteria and Firmicutes. The principal component analysis suggests a separation between three groups of compartments: 1) soil 2) crops, feeds and drinking water 3) farmers, pigs, wild animals, manure and wastewater, which may show e.g. a smaller shifting between the microorganisms found globally in the compartments of these three groups. No significant differences were detected between soil amended with manure and without it, which may be due to an history of exposure to manure in the preceding years. ExDNA and total DNA were also subjected to target enrichment (equivalent to gene capture) to characterize and quantify ARGs in the different compartments. The analysis started with samples obtained from the wild animal compartment where we detected ARG conferring resistance to about 30 antibiotic classes, where β-lactams, tetracyclines, aminoglycosides and fluoroquinolones were the most prevalent. In the same compartment, we identified e.g. 11 ARG-types conferring resistance to fluoroquinolones. As for the competent bacteria the highest loads were observed in feed (exDNA), groundwater (total DNA) and field drainage (total DNA). We can conclude that bacteria known to be able to take up extracellular DNA are present in all environmental compartments and may serve as receptorsfor ARGs that are harboured on free exDNA. We also characterized genetically (WGS-based typing) and phenotypically (antimicrobial susceptibility testing) more than 500 cultivable bacteria. In all countries, isolates retrieved from wastewater, pig manure and pigs were those carrying more ARGs, including extended-spectrum beta-lactamases, but also ARGs associated to antimicrobials used usually in animal husbandry such as aminoglycosides, tetracyclines or sulfonamides. However, AMR data in our study (including those from metagenomics) are not thought to be a direct result of antimicrobial drug use, because 'globally' there is no direct linkage of AMR (associated with the antibiotics from the classes identified in WP4) across the various compartments and countries, therefore, there will be other risk factors (to be identified yet, if achievable). WP3 aimed at elucidating the role of Clostridioides difficile as a pathogen/ARGs transfer platform over ecosystems boundaries and the genetic overlap between human and non-human zoonotic reservoirs. At the farm level, dominant clones in environmental compartments associated with pig production were identified, suggesting a transmission chain between compartments involving these animals. The results contributed to unveil the role played by animal and environmental reservoirs within the C. difficile epidemiology. The studies assessing genetic overlap between human and non-human C. difficile lineages at different One Health settings support the zoonotic relevance of C. difficile and its presence in novel reservoirs. In WP4 we aimed at determining the selection pressures (antimicrobials, elements and herbicides) for antimicrobial resistance in environmental ecosystems (soil, water, faeces, manure, plants, feeds). We analysed samples collected by WP2. The highest concentrations of antimicrobials were detected in manure and faeces and are likely to select bacteria resistant to these compounds. The results of WP4 will be used for an impact evaluation of the analysed substances on the prevalence and quantities of ARGs encoded on extracellular DNA in exposed bacterial populations residing in the tested environmental compartments. In WP5 we successfully established and optimised (pH, temperature, flow rates and volumes) an in vitro model of the pig large intestine using pig faeces and conditions to represent the natural complex microbial community of the gut. We demonstrated sufficient growth and maintenance of an E. coli J53 strain to serve as a recipient of exDNA to study the impact of antibiotics and/or trace elements on the efficiency of natural transformation within the gut. Also, a soil microcosm setting was created using non-manured agricultural soil and Acinetobacter baylyi ADP1 as receptor of extracellular DNA. The results showed a repression of the mRNA levels of two competence genes (comA and dprA) during the first 6 hours after spiking if the microcosm was incubated at 35°C compared to 20°C. These results indicate that elevated environmental temperatures may diminish the ability of naturally competent soil bacteria to take up extracellular DNA and may reduce AMR spread by bacterial transformation. WP6 focused on generating a protocol for a systematic review on factors influencing the prevalence of antibiotic resistance in the environment and on formulating and validating a mathematical model for the dynamics of microbial communities. The protocol was registered in a public repository and accepted in Dec/2022 and published in Jan/2023 in an international journal (Environment International: https://doi.org/10.1016/j.envint.2022.107707). With respect to the task on modelling microbial communities, we have formulated a new model and validated it with in-silico data purposely developed. FED-AMR has a societal, policy and economic impact, as well as scientific impact through cross-sector communication of data contributing to the advancement of science. This project used the true concept of One Health, namely with an important environmental component (farmers, pigs, wild animals, manure, air of pig barns, feeds, crops, soil, water).