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The Project #FED-AMR

Start: 1 January 2020
Duration: 2.5 Years
Domain: Antimicrobial Resistance
Keywords: Bacterial transformation, free extracellular DNA, antimicrobial resistance, horizontal gene transfer, ecosystem boundaries
Contact: Werner Ruppitsch (AGES)

FED-AMR: The role of free extracellular DNA in dissemination of antimicrobial resistance over ecosystem boundaries along the food/feed chain

Antimicrobial resistance (AMR) is a global threat to the environment and a public health issue. Risk evaluation of pathogens, antimicrobial resistance genes (ARGs), as well as mobile genetic elements (MGEs) in diverse environments and matrixes are mainly based on total DNA (tDNA) data. The role of the extracellular free DNA (exDNA) is still neglected in most environmental studies.

Overall, the FED-AMR project contributed:

  • To improve the methods and knowledge in environmental exDNA research and its role in AMR transmission.
  • To collect and analyse large amounts of data and metadata from an agricultural environment, which outcomes have been presented to stakeholders.
  • To underline the importance of the role of exDNA as a source of diverse antimicrobial resistances in the environment, through the results of the FED-AMR project, which, in combination with the extremely long persistence and its important role as a structural component of biofilms, might represent a serious future public health threat that is currently neglected.
  • To still perform data analysis with the metadata produced and to combine all data i.e. exDNA, tDNA, pesticides, antibiotics, heavy metals, microbiomes, as well as to identify clinically relevant bacterial species, which may help to elucidate the transfer and development of resistances in microbial communities in an agricultural environment.
  • To have identified new potential sources of emerging resistant microorganisms along the feed/food chain with a focus on the environment and wildlife as a typically neglected component of One Health.
  • To confirm that C. difficile can spread clonally across the different compartments (animal and environment); the compartments soil and wastewater showed greater diversity in terms of C. difficile ribotypes, while faecal and manure samples indicating the dominant strain; this suggests a unidirectional spreading out of the animal compartments into the environment, while the soil and wastewater compartments seem to act as a reservoir for different input pathways beyond the animal compartments. Of particular interest is the possibility that C. difficile is transferred back to animal compartments via groundwater and surface water. The introduction of antibiotics or other substances that promote the development of antimicrobial resistance in C. difficile into any of the compartments can have an impact on the other compartments.
  • To demonstrate the genetic overlap between human and non-human C. difficile lineages at different One Health settings, supporting the high zoonotic importance of this human pathogen.
  • To develop new ongoing projects such as HERA NGS II, funded by the EU Commission, which will incorporate and strengthen the knowledge acquired in this project.
  • To understand the need to further improve any new method and new technologies, dynamically, despite the promising results we may have, as in the case of the FED-AMR.; therefore, with FED-AMR further research is essential before implementation into regional/ national surveillance systems.
  • To a private company working on machine learning for detection of AMR from WGS data and a university institute working with exDNA, with whom a collaboration was established and further scientific exchanges in regards exDNA can occur in the future.
  • To networking and communication between partners and reference laboratories from different backgrounds, sectors and countries, leading to new knowledge (such as at the level of the design of new protocols, or the implementation of new pipelines, or still for analysing data in the interface of genomics and informatics).

Project Outputs, Outcomes and Impact

FED-AMR project has implemented laboratory protocols related to the collection and genetic and phenotypic characterisation of clinically-relevant bacteria present in different environmental compartments. These were identified by the WHO for their relevance in terms of their resistance to last line antibiotics.

The HOALs, which correspond to real experimental open-air laboratories, will be regarded as an important model for in-depth studies that intend to continue to evaluate the spread of AMR.

FED-AMR project has developed important strategies for 16S and AMR metagenomic analysis, which are being disseminated to the scientific community through scientific publications.

FED-AMR project has also highlighted the harmonization and dissemination of protocols for C. difficile collection and isolation in animal, food and environmental samples, and the development of a pipeline for identification of MGEs in C. difficile (ClosTyper). All these tools are already in use by the partners and were or will be disseminated externally through scientific publications.

Selection pressures (antimicrobials, elements and herbicides) for antimicrobial resistance in environmental ecosystems (soil, water, faeces, manure, plants, feeds) were determined. Most of the samples showed low or usual concentrations of the analysed substances, but in some samples (soil, faeces and manure) antibiotics from two antimicrobial classes (fluoroquinolones and tetracyclines) were detected at or above the minimum selective concentrations for bacteria. The results will be used for an impact evaluation of antimicrobials, elements and herbicides on the prevalence and quantities of ARGs encoded on extracellular DNA in the tested environmental compartments.

Additionally to the eight published manuscripts, the Code developed by WP6 will be available in GitHub and all the FASTQ files from the 16S sequencing and the ARG profiling will be available on ENA once the results are published. Due to the limited amount of studies on gene capture for AMR, our project provides valuable datasets that can be used by other researchers interested in using similar methodologies for deep characterisation of AMR gene diversity.

The scientific impact of FED-AMR:

  • By a study capitalised on advancements in high-throughput sequencing methods and analytical tools, as it provides the large scalability necessary to investigate bacterial communities, in a way to explore and mapping AMR in exDNA, either in human, animal, and environmental settings, and in several sample matrices.
  • By sampling campaigns that provided basic information for establishing ARG monitoring in environmental compartments, which is recommended by EFSA and has the potential to become compulsory for EU MS.
  • By using 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).

The societal, policy-making and economic impact of FED-AMR:

  • Impactful research that adds value to a European, national and international level.
  • Decisive for assessing the potential of exDNA to serve as a high-risk source of resistance determinants in agricultural soils and along the food/feed chain.
  • Impact on strategies to improve and/or upgrade wastewater treatment plants, as it is decisive for WWTP engineers to know if they have to design devices that only kill bacteria or if strategies to eliminate bacterial DNA from the waste streams would have to be applied.
  • As reliable and accurate surveillance is fundamental to characterize the risk of AMR in a given region, the results obtained show how essential it is to track the spread of specific ARGs geographically and over time, identify new ARGs and support preventive measures and interventions against AMR pathogens.
  • Through a systematic evidence map, we will gather and collate data to inform future research, policy-relevant systematic review questions, and future funding strategies, concerning risk mitigation for antibiotic resistance emerging in the environment. As such, we expect that the evidence map will be relevant to e.g. WHO, ECDC and other EU agency, and national public health agencies. We expect that the evidence will contribute to the current debate about the role of environmental fluctuations always occurring in nature on the emergence and dynamics of antimicrobial resistance.

Project Assets

Dost, I., Abdel-Glil, M., Schmoock, G., Menge, C., Berens, C., González-Santamarina, B., Wiegand, E., Neubauer, H., Schwarz, S., Seyboldt, C. (2023). Clostridioides difficile in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance. Antibiotics. 12(1), 86. DOI:  https://doi.org/10.3390/antibiotics12010086

Alves, F., Castro, R., Pinto, M., Nunes, A., Pomba, C., Oliveira, M., Silveira, L., Gomes, J. P., Oleastro, M. (2023). Molecular epidemiology of Clostridioides difficile in companion animals: Genetic overlap with human strains and public health concerns. Frontiers in Public Health. 10, 1070258. DOI: https://doi.org/10.3389/fpubh.2022.1070258

Gardner, B., Betson, M., Cabal Rosel, A., Caniça, M., Chambers, M. A., Contadini, F. M., Gonzalez Villeta, L. C., M. M., La Ragione, R. M., de Menezes, A., Messina, D., Nichols, G., Olivença, D. V., Phalkey, R., Prada, J. M., Ruppitsch, W., Santorelli, L. A., SelemetasN., Tharmakulasingam, M., van Vliet, A. H. M.,  Woegerbauer, M., Deza-Cruz, I., Lo Iacono, G. (2023). Mapping the evidence of the effects of environmental factors on the prevalence of antibiotic resistance in the non-built environment: Protocol for a systematic evidence map. Environment International. 171, 107707. DOI: https://doi.org/10.1016/j.envint.2022.107707

Gardner, B., Betson, M., Cabal Rosel, A., Caniça, M., Chambers, M., Contadini, F. M., Gonzales Villeta, L. C. Hassan, M. M., La Ragione, R., De Menezes, A., Messina, D. Nichols, G., Olivenca, D. V., Phalkey, R., Prada, J. M., Ruppitsch, W., Santorelli, L. A., Selemetas, N., Tharmakulasingam, M., van Vliet, A. H. M., Wögerbauer, M., Deza-Cruz, I., Lo Iacono, G. (2022) Factors associated with the prevalence of antibiotic resistance in the environment from a One Health perspective: Protocol for a systematic evidence map. Center for Open Science. DOI: https://doi.org/10.17605/OSF.IO/A8GV6

Alves, F., Cano, M., Brondani, G., Nunes, A., & Oleastro, M. (2022). Airborne spores’ dissemination of a swine associated Clostridioides difficile clone. Anaerobe. 78, 102651. DOI: https://doi.org/10.1016/j.anaerobe.2022.102651

Alves, F., Nunes, A., Castro, R., Sequeira, A., Moreira, O., Matias, R., Rodrigues, J. C., Silveira, L., Gomes, J. P., & Oleastro, M. (2022). Assessment of the Transmission Dynamics of Clostridioides difficile in a Farm Environment Reveals the Presence of a New Toxigenic Strain Connected to Swine Production. Frontiers in Microbiology. 13, 858310. DOI: https://doi.org/10.3389/fmicb.2022.858310

Cabal, A., Rab, G., Daza-Prieto, B., Stöger, A., Peischl, N., Chakeri, A., Mo, S.S., Bock, H., Fuchs, K., Sucher, J., Rathammer, K,, Hasenberger. P,, Stadtbauer. S,, Caniça. M,, Strauß. P,, Allerberger. F,, Wögerbauer. M,, Ruppitsch. W. (2022). Characterizing Antimicrobial Resistance in Clinically Relevant Bacteria Isolated at the Human/Animal/Environment Interface Using Whole-Genome Sequencing in Austria. International Journal of Molecular Sciences. 23(19), 11276. DOI: https://doi.org/10.3390/ijms231911276

Gardner, B., Hassan, M. M., Chambers, M., La Ragione, R. M., & Lo Iancono, G. (2022). Modelling the dynamics and long-term stability of perturbed gut microbiota. Poster presentation at ONE Conference 2022, Brussels, Belgium. 21-24 June 2022. DOI: https://doi.org/10.5281/zenodo.6860460

Cabal Rosel, A., Peischl, N., Daza, B., Stöger, A., Rab, G., Rathammer, K., Allerberger, F., Wögerbauer, M., & Ruppitsch, W. (2022). Antimicrobial resistance and genetic relatedness among Escherichia coli isolates across the animal-human-wildlife interface in Austria. Poster presentation at 32nd European Congress of Clinical Microbiology and Infectious Diseases, Lisbon, Portugal. 23-26 April 2022. DOI: https://doi.org/10.5281/zenodo.6641985

Kořínková M, Štěpánková M, Drahošová Z, Matoušková N, Matějů L, Hochmalová K, Zimová M. (2021). International project to map the potential for horizontal transfer of antibiotic resistance genes across different ecosystems. Project presentation by SZU during an Ekomonitor seminar on Waste Analytics. DOI: https://doi.org/10.5281/zenodo.5770032

Cabal A, Peischl N, Rab G, Stöger A, Springer B, Sucher J, Allerberger F, Ruppitsch W. (2021). Draft genome sequence of a multidrug-resistant Escherichia coli sequence type 1193 pandemic clone isolated from wastewater in Austria. Microbiol Resource Announcements 10:e00762-21. DOI: https://doi.org/10.1128/MRA.00762-21.

D1.1- Scientific Supervisory Board and local Administrative representatives nomination

D1.2- Unified sampling and experimental protocols, which are core part of the project

D1.3- Data and Protocol Management Plan

D1.4- Webinars

D1.4 – Annex 1

D1.5- 12 Month Report Y3

D1.6 – Interim project report

D1.7- Final Report Y5

D2.1- 9 Month Report Y3

D2.1 List of sampling compartments, points and European test areas and harmonised protocols in alignment with other project protocols available in data repository

D2.1- Annex 1

D2.1- Annex 2

D2.1- Annex 3

D2.1- Annex 4

D2.1- Annex 5

D2.1- Annex 6

D2.1- Annex 7

D2.1- Annex 8

D2.1- Annex 9

D2.1- Annex 10

D2.1- Annex 11

D2.1- Annex 12

D2.1- Annex 13

D2.1- Annex 14

D2.1- Annex 15

D2.1- Annex 16

D2.1- Annex 17

D2.1- Final Report (Final Version)

 D2.2- Preliminary data collection on ARG prevalence and ARG background load in the compartments analysed so far

D2.3- Annual report Y3 of Work Package 2

D2.4 – Determination of naturally transformable bacteria in tested environmental compartments (T2.5) – Version 3

D2.5- Shotgun Sequencing: ARG Diversity in Tested Environmental Compartments 

D2.5 Annex 1.2

D2.6 – 16S metagenomics results: Microbial biodiversity and phylogenetic relationships of ARBs over ecosystem boundaries 

D2.7- Quantity and stability of free extracellular DNA observed in environmental compartments tested so far

D2.8 – Annual Report Y4 (WP2)

D2.9- ARG dynamics in an agricultural testing area: Response of ARG concentrations according to different fertilisation techniques and crops over an annual growth period

D2.10- Final report on WP2 and draft version of peer-reviewed publication 

D2.10- Annex 1

D3.1- Database of zoonotic C. difficile isolates across participants countries

D3.1- Annex 1

D3.1- Annex 2

D3.2- Overview of genetic overlap between human and non-human Clostridiodes difficile isolates

D3.3- Classification of pig farm compartments according to their role in the epidemiology of Clostridioides difficile

D4.1- Standardised Protocol for sampling and testing of environmental samples

D4.1- Annex 1

D4.1- Annex 2

D4.1- Annex 3

D4.2- Quantitative results of antibiotics in water

D4.3- Quantitative results of antibiotics in manure

D4.4- Quantitative results of antibiotics in faeces

D4.5- Quantitative results of antibiotics in soil

D4.6- Quantitative results of herbicides in environmental sample

D4.7 – Quantitative results of trace elements in environmental samples

D5.1- E. coli strains demonstrated to be suitable for transformation

D5.2- Optimal growth parameters for cultivating E. coli within the porcine gut model

D5.13- Results of the soil microcosm experiments regarding environmental effects on competence gene expression. 

D6.1- Protocol for a systematic review on environmental factors influencing the prevalence of antibiotic resistance in the environment

D6.2- Findings presented at one international conference and one national conference

D6.3- Update of codes and documentations in public repository e.g. GitHub

D6.4- Submission/publication of 1/2 paper(s) on how resilience of microbial communities depends on external environmental drivers and richness and diversity of the community

D6.5- Main code for the mathematical modelling made available in public repository (e.g. GitHub) with associated documentation (which can be used as “Material and Method” section of the forthcoming publications)



Project Events

One Health EJP Project Kick-off meeting- 13th November 2019, Berlin, Germany

FED-AMR Kick-off meeting- 27th January 2020, Vienna, Austria

FED-AMR progress meeting- 21-22nd April 2022, Lisbon, Portugal & online

FED-AMR closure meeting- 21st December 2022, online

Scientific webinars

Environmental reservoirs of antimicrobial resistance genes- 21st July 2020, speaker: Prof. Elizabeth M H Wellington

ARES Genetics analysis results- 9th September 2020, speakers: Dr. Andreas E. Posch & Dr. Sarah Lepuschitz

Extracellular DNA in natural environments: a neglected source for antibiotic resistance?- 24th September 2020, speaker: Dr. Markus Wögerbauer

Biotechnology and Safety: Tracking and Analysing Free-floating Extracellular DNA across Urban Waterways- 10th December 2020, speakers: Prof. David Weissbrodt & Dr. David Calderón Franco

C. difficile – the environmental perspective- 12th May 2021, speaker: Prof. Dr. Maja Rupnik

Metagenomics – ecological surveillance- 6th October 2021, speaker: Dr. Ana Sofia Ribeiro Duarte

Project Updates

FED-AMR also have a project specific webpage on the AGES website.

English abstract of the Master Thesis compiled within the FED-AMR project: Coelho, R. G., Manageiro, V. M. M. G., & Dias, D. M. A.. (2023). Master Thesis: Characterization of antibiotic resistance in strains isolated from different environmental reservoirs. DOI: https://doi.org/10.5281/zenodo.7584901



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