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Periodic Report Summary 3 - EFFORT (Ecology from Farm to Fork Of microbial drug Resistance and Transmission)

Project Context and Objectives:
The EFFORT project studies the complex epidemiology and ecology of antimicrobial resistance and the interactions between bacterial communities of commensals and pathogens in animals, the food chain and the environment.

This is conducted by a combination of epidemiological and ecological studies using newly developed molecular and bio-informatics technologies. EFFORT includes an exposure assessment of humans from animal and environmental sources. The ecological studies on isolates are verified by in vitro and in vivo studies. Moreover, real-life intervention studies are conducted with the aim to reduce the use of antimicrobials in veterinary practice. Focus is to understand the eco-epidemiology of antimicrobial resistance from animal origin and based on this, to predict and limit the future evolution of AMR and exposure to humans of the most clinically important resistance determinants by the inclusion of different sources of information in our prediction models.

Through its results, EFFORT provides scientific evidence and high-quality data that will inform decision makers, the scientific community and other stakeholders about the consequences of Antimicrobial Resistance (AMR) in the food chain, in relation to animal health and welfare, food safety and economic aspects. These results can be used to support political decisions and to prioritise risk management options along the food chain.

Please find below the main objectives and important innovative aspects of EFFORT:

- The characterization of the resistome of production animals as determined by metagenomics and assessing the added value of genomic analysis of isolates and metagenomic analysis of bacterial communities in comparison with the conventional used methods for EU based surveillance of antimicrobial resistance using indicator organisms.
- The quantification and characterisation of herd level antimicrobial usage in different animal species.
- The comprehensive and multinational/multispecies datasets that will be collected and built will allow determination of the complex associations between risk factors including antimicrobial usage, and the occurrence of resistance (resistome and conventional data).
- An estimation of the relative contribution of different sources and various transmission routes on antimicrobial resistance in humans in the general population as well as in selected occupational risk groups. This will also allow for an estimation of the effect of reduced antimicrobial use and other specific interventions on human exposure.
- The determination of genetic characteristics involved in the success of high-risk clones and mobile genetic elements in the epidemiology of AMR and the estimation of the relative impact on human infection risks caused by transfer of antimicrobial resistance determinants between commensals and pathogenic organisms.
- The implementation of on-farm interventions (e.g. restricted usage of antimicrobials) in multiple European countries and animal species following a common approach, including an analysis of the economic effects, animal welfare consequences and resistance levels.
- The use of novel statistical analytical approaches for rich meta-genomic data to obtain a “fingerprint” of resistance patterns for different populations (humans, animals) and the environment. Simultaneously these fingerprint patterns will be associated to determinants of antimicrobial resistance of relevance for human and animal health.

Project Results:
Through its results, EFFORT provides scientific evidence and high-quality data that will inform decision makers, the scientific community and other stakeholders about the consequences of AMR in the food chain, in relation to animal health and welfare, food safety and economic aspects. These results can be used to support political decisions and to prioritise risk management options along the food chain.

Progress is as follows:

WP1 “Integrated evidence base for the food chain”
WP1 will assure the use of standardized, efficient data collection methods along diverse food chains and integration of evidence in an accessible format that facilitates further analysis and comparison across food systems. This will include phenotypic resistance data, antimicrobial consumption data and relevant meta-data. All data has been collected and quality checks have been performed. Furthermore, this WP will together with WP2 ensure common data collection formats and create a searchable web-accessible database for meta-, phenotypic and genotypic data.
During period 3, the data on animal welfare for all participating countries is now available and has been analyzed, and the results are currently prepared as an article. Also, publications on determinants for AMR at farm level are under preparation. In these publications a meta-analysis is done on the AMR levels determined by metagenomics and farm characteristics. All MIC data was analyzed in relation to antibiotic use, and data by animal species and antibiotic use data was analyzed and quantified by animal species (pigs and poultry) and submitted to the Journal of Antimicrobial Chemotherapy in 2018. Analysis of AMU for companion animals, veal calves, fish and turkeys is currently being conducted.
The dataset compiled in this WP is unique for its coverage of (nine) countries, quality and broadness of data (antimicrobial usage, phenotypic and genotypic data on antimicrobial resistance, welfare data, and other relevant meta-data of farms).

WP2 “Molecular approaches for determining the molecular ecology and epidemiology of AMR genes”
Different molecular techniques are used in EFFORT: metagenomics (sequencing all DNA in faecal samples and a selection of dust samples), qPCR for the quantitative detection of 5 single genes in DNA samples isolated from faecal, meat, slaughterhouse (e.g. gloves) and environmental samples (e.g. gloves), and whole genome sequencing of single bacterial isolates.
Earlier in the project, metagenomics of pigs and poultry samples were performed. In period 3, turkey, companion animals and wild life samples were sequenced for metagenomic analysis.
Based on the results of metagenomic data analysis of pigs and poultry samples, the selection of genes ermB, aph3`-III, sul2 (and vanA/B) for the qPCR assay was development and validated. This development and validation of all assays was successfully finished. Data acquisition for one qPCR target is slightly delayed and expected to be completed within 3 months.
DNA was isolated from samples from the in-depth study (samples from carcasses, gloves from meat end products) to be able to do qPCR analysis.
Within period 3, WP2 has been sequencing single isolates from WP1. A protocol has been established and within 5 countries (NL, DK, GE, PL, ES) a selection of 50 single E. coli isolates from pigs and poultry was made and currently being analysed by Whole Genome Sequencing (WGS). Additionally, 50 veal calf isolates were analysed as well. The purpose of this approach is to compare the WGS output (in particular presence of genetic resistance markers) with the MICs of these isolates and the metagenome (MG) analysis of the pooled fecal samples these isolates were collected from.

WP3 “Ecology and transfer of resistance mechanisms”
WP3 will review and identify current knowledge and knowledge gaps that link biophysical processes during the complete food chain and transfer of antimicrobial resistance genes between bacteria. Further, these parameters are analysed, and tested in vitro in different laboratories and bacterial model systems, to identify the processes that hinder/enhance transfer of antimicrobial resistance genes along the food chain, in order to propose interventions to hinder transfer events.
During period 2, WP3 focused on a pre-trial broiler chicken infection model with S. Corvallis and S. Paratyphi B (dt+) as donor and recipient strain, respectively. Also, WP3 focused on the characterization of resistance plasmids that are found in various animal species and humans but are especially abundant in poultry. Phylogenetic analysis and experiments targeting phenotypic traits have provided new insights in the dissemination and fitness cost of these plasmids carried by Enterobacteriaceae. Within period 3, WP3 focused on the animal trials. A novel plasmid-host adaptation mechanism has been identified and is based on acquisition of Insertion sequences from the chromosome of the bacterial host by the plasmid. The relevance of multicopy plasmids as gene capture platforms has been shown in single isolates and metagenomic samples.

WP4 “Epidemiological analysis of antimicrobial resistance patterns in humans and the environment”
During period 2, meta-data for pigs and poultry was compiled and a common database was created. Additionally, a database for the data originated from the in-depth study was assigned. Extensive evaluation of data consistency and data quality was performed, and first preliminary exploratory analysis of the data was carried out. During P3 data on resistance has been collected in a range of environmental compartments (in farm dust and air filters around farms, in slaughterhouse gloves and air filters of slaughterhouse workers). The samples have been analysed by qPCR and partly by metagenomics (dust samples). The results have been made available to the consortium as data sets.
A third poultry slaughterhouse in Germany could be encouraged to participate. Additional 120 glove samples, 101 stool samples from slaughterhouse workers, 6 air samples, 24 carcass samples, as well as 12 poultry fecal samples were collected. These samples were processed in the lab immediately to be stored at -80°C as pellets and the DNA pellets will be tested by qPCR. The results of the qPCR were provided in M55 and are currently analyzed.

WP5 “Relationship between farming practices, antimicrobial usage, animal health and resistance”
The aim of WP5 is to study the relationship between farming practices (including biosecurity), antimicrobial usage, animal health and antimicrobial resistance. In P2, quantification of antimicrobial consumption data and the further necessary assumptions were made based on scientific literature. Data verification took place and treatment incidences (TI) were calculated. Furthermore, based on the verified data, antimicrobial consumption data for pigs and poultry are further explored looking at: 1) Antimicrobial consumption per animal category (pigs) and country for group treatments and purchased products; 2) Antimicrobial consumption per antimicrobial class based on used and purchased active substance (mg/kg animal); 3) The relation between TI based on group treatments on the one hand and purchased antimicrobials on the other hand; 4) The relation between TI for the different animal categories (pigs), both for group treatments and purchased antimicrobials.
During period 3, the analysis on AMU are finalized. A full description of AMU, biosecurity (and welfare) for several animal species (except for fish) is ready. For broilers the results are described in a paper, which is submitted to a scientific journal. Also, for pigs the submission of a paper on AMU in pigs is done. Identification of health, welfare and production related risk factors influencing antimicrobial consumption for pigs and broilers was done as well as the identification of antimicrobial consumption related risk factors at animal and herd level influencing antimicrobial resistance for pigs and broilers.
All these results are now being translated into scientific papers.

WP6 “Intervention studies aiming at reducing AM usage and resistance in pig and poultry production”
During period 2 the ADS tool and study protocols have been applied. Farms were recruited (in France and the Netherlands for pigs, and in France, Belgium and Spain for poultry). The Danish partner (O-VET) left the consortium. Visits and audits were performed, and data were collected and uploaded in a central database. For all intervention farms action plans were made and follow up visits were done. Initial sampling for further bacteriological analysis was performed in all farms.
During Period 3, poultry action plans were analyzed and show a large range of intervention recommended by veterinarians. The preliminary statistical analysis of the data has shown a reduction of antimicrobial usage in all farms (control and intervention) in the 3 countries with differences in the retrospective average level and the reduction trends observed in the different countries.
In France and the Netherlands, the study was carried out in pig farms, more than 60 pig farms were recruited. Action plans, AMU and performance data were collected for further analysis. Through EFFORT, the electronic ADS tool was implemented in the Netherlands to test the feasibility to collect and review data by veterinarians and farmers. It allows collecting information through smartphones and/or computer and to share information and knowledge.

WP7 “Quantification of exposure to AMR through different transmission routes from animals to humans”
The work in WP7 relies mainly on data collected in other WPs and was therefore limited during the first project period. In period 2, a model framework was developed. For each possible transmission route, equations to estimate exposure were defined and required input data were identified. A database was created to collect data from the literature on specific model parameters.
In period 3, a model was developed to quantify the exposure of European consumers to resistance genes via meat consumption. The model was parameterized with data from a range of public data sources (e.g. EFSA food consumption survey, trade statistics) as well as with results from EFFORT research. qPCR results were available for selected resistance genes and these were used to develop a first running version of the model.
As no metagenomic results are available for all sources of occupational exposure (only for human samples) qPCR data will be used instead for analysis. This has considerable impact on the initial plan to develop a machine learning model using metagenomic results. Close collaboration and discussion is ongoing between partners how to handle these and to find a solution for these analyses. As soon as all data are available analysis can be finalized.

WP8 “Economic impact analysis”
During the second period of the project, the animal-oriented parameters for the Herd Health and Welfare Index were collected. Also, bio-economic modeling of broiler and pig (fattening) farms was completed and elaboration of the theoretical foundation for analysis of the Economic Value of AM, as well as Economic Efficiency of the farm (subject to various management routines, amongst (changes in) use of AM took place. This resulted in a 3-stage modeling approach focused on economic analysis which will be applied to various datasets that become available within the EFFORT project.
During period 3 the Herd Health and Welfare Index (HHWI) was calculated for all nine countries in the EFFORT consortium. A major improvement in the economic value of AMU at the broiler farm level has been achieved. These qualitative and quantitative insights will be used in the development of guidelines for policy makers involved in reducing the use of AM and the supportive role of intervention. A set of decision options of measures to reduce the microbiological (and hence AM) load of both broilers and pigs in the off-farm stages were obtained; this was done through a cost-effectiveness analysis. These decision options are currently discussed with actors in the field on (im-) possibilities for implementation.

WP9 “Project dissemination and training”
WP9, responsible for dissemination activities, is taking care of disseminating widely news and outputs, which have been produced within the EFFORT project. To ensure consistency and quality of this process, a project dissemination plan has been implemented and is followed for all dissemination actions. A strong online presence can be expected, via the EFFORT public website (, the Twitter account (@effortamr) and a LinkedIn group (EFFORT: Ecology from Farm to Fork of microbial drug resistance and transmission). To inform relevant stakeholders and to obtain feedback on the project’s workplan, a series of online policy round tables have been organised. Members of the EFFORT consortium have also attended scientific conferences and meetings with particularly relevant stakeholders such as the European Medical Agency (EMA).
Within period 3, a workshop consisting of lectures and hands-on exercises on Metagenomics applied to surveillance of pathogens and AMR was developed. The workshop was successfully delivered on 19-20th March 2018. The workshop was part of a blended training, which also included an e-learning component. The related e-learning course was successfully delivered to all workshop participants one month before the workshop (Tallinn, March 19-20). Subsequently, it was adapted to a Massive Online Open Course, which was released to public on the platform Coursera, by May 31st, 2018 ( with a massive participation.
The dates for the final EFFORT international conference are set for November 26-28th 2018. The conference will be held in Utrecht The Netherlands. A conference website ( is made and registration is open.

Potential Impact:
The expected final results and their potential impact and use

The EFFORT research will provide scientific evidence and high-quality data that will inform decision makers, the scientific community and other stakeholders about the consequences of AMR in the food chain, in relation to animal health and welfare, food safety and economic aspects. The project’s research will also contribute to the reduction of the transfer and spread of antimicrobial resistance by using evaluated intervention strategies that are targeting a range of possible intervention points along the food chain.

Specifically, EFFORT will strive to answer the following fundamental, but complex questions demanded by risk managers:
What is the association between antimicrobial usage in food-producing animals and human exposure to AMR determinants?
What is the relative contribution of transmission routes and sources of human exposure to AMR determinants?
How can human exposure to AMR determinants through food-producing animals be reduced?
What is the most cost-effective way of monitoring antimicrobial resistance occurrence in food-producing animals and in the food chain?
The answers are expected to support political decisions and to prioritise risk management options along the food chain both on the short and long-term horizon. In addition, the results can inspire and guide future research initiatives.

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