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FARMED: Fast Antimicrobial Resistance and Mobile-Element Detection using metagenomics for animal and human on-site tests

01/2020-06/2022

Funding programme / funding institution: Horizon 2020

Project homepage: https://onehealthejp.eu/

Project description:

Antimicrobial resistance (AMR) threatens some of the greatest medical advances in modern times. Current AMR and pathogen detection is primarily reliant on classical culturing techniques, which may be slow. The development of new tools for real-time detection of resistant pathogens is a priority topic of the EJP. On-site and real-time analysis requires independence from culturing techniques to facilitate rapid interventions, especially during outbreaks. Furthermore, it requires robust protocols using minimal technical equipment that can be used outside of the normal laboratory environment. Metagenomic sequencing using short-read data can detect the composition of microbial communities for the assessment of potential pathogens and AMR or virulence genes, which could become an invaluable diagnostic tool. However, short-read technologies cannot reliably associate individual genes in a community to specific organisms and is a potential limitation for detecting AMR in pathogens.

The FARMED project aims to address these issues by using the ONT MinION, comparing this technology to other metagenomic sequencing technologies, and assessing its ability for diagnostic use on a range of sample matrices within both the laboratory and the field. Using long-read metagenomic sequencing, the local genetic context of AMR genes can be derived, and as such, the presence of the AMR genes can be attributed to specific species and plasmids, within the bacterial community. This technology will enable the identification of a plethora of bacterial species and linkage of particular species to a range of AMR genes. We will create harmonised protocols for on-site metagenomic DNA extraction and sequencing library preparation on the MinION. We will simultaneously adapt existing methods for DNA extraction with the ONT VolTRAX system. Further, we will develop efficient real-time mapping strategies that identify the origin of the genetic context of AMR genes, identify the host bacterial species to enable specific pathogen detection.

In addition to on-site metagenome analysis, we will investigate and establish best practice for ‘off-site’ analysis of plasmids sequences by comparing with purified isolates. We will compare the use of long-read and short-read data, as well as a combination of the technologies such as Hi-C, to assemble complete plasmids from metagenomics data. Hi-C sequencing is a novel method that uses proximity ligation of DNA molecules to determine the genetic context of AMR genes in relation to linkage to the bacterial host chromosome. Defined communities, containing isolates harbouring known plasmids, will be used to determine whether plasmid sequences can be derived from these metagenomic data.

We will provide an interactive workshop to transfer our extensive wet- and dry-lab experiences, including molecular and bioinformatic expertise, gained from this project. We predict that the methodologies and tools emerging from FARMED will be highly attractive and imminently applicable to a wide range of users beyond the consortium, to rapidly monitor AMR present in pathogens in many different environments. We envisage this technique, will be especially useful in low or middle-income countries where the burden of pathogen occurrence and AMR may be high but there is limited access to specialist or even ‘standard’ laboratory equipment for diagnosis.

Antimicrobial resistance (AMR) threatens some of the greatest medical advances in modern times. Current AMR and pathogen detection is primarily reliant on classical culturing techniques, which may be slow. The development of new tools for real-time detection of resistant pathogens is a priority topic of the EJP. On-site and real-time analysis requires independence from culturing techniques to facilitate rapid interventions, especially during outbreaks. Furthermore, it requires robust protocols using minimal technical equipment that can be used outside of the normal laboratory environment. Metagenomic sequencing using short-read data can detect the composition of microbial communities for the assessment of potential pathogens and AMR or virulence genes, which could become an invaluable diagnostic tool. However, short-read technologies cannot reliably associate individual genes in a community to specific organisms and is a potential limitation for detecting AMR in pathogens.

The FARMED project aims to address these issues by using the ONT MinION, comparing this technology to other metagenomic sequencing technologies, and assessing its ability for diagnostic use on a range of sample matrices within both the laboratory and the field. Using long-read metagenomic sequencing, the local genetic context of AMR genes can be derived, and as such, the presence of the AMR genes can be attributed to specific species and plasmids, within the bacterial community. This technology will enable the identification of a plethora of bacterial species and linkage of particular species to a range of AMR genes. We will create harmonised protocols for on-site metagenomic DNA extraction and sequencing library preparation on the MinION. We will simultaneously adapt existing methods for DNA extraction with the ONT VolTRAX system. Further, we will develop efficient real-time mapping strategies that identify the origin of the genetic context of AMR genes, identify the host bacterial species to enable specific pathogen detection.

In addition to on-site metagenome analysis, we will investigate and establish best practice for ‘off-site’ analysis of plasmids sequences by comparing with purified isolates. We will compare the use of long-read and short-read data, as well as a combination of the technologies such as Hi-C, to assemble complete plasmids from metagenomics data. Hi-C sequencing is a novel method that uses proximity ligation of DNA molecules to determine the genetic context of AMR genes in relation to linkage to the bacterial host chromosome. Defined communities, containing isolates harbouring known plasmids, will be used to determine whether plasmid sequences can be derived from these metagenomic data.

We will provide an interactive workshop to transfer our extensive wet- and dry-lab experiences, including molecular and bioinformatic expertise, gained from this project. We predict that the methodologies and tools emerging from FARMED will be highly attractive and imminently applicable to a wide range of users beyond the consortium, to rapidly monitor AMR present in pathogens in many different environments. We envisage this technique, will be especially useful in low or middle-income countries where the burden of pathogen occurrence and AMR may be high but there is limited access to specialist or even ‘standard’ laboratory equipment for diagnosis.

Project Partners

• Sciensano
• Animal and Plant Health Agency
• DTU Fødevareinstituttet (National Food Institute)
• Wageningen Bioveterinary Research

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