HAM-ART: An optimised culture-free Hi-C metagenomics pipeline for tracking antimicrobial resistance genes in complex microbial communities

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Abstract Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli . Author Summary Antimicrobial resistance (AMR) is one of the biggest global health threats humanity is facing. Understanding the emergence and spread of AMR between different bacterial species is crucial for the development of effective countermeasures. In this paper we describe a user-friendly, affordable and comprehensive (laboratory and bioinformatics) workflow that is able to identify, associate and track AMR genes in bacteria. We demonstrate the efficiency and reliability of the method by comparing 50 faecal microbiomes from pig farms with high-antibiotic use (conventional farms), and 50 faecal microbiomes from pig farms with low-antibiotic use (organic farms). Our method provides a novel approach to resistance gene tracking, that also leads to the generation of high quality metagenomic assembled genomes that includes genes on mobile genetic elements, such as plasmids, that would not otherwise be included in these assembled genomes.

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