Application of hybridisation capture and target enrichment of antibiotic resistance genes in aquaculture foods

Aquaculture is a form of intense food production and for some conventional open-system aquaculture high use of antimicrobials is reported. Intensive use of antimicrobials is leading to high rates of antimicrobial resistance (AMR). Spread of pathogens, resistant bacteria as well as drug residues into the environment is easily possible. In contrast, in recirculating aquaculture system (RAS) and aquaponics there is little exchange with the environment and the use of antimicrobial agents is very limited due to biological filter systems. The knowledge on the presence and distribution of antibiotic resistances in these systems is scarce.
Selective isolation of antibiotic resistant bacteria is time and cost intensive and not a feasible approach without clear target genes and/ or bacteria. So, for investigations on AMR in RAS and aquaponics, a broader approach was needed. Two approaches were chosen to compare its applicability in AMR gene detection: isolation of plasmids with subsequent sequencing or bait-based enrichment of metagenomic DNA followed by sequencing. It was shown before that hybridisation capture and target enrichment allows cost efficient, directed and more in-depth sequencing of target genes compared to metagenome sequencing. For evaluation of the methods, different positive controls were created. First, a mock community was designed, containing Escherichia coli, Salmonella enterica and Staphylococcus hyicus. Secondly, frozen fish produced in aquaculture were purchased from supermarket, thawed and enriched in buffered peptone water. Plasmid DNA was extracted from enrichments using different plasmid DNA extraction kits and sequenced using Illumina NextSeq 550 System. Further, genomic DNA was extracted using three different DNA extraction kits. AMR genes were enriched using predesigned hybridisation capture kit (Daicel Arbor Biosciences) containing 3565 different AMR gene probes and then sequenced. Results showed, that bait-based enrichment resulted in detection of a larger variety of AMR genes compared to plasmid extraction. Both methods are limited in determination of gene variants differing only in few nucleotides (e.g. blaTEM). Although, further evaluations on run parameters have to be carried out to define the complete workflow, it was shown, that this approach will be useful to collect more precise information on AMR in RAS and aquaponics.