Metagenomic characterization of genetically modified Bacillus contaminations in commercial food enzyme products

Genetically modified microorganisms (GMM) are frequently employed for manufacturing of mi-crobial fermentation products such as food enzymes or vitamins. Although the fermentation product is required to be pure, GMM contaminations have repeatedly been reported in diverse commercial microbial fermentation produce types, leading to several rapid alerts at European level. The aim of this study was to explore the potential of shotgun metagenomic high-throughput sequencing in order to detect DNA contaminations in fermentation product with a special focus on genomic characterization of unauthorized GMM. By combining short- and long-read metagenomic sequencing, we could characterize two transgenic constructs with inser-tions of amylase genes originating from B. amyloliquefaciens and B. licheniformis, respectively, which were both present in all four investigated sampels. Additionally, the samples were con-taminated with up to three unculturable Bacillus strains, carrying genetic modifications that may hamper their ability to sporulate, and several samples contained viable Bacillus strains. Altogether these contaminations constitute a considerable load of antimicrobial resistance genes, representing a potential public health risk. In conclusion, our study showcases the added value of metagenomics to investigate the quality and safety of microbial fermentation products.