Direct identification and molecular characterization of zoonotic hazards in raw milk by metagenomics using Brucella as a model

Metagenomics is a valuable diagnostic tool to enhance microbial food safety because (i) it enables the untargeted detection of pathogens, (ii) it is fast since primary isolation of microorganisms is not required, and (iii) it has high resolving power on nucleic acid level allowing for a detailed characterization of pathogens. For shotgun metagenomics, total nucleic acids are isolated from complex samples such as foodstuff. Along with microbial NAs high amounts of matrix NAs are extracted that in turn might outcompete microbial NAs during next-generation sequencing and lead to a decreased sensitivity for the detection of low abundant microorganisms. Sensitive laboratory methods are particularly indispensable to detect highly-pathogenic foodborne bacteria like Brucella spp. because only a low infectious dose is needed to cause human disease through the consumption of contaminated dairy or meat products. In our study, we applied shotgun metagenomics sequencing for the identification and characterization of Brucella spp. in artificially and naturally contaminated raw milk from various ruminants. With the depletion of eukaryotic cells prior to DNA extraction, Brucella was detectable at very low numbers while at the same time microbiological culture and isolation of the fastidious bacteria commonly failed. Simultaneously, we detected several other zoonotic pathogens, antimicrobial resistance genes and virulence factors in the examined raw milk samples. Last but not least, we were able to retrieve the genotype of a Brucella isolate from a metagenomics dataset, indicating the potential of metagenomics for outbreak investigations using SNP and cgMLST typing. In this way, we developed a new approach for strain prediction based on SNPs to identify the correct species and define a specific strain with only low amounts of genus-specific reads per sample. In summary, our study showed that metagenomics is a highly sensitive tool for biological risk assessment of foodstuffs, particularly when pathogen isolation is hazardous or challenging.