Strain-Level Metagenomic Analysis of the Fermented Dairy Beverage Nunu Highlights Potential Food Safety Risks

Abstract

The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole-metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on wholemetagenome assembly, which is a computationally demanding process. Here we demonstrated that three short-read-alignment-based methods, i.e., MetaMLST, PanPhlAn, and StrainPhlAn, could accurately and rapidly identify pathogenic strains in spinach metagenomes that had been intentionally spiked with Shiga toxin-producing Escherichia coli in a previous study. Subsequently, we employed the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product that is produced by the spontaneous fermentation of raw cow milk. We showed that nunu samples were frequently contaminated with bacteria associated with the bovine gut and, worryingly, we detected putatively pathogenic E. coli and Klebsiella pneumoniae strains in a subset of nunu samples. Ultimately, our work establishes that short-read-alignmentbased bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilization.IMPORTANCE Foodborne pathogens are responsible for millions of illnesses each year. Here we demonstrate that short-read-alignment-based bioinformatics tools can accurately and rapidly detect pathogenic strains in food products by using shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly; therefore, they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that wholemetagenome sequencing can be used as a practical food safety tool to prevent diseases or to link outbreaks to specific food products.KEYWORDS fermentation, food-borne pathogens, metagenomics I n recent years, high-throughput sequencing (HTS) has become an important tool in food microbiology (1). HTS enables in-depth characterization of food-related microbial isolates, via whole-genome sequencing (WGS), and it facilitates cultureindependent analysis of mixed microbial communities in foods, via metagenomic sequencing.WGS has provided invaluable insights into the genetics of starter cultures (2, 3), and it is routinely used in epidemiology to identify outbreak-associated foodborne pathogens isolated from clinical samples, through comparison of the single-nucleotide polymorphism (SNP) profiles of outbreak strain genomes versus nonoutbreak strain

The Present and Future of Whole Genome Sequencing (WGS) and Whole Metagenome Sequencing (WMS) for Surveillance of Antimicrobial Resistant Microorganisms and Antimicrobial Resistance Genes across the Food Chain

“…In a follow-up study by the same research team [ 103 ], spinach was spiked with different strains of Shiga toxin-producing E. coli at a low concentration of 0.1 CFU/g and then subjected to WMS analyses, which successfully achieved strain level identification of the pathogenic strains despite the presence of indigenous E. coli strains. In a survey on the safety of nunu, a traditional Ghanaian fermented milk product, Walsh et al [ 10 ] applied three short-read alignment-based bioinformatics methods (MetaMLST, PanPhlAn, and StrainPhlAn) in order to identify pathogenic strains. They detected putative pathogenic E. coli and Klebsiella pneumoniae strains in some nunu samples, indicating fecal contamination, poor hygiene in the production process and a potential hazard to the health of consumers.…”

“…Several recent studies have demonstrated the potential of routine whole genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control [ 7 ]. Metagenomics is also a powerful tool that allows for the culture-independent analysis of complex microbial communities, and has potential applications in AMR surveillance [ 8 , 9 , 10 , 11 , 12 ]. Indeed, it can provide access to all the genetic resources in a given environmental niche, which is essential for accessing the genomes of difficult-to-culture or non-cultivable microorganisms, and therefore could assist the tracking of AMR genes and mobile genetic elements, providing the necessary information to implement quantitative risk assessments to identify hotspots and routes of transmission of AMR across the food chain.…”

Tracking the Dairy Microbiota from Farm Bulk Tank to Skimmed Milk Powder

“…Therefore, there is an insufficient understanding of the functional potential of the microbial communities and, indeed, characterization of nonbacterial microbial contamination along the dairy chain. Recently, shotgun metagenomic sequencing, which overcomes these issues, has been used to study dairy products (16)(17)(18). Here, 16S rRNA gene amplicon and shotgun metagenomic analyses are used together to facilitate an in-depth study of the dairy microbiome from the farm through transportation and processing to a skimmed milk powder (SMP) and, in the process, provide valuable information regarding the impacts of collection, storage, and processing on this.…”

DNA extraction and host depletion methods significantly impact and potentially bias bacterial detection in a biological fluid

“…Despite the challenges associated with nucleic acid extraction from milk and the amount of host DNA present, several investigations have successfully used milk (13)(14)(15) or other dairy products (16) as their sample of interest in targeted and untargeted HTS studies, highlighting the potential for application of HTS technologies in food production settings. Total RNA sequencing can be more informative than untargeted DNA sequencing as it has the potential to provide gene expression (e.g.…”