Mesophilic Sporeformers Identified in Whey Powder by Using Shotgun Metagenomic Sequencing

Abstract

Spoilage and pathogenic spore-forming bacteria are a major cause of concern for producers of dairy products. Traditional agar-based detection methods employed by the dairy industry have limitations with respect to their sensitivity and specificity. The aim of this study was to identify low-abundance sporeformers in samples of a powdered dairy product, whey powder, produced monthly over 1 year, using novel culture-independent shotgun metagenomics-based approaches. Although mesophilic sporeformers were the main target of this study, in one instance thermophilic sporeformers were also targeted using this culture-independent approach. For comparative purposes, mesophilic and thermophilic sporeformers were also tested for within the same sample using culture-based approaches. Ultimately, the approaches taken highlighted differences in the taxa identified due to treatment and isolation methods. Despite this, low levels of transient, mesophilic, and in some cases potentially pathogenic sporeformers were consistently detected in powder samples. Although the specific sporeformers changed from one month to the next, it was apparent that 3 groups of mesophilic sporeformers, namely, ,/ , and a third, more heterogeneous group containing, dominated across the 12 samples. Total thermophilic sporeformer taxonomy was considerably different from mesophilic taxonomy, as well as from the culturable thermophilic taxonomy, in the one sample analyzed by all four approaches. Ultimately, through the application of shotgun metagenomic sequencing to dairy powders, the potential for this technology to facilitate the detection of undesirable bacteria present in these food ingredients is highlighted. The ability of sporeformers to remain dormant in a desiccated state is of concern from a safety and spoilage perspective in dairy powder. Traditional culturing techniques are slow and provide little information without further investigation. We describe the identification of mesophilic sporeformers present in powders produced over 1 year, using novel shotgun metagenomic sequencing. This method allows detection and identification of possible pathogens and spoilage bacteria in parallel. Strain-level analysis and functional gene analysis, such as identification of toxin genes, were also performed. This approach has the potential to be of great value with respect to the detection of spore-forming bacteria and could allow a processor to make an informed decision surrounding process changes to reduce the risk of spore contamination.

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

“…However, this approach increases the overall cost. Another approach could be to enrich for specific subpopulations prior to DNA extraction (30). Developments such as these, combined with advances in portable sequencing technologies and further advances in the speed and accuracy of in silico tools, have the potential to greatly assist decision-making in this and other food chains.…”

“…Fat was removed (from raw-milk samples), and the supernatant was discarded. From there, pellets were washed and subjected to lysozyme treatment as previously reported (30). Twenty-eight microliters of proteinase K was added, and the mixture was incubated at 55°C for 15 min.…”

“…Adonis from the R vegan package was used to determine differences in beta diversity. Shotgun metagenomic data were processed as previously described (30). Briefly, raw metagenomic reads were checked for the presence of bovine reads, which were removed; filtered based on the presence of quality and quantity; and trimmed to 170 bp with a combination of Picard tools and SAMtools (39).…”

Microbiome-based environmental monitoring of a dairy processing facility highlights the challenges associated with low microbial-load samples

“…DNA sequencing methods have recently been applied to dairy and environmental samples to determine the microbial population composition and enable source tracking 7,[12][13][14] . High throughput metagenomic sequencing can provide greater insights into the taxonomic composition of populations present in these environments than culture based methods.…”

Microbiome-based environmental monitoring of a dairy processing facility highlights the challenges associated with low microbial-load samples

“…DNA sequencing methods have recently been applied to dairy and environmental samples to determine the microbial population composition and enable source tracking (Doyle et al ., 2017; McHugh et al ., 2018; Fretin et al ., 2018; Cho et al ., 2018, McHugh et al ., 2020). High throughput metagenomic sequencing can provide greater insights into the taxonomic composition of populations present in these environments than culture based methods.…”