The Use of Shotgun Metagenomic Sequencing for Mesophilic Spore-formers Identified in Whey Powder

Spore formers can cause spoilage in the dairy industry. Current agar based methodology are slow, and have limitations in sensitivity and specificity. Shotgun metagenomic sequencing approach was tested as an alternative. It allows to broadly sampling all genes in all organisms present in a given sample. This method allows detection and identification of possible pathogens and spoilage bacteria in parallel. Shotgun metagenomics also provides a means to study unculturable microorganisms that are otherwise difficult or impossible to analyze. The specific spore-formers population changed from month-to-month. However, 3 groups of mesophilic spore-formers, (Bacillus cereus, Bacillus licheniformis/Bacillus paralicheniformis, and Brevibacillus brevis), dominated throughout the year. Total thermophilic spore-former taxonomy was considerably different from mesophilic taxonomy. The study shows potential for this technology, to facilitate the detection of spore-formers present in dairy powders, allowing informed decisions surrounding process changes to reduce the risk of spore contamination. @ https://aem.asm.org/content/84/20/e01305-18" rel="nofollow noopener noreferrer" target="_blank">https://aem.asm.org/content/84/20/e01305-18
Mesophilic Sporeformers Identified in Whey Powder by Using Shotgun Metagenomic Sequencing

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, Bacillus cereus, Bacillus licheniformis/Bacillus paralicheniformis, and a third, more heterogeneous group containing Brevibacillus brevis, 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.

IMPORTANCE 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.

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