Application of Shotgun Metagenomic Sequencing for the Investigation of Populations of Spore-formers in Whey Powders

Spore-forming bacteria are a cause for concern in the dairy industry for their spoilage and pathogenic potential. Traditional culturing and enumeration methods provide little information about the taxonomy of low level spore-formers or their relationship dynamics. Shotgun metagenomic sequencing provides a potential solution and allows analysis of both the constituents of a community as well as their functional potential. In order to bypass the biases of difficult-to-extract spore DNA, and potential presence of DNA from dead cells, we first enriched rehydrated dairy powders for populations of spore-formers. This allowed spore-formers to germinate and proliferate as vegetative cells, thereby increasing the proportion of DNA from living cells to that from any dead cells that may be present. Shotgun metagenomic sequencing was then applied to DNA extracted from these enriched samples and, thus, the approach facilitated an analysis of the spore-formers that could potentially proliferate in the rehydrated powder if exposed to favorable temperatures for extended periods.Results showed different populations exist depending on the enrichment and isolation method used. Among those present were potentially pathogenic species, which were identified on the basis of the associated toxin-encoding genes. Genes were found that could possibly shape the dynamics of populations in the powder including bacteriocin genes. Ultimately, this method has the potential to be utilized for detecting spore-forming bacteria in dairy powder, and examine their relationships in the food processing environment but brings with it the need for guidelines regarding the interpretation of results, including the determination of the level of risk that accompanies the identification of particular species.