Nanopore-based amplicon sequencing for rapid detection and identification of <i>Bacillus</i> spp. in plant-based products

<i>Bacillus</i> contamination in plant-based food products is a significant concern due to heat-resistant spores that can survive heating and proliferate during storage or handling, possibly leading to foodborne illnesses. Given the potential of high-throughput DNA sequencing to enhance microbial monitoring, we used a targeted approach by combining amplification of the <i>Bacillus tuf</i> gene with Oxford Nanopore sequencing. The ability of the MinION-based protocol to detect and identify closely related <i>Bacillus</i> species was first assessed using plant-based food samples spiked with spore suspensions of five representative <i>Bacillus</i> strains. A DNA extraction method, relying on food enzymatic pre-processing combined with mechanical cell lysis, was implemented to maximize <i>Bacillus</i> spore DNA recovery, and a droplet digital PCR (ddPCR) assay was used to evaluate extraction efficiency. Afterwards, the approach was applied to 72 different commercial plant-based foods and supplements to compare nanopore-based <i>tuf</i> profiling with established nanopore-based 16S rRNA analysis and culture-based methods. ddPCR analysis showed the high efficiency of the DNA extraction procedure for <i>Bacillus</i> spores from spiked samples. Sequencing of the <i>tuf</i> gene with the MinION device successfully differentiated the five different <i>Bacillus</i> species selected as reference strains for the artificial inoculation of food, when the resulting sequences were aligned against the custom-made BacTufDB database. Tests on commercial products confirmed the <i>tuf</i> gene ability (over the 16S rRNA gene) to highlight the presence of <i>Bacillus</i> species: <i>Bacillus</i> and closely related genera were detected in 35 of the tested plant-based products, out of which seven were contaminated by <i>Bacillus cereus</i> group. The study demonstrated that the <i>tuf</i>-based methodology more effectively detects <i>Bacillus</i> species in plant-based products, offering potential applications in food safety and quality control.