Molecular Characterization of Bacillus spp. Isolated from Ambient Gnocchi and Evaluation of a Biopreservation Approach

Bacillus spp. can be a risk for consumers and cause food spoilage. Fifty-four strains of Bacillus spp. were isolated from Italian ambient gnocchi in different storage conditions and formulations. The strains were identified and characterized by Whole Genome Sequencing (WGS), treated with Thymus vulgaris essential oil (TOE) and the effects were evaluated by the expression of some genes involved in stress response. Method: The genomes of the 54 Bacillus strains, obtained by WGS, were identified and characterized by Ribosomal MLST tool (rMLST) and k-SNP3 analysis. Phylogenetic trees based on the housekeeping genes gyrB, rpoB, and tuf were also realized to confirm species identification. Genes distances were determined from alignments through the Jukes-Cantor method, while the dendrogram was obtained with the Neighbor- Joining method. After that, virulence factors, antibiotic resistance, and bacteriocin production genes were retrieved by the genomes by the Basic Local Alignment Search Tool (BLAST), to highlight potential negative or positive roles of the isolates in gnocchi. Then, two isolates of the most found species (Bacillus cereus 11 and Bacillus subtilis 58L) were exposed to Thymus vulgaris essential oil (TOE), to evaluate the Minimum Inhibitory Concentration (MIC). Subsequently, real-time RT-PCR SYBR green was used to investigate the expression of some genes involved in the stress mechanisms induced by sublethal concentrations of TOE at increasing exposure times. Results: Phylogenetically, close Bacillus spp. strains were isolated from both ambient gnocchi and their ingredients, especially rice flour, starch, and turmeric, showing how the contamination of the raw materials is crucial for the safety and quality of the finished product. The pathogenic species B. cereus, B. thuringiensis, and B. paranthracis were isolated from not-spoiled gnocchi, and this finding represents a risk for consumers. The presence of virulence and antibiotic resistance genes in the genomes of these isolates confirms their pathogenic potential. In detail, one isolate of B. cereus and one of B. thuringiensis harbored the cescC gene, linked to the production of cereulide, a peptide which constitutes the emetic toxin of B. cereus, responsible for emetic syndrome and liver failure. Instead, B. subtilis was the most isolated species, also from gnocchi with evident spoilage (slime and reddish discoloration), and this demonstrates its ability to spoil gnocchi and grow even in non-permissive conditions. In fact, isolation from gnocchi with lactic acid in the formulation demonstrates the resistance of B. subtilis strains to low pH. Furthermore, some B. subtilis isolates retrieved genes for the bacteriocin subtilin biosynthesis, revealing potential positive properties. Regarding the antimicrobial effect of TEO, MIC value was 1.25 μL/mL for B. cereus 11 and B. subtilis 58L. At sublethal concentrations (0.31 μL/mL and 0.63 μL/mL) TEO lengthened the lag phase of microorganisms and determined the upregulation of genes involved in mechanisms of response to stress due to exposure to the antimicrobial agent, such as repair of membrane damage, restoration of quorum sensing mechanisms, biofilm formation, and mobility (pbpF, spoA, plcR, sinR). Discussion: Our data provide new insights on the spoilage and pathogenic potential of Bacillus spp. in ambient gnocchi, a product whose market penetration is increasing in Italy. In view of the elimination of preservatives, TEO provides a stressful effect, capable of triggering defense mechanisms. Therefore, these results are promising for future applications in food environments.