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Processed cheese is a commercial product characterized by high microbiological stability and extended shelf life obtained through the application of severe heat treatment. However, spore-forming bacteria can survive through thermal processes. Among them, microorganisms belonging to  Bacillus  genus have been reported. In this study, we examined the microbiological population of the first hours’ production of processed cheeses in an Italian dairy plant during two seasons, between June and October 2020. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify bacteria colonies, allowing the isolation of  Bacillus cereus  and  Bacillus   subtilis  strains. These results were further confirmed by amplification and sequencing of 16 rRNA bacterial region. A multi-locus sequence type (MLST) analysis was performed to assess the genetic similarity among a selection of isolates. The fourteen  B. cereus  strains showed two sequence types: ST-32 was observed in only one strain and the ST-371 in the remaining thirteen isolates. On the contrary, all twenty-one  B. subtlis  strains, included in the study, showed a new allelic profile for the  pycA  gene, resulting in a new sequence type: ST-249. For  B. cereus  strains, analysis of toxin genes was performed. All isolates were positive for  nheABC ,  entFM , and  cytK , while  hblABCD ,  bceT , and  ces  were not detected. Moreover, the biofilm-forming ability of  B. cereus  and  B. subtilis  strains was assessed, and all selected isolates proved to be biofilm formers (most of them were stronger producers). Considering the genetical similarity between isolates, jointly with the capacity to produce biofilm, the presence of a recurring  Bacillus  population could be hypothesized.

Characterization of Vegetative Bacillus cereus and Bacillus subtilis Strains Isolated from Processed Cheese Products in an Italian Dairy Plant