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

ABSTRACT: 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 Bacillussubtilis 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.

SUBMITTER: Catania AM

PROVIDER: S-EPMC8622485 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in B. subtilis were found to be members of the If subfamily. These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of B. subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in B. subtilis, ybgE and ywaA, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of B. cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms. In both B. cereus and B. anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from B. cereus, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of B. subtilis YbgE and also inhibited growth of B. subtilis and B. cereus in culture.

Project description:While some species in the Bacillus cereus group are well-characterized human pathogens (e.g., B. anthracis and B. cereus sensu stricto), the pathogenicity of other species (e.g., B. pseudomycoides) either has not been characterized or is presently not well understood. To provide an updated characterization of the pathogenic potential of species in the B. cereus group, we classified a set of 52 isolates, including 8 type strains and 44 isolates from dairy-associated sources, into 7 phylogenetic clades and characterized them for (i) the presence of toxin genes, (ii) phenotypic characteristics used for identification, and (iii) cytotoxicity to human epithelial cells. Overall, we found that B. cereus toxin genes are broadly distributed but are not consistently present within individual species and/or clades. After growth at 37°C, isolates within a clade did not typically show a consistent cytotoxicity phenotype, except for isolates in clade VI (B. weihenstephanensis/B. mycoides), where none of the isolates were cytotoxic, and isolates in clade I (B. pseudomycoides), which consistently displayed cytotoxic activity. Importantly, our study highlights that B. pseudomycoides is cytotoxic toward human cells. Our results indicate that the detection of toxin genes does not provide a reliable approach to predict the pathogenic potential of B. cereus group isolates, as the presence of toxin genes is not always consistent with cytotoxicity phenotype. Overall, our results suggest that isolates from multiple B. cereus group clades have the potential to cause foodborne illness, although cytotoxicity is not always consistently found among isolates within each clade.IMPORTANCE Despite the importance of the Bacillus cereus group as a foodborne pathogen, characterizations of the pathogenic potential of all B. cereus group species were lacking. We show here that B. pseudomycoides (clade I), which has been considered a harmless environmental microorganism, produces toxins and exhibits a phenotype consistent with the production of pore-forming toxins. Furthermore, B. mycoides/B. weihenstephanensis isolates (clade VI) did not show cytotoxicity when grown at 37°C, despite carrying multiple toxin genes. Overall, we show that the current standard methods to characterize B. cereus group isolates and to detect the presence of toxin genes are not reliable indicators of species, phylogenetic clades, or an isolate's cytotoxic capacity, suggesting that novel methods are still needed for differentiating pathogenic from nonpathogenic species within the B. cereus group. Our results also contribute data that are necessary to facilitate risk assessments and a better understanding as to which B. cereus group species are likely to cause foodborne illness.

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

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