Genetic and phenotypic assessments for the safety of probiotic Bacillus clausii 088AE.

In this study, we report on whole genome sequence analysis of clinically documented, commercial probiotic Bacillus clausii 088AE and genome features contributing to probiotic properties. The whole genome sequence of B. clausii 088AE generated a single scaffold of 4,598,457 bp with 44.74 mol% G + C. This assembled genome sequence annotated by the RAST resulted in 4371 coding genes, 75 tRNAs, and 22 rRNAs. Gene ontology classification indicated 39.5% proteins with molecular function, 44.24% cellular component, and 16.25% proteins involved in biological processes. In taxonomic analysis, B. clausii 088AE shared 99% identity with B. clausii DSM 8716. The gene sequences related to safety and genome stability such as antibiotic resistance (840), virulence factors (706), biogenic amines (1), enterotoxin (0), emetic toxin (0), lanthipeptides (4), prophage (4) and clustered regularly interspaced short palindromic repeats (CRISPR) sequences (11), were identified and evaluated for safety and functions. The absence of functional prophage sequences and the presence of CRISPR indicated an advantage in genome stability. Moreover, the presence of genome features contributing to probiotic characteristics such as acid, and bile salt tolerance, adhesion to the gut mucosa, and environmental resistance ensure the strains survivability when consumed as a probiotic. In conclusion, the absence of risks associated with sequences/genes in the B. clausii 088AE genome and the presence of essential probiotic traits confirm the strain to be safe for use as a probiotic.

Impact of a Gastrointestinal Stable Probiotic Supplement Bacillus coagulans LBSC on Human Gut Microbiome Modulation.

Bacillus coagulans LBSC showed stability in acidic pH, bile and simulated human gastrointenstinal juices. Under static gut model, when passed through oral, gastric and intestinal phases, B. coagulans LBSC was found to be stable as free viable spores and also with various foods such as milk and baby foods, as well as American and European diets. In human studies, modulation of gut microbiota by B. coagulans LBSC was comprehended by whole genome metagenome analysis of fecal samples obtained from pre- and post-treatment of irritable bowel syndrome (IBS) patients. B. coagulans LBSC treatment showed positive modulation in gut microbiota, especially up regulation of phyla such as Actinobacteria and Firmicutes, whereas down regulation of Bacteroids, Proteobacteria, Streptophyta and Verrucomicrobia. Simultaneously, it has altered various microbiota associated metabolic pathways to create the normalcy of gut microenvironment.

Genome based safety assessment for Bacillus coagulans strain LBSC (DSM 17654) for probiotic application.

The present study on Bacillus coagulans strain LBSC (DSM 17654) describes the use of whole genome sequencing, in correlation with the phenotypic properties to assess the safety of the strain. Analysis of the 16S rRNA sequence of the B. coagulans strain LBSC (DSM 17654), showed 100% homology with 99% coverage with B. coagulans strain HM-08. BLAT (BLAST Like Analysis Tool) analysis for whole genome comparison with B. coagulans ATCC 7050, B. coagulans HM-08 and B. coagulans Slac showed 96%, 99% and 99% sequence identity respectively. Whole genome sequencing results demonstrated a single scaffold of 36,35,902 bp and 3331 coding sequences. Gene ontology segregated the proteins as those with molecular function, cellular component and biological process of the predicted genes from assembled genome. Risk associated sequences like antibiotic resistance genes, biogenic amine producing genes, virulence factor genes and other safety related genes were identified with focus on horizontal gene transfer and its non-functionality. The absence of mobile elements in the vicinity of the genes, render it non-transferable and non-toxic phenotypic properties confirm the non-functionality of the genes. Absence of functional genes of concern and confirmation of absence of mobile elements in the vicinity of other non-clinically significant genes indicated no safety concern. The absence of complete and functional prophage sequences which are deleterious for the genome stability and presence of CRISPR system which are advantageous for genome stability by acting as a barrier to entry of foreign DNA elements indicated the stability of the genome. The molecular approach used in this study satisfies the requirements for the safety assessment of the probiotic strain which could indicate it to be potentially safe.

A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

Trichoderma species are widely used as production hosts for industrial enzymes. Identification of Trichoderma species requires a complex molecular biology based identification involving amplification and sequencing of multiple genes. Industrial laboratories are required to run identification tests repeatedly in cell banking procedures and also to prove absence of production host in the product. Such demands can be fulfilled by a brief method which enables confirmation of strain identity. This communication describes one step identification method for two common Trichoderma species; T. citrinoviride and T. reesei, based on identification of polymorphic region in the nucleotide sequence of translation elongation factor 1 alpha. A unique forward primer and common reverse primer resulted in 153 and 139 bp amplicon for T. citrinoviride and T. reesei, respectively. Simplification was further introduced by using mycelium as template for PCR amplification. Method described in this communication allows rapid, one step identification of two Trichoderma species.