We determined the complete genome sequence of Lactobacillus brevis KB290, a probiotic lactic acid bacterium isolated from a traditional Japanese fermented vegetable. The genome contained a 2,395,134-bp chromosome that housed 2,391 protein-coding genes and nine plasmids that together accounted for 191 protein-coding genes. KB290 contained no virulence factor genes, and several genes related to presumptive cell wall-associated polysaccharide biosynthesis and the stress response were present in L. brevis KB290 but not in the closely related L. brevis ATCC 367. Plasmid-curing experiments revealed that the presence of plasmid pKB290-1 was essential for the strain's gastrointestinal tract tolerance and tendency to aggregate. Using next-generation deep sequencing of current and 18-year-old stock strains to detect low frequency variants, we evaluated genome stability. Deep sequencing of four periodic KB290 culture stocks with more than 1,000-fold coverage revealed 3 mutation sites and 37 minority variation sites, indicating long-term stability and providing a useful method for assessing the stability of industrial bacteria at the nucleotide level.

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. Isolation stress for 30 days alters hepatic gene expression profiles, especially with reference to lipid metabolism in mice.

We examined the survivability of Lactobacillus brevis KB290 and derivative strain KB392 in artificial digestive juices and bile salts. The strains have similar membrane fatty acids but different amounts of cell-bound exopolysaccharides (EPS). In artificial digestive juices, KB290 showed significantly higher survivability than KB392, and homogenization, which reduced the amount of EPS in KB290 but not in KB392, reduced the survivability only of KB290. In bile salts, KB290 showed significantly higher survivability than KB392, and cell-bound EPS extraction with EDTA reduced the survivability of only KB290. Transmission electron microscopy showed there to be a greater concentration of cell-bound EPS in KB290 than in either KB392 or EDTA-treated or homogenized KB290. We conclude that KB290's cell-bound EPS (which high performance liquid chromatography showed to be made up of glucose and N-acetylglucosamine) played an important role in bile salt tolerance.

Our purpose was to investigate the safety of the probiotic strain Lactobacillus brevis KB290. The European Qualified Presumption of Safety (QPS) evaluation approach was applied to the strain. We determined the strain's antibiotic resistance, verified it at the genetic level, and determined whether it could be transferred to intestinal microflora. Of 14 antibiotics tested, 11 showed MICs within the limits of the QPS criteria. However, the L. brevis KB290 MICs of ciprofloxacin (a fluoroquinolone), tetracycline, and vancomycin were two, four, and eight times, respectively, the breakpoint MICs suggested by the European Scientific Committee on Animal Nutrition, and the MIC of tetracycline was eight times the breakpoint MIC suggested by the European Scientific Panel on Additives and Products or Substances Used in Animal Feed. Using analysis of gapped-genome sequences, we found no known transferable determinants for tetracycline or vancomycin resistance, and we found no mutations in the quinolone resistance-determining regions of the genes encoding GyrA or ParC for ciprofloxacin resistance associated with insertion sequences, integrons, or transposons. These data were confirmed by using PCR primers specific for the respective genes. We assessed the transferability of the resistance traits in conjugation experiments with enterococci and obtained no transconjugants, strongly suggesting that the resistance traits were not transferable. This study demonstrated that the antibiotic resistance observed in L. brevis KB290 was due not to dedicated mechanisms but to intrinsic resistance. According to the QPS criteria, these results provide safety assurance for the ongoing use of L. brevis KB290 as a probiotic.