# Identification and Safety Assessment of Enterococcus casseliflavus KB1733 Isolated from Traditional Japanese Pickle Based on Whole-Genome Sequencing Analysis and Preclinical Toxicity Studies

## TL;DR

This study confirms the safety of Enterococcus casseliflavus KB1733, isolated from Japanese pickles, using genome sequencing and toxicity tests.

## Contribution

The novelty lies in the comprehensive safety assessment of KB1733 using whole-genome sequencing and preclinical toxicity studies.

## Key findings

- Genome analysis confirms KB1733 is E. casseliflavus with no virulence factor genes.
- Preclinical toxicity tests showed no adverse effects in rats at high doses.
- No increase in revertant colonies was observed in bacterial reverse mutation tests.

## Abstract

The present study involves the precise identification and safety evaluation of Enterococcus casseliflavus KB1733, previously identified using 16S rRNA analysis, through whole-genome sequencing, phenotypic analysis, and preclinical toxicity studies. Analyses based on the genome sequencing data confirm the identity of KB1733 as E. casseliflavus and show that the genes related to vancomycin resistance are only present on the chromosome, while no virulence factor genes are present on the chromosome or plasmid. Phenotypic analyses of antibiotic resistance and hemolytic activity also indicated no safety concerns. A bacterial reverse mutation test showed there was no increase in revertant colonies of heat-killed KB1733. An acute toxicity test employing heat-killed KB1733 at a dose of 2000 mg/kg body weight in rats resulted in no deaths and no weight gain or other abnormalities in the general condition of the animals, with renal depression foci and renal cysts only occurring at the same frequency as in the control. Taking the background data into consideration, the effects on the kidneys observed in the current study were not caused by KB1733. Our findings suggest that KB1733 is non-pathogenic to humans/animals, although further studies involving repeated oral toxicity tests and/or clinical tests are required.