In Vitro and In Vivo Safety Evaluation of Streptococcus salivarius eK12, a Genetically Modified Dietary Probiotic Derived from the Oral Probiotic S. salivarius K12.

Group A Streptococcus (GAS) pharyngitis is a common and recurrent childhood illness, usually treated with antibiotics. While effective, repeated antibiotic use contributes to antimicrobial resistance, disrupts host microbiota, and increases adverse effects. Streptococcus salivarius K12 is an extensively studied probiotic for oral health, particularly for reducing the incidence of GAS pharyngotonsillitis. A modified version, S. salivarius eK12, has recently been developed from the wild-type K12 through genetic modification to enhance anti-GAS activity. However, its safety and tolerability have not previously been evaluated, and this study provides the first systematic assessment of eK12 under OECD-guided toxicological frameworks. Genotoxicity was assessed through the bacterial reverse mutation assay (OECD 471, Ames test) in five Salmonella typhimurium strains, with and without metabolic activation. Acute oral toxicity was examined in female Wistar rats at a limit dose of 2,000 mg/kg (~2 × 1011 CFU/kg) (OECD 423). Sub-chronic safety was investigated in a 90-day repeated-dose oral toxicity study (OECD 408) using Sprague Dawley rats randomized to vehicle, low, mid, or high eK12 dose groups, with additional recovery cohorts (vehicle, high dose) observed for 28 days post-treatment. Results showed no mutagenic activity, no mortality, and no treatment-related abnormalities in clinical, hematological, biochemical, or histopathological parameters. Minor fluctuations were incidental and non-dose dependent. Recovery groups confirmed the absence of delayed or persistent toxicity. These findings indicate that eK12 is potentially non-mutagenic, non-toxic, and well tolerated, with a NOAEL of 2,000 mg/kg/day (~2 × 1011 CFU/kg/day), retaining the favorable safety profile of its parental strain K12 and supporting its further development.