Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR-based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain-specific quantitative real-time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA-intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 105-109 cells per g of faeces was enumerated. After 11 healthy subjects ingested 10 center dot 7 log CFU of BbrY daily for 10 days, 6 center dot 9 (+/- 1 center dot 5) log CFU g-1 [mean (+/- SD)] of BbrY was detected in faeces by using strain-specific transgalactosylated oligosaccharide-carbenicillin (T-CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7 center dot 5 (+/- 1 center dot 0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8 center dot 1 (+/- 0 center dot 8) log cells per g. Conclusions: Strain-specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain-specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.