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

Abstract

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 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) 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 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) 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.

A randomised controlled trial of the probiotic Bifidobacterium breve BBG-001 in preterm babies to prevent sepsis, necrotising enterocolitis and death: the Probiotics in Preterm infantS (PiPS) trial

“…A strain-specific quantitative real-time PCR method previously reported by Fujimoto et al in 2011 70 was used to detect B. breve BBG-001. The forward PCR primer sequence was 5′-ATGGCAAAACCGGGCTGAA-3′ and the reverse 5′-CCCACCTCTCATCCGC-3′ to give a 313-bp PCR product.…”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…The quantification was carried out on faecal samples spiked with BbrY strain, on the BbrY culture and on the faecal samples collected from the healthy volunteers who ingested a commercially available fermented milk product containing BbrY, once daily for 10 days. They confirmed the use of a combination of qPCR with PMA treatment and BbrY-specific primers as a quick and accurate method for quantification of viable BbrY in faecal samples (Fujimoto et al, 2011).…”

“…The treatment of bacteria with EMA as a promising tool of DNA-based differentiation between viable and dead pathogenic bacteria was first proposed by Nogva et al in 2003(Nogva et al, 2003. In the following years several applications of this approach have been reported, where the method was optimised for different complex media such as faeces, fermented milk and environmental samples (Garcia-Cayuela et al, 2009;Fittipaldi et al, 2011;Fujimoto et al, 2011). Since ethidium monoazide has been suggested as being toxic to some viable cells, PMA has been proposed as a more appropriate alternative to EMA (Nocker et al, 2006;Fujimoto et al, 2011).…”

“…A similar approach was applied to B. breve strain Yakult (BbrY) by Fujimoto et al (2011). The specificity of the BbrY-specific primer set was confirmed by PCR using DNA from 112 bacterial strains belonging to B. breve species, of other Bifidobacterium species and representatives of 11 other genera.…”

“…The specificity of the BbrY-specific primer set was confirmed by PCR using DNA from 112 bacterial strains belonging to B. breve species, of other Bifidobacterium species and representatives of 11 other genera. The BbrY-specific primers were used in a real-time PCR with PMA treatment to measure the number of BbrY in the faeces of subjects who drank a fermented milk product containing BbrY (Fujimoto et al, 2011).…”