Quantitative Detection of Viable Bifidobacterium bifidum BF-1 Cells in Human Feces by Using Propidium Monoazide and Strain-Specific Primers

Abstract

We developed a PCR-based method to detect and quantify viable Bifidobacterium bifidum BF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 10 10 to 10 6 cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4=,6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 10 5.3 to 10 10.3 cells/g feces (wet weight) (r > 0.99, P < 0.001). After 12 healthy subjects ingested 10 10.3 to 10 11.0 CFU of BF-1 in a fermented milk product daily for 28 days, 10 4.5 ؎ 1.5 (mean ؎ standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 10 6.2 ؎ 0.4 viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 10 7.6 ؎ 0.7 BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (P < 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces.

Continuous consumption of fermented milk containing <i>Bifidobacterium bifidum</i> YIT 10347 improves gastrointestinal and psychological symptoms in patients with functional gastrointestinal disorders

“…In the present study, improvement of some symptoms was observed at CL, that is, after only 4 weeks of consumption of fermented milk. Because B. bifidum YIT 10347 is known to remain alive in the intestines [ 24 ], we speculate that consumption of B. bifidum YIT 10347 fermented milk ameliorates the intestinal environment, resulting in increased stress tolerance and improved gastrointestinal and psychological symptoms. Ongoing maintenance of this amelioration is a probable reason for the improvement seen after 4 weeks of consumption of fermented milk in this study.…”

Review: Diversity of Microorganisms in Global Fermented Foods and Beverages

“…Both culturable and non-culturable microorganisms from any fermented food and beverage may be identified using culture-dependent and -independent methods to document a complete profile of microorganisms, and also to study both inter- and intra-species diversity within a particular genus or among genera (Ramos et al, 2010 ; Greppi et al, 2013a , b ; Yan et al, 2013 ). A combination of Propidium MonoAzide (PMA) treatment on samples before DNA extraction and molecular quantifying method can be used to accurately enumerate the viable microorganisms in fermented foods (Desfossés-Foucault et al, 2012 ; Fujimoto and Watanabe, 2013 ).…”

Optimization of Viability Treatment Essential for Accurate Droplet Digital PCR Enumeration of Probiotics

“…Ethidium monoazide (EMA) and propidium monoazide (PMA) are the DNA-intercalating dyes developed for v-PCR and several articles describe their individual use to quantify viable strains of lactic acid bacteria and probiotics (Fujimoto and Watanabe, 2013;Gobert et al, 2018;Kramer et al, 2009;Meng et al, 2010). Despite this, there is evidence that both PMA and EMA present limitations in their application as viability dyes.…”