Quantitative detection of viable Bifidobacterium bifidum BF-1 cells in human feces by using propidium monoazide and strain-specific primers.
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.
Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan.
Junji
J
Fujimoto
Koichi
K
Watanabe
D001386:Azides / Q000378:metabolism; D058491:Bacterial Load; D001644:Bifidobacterium / Q000302:isolation & purification* / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000378:metabolism; D005243:Feces / Q000382:microbiology*; D006801:Humans; D050296:Microbial Viability; D011419:Propidium / Q000031:analogs & derivatives / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D001386:Azides; D004791:Enzyme Inhibitors; C533957:propidium monoazide; D011419:Propidium