A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus-and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10 6 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10 6 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group-or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10 ؊3 cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10 0 to 10 5 CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4,6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n ‫؍‬ 38) at a level of 10 3 cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.For almost a century, culture techniques have been recognized as the "gold standards" for determining viable bacterial counts. As the human fecal flora has been reported to consist of approximately 400 bacterial species (12, 35) and these species are present at a concentration of 10 11 viable microorganisms per g of contents (42), conventional culture techniques for enumeration of different populations involve the use of selective microbiological media, followed by isolation of pure cultures and the use of confirmatory biochemical tests. Recently, a number of molecular methods based on immunological and genotypic techniques have been developed (41,48). In analyses of the gut microflora, a number of molecular methods have been used in place of cultivation-based techniques. Techniques such as the clone library method (42, 46), denaturing gradient gel electrophoresis (13), and terminal restriction fragment length polymorphism (31, 36) allow analysis of predominant bacteria that are difficult to culture. The fluorescent in situ hybridization method (18,43) and the quantitative PCR (qPCR) method with rRNA-targeted oligonucleotide probes or primers have also been used as culture-independent methods. Among these, PCR methods targeting mainly well-conserved 16S rRNA genes have prevailed for rapid quantification of bacteria and are recognized as having two advant...

Forty healthy individuals with a low defecation frequency were selected, and the effects of intake of a fermented milk beverage that contains Lactobacillus casei strain Shirota (LcS) at 4 × 10 9 bacteria/bottle for 2 weeks (1 bottle/day) on the defecation frequency and intestinal microflora were evaluated by the placebo-controlled double-blind cross-over scheme. Defecation frequency both in times per week and days per week significantly increased in the LcS beverage period compared with the frequency before the beginning of intake. The differences were more notable in those with a stronger tendency to constipation (frequency of defecation before intake ≤ 4.0 times/week, n=21), and the frequency of defecation in the LcS beverage period was significantly higher than in the placebo period. The stool smell and feeling of completion of voiding improved significantly in the LcS beverage period compared with the placebo period, and in those with a stronger tendency to constipation, the stools were significantly softened compared with the state before intake. The number of bifidobacteria and their percentage in the total number of fecal bacteria in the LcS beverage period increased significantly compared with the levels before intake and were significantly higher than the values in the placebo period. No marked change due to the intake of the LcS beverage was observed in the other components of the microflora, the organic acid contents, stool pH, water content, or contents of putrefactive metabolites. These results suggest that intake of the probiotic fermented milk beverage conditions the intestines by improving the state of bowel movements and stool quality and increasing the fecal population level of bifidobacteria.

We used sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) to quantify the Clostridium coccoides group, which is a major anaerobic population in the human intestine. For this purpose, the C. coccoides group was classified into 3 subgroups and 19 species for expediency in accordance with the existing database, and specific primers were newly developed to evaluate them. Population levels of the C. coccoides group in human feces determined by RT-qPCR were equivalent to those determined by fluorescence in situ hybridization. RT-qPCR analysis of fecal samples from 96 volunteers (32 young children, 32 adults and 32 elderly) by using the 22 new primer sets together with the C. coccoides group-specific primer setm revealed that (i) total counts obtained as the sum of the 3 subgroups and 19 species were equivalent to the results obtained by using the C. coccoides group-specific primer set; (ii) total C. coccoides-group counts in the elderly were significantly lower than those in young children and adults; (iii) genus Blautia was the most common subgroup in the human intestinal C. coccoides-group populations at all age populations tested; (iv) the prevalences of Fusicatenibacter saccharivorans and genus Dorea were significantly higher in adults than in young children and the elderly; and (v) the prevalences of C. scindens and C. hylemonae, both of which produce secondary bile acid in the human intestine, were significantly higher in the elderly than in young children and adults. Hierarchical clustering and principal component analysis showed clear separation of the bacterial components between adult and elderly populations. Taken together, these data suggest that aging plays an important role in the diversity of C. coccoides-group populations in human intestinal microbiota; changes in this diversity likely influence the health of the host.

Lactobacillus equi sp. nov. is described on the basis of 18 strains isolated as one of the predominant intestinal lactobacilli from horse faecal specimens. These 18 strains were isolated from 10 horses of 6 different farms out of 20 horses of 10 farms examined. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming, non-motile, lactic-acidhomofermentative rods. The DNA GMC content was 389 O08 mol %. DNA-DNA hybridization failed to associate these strains closely with any of the validly described type strains used. Analysis of the 16S rRNA gene sequence of representative strain YIT 0455 T revealed that the new isolates represent a new Lactobacillus species, for which the name Lactobacillus equi is proposed. The type strain is YIT 0455 T (l ATCC BAA-261 T l JCM 10991 T ).