Digoxigenin-Labeled Deoxyribonucleic Acid Probes for the Enumeration of Bifidobacteria in Fecal Samples

Kaneko, Tsutomu; Kurihara, Hiroko

doi:10.3168/jds.s0022-0302(97)76054-5

Cited by 15 publications

(10 citation statements)

References 24 publications

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“…The advantage of whole-cell hybridization for the detection of bacteria in contrast to other molecular methods such as PCR (19,27,50) and dot blot hybridization (12,25,52), lies in the fact that the former method also allows the distinction of bacteria based on their morphology, as cells can be visualized in the microscope.…”

Section: Discussionmentioning

confidence: 99%

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Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

Schwiertz

Blay

Blaut

2000

Appl Environ Microbiol

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Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10 7 cells g (dry weight) of feces ؊1 . The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.The genus Eubacterium (41) contains anaerobic, non-sporeforming, gram-positive rods which are distinguished from other genera mainly on the basis of negative metabolic characteristics (37). In the human intestinal tract, Eubacterium is the second most common genus after the genus Bacteroides and is more common than the genus Bifidobacterium (16). The importance of members of the genus Eubacterium has been reported previously (10,24,38,48). Since the identification of Eubacterium species based on phenotypic traits requires experience and is time-consuming (5,15,16,36,44), many studies involving human fecal flora composition have refrained from looking at this genus (13,20,34,42).Considerable effort has been invested in the application of molecular techniques such as PCR (19,27,50) and hybridization (12, 25, 52) for the identification of fecal bacteria. However, the extraction, purification, and amplification of nucleic acids by PCR from fecal samples are often selective and limited. In contrast, whole-cell hybridization with fluorescently labeled, 16S rRNA-targeted oligonucleotide probes allows the determination of the numerical abundance of bacteria, including unculturable strains (39), in various ecosystems such as water (1,26,32), sludge (23), and fecal samples (17,18,29).In contrast to the genus Bifidobacterium, for example, which is a phylogenetically and phenotypically well-defined taxon, Eubacterium species (9, 31) are phylogenetically diverse and thus it is not possible to design a genus-specific probe for Eubacterium spp. Therefore, probes for phylogenetic clusters or species have to be considered. The development and validation of a probe for the detection of a Eubacterium species has been reported recently (45).The purpose of this study was to develop and apply 16S rRNA-targeted oligonucleotide probes to human feces for the detection of numerically dominant fecal Eubact...

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Section: Discussionmentioning

confidence: 99%

“…Considerable effort has been invested in the application of molecular techniques such as PCR (19,27,50) and hybridization (12,25,52) for the identification of fecal bacteria. However, the extraction, purification, and amplification of nucleic acids by PCR from fecal samples are often selective and limited.…”

mentioning

confidence: 99%

Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

Schwiertz

Blay

Blaut

2000

Appl Environ Microbiol

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“…Stool samples for the bacteriologic analysis were collected on day 7 of each experimental period. The preparation of P. freudenreichii culture and quantitative bacteriologic analyses were carried out by the procedures described in our previous reports (18,19,33). The results were statistically analyzed with the non-parametric Wilcoxon signed-ranks test (Table 3).…”

Section: B Placebo-controlled Human Studymentioning

confidence: 99%

A Novel Bifidogenic Growth Stimulator Produced by Propionibacterium freudenreichii

Kaneko

1999

Bioscience Microflora

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A novel bifidogenic growth stimulator (BGS) was present in the cell-free filtrate and in methanol extract fraction of the starter (Propionibacterium freudenreichii) cells for the manufacture of Swiss-type cheese. After purification of the BGS isolated from the lyophilized cells, the mass (217.037) was determined by high-resolution electron impact mass spectrometry (MS) and liquid chromatography-MS spectra. Various experimental analyses indicated that the chemical structure of the BGS was 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ). We examined the activity of the P. freudenreichii ET culture containing the BGS on a gut bacterial composition using an anaerobic continuous culture system and found that the BGS seems to enhance the selective utilization of oligosaccharides by bifidobacteria. Furthermore, a placebo-controlled study of the effects of BGS on fecal flora and stool frequency was carried out in healthy human subjects. A drink with the sterilized ET-3 culture was administered once a day for 7 days. Bifidobacterium percentage in the fecal flora and stool frequency were significantly increased by administration of the P. freudenreichii culture. The ACNQ exhibited growth stimulation of bifidobacteria at an extremely low concentration and enhanced the activities of NADH oxidase and NADH peroxidase in bifidobacteria. It was revealed that ACNQ works as a good electron acceptor of NAD(P)H diaphorase. The ACNQred was easily auto-oxidized and also acted as a better electron donor of NAD(P)H peroxidase. These ACNQ-mediated reactions seem to play roles in NAD(P)+-regeneration processes and seem to be responsible for the growth stimulation of bifidobacteria.

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“…The distribution of Bifidobacterium species is highly variable: some are associated exclusively with humans and others are exclusive to other animals (4,27). One molecular approach to species identification in ecological and taxonomic studies is the analysis of the 16S rRNA molecule and its gene as a target (5,14). Sequence-specific primers or probes based on its sequence have been effective in the detection and identification of the genus (15) or individual species of Bifidobacterium in mixed populations, which are difficult and sometimes impossible by phenotypic characterization (24,26,30,31).…”

mentioning

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Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

Bonjoch

Blanch

2004

Appl Environ Microbiol

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Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

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Digoxigenin-Labeled Deoxyribonucleic Acid Probes for the Enumeration of Bifidobacteria in Fecal Samples

Cited by 15 publications

References 24 publications

Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

A Novel Bifidogenic Growth Stimulator Produced by Propionibacterium freudenreichii

Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

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