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Applied and Environmental Microbiology, October 1999, p. 4506-4512, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Distribution of Bifidobacterial Species in Human Intestinal
Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific
Primers
Takahiro
Matsuki,1,2,*
Koichi
Watanabe,1
Ryuichiro
Tanaka,1
Masafumi
Fukuda,3 and
Hiroshi
Oyaizu2
Yakult Central Institute for Microbiological
Research, 1796 Yaho, Kunitachi, Tokyo 186-8650,1
Graduate School of Agriculture and Agricultural Life Science,
The University of Tokyo, Bunkyo-ku, Tokyo
113-8657,2 and School of Medicine,
Nagasaki University, 1-12-4 Sakamoto, Nagasaki-shi, Nagasaki
852-8523,3 Japan
Received 8 March 1999/Accepted 10 July 1999
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ABSTRACT |
In order to clarify the distribution of bifidobacterial species in
the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal
samples obtained from 48 healthy adults and 27 breast-fed infants. To
cover all of the bifidobacterial species that have been isolated from
and identified in the human intestinal tract, species-specific primers
for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and
used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and
the B. catenulatum group (B. catenulatum and
B. pseudocatenulatum) that were developed in a previous
study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS
Microbiol. Lett. 167:113-121, 1998). The specificity of the nine
primers was confirmed by PCR, and the species-specific PCR method was
found to be a useful means for identifying Bifidobacterium
strains isolated from human feces. The results of an examination of
bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in
44 of 48 samples [92%]), followed by B. longum and
B. adolescentis, in the adult intestinal bifidobacterial
flora and that B. breve, B. infantis, and
B. longum were frequently found in the intestinal tracts of
infants. The present study demonstrated that qualitative detection of
the bifidobacterial species present in human feces can be accomplished
rapidly and accurately.
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INTRODUCTION |
The human intestinal tract harbors a
large, active, and complex community of microbes. The intestinal
microflora plays several significant roles in the digestion of food,
the metabolism of endogenous and exogenous compounds, the production of
essential vitamins, immunopotentiation, and the prevention of
colonization by pathogens in the gastrointestinal tract and hence is
involved in maintaining human health (7, 8).
Members of the genus Bifidobacterium are some of the most
common organisms in the human intestinal tract (26). It has
been suggested that Bifidobacterium species are important in
maintaining general health because they contribute to a beneficial
microflora in the intestinal tract and that the diversity and number of
Bifidobacterium species provide a marker for the stability
of the human intestinal microflora (28). Therefore, many
attempts have been made to increase the number of
Bifidobacterium cells in the intestinal tract by supplying
certain bifidobacterial strains and food ingredients that stimulate the
growth of bifidobacteria as food additives (7, 8, 11, 15).
Hence, the distribution of bifidobacteria in the human intestinal
microflora is of major interest. Using classical culture methods,
workers have found that Bifidobacterium adolescentis and
B. longum are major bifidobacterial species in the adult
intestinal microflora (4, 5, 17, 19, 20) and that B. infantis and B. breve are predominant species in the intestinal tracts of human infants (2, 3, 17, 20). In addition, B. catenulatum, B. pseudocatenulatum,
B. angulatum, and B. dentium have been also
reported to be human intestinal bifidobacteria (24, 25), and
B. gallicum has been reported to be a rarely isolated
species (14). However, the classical culture methods,
including isolation, identification, and enumeration of these species,
are labor-intensive and time-consuming. Moreover, identification based
on phenotypic traits does not always provide clear-cut results and is
sometimes unreliable.
For some years, 16S rRNA sequence comparison has attracted attention as
a reliable method for classification and identification of several
bacterial species (22, 31). 16S rRNA-targeted hybridization probes or PCR primers enable rapid and specific detection of a wide
range of bacterial species, and procedures in which these probes and
primers are used have become key procedures for detecting microorganisms (6, 10, 12, 23, 30, 32).
In order to develop an accurate and convenient method for
characterization of bifidobacteria in the intestinal microflora, we prepared 16S rRNA-gene (rDNA)-targeted species-specific and group-specific primers for all known species of bifidobacteria that
inhabit the human intestinal tract. In the present study, a
species-specific PCR technique performed with fecal DNA was also
used to investigate the distribution of bifidobacteria in the intestinal microflora of human adults and infants.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
The strains listed
in Table 1 were obtained from the
American Type Culture Collection (Rockville, Md.), the Japan Collection of Microorganisms (Wako, Japan), the German Collection of
Microorganisms and Cell Cultures (Braunschweig, Germany), the National
Collection of Food Bacteria (Reading, United Kingdom), the National
Collection of Type Cultures (London, United Kingdom), the National
Institute of Biosciences and Human Technology (Tsukuba, Japan), and the Yakult Central Institute for Microbiological Research (Tokyo, Japan).
Most of the strains were cultured anaerobically in GAM broth (Nissui
Seiyaku, Tokyo, Japan) supplemented with 1% glucose at 37°C
overnight; Escherichia coli was cultured aerobically in Trypticase soy broth (Difco, Detroit, Mich.) at 37°C overnight. Direct microscopic counts of pure cultured bifidobacteria were obtained
by using duplicate smears of 0.01 ml of a 102-fold dilution
spread over 1 cm2 of a glass slide. The smears were
heat fixed and gently Gram stained. Six edge fields and four center
fields were counted, and the counts were then correlated with the
actual sample size (9).
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TABLE 1.
Bacterial strains and results of PCR assays in which
species-specific primers BiLON, BiINF, BiDEN, and BiGAL
were useda
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Development of 16S rDNA-targeted species-specific primers.
Using 31 bifidobacterial 16S rDNA sequences whose accession numbers
were described previously (16), we prepared a multiple alignment with the program Clustal W (29). Then potential
primer target sites for species-specific detection were identified for all species except B. catenulatum and B. pseudocatenulatum, which were treated as the B. catenulatum group. We then designed eight species-specific primers
and a group-specific primer for Bifidobacterium species that
have been detected in human intestinal tracts (Table 2). These primers were synthesized
commercially by Greiner Japan or Rikaken (Tokyo, Japan).
PCR amplification.
Each PCR mixture (25 µl) was composed
of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, each
deoxynucleoside triphosphate at a concentration of 200 µM, each
species-specific primer (Table 2) at a concentration of 0.25 µM,
template DNA, and 0.9 U of Taq DNA polymerase (Perkin-Elmer,
Norwalk, Conn.). The PCR was carried out with a Touchdown thermal
cycler (Hybaid, Middlesex, United Kingdom). The following amplification
program was used: one cycle consisting of 94°C for 5 min, followed by
35 cycles consisting of 94°C for 20 s, 55°C for 20 s, and
72°C for 30 s and finally one cycle consisting of 72°C for 5 min. The amplification products were subjected to gel electrophoresis
in 1% agarose, followed by ethidium bromide staining.
Isolation and identification of Bifidobacterium
strains.
Isolation of the Bifidobacterium strains
listed in Table 3 from human feces and
identification based on DNA-DNA homology tests were carried out by
using the methods described previously (16). Carbohydrate
fermentation patterns were determined by using the API 50 CHL system
(API, La Balme les Grottes, France).
Fecal sampling.
The fecal samples used in this study were
obtained from 51 healthy male adults ranging from 23 to 54 years old
(mean, 38.8 ± 8.9 years) and 27 healthy breast-fed babies ranging
from 22 to 46 days old (mean, 31.2 ± 4.5 days). The babies were
born in Nagasaki University Hospital and had been delivered by the
vaginal route. The wet weight of each fecal sample was 10 mg. Each
sample was washed three times by suspending it in 1.5 ml of distilled water and centrifuging it at 15,000 rpm in order to reduce the amount
of PCR inhibitors, and the pellets were then stored at 
70°C until
they were used for DNA preparation.
DNA extraction from fecal samples.
DNA was extracted from
fecal samples essentially by the methods of Zhu et al. (33).
Briefly, a fecal sample was suspended in a solution containing 250 µl
of extraction buffer (100 mM Tris-HCl, 40 mM EDTA; pH 9.0) and 50 µl
of 10% sodium dodecyl sulfate and then subjected to freeze-thawing.
Benzyl chloride (150 µl) was added to the suspension, and the mixture
was vortexed vigorously at 50°C for 30 min by using a MicroIncubator
M-36 apparatus (TAITECH, Tokyo, Japan). Then 150 µl of 3 M sodium
acetate was added, and the mixture was cooled on ice for 15 min. After
centrifugation at 15,000 × g for 10 min, the supernatant
was collected, and DNA was obtained by isopropanol precipitation.
Finally, the DNA was suspended in 100 µl of TE (10 mM Tris-HCl, 1 mM
EDTA; pH 8.0). Contamination with PCR inhibitors was checked for by PCR
amplification by using a mixture containing 10 ng of DNA extracted from
B. gallicum JCM 8224T, the specific BiGAL
primers, and 1 µl of fecal DNA solution. When the extracted DNA was
found to be contaminated by PCR-inhibiting substances, further
purification was performed by using a MicroSpin S-400 column (Amersham
Pharmacia Biotech, Uppsala, Sweden) as recommended by the manufacturer.
Routinely, 1 µl of a fecal DNA solution was used for PCR analysis.
Enumeration of the bifidobacterial population by classical
culture methods.
The population of Bifidobacterium
species in fecal samples was enumerated as follows. Fecal samples from
adults were collected anaerobically, and serial 10-fold dilutions were
prepared with prereduced dilution buffer with vigorous shaking
(9). Then, 0.05-ml samples of the 105 to
108 dilutions were plated onto
Bifidobacterium-specific TOS agar (27). This
medium contained (per liter) 10 g of Trypticase (BBL Microbiology
Systems, Cockeysville, Md.), 1 g of yeast extract (Difco), 3 g of KH2PO4, 4.8 g of
K2HPO4, 3 g of
(NH4)2SO4, 0.2 g of
MgSO4, 0.5 g of L-cysteine, 10 g of
TOS-S (Yakult Honsha Co., Tokyo, Japan), and 15 g of powdered agar
(Difco). The plates were incubated anaerobically at 37°C for 3 days,
and the colonies that appeared on the samples with the highest dilution
were picked in succession into GAM broth (Nissui Seiyaku, Tokyo,
Japan). The isolates were identified by using species-specific PCR
primers. The total population level of each species was calculated by
determining the number of CFU per gram of feces.
Statistical analysis.
To determine the statistical
significance of the results, Student's t test was used.
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RESULTS |
Specificity of primers.
Figure 1
shows the electrophoresis patterns of the PCR products obtained for 10 Bifidobacterium species when their specific primers were
used. The specificity of the primers was confirmed by PCR in which we
used both chromosomal DNAs extracted from 50 different strains
belonging to 31 Bifidobacterium species and DNAs extracted
from 15 non-Bifidobacterium species which are commonly found
in the human intestinal microflora (Table 1). The PCR specificity of
other primers, such as BiADO, BiANG, BiBIF, BiBRE, and BiCATg, has been
reported previously (16). Most of the primers detected the
target species specifically; the only exceptions were the BiLON
primers, which cross-reacted with B. suis.
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FIG. 1.
PCR products obtained for 10 Bifidobacterium
species with their specific primers. Lane M, DNA size markers (sizes
[in bases] are indicated on the left); lane 1, B. adolescentis ATCC 15703T; lane 2, B. angulatum ATCC 27535T; lane 3, B. bifidum
ATCC 29521T; lane 4, B. breve ATCC
15700T; lane 5, B. catenulatum ATCC
27539T; lane 6, B. pseudocatenulatum JCM
1200T; lane 7, B. longum ATCC
157071T; lane 8, B. infantis ATCC
15697T; lane 9, B. dentium ATCC
27534T; lane 10, B. gallicum JCM
8224T; lane 11, negative control (PCR performed with primer
BiADO and E. coli ATCC 11775T).
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Identification of isolated Bifidobacterium
strains.
The species-specific PCR technique was used to identify
Bifidobacterium strains isolated from human feces. As shown
in Table 3, 13 isolates were clearly identified as 11 strains of
B. longum (MC-10, MC-11, MC-12, MC-23, MC-24, MC-25, MC-26,
MC-27, MC-28, MC-29, and MC-30) and 2 strains of B. infantis
(MC-8 and MC-9) by using the newly developed BiLON and BiINF primers.
Using DNA-DNA hybridization tests, we identified these strains as
members of B. longum or B. infantis, but it was
difficult to distinguish the two species because the levels of homology
of each isolate to the reference strains (B. longum ATCC
15707T and B. infantis ATCC 15697T)
were similar, ranging from 60 to 95%. However, identification of these
strains was possible because all of the B. longum strains fermented arabinose and melezitose, whereas no B. infantis
strain fermented these sugars (26). Thus, identification of
these strains based on DNA-DNA homology data and carbohydrate
fermentation patterns gave the same results as identification by the
species-specific PCR technique.
DNA preparation.
The benzyl chloride extraction method
provided sufficient amounts of DNA to perform PCR amplification for
both pure cultures of bifidobacteria and fecal samples. The washing
steps effectively removed the PCR inhibitors from all 27 infant fecal
samples and 36 of 51 adult fecal samples. However, the DNA extracted
from 15 samples was still contaminated by PCR-inhibiting substances even after the washing steps. Therefore, further purification was
performed with the MicroSpin S-400 column, and the PCR inhibitors were
removed from 12 of the 15 samples. Finally, the DNAs extracted from 48 adult and 27 infant fecal samples were subjected to the distribution
analysis described below.
Detection limits of the species-specific PCR methods.
In order
to determine the detection limit of the species-specific PCR,
approximately 10 mg (wet weight) of a fecal sample that did not contain
B. angulatum was mixed with various amounts of B. angulatum ATCC 27535T cells (108 to
102 cells per 10 mg), and DNAs were isolated from these
mixtures. Figure 2 shows that the target
species was detected by this procedure at a concentration of
102 cells per PCR assay mixture (equivalent to
104 cells per 10 mg of feces or 106 cells per g
of feces). The same results were obtained when diluted samples of the
DNA extracted from 109 cells of the B. angulatum
strain were used as the template DNA (data not shown). As other
Bifidobacterium species are usually present in human fecal
samples, the detection limit was examined by using diluted DNA
extracted from 109 cells of pure cultured bifidobacteria.
The results obtained for B. adolescentis ATCC
15703T and NCFB 2229, B. bifidum ATCC
29521T, B. breve ATCC 15700T,
B. catenulatum ATCC 27539T, B. pseudocatenulatum JCM 1200T, and B. dentium
ATCC 27534T were the same as the results obtained for
B. angulatum (data not shown). On the other hand, B. longum ATCC 157071T, B. infantis ATCC
15697T, and B. gallicum ATCC 15697T
were detected when they were present at a concentration of
103 cells per PCR mixture (data not shown).
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FIG. 2.
Detection limits of the species-specific PCR methods, as
determined by using DNAs extracted from 10-mg fecal samples mixed with
various amounts of B. angulatum ATCC 27535T.
Lane M, DNA size markers (sizes [in bases] are indicated on the
left); lane 1, 106 cells per PCR mixture; lane 2, 105 cells; lane 3, 104 cells; lane 4, 103 cells; lane 5, 102 cells; lane 6, 10 cells;
lane 7, 1 cell; lane 8, no cells (negative control).
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Distribution of Bifidobacterium species in the feces of
healthy adults and infants.
The distributions of bifidobacterial
species in 48 healthy adults and 27 breast-fed infants are shown in
Tables 4 and
5, respectively. Table
6 summarizes the bifidobacterial species found, their frequencies, and the numbers of species detected in
individuals. In adult intestinal tracts, the B. catenulatum group was the most common taxon (detected in 44 samples [92%]), followed by B. longum (31 samples [65%]) and B. adolescentis (29 samples [60%]). B. bifidum (18 samples [38%]) and B. breve (6 samples [13%]) were
subdominant species. B. dentium (three samples [6.3%])
and B. angulatum (two samples [4.2%]) were minor species. No B. infantis or B. gallicum was detected
in this study. In breast-fed infants, B. breve (19 samples [70%]) was the most frequently found species, followed by
B. infantis (11 samples [41%]) and B. longum (10 samples [37%]). B. bifidum (six samples
[22%]), the B. catenulatum group (five samples [19%]),
and B. dentium (three samples [11%]) were detected
occasionally. B. adolescentis (two samples [7.4%]) and B. angulatum (one sample [3.7%]) were rare species in
infants. B. gallicum was not detected. In one adult and
three infant fecal samples, no bifidobacterial species were detected.
The average numbers of species detected per individual were 2.8 ± 1.2 in adults and 2.1 ± 1.6 in infants. The intestinal
Bifidobacterium flora of adults was more complex and diverse
than that of infants (P < 0.05).
Comparison of the species-specific primer method with the culture
method.
The bifidobacterial composition obtained with the new
primer method was compared with results obtained with the classical culture method (Table 7). All of the
species isolated and identified by the culture method were also
detected by the species-specific PCR technique. It should be noted that
there were some species that were detected by the PCR method but not by
the culture method. This indicates that the PCR method is able to
detect a wider range of species than the culture method.
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TABLE 7.
Comparison of the species-specific primer method with the
classical culture method when the same samples were used
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DISCUSSION |
In this study, we developed molecular methods to investigate the
distribution of bifidobacterial species in human intestinal tracts by
using DNAs extracted from fecal samples. We have previously described
five different species- and group-specific primers for B. adolescentis, B. angulatum, B. bifidum,
B. breve, and the B. catenulatum group
(16). In addition to these primers, 16S rDNA-targeted species-specific primers for B. longum, B. infantis, B. dentium, and B. gallicum were
developed and used in this study. The nine different pairs of primers
cover all of the bifidobacterial species that have been isolated and
identified in the human intestinal tract, and therefore they provide an
effective way to analyze Bifidobacterium species that
inhabit the human intestinal tract. Although the BiLON primers did not
distinguish B. longum and B. suis, we believe
that B. suis should be taxonomically combined with B. longum, because the two species are closely related based on a
DNA-DNA homology value of 75 to 78% (13) and on the level of 16S rDNA similarity (more than 99%) (18). We also
believe that B. catenulatum and B. pseudocatenulatum should be treated as the B. catenulatum group due to their similarity in the DNA-DNA homology
test, murein type, and 16S rDNA sequences, as discussed previously
(16). The newly developed BiLON and BiINF primers distinguished B. longum and B. infantis, even
though these taxa are closely related species (13, 18).
These primers are effective for identification of the two species, as
confirmed with isolated bifidobacteria (Table 3).
The benzyl chloride extraction used in this study was very simple and
provided sufficient amounts of DNA for PCR amplification. It has been
reported that PCR-based analysis of fecal samples is difficult to
perform due to the presence of multiple inhibitors of the polymerase
enzyme reaction (23). In the present study, the washing
steps and purification with the MicroSpin S-400 column were effective
in reducing the amounts of PCR inhibitors found in the fecal DNA
solutions for most of the samples. However, additional improvements in
the preparation procedures for fecal DNA are required, since 3 of 51 samples still contained inhibitors. Targeted Bifidobacterium species were detected when they were present at a concentration of at
least 102 or 103 cells per PCR mixture,
indicating that the detection limit for the procedures used was
106 or 107 cells per g of feces. In contrast,
the sensitivity of the analysis based on the conventional culture
method in which Bifidobacterium-specific selective medium is
used is limited because it is difficult to detect minor species from a
cultivated plate among the numerous predominant species. As the
predominant Bifidobacterium species are usually present at a
level of 109 to 1010 cells per g of human feces
(2, 5, 17, 19, 20), the detection limit of the culture
method for minor bifidobacterial species is about 108 cells
per g. Therefore, the differences between the PCR method and the
culture method shown in Table 7 account for the different detection
limits. The present species-specific PCR detection method for
bifidobacteria is about 10 to 100 times more sensitive than the
classical culture method.
Examination of the bifidobacterial species distribution in the human
intestinal tract revealed that the B. catenulatum group is
the most common taxon inhabiting the human adult intestinal tract. This
is a notable finding because it has frequently been reported that
B. adolescentis is the most common species (5, 17, 19,
20). The difference may be due to the use of different identification techniques. It has been reported that it is difficult to
differentiate B. adolescentis, B. catenulatum,
and B. pseudocatenulatum based on the usual carbohydrate
fermentation pattern (24, 25). Therefore, the B. catenulatum group may have been confused with B. adolescentis in some studies. On the other hand, some
previous studies showed that B. catenulatum and B. pseudocatenulatum are members of the human adult intestinal
microflora (4, 24, 25), but the frequencies in these studies
were not as high as the frequencies in our study. The difference may be
due to the difference in detection limits between the conventional
culture method and the 16S rDNA-targeted PCR technique or to regional differences in microfloras. B. infantis has a unique host
specificity, even though this species is closely related to B. longum, as indicated by DNA-DNA homology and 16S rDNA sequence
similarity (13, 18). It is interesting that B. breve was detected in adult fecal samples even though it has been
recognized as a typical infantile bifidobacterial species (4, 5,
17, 19, 20). This may have been due to the difference in the
detection limits of the techniques used, as described above. B. gallicum was not detected in this study, suggesting that B. gallicum should not be recognized as a member of the human
intestinal microflora because the type strain is the only strain that
has been isolated from human feces so far (14).
In the present study, the distributions of bifidobacterial species were
basically consistent with the results obtained by the classical culture
methods (2-5, 17, 19, 20) except for the B. catenulatum group, suggesting that the species-specific PCR method
is a reliable technique for investigating intestinal floral components.
For further investigation, an improved quantitative PCR method is
necessary. In the near future, the quantitative PCR method combined
with the species-specific primers for intestinal floral components is
expected to lead to new opportunities for noncultivation studies of
intestinal microflora.
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ACKNOWLEDGMENTS |
We thank T. Mitsuoka, University of Tokyo, for his valuable advice.
This work was supported by the Yakult Bio-Science Foundation (Tokyo, Japan).
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FOOTNOTES |
*
Corresponding author. Mailing address: Yakult Central
Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. Phone: 81 (42) 577 8960. Fax: 81 (42) 577 3020. E-mail: matsukit{at}rd5.so-net.ne.jp.
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0099-2240/99/$04.00+0
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Abstract
Introduction
