The human intestinal microbiota have traditionally
been compared by analyzing isolates using an anaerobic
culture-based method. Consequently, it has been docu-
mented that more than 1012bacteria cells per g of content
(dry matter) (11, 12, 19, 28) and estimated that 400–500
species inhabit in the intestinal tract (22). The compo-
sition and activity of indigenous intestinal microbiota are
of paramount importance in human immunology, nutri-
tion, and pathological processes and hence the health of
the individual (31). However, the cultivable bacteria
were 20 to 30% of the total because of the strict anaer-
obic and complex environment (12, 19, 28). Thus, our
understanding of the microbiota in human intestinal
tract is incomplete due to the culture-based method.
The culture-independent approach based on molecu-
lar-biological techniques has revealed a great diversity of
microbiota in environmental samples (1). Phylogenetic
analysis based on the PCR cloning strategy has been
used to characterize human fecal microbiota (15, 28,
32). Recently, we reported that the human fecal micro-
biota could be analyzed by 16S rDNA libraries and a
strictly anaerobic culture-based method (12, 13). We
detected many novel phylotypes and species that have not
yet been characterized and showed phylogenetic corre-
lation between newly isolated strains and 16S rRNA
sequences (16S rDNA). In addition, major interindi-
vidual differences were evident in the composition of
intestinal microbiota as determined by two independent
approaches. The culture-based method is inadequate
for the understanding of the whole human intestinal
microbiota; thus it is necessary to use molecular-bio-
logical techniques like the 16S rDNA library.
According to the analysis of culture-based methods,
the composition of the human fecal microbiota changes
with age (3, 21). Mitsuoka and Hayakawa (21) reported
a significant increase in the number of lactobacilli and
clostridia and a significant decrease in the number of bifi-
dobacteria in the elderly compared with younger indi-
viduals. Recently, Hold et al. (15) recently used the
Molecular Analysis of Fecal Microbiota in Elderly
Individuals Using 16S rDNA Library and T-RFLP
Hidenori Hayashi*, Mitsuo Sakamoto, Maki Kitahara, and Yoshimi Benno
Japan Collection of Microorganisms, RIKEN, Wako, Saitama 351–0198, Japan
Received January 31, 2003; in revised form, May 16, 2003. Accepted May 22, 2003
Abstract: Fecal microbiota in six elderly individuals were characterized by the 16S rDNA libraries and ter-
minal restriction fragment length polymorphism (T-RFLP) analysis. Random clones of 16S rRNA gene
sequences were isolated after PCR amplification with universal primer sets from total genomic DNA
extracted from feces of three elderly individuals. These clones were partially sequenced (about 500 bp). T-
RFLP analysis was performed using 16S rDNA amplified from six subjects. The lengths of the terminal
restriction fragment (T-RF) were analyzed after digestion by HhaI and MspI. Among 240 clones obtained,
approximately 46% belonged to 27 known species. About 54% of the other clones were 56 novel “phylo-
types” (at least 98% homology of clone sequence). These libraries included 83 species or phylotypes. In
addition, about 13% (30 phylotypes) of these phylotypes were newly discovered in these libraries. A large
number of species that are not yet known exist in the feces of elderly individuals. 16S rDNA libraries and
T-RFLP analysis revealed that the majority of bacteria were Bacteroides and relatives, Clostridium rRNA
cluster IV, IX, Clostridium rRNA subcluster XIVa, and “Gammaproteobacteria”. The proportion of
Clostridium rRNA subcluster XIVa was lower than in healthy adults. In addition, although Ruminococcus
obeum and its closely related phylotypes were detected in high frequency in healthy young subjects,
hardly any were detected in our elderly individuals. “Gammaproteobacteria” were detected at high frequency.
Key words: Elderly persons’ fecal microbiota, 16S rDNA library, T-RFLP, Phylogenetic analysis
Microbiol. Immunol., 47(8), 557–570, 2003
Abbreviations: DDBJ, DNA Data Bank of Japan; EMBL,
European Molecular Biology Laboratory; PCR, polymerase chain
reaction; RDP, Ribosomal Database Project; T-RF, terminal
restriction fragment; T-RFLP, terminal restriction fragment length
*Address correspondence to Dr. Hidenori Hayashi, Japan Col-
lection of Microorganisms, RIKEN, 2–1 Hirosawa, Wako, Saita-
ma 351–0198, Japan. Fax: ?81–48–462–4619. E-mail: hayashi
16S rDNA library method to compare the bacterial
species and phylotypes in colonic tissue samples of
elderly persons. Although some novel phylotypes were
detected from colonic samples, the composition of fecal
microbiota resembled those described previously (15, 28).
However, results of analysis of intestinal microbiota in
feces of elderly individuals have not yet been published.
Terminal restriction fragment length polymorphism (T-
RFLP) is a very convenient tool for comparing microbial
communities (17). T-RFLP can rapidly recognize com-
plex bacterial communities as terminal restriction frag-
ments (T-RFs) patterns or profiles. In addition, although
T-RFLP does not have a high resolution like the 16S
rDNA library, the distribution of microorganisms can be
divided into clusters or groups. We have recently used
this method to characterize the fecal microbiota in
healthy humans and a strict vegetarian (13, 27).
In the present study, we describe the fecal microbiota
in elderly individuals based on the analysis of the 16S
rDNA library and T-RFLP analysis of fecal samples.
We also deal with the many novel phylotypes discovered
in this study.
Materials and Methods
DNA extraction. Fecal samples were collected from
six elderly individuals (A: 94-year-old male, B: 88-year-
old female, C: 75-year-old female, D: 84-year-old female,
E: 74-year-old female, F: 79-year-old female) who lived
in a home for elderly people. Each fecal sample (60 mg)
was suspended in 5 ml of buffer A (10 mM Tris-HCl and
1 mM EDTA, pH 7.5), and washed four times using
buffer A. Bacterial DNA was extracted from the fecal
sample with a soil DNA kit (Mo Bio Laboratories, Inc.,
Calif., U.S.A.) using a FastPrepTMinstrument (Bio 101,
Vista, Calif., U.S.A.) (6).
PCR amplification and cloning. Two-universal
primers 27F (5' AGAGTTTGATCCTGGCTCAG 3')
and 1492R (5' GGTTACCTTGTTACGACTT 3') (18)
were used in PCR to amplify the 16S rRNA gene coding
region. Amplification reactions were performed in a
total volume of 100 µl containing 250 ng of DNA
extracted from three fecal samples (Samples A, B, and
C). PCR amplifications were performed using the fol-
lowing program: 95 C for 3 min, followed by 15 cycles
consisting of 95 C for 30 sec, 50 C for 30 sec, 72 C for
1.5 min, and a final extension period of 72 C for 10
min. The amplified 16S rDNA was purified using an
UltraClean PCR Clean-up kit (Mo Bio Laboratories). A
purified amplicon from the fecal sample was ligated
into the plasmid vector pCR®2.1, then transformed into
One Shot®INVαF' competent cells using the Original TA
Cloning kit (Invitrogen, San Diego, Calif., U.S.A.).
Plasmid DNA of selected transformants was purified
using MultiScreen 96-well filter plates (Millipore, Bed-
ford, Mass., U.S.A.).
DNA sequencing and phylogenetic analysis. Plas-
mid DNAs from 16S rDNA libraries and the amplicon of
DNA from colonies were used as templates for sequenc-
ing. An equal portion (about 500 bp) of 16S rDNA
(Escherichia coli position 27 to 520) was used for
sequence analysis. The dideoxy chain termination reac-
tion was conducted with a double-stranded DNA tem-
plate and 27F or 520R (5' ACCGCGGCTGCTGGC 3')
(18) primer using the BigDye Terminator Cycle Sequenc-
ing Kit (Applied Biosystems, Foster City, Calif., U.S.A.),
and products were analyzed on a model ABI PRISM
3700 DNA analyzer system (Applied Biosystems).
Nucleotide sequences were analyzed with FASTA search
(25). All sequences were examined for possible chimeric
artifacts by the CHECK CHIMERA program of the
Ribosomal Database Project (RDP) (20).
Sequence data were aligned with the CLUSTAL W
(30) package and corrected by manual inspection.
Nucleotide substitution rates (Knucvalues) were calculated
(16) and the phylogenetic trees were constructed using
the neighbor-joining method (26). Bootstrap resam-
pling analysis (10) of 100 replicates was performed to
estimate the confidence of tree topologies. The term
“phylotype” has been used for clusters of clone
sequences that differed from known species by about 2%
and were at least 98% similar to members of their clus-
T-RFLP analysis. The operation was carried out as
described previously (13, 27). 27F and 1492R primers
were used for T-RFLP. 27F was labeled with 6-FAM (6-
carboxyfluorescein, Applied Biosystems). PCR condi-
tions were the same as those used for amplification of
16S rDNA sequences from fecal samples except for the
extension reaction, which was performed for 30 cycles.
PCR products were purified by polyethylene glycol
(PEG 6000) (14) and redissolved in 20 µl of sterile dis-
tilled water. Purified PCR products were digested with
HhaI or MspI. The restriction digest product (1 µl) was
mixed with 12 µl of deionized formamide and 1 µl of
DNA fragment length standard. GS-500 ROX and GS-
1000 ROX (Applied Biosystems) were used as the inter-
nal standard markers. The length of T-RFs were ana-
lyzed by electrophoresis on a model ABI PRISM 310
Genetic Analyzer (Applied Biosystems) in Genescan
mode. Fragment sizes were estimated by the local
Southern Method in GenScan 3.1 software (Applied
Biosystems). T-RFs with a peak height less than 25
fluorescence units were excluded from the analysis.
Predicted T-RFLP patterns of the 16S rDNAs of species
and phylotypes were obtained using the GENETYX-
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