Koji NAGASHIMA 1 *, Jun MOCHIZUKI 2, Takayoshi HISADA 2, Shuji SUZUKI 2 and Kengo SHIMOMURA 2#

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1 Full Paper Bioscience Microflora Vol. 25 (3), , 2006 Phylogenetic Analysis of 16S Ribosomal RNA Gene Sequences from Human Fecal Microbiota and Improved Utility of Terminal Restriction Fragment Length Polymorphism Profiling Koji NAGASHIMA 1 *, Jun MOCHIZUKI 2, Takayoshi HISADA 2, Shuji SUZUKI 2 and Kengo SHIMOMURA 2# 1 Hokkaido Food Processing Research Center, Midorimachi, Bunkyodai, Ebetsu , Japan 2NCIMB division, TechnoSuruga Co., Ltd., 330 Nagasaki, Shimizu, Shizuoka , Japan #Present address: Graduate School of Fisheries Sciences, Hokkaido University, Minatomachi, Hakodate , Japan Received October 24, 2005; Accepted March 16, 2006 We have developed a terminal restriction fragment length polymorphism (T-RFLP) method for determining the structure and dynamics of the microbial gut community. In this paper, the improved T-RFLP method in combination with an analysis of the fecal 16S rdna (ribosomal RNA gene) clone libraries from six individuals is described. A total of 418 different partial sequences of 16S rdna were determined and subjected to a phylogenetic analysis and homology examination. We found that the sequences were roughly divided into six phylogenetic groups containing seven subgroups and were related to 71 known species with over 90% similarity. With the exception of a few cases, we found that in silico BslI-digestion of the sequences belonging to the same bacterial group or subgroup generated terminal restriction fragments of similar lengths. We concluded that human intestinal microbiota predominantly consists of the members of approximately ten phylogenetic bacterial groups and that these bacterial groups are effectively distinguished by our T-RFLP system. Key words: human fecal microbiota; 16S rdna clone library; T-RFLP INTRODUCTION The human intestinal microbiota plays a crucial role in the health of the host through its effects on nutrition, pathogenesis and the immune system. However, due to its complexity, the microbial community in the human gut is poorly understood. To overcome this obstacle, a high throughput system that would allow collection of data concerning the structure and dynamics of the microbial community in a large number of individuals is needed. Several 16S rrna-based molecular tools (2 10, 13, 17 20) have been developed that are superior to culture-based methods with respect to detection efficiency of microbiota and facility of the procedure. Of these, T-RFLP (terminal restriction fragment length polymorphism) seems to be the most effective due to its higher throughput and reproducibility (15). Since the conventional T-RFLP (10) targets the variable regions 1 to 3 of the 16S rrna gene (rdna) for analysis and employs several restriction enzymes that generate a large number of terminal restriction fragments (T-RFs), it *Corresponding author. Mailing address: Hokkaido Food Processing Research Center, Midorimachi, Bunkyodai, Ebetsu, , Japan. Phone: Fax: nagashima_koji@foodhokkaido.gr.jp. allows analysis of bacterial communities at the level of species. On the other hand, it is laborious to arrange data from T-RFLP and to assign each T-RF to bacterial species based on size, because a number of T-RFs stand close to each other. For rapid and easy analysis of fecal bacterial communities with T-RFLP, we recently developed a method different from the conventional T- RFLP which uses a primer-enzyme combination (14), in this method, the fluorescently labeled 516f and 1510r primers (Escherichia coli positions 516 to 532 and positions 1510 to 1492, respectively) are used for the amplification of the 16S rdna, and BfaI in combination with RsaI or BslI are used as the restriction enzymes. This method reduces the complexity of the T-RF profile so that data analysis can be carried out more readily and makes it possible to recover T-RFs by agarose gel electrophoresis, which allows cloning and sequencing of them. In addition, the predominant operational taxonomic units (OTUs, which correspond to either T- RFs or T-RF clusters) that were detected in the T-RFLP profiles from eight individuals roughly corresponded to the predominant bacterial groups in human feces, including the genera Bacteroides, Bifidobacterium, Clostridium, Enterococcus, Eubacterium, Fusobacterium, Lactobacillus, Prevotella, 99

2 100 K. NAGASHIMA, et al. Ruminococcus, Streptococcus, and Veillonella, on the basis of the T-RF length. The T-RF lengths were calculated from the sequences in the Ribosomal Database Project and in our fecal 16S rdna clone libraries from two individuals. In order to further understand the structure of human gut microflora and to improve the utility of our T-RFLP method, in the present study, we determined the fecal 16S rdna sequences from an additional four individuals, and performed phylogenetic clustering and in silico digestion by BslI, of these sequences. As a result, we confirmed the results of analysis of the human fecal 16S rdna clone library reported by other researchers (5 7, 18, 19) and demonstrated a more unambiguous correspondence between OTUs and the phylogenetic bacterial groups. MATERIALS AND METHODS Fecal samples, DNA extraction and T-RFLP analysis Total fecal DNA was isolated from samples from 6 healthy individuals (A, 48-year-old male; B, 49-year-old female; C, 38-year-old male, G, two-month-old female; I, 5-year-old male; J, 48-year-old male) as previously described (14). Briefly, the fecal samples were suspended in a solution containing 100 mm Tris-HCl (ph 9.0) and 40 mm EDTA after washing three times with sterile distilled water (corresponding to about 10 mg of wet weight), then beaten in the presence of glass beads using a mini-bead beater (BioSpec Products). Thereafter, DNA was extracted from the bead-treated suspension using benzyl chloride as described by Zhu et al. (21) and then purified using a GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences). The amplification of the fecal 16S rdna, restriction enzyme digestion, size-fractionation of T-RFs and the T- RFLP data analysis were previously described (14). Briefly, PCR was performed using the total fecal DNA and the primers of 5' HEX-labeled 516f (5'- TGCCAGCAGCCGCGGTA-3'; E. coli positions 516 to 532) and 1510r (5'-GGTTACCTTGTTACGACTT-3'; E. coli positions 1510 to 1492) The resulting 16S rdna amplicons were treated with 2 U of BslI (New England BioLabs) for 1 h and the digestives were fractionated using an automated sequence analyzer (ABI PRISM 310 Genetic Analyzer, Applied Biosystems) in GeneScan mode (the injection time was 20 s and the run-time was 40 min). Clone library analysis The fecal 16S rdna clone libraries were constructed using the TOPO TA Cloning Kit (Invitrogen) and the sequences of both strands of the cloned DNA (E. coli positions 516 to 1510) were determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) as previously described (14). The sequence data were subjected to homology searches with the BLAST or FASTA program, the phylogenetic tree was constructed with the CLUSTAL W program and TreeView software (these programs are at the DDBJ web site, and in silico restriction enzyme digestion was analyzed with the MacVector software (Oxford Molecular Ltd). The sequence data for individuals C and G are the same as that reported previously (14). Reference strains used for phylogenetic analysis are listed in Table 1. Nucleotide sequence accession numbers All sequences, in which primer sequences were removed, from the clone libraries were deposited in DDBJ with the following accession numbers: AB to AB094188, AB094344, AB and AB to AB RESULTS Phylogenetic analysis of 16S rdna sequences from human fecal bacteria Prior to the clone library construction, we selected the fecal samples to give the largest number of OTUs in T- RFLP. The T-RFLP profiles are shown in Fig. 1. A total of 418 different partial sequences (E. coli positions 533 to 1491) of the 16S rdna from the selected six fecal samples were determined and examined for homology by comparison with the BLAST or FASTA databases, and phylogenetic trees were constructed. The sequences showed homology predominantly to Bifidobacterium, Bacteroides, Enterobacteriales (Escherichia), Prevotella, Megamonas, Lactobacillus, Streptococcus, and Clostridium rrna groups IV, IX, XI, XIVa, and XVIII, although the sequence distribution in the clone library was different among individuals (Table 2). Phylogenetically, the sequences were divided into six groups containing seven subgroups (see Fig. 2 and Supplement file 1, available online at (21)) and each of these showed a similarity of over 90% to that of known species (see Supplement file 2, available online at (22)). Group I consisted of two subgroups, subgroups A and B, that contained 15 sequences from three individuals and 155 sequences from six individuals, respectively. The sequences in subgroup A were related to the members of Clostridium rrna cluster XI (1), including

3 T-RFLP OF HUMAN FECAL MICROBIOTA 101 Table 1. List of reference strains used for phylogenetic analysis Strains Accession numbers Bacteroides acidifaciens A40 T* AB Bifidobacterium breve ATCC15700 T AB Bifidobacterium infantis ATCC15697 D86184 Butyrivibrio fibrisolvens ATCC19171 T U41172 Catenibacterium mitsuokai JCM10609 T AB Clostridium amygdalinum BR-10 T AY Clostridium bartlettii WAL16138 AY Clostridium clostridioforme ATCC25537 T M59089 Clostridium cocleatum DSM1551 T Y18188 Clostridium glycolicum DSM1288 T X76750 Clostridium indolis DSM755 T Y18184 Clostridium lactatifermentans G17 T AY Clostridium leptum DSM753 T AJ Clostridium nexile DSM1787 T X73443 Clostridium orbiscindens DSM674 T Y18187 Collinsella aerofaciens JCM10188 T AB Eggerthella lenta ATCC25559 T AF Enterococcus malodoratus ATCC43197 T AF Escherichia coli V00348 Eubacterium eligens ATCC27750 T L34420 Eubacterium hallii ATCC27751 T L34621 Lactobacillus casei JCM1134 T D16551 Megamonas hypermegale DSM1672 T AJ Phascolarctobacterium faecium ACM3679 T X72865 Prevotella melaninogenica ATCC25845 T AY Roseburia intestinalis L1-82 T AJ Ruminococcus bromii X85099 Ruminococcus gnavus ATCC29149 T X94967 Ruminococcus parasangius ATCC15912 F Ruminococcus schinkii B T X94965 Streptococcus pasteuri ACM3611 T X94337 Streptomyces albus DSM40313 T AJ Thermus thermophilus HB8 X07998 Veillonella ratti ATCC17746 T AF * The letter T indicates the type strain. C. bartlettii, C. glycolicum and C. lituseburense, with sequence similarities of 97 98%. The sequences in subgroup B were related to the members of Clostridium rrna subcluster XIVa (Clostridium coccoides subgroup (18)), including Butyrivibrio fibrisolvens, Clostridium amygdalinum, C. bolteae, C. clostridioforme, C. hathewayi, C. indolis, C. lactatifermentans, C. nexile, C. saccharolyticum, Desulfotomaculum guttoideum, Eubacterium eligens, E. hallii, E. ramulus, E. rectale, E. ventriosum, Hespellia stercorisuis, Lachnospira pectinoschiza, Roseburia intestinalis, Ruminococcus gnavus, R. hydrogenotrophicus, R. obeum, R. productus, R. schinkii and R. torques, with similarities of 91 99%. Group II contained 26 sequences from four individuals that were related to the members of Clostridium rrna cluster IV (Clostridium leptum subgroup (18)), including C. orbiscindens, Eubacterium desmolans, Faecalibacteruim prausnitzii and Ruminococcus bromii, with similarities of 94 99%. Group III consisted of three subgroups, subgroups C, D and E, that contained 40 sequences from five individuals, 14 sequences from three individuals and 14 sequences from three individuals, respectively. The sequences in subgroup C were related to the members of the class Bacilli, including Abiotrophia elegans, Bacillus fumarioli, Enterococcus malodoratus, Lactobacillus casei, L. cellobiosus, Streptococcus parasanguinis, S. pasteuri, S. pseudopneumoniae, S. salivarius, S. thermophilus and S. vestibularis, with similarities of %. The sequences in subgroup D were related to the members of Clostridium rrna cluster XVI, XVII and XVIII, including C. cocleatum, C. innocuum, Eubacterium biforme, and Catenibacterium mitsuokai, with similarities of 93-99%. The sequences in subgroup

4 102 K. NAGASHIMA, et al. Fig. 1. T-RFLP profiles derived from six fecal samples and OTU distribution profiles of 16S rdna clone libraries. The number of the 16S rdna clones that yielded the indicated OTU with BslI-digestion is presented beneath each T-RFLP profile of individuals A to J.

5 T-RFLP OF HUMAN FECAL MICROBIOTA 103 Table 2. Classification of clones in fecal 16S rdna library into bacterial groups No. of clones that habor the sequences derived from indicated bacterial groups, in each individual (%) Bacterial groups A B C G I J Overall Gram-positive bacteria Low G+C subclass Clostridaleas Clostridium cluster IV 10 (13.3) 8 (11.6) 7 (11.1) 1 (1.3) 0 (0.0) 0 (0.0) 26 (5.8) Clostridium cluster IX 1 (1.3) 1 (1.4) 0 (0.0) 5 (6.3) 0 (0.0) 8 (10.7) 15 (3.3) Clostridium cluster XI 1 (1.3) 2 (2.9) 0 (0.0) 0 (0.0) 12 (13.8) 0 (0.0) 15 (3.3) Clostridium subcluster XIVa 16 (21.3) 29 (42.0) 27 (42.9) 6 (7.6) 42 (48.3) 39 (52.0) 159 (35.5) Clostridium cluster XVI 1 (1.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.4) Clostridium cluster XVII 3 (4.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 3 (0.7) Clostridium cluster XVIII 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 9 (10.3) 0 (0.0) 9 (2.0) Bacillales Bacillus 0 (0.0) 2 (2.9) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.4) Lactobacillales Enterococcus 0 (0.0) 0 (0.0) 0 (0.0) 2 (2.5) 1 (1.1) 0 (0.0) 3 (0.7) Lactobacillus 1 (1.3) 4 (5.8) 0 (0.0) 0 (0.0) 3 (3.4) 0 (0.0) 8 (1.8) Streptococcus 15 (20.0) 1 (1.4) 1 (1.6) 8 (10.1) 1 (1.1) 0 (0.0) 26 (5.8) Others 0 (0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.1) 0 (0.0) 1 (0.2) High G+C subclass Actinomycetales Bifidobacterium 1 (1.3) 13 (18.8) 6 (9.5) 46 (58.2) 14 (16.1) 0 (0.0) 80 (17.9) Others 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.3) 0 (0.0) 0 (0.0) 1 (0.2) Gram-negative bacteria Bacteroidales Bacteroides 0 (0.0) 8 (11.6) 22 (34.9) 7 (8.9) 4 (4.6) 13 (17.3) 54 (12.1) Prevotella 18 (24.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 18 (4.0) Megamonas 8 (10.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 8 (1.8) Enterobacteriales Escherichia 0 (0.0) 0 (0.0) 0 (0.0) 2 (2.5) 0 (0.0) 15 (20.0) 17 (3.8) Others 0 (0.0) 1 (1.4) 0 (0.0) 1 (1.3) 0 (0.0) 0 (0.0) 2 (0.4) Total E were related to members of Clostridium rrna cluster IX, including Dialister invisus, Megasphaera elsdenii, Phascolarctobacterium faecium and Veillonella ratti, with similarities of %, and to Megamonas hypermegale, which is a member of the order Bacteroidales, with similarities of 95%. Group IV contained 70 sequences from five individuals that were related to the members of the genus Bifidobacterium, including B.adolescentis, B. breve, B. catenulatum, B. infantis, B. longum and B. pseudocatenulatum, with similarities of %. Group V contained 17 sequences from two individuals that were most closely related to Escherichia coli, which is a member of the order Enterobacteriales, with similarities of %. Group VI was a cluster of the order Bacteroidales and consisted of two subgroups, subgroup F and G, that contained 54 sequences from five individuals and 18 sequences from one individual, respectively. The sequences in subgroup F were related to members of the genus Bacteroides, including B. acidifaciens, B. distasonis, B. fragilis, B. merdae, B. thetaiotaomicron, B. uniformis and B. vulgatus, with similarities of 91 99%. The sequences in subgroup G were related to members of the genus Prevotella, including P. melaninogenica and P. ruminicola, with similarities of 90 94%. Correspondence of T-RFs generated by in silico restriction enzyme digestion of 16s rdna sequences and phylogenetic bacterial groups The 16S rdna sequences (E. coli positions 516 to 1510) derived from human fecal microbiota were digested in silico by BfaI together with RsaI or by BslI. When the lengths of T-RFs generated by BslI digestion were sorted, it was found that T-RFs of similar length generally corresponded to the same bacterial group or subgroup (see (22)). As summarized in Table 3, with the exception of several T-RFs, it was possible to correspond the approximately 115-nucleotide (nt) T-RF to group IIIsubgroup E; the 172-, approx and approx. 750-nt T-RFs corresponded to group II; the approx. 128-nt T- RF corresponded to group IV; the 317-nt T-RF

6 104 K. NAGASHIMA, et al. Fig. 2. Phylogenetic clustering diagram for the 16S rdna sequences from six fecal samples. Numbers at the internal branches indicate bootstrap values (expressed as percentage of 1000 replications) for clusters. The scale bar represents 0.1 substitutions per nucleotide position. corresponded to group VI-subgroup G; the 328-, 517- and approx. 663-nt T-RFs corresponded to group IIIsubgroup C; the 335-nt T-RF corresponded to group I- subgroup A; the approx and approx. 468-nt T-RFs corresponded to group VI-subgroup F; the approx. 491-, approx. 502-, 514-, approx. 757-, approx. 959-, and 995-nt T-RFs corresponded to group I-subgroup B; the 643- and 654-nt T-RFs corresponded to group IIIsubgroup D; and the approx. 923-nt T-RF corresponded to group I. These sequence/group correspondences, however, did not apply to the following clones: I48, G32, A74, B71, I26, A33, I33, C1, C32, and B88 (see (22)). The approx. 940-nt T-RF predominantly represented the two phylogenetically different bacterial groups of subgroup B and group V. Since the sequence of group V (Enterobacteriales) yielded a unique 191-nt T-RF by MboI digestion (data not shown), it was possible to discriminate it from the other sequences. In contrast, a distinct correspondence between the T-RFs generated by BfaI-RsaI double-digestion and bacterial groups was not observed, with the exception of the approx and 254-nt T-RFs, which corresponded to Bifidobacterium of group IV and Megamonas sp. of group III-subgroup E, respectively (see (22)).

7 T-RFLP OF HUMAN FECAL MICROBIOTA 105 Table 3. Correspondence of T-RFs, OTUs and phylogenetic bacterial groups T-RFs (nt) a OTUs Phylogenetic groups d c Group I - subgroup B / Clostridium subcluster XIVa 113, 114, Group III - subgroup E / Clostridium cluster IX, Megamonas 125, 126, 127, Group IV / Bifidobacterium 141 NA b Group VI - subgroup G / Prevotella NA 144 NA Group II / Clostridium cluster IV Group VI -subgroup G / Prevotella Group III - subgroup C / Lactobacillales Group I - subgroup A / Clostridium cluster XI 365, Group VI - subgroup F / Bacteroides 369, Group II / Clostridium cluster IV Group III -subgroup D / Clostridium cluster XVIII 466, 467, Group VI - subgroup F / Bacteroides 488, 489, 490, Group I - subgroup B / Clostridium subcluster XIVa 501, C Group I - subgroup B / Clostridium subcluster XIVa Group I - subgroup B / Clostridium subcluster XIVa Group III - subgroup C / Lactobacillales 643 NA Group III - subgroup D / Clostridium cluster XVII Group III -subgroup D / Clostridium cluster XVIII 659, 661, 662, Group III - subgroup C / Lactobacillales 747, 748, 749, Group II / Clostridium cluster IV 753, 754, 755, 756, Group I - subgroup B / Clostridium subcluster XIVa Group VI - subgroup F / Bacteroides 919, 920, Group I / Clostridium cluster XI, subcluster XIVa 935, 937, 938, 939, Group I - subgroup B / Clostridium subcluster XIVa Group V / Enterobacteriales 954, 955, 956, 957, Group I - subgroup B / Clostridium subcluster XIVa Group I - subgroup B / Clostridium subcluster XIVa a Generated by in silico Bsl I-digestion of the 16S rdna sequences from six fecal samples. nt, nucteotides. b NA, not applicable. cthese OTUs have been detected in the T-RFLP profiles in another. d Refer to Fig. 1. DISCUSSION We randomly selected clones from each fecal 16S rdna library derived from six individuals and determined their sequences. These sequences were mainly derived from bacteria related to Bifidobacterium, Bacteroides, Enterobacteriales (Escherichia), Prevotella, Megamonas, Lactobacillus, Streptococcus, and Clostridium rrna groups IV, IX, XI, XIVa and XVIII. Similar results were obtained in other studies on equivalent clone libraries (5 7, 18, 19). Although we detected only a few sequences related to Clostridium rrna clusters XVI and XVII in the two clone libraries, in the other studies many sequences were detected in the clone library derived from an individual (5). The clones we examined were related to 71 known species overall (13 to 31 species per individual), with over 90% sequence similarity. Hold et al. (7) analyzed the clone libraries of colonic tissue samples derived from three elderly individuals and related the detected clones to 41 type strains overall (19 to 21 species per individual), with over 88% sequence similarity. Meanwhile, Suau et al. (18) detected 82 molecular species in one subject that were 98% similar. Similarly, Hayashi et al. (5, 6) detected 130 molecular species overall in three adult subjects (48 to 65 species per individual), and 83 molecular species overall in three elderly subjects (23 to 38 species per individual). In general, it is considered that intestinal microbiota in an individual predominantly consist of several tens of species belonging to several phylogenetic groups. Although Megamonas sp. are anaerobic gramnegative bacilli and are taxonomically classified into the family of Bacteroidaceae, it is phylogenetically related to a member of Clostridium rrna cluster IX. We detected eight clones related to Megamonas in one subject (Table 2) and Hayashi et al. (5) also detected many clones related to Megamonas in two subjects. It is not known whether this bacterium functions as Bacteroides or Clostridium in the intestine. A total of 20 Bifidobacteria-related sequences were detected in adult subjects, which are derived from species

8 106 K. NAGASHIMA, et al. closely related to B. pseudocatenulatum, B. longum, B. adolescentis and B. catenulatum; while 46 Bifidobacteria-related sequences were detected in an infant subject, which are derived from species closely related to B. longum, B. breve and B. infantis. A similar difference in Bifidobacterium species distribution has also been observed with respect to the detection frequency of these species in adult and infant samples (11). We here showed that the T-RFs of similar lengths were produced from the 16S rdna sequences from bacteria belonging to the same phylogenetic group or subgroup, with respect to in silico BslI-digestion, although there were several exceptions. When these T-RFs are distinctly resolved, it will be possible to obtain detailed information on human intestinal microbiota. However, since the fragment analysis tool we previously developed (14) does not have a sufficiently high resolution or accuracy, especially in the high molecular weight regions, it was difficult to distinguish OTUs that were of similar size. Consequently, we constructed a set of sizemarkers for the OTUs that were generated by PCR using the cloned 16S rdna representing each OTU (clone A05, A24, A81, A91, B02, B07, B10, B11, B41, B50, B61, B88, B84, C22, C93, I06, I13, I19, I20, I31, I35, I82, J38, J51, or J72 shown in Supplement file 2) as a template, followed by in silico BslI-digestion. With this method, we can detect T-RFs with a considerably higher resolution and accuracy than the earlier method, allowing the detection of correspondence between T-RFs generated by the in silico digestion and OTUs detected in T-RFLP analysis as shown in Table 3. Sakamoto et al. (16) reported the application of the conventional T-RFLP to analysis of human fecal microbiota and demonstrated that the T-RFLP analysis is useful for assessment and comparison of the structure of fecal bacterial communities. Thereafter, to rapidly predict the species composition of fecal microbiota from T-RFLP profile data, they developed a novel phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM) (12). On the other hand, our T-RFLP allows more rapid analysis of the bacterial community by lowering its resolution to the level of genus or lower-order group. Therefore, the PAD-HCM and our method seem to be complementary. It has been said that T-RFLP and random cloning methods are complementary tools for the analysis of bacterial communities but they give a different result with respect to richness and abundance of bacterial species. Therefore, in this study, we investigated the correlation of the T-RFLP profile and the OTU distribution of the 16S rdna clones. As shown in Fig. 1, there was not always a correlation between them. This is considered to be due to the so-called cloning bias (20) since the T-RFLP and the clone library analyses were performed using products amplified under the same PCR conditions, although the results of the two analyses appeared to correlate in the cases of the 124-OTU (corresponded to Bifidobacterium), the 494-OTU (corresponded to Clostridium rrna subcluster XIVa) and the 940-OTU (corresponded to Clostridium rrna subcluster XIVa and Enterobacriales). Bacterial communities in the human intestine are very complex and consequently the development of a new technology to analyze them is necessary. We have developed a novel T-RFLP system for the analysis of the bacterial communities and here attempted to improve the utility of this technique by combination with analysis of the fecal 16S rdna clone libraries from six individuals. The results showed that human intestinal microbiota are predominantly constructed of the members of approximately ten phylogenetic bacterial groups and that these bacterial groups could be effectively distinguished by our T-RFLP system. Acknowledgement. We thank Ms. Kimiko Minamida (Northern Advancement Center for Science and Technology, Sapporo, Japan) for her technical assistance. REFERENCES (1) Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, Cai J, Hippe H, Farrow JAE The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44: (2) Franks AH, Harmsen HJM, Raangs GC, Jansen GJ, Schut F, Welling GW Variation of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rrnatargeted oligonucleotide probes. Appl Environ Microbiol 64: (3) Gong J, Forster RJ, Yu H, Chambers JR, Sabour PM, Wheatcroft R, Chen S Diversity and phylogenetic analysis of bacteria in mucosa of chicken ceca and comparison with bacteria in the cecal lumen. FEMS Microbiol Lett 208: 1 7. (4) Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, Welling GW Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 30: (5) Hayashi H, Sakamoto M, Benno Y

9 T-RFLP OF HUMAN FECAL MICROBIOTA 107 Phylogenetic analysis of the human gut microbiota using 16S rdna clone libraries and strictly anaerobic culture-based methods. Microbiol Immunol 46: (6) Hayashi H, Sakamoto M, Kitahara M, Benno Y Molecular analysis of fecal microbiota in elderly individuals using 16S rdna library and T-RFLP. Microbiol Immunol 47: (7) Hold GL, Pryde SE, Russell VJ, Furrie E, Flint HJ Assessment of microbial diversity in human colonic samples by 16S rdna sequence analysis. FEMS Microbiol Ecol 39: (8) Langendijik PS, Schut F, Jansen GJ, Raangs GC, Kamphuis GR, Wilkinson MHF, Welling GW Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rrnatargeted probes and its application in fecal samples. Appl Environ Microbiol 61: (9) Leser TD, Lindecrona RH, Jensen TK, Jensen BB, Møller K Changes in bacterial community structure in the colon of pig fed different experimental diets and after infection with Brachyspira hyodysenteriae. Appl Environ Microbiol 66: (10) Liu W, Marsh TL, Cheng H, Forney LJ Characterization of microbial diversity by determining terminal restriction fragment length polymorphism of genes encoding 16S rrna. Appl Environ Microbiol 63: (11) Matsuki T, Watanabe K, Tanaka R, Fukuda M, Oyaizu H Distribution of bifidobacterial species in human intestinal microflora examined with 16S rrna-gene-targeted species-specific primers. Appl Environ Microbiol 65: (12) Matsumoto M, Sakamoto M, Hayashi H, Benno Y Novel phyligenetic assignment database for terminal-restriction length polymorphism analysis of human colonic microbiota. J Microbiol Method 61: (13) McCracken VJ, Simpson JM, Mackie RI, Gaskins HR Molecular ecological analysis of dietary and antibiotic-induced alterations of the mouse intestinal microbiota. J Nutr 131: (14) Nagashima K, Hisada T, Sato M, Mochizuki J Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces. Appl Environ Microbiol 69: (15) Osborn AM, Moore ER, Timmis KN An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ Microbiol 2: (16) Sakamoto M, Hayashi H, Benno Y Terminal retriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities. Microbiol Immunol 47: (17) Simpson JM, McCracken VJ, White BA, Gaskins HR, Mackie RI Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota. J Microbiol Methods 36: (18) Suau A, Bonnet R, Sutren M, Godon J, Gibson GR, Collins MD, Doré J Direct analysis of genes encoding 16S rrna from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 65: (19) Wilson KH, Blitchngton RB Human colonic biota studied by ribosomal DNA sequence analysis. Appl Environ Microbiol 62: (20) Zoetendal EG, Akkermans ADL, de Vos WM Temperature gradient gel electrophoresis analysis of 16S rrna from human fecal samples reveals stable and host-specific communities of active bacteria. Appl Environ Microbiol 64: (21) Zhu H, Qu F, Zhu L Isolation of genomicdnas from plants, fungi and bacteria using bezyl chloride. Nucleic Acid Res 21: (22) Supplement file 1. Phylogenetic tree of 16S rdna sequences from fecal samples. The tree was rooted with an out-group species (Thermus thermophilus). Numbers at the internal branches indicate bootstrap values (expressed as percentage of 1000 replications) for groups. The scale bar represents 0.05 substitutions per nucleotide position. The figure after the clone number represents the size of the T-RF generated by computer model BslI-digestion of the sequence. (23) Supplement file 2. List of 16S rdna clones. Accession numbers in the DDBJ database, size of T- RF generated by BslI- or BfaI-RsaI double-digestion, closely related species together with homology percentage and taxonomic position of the clone are presented. The clones are arranged in order of the phylogenetic tree (Table A1) or the T-RF size (Table A2 and A3).