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1Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Saitama 351-0198, Japan 2Departments of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan 3Department of Medicine, Shiga Health Insurance Hospital, Otsu, Shiga 520-0846, Japan
Correspondence Hidenori Hayashi hayashi{at}jcm.riken.jp
Received October 20, 2004
Accepted July 20, 2005
Microbiota in gut contents of jejunum, ileum, caecum and recto-sigmoid colon obtained from three elderly individuals at autopsy were compared using 16S rRNA gene libraries and terminal restriction fragment length polymorphism (T-RFLP). Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets of total genomic DNA extracted from each sample of gut contents. An average of 90 randomly selected clones were partially sequenced (about 500 bp). T-RFLP analysis was performed using the 16S rRNA gene amplified from each sample. The lengths of the terminal restriction fragments were analysed after digestion with HhaI and MspI. The jejunal and ileal microbiota consisted of simple microbial communities of streptococci, lactobacilli, ‘Gammaproteobacteria', the Enterococcus group and the Bacteroides group. Most of the species were facultative anaerobes or aerobes. The Clostridium coccoides group and the Clostridium leptum subgroup, which are the most predominant groups in human faeces, were not detected in samples from the upper gastrointestinal tract. The caecal microbiota was more complex than the jejunal and ileal microbiota. The C. coccoides group, the C. leptum subgroup and the Bacteroides group were detected in the caecum. The recto-sigmoidal colonic microbiota consisted of complex microbial communities, with numerous species that belonged to the C. coccoides group, the C. leptum subgroup, the Bacteroides group, ‘Gammaproteobacteria', the Bifidobacterium group, streptococci and lactobacilli, and included more than 26 operational taxonomic units. The results showed marked individual differences in the composition of microbiota in each region.
The GenBank/EMBL/DDBJ accession numbers for the novel 16S rRNA gene sequences detected by this study are AB189894–AB189908.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The small-intestinal microbiota have been compared elsewhere by analysing isolates using anaerobic culture-based methods (Bentley et al., 1972; Corrodi et al., 1978; Finegold et al., 1983; Gorbach et al., 1967; Thadepalli et al., 1979). Corrodi et al. (1978) sampled aspirates of both jejunum and distal ileum contents in patients about to undergo intestinal bypass for morbid obesity. These samples were collected by direct needle at the time of surgery. The jejunal microbiota were sterile or contained low counts of predominantly aerobic bacteria. On the other hand, many aerobes and anaerobes existed in the distal ileum. In addition, anaerobes such as species of Bacteroides, Clostridium, Fusobacterium and Eubacterium were detected from ileal specimens. Bacterial numbers were greater in the ileum than the jejunum. Thadepalli et al. (1979) collected small-intestinal contents at the time of exploratory laparotomy for intra-abdominal trauma. More than half of the jejunal and ileal samples were sterile. Recently, Wang et al. (2003) used the 16S rRNA gene library method to analyse the terminal ileal microbiota. Twenty-seven OTUs were identified, less than the number in the colon. However, the results of analysis of small-intestinal microbiota have not yet been published.
The composition of the caecal microbiota is also poorly understood. Marteau et al. (2001) used dot-blot hybridization to compare the communities in caecal microbiota using six rRNA-targeted probes. They demonstrated lower numbers of the Bifidobacterium group, the Bacteroides group, the C. coccoides group and the C. leptum subgroup bacteria in caecal contents compared with faeces. Facultative anaerobes, however, were more prominent (higher proportion of total) in the caecum.
We analysed the jejunal, ileal, caecal and recto-sigmoidal colonic microbiota (which contain many unexploited bacteria) in elderly individuals based on 16S rRNA gene libraries and T-RFLP analysis. In addition, the composition of the small-intestinal microbiota was clarified by molecular methods.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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PCR amplification and cloning.
Two universal primers, 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGT TACGACTT-3') (Lane, 1991), were used to amplify the 16S rRNA gene sequences. PCR amplification was performed using the following program: 95 °C for 3 min, followed by 15 cycles consisting of 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1.5 min, and a final extension period of 72 °C for 10 min. The amplified 16S rRNA genes were purified using the UltraClean PCR clean-up kit (Mo Bio Laboratories). The purified amplicons from faecal samples were ligated into the plasmid vector pCR2.1. One Shot INV
F' competent cells (Invitrogen) were transformed with the ligation mixture. Plasmid DNA of selected transformants was purified using MultiScreen 96-well filter plates (Millipore).
DNA sequencing and phylogenetic analysis.
Plasmid DNAs from the 16S rRNA gene libraries were used as templates for sequencing. An equal portion (about 500 bp) of 16S rRNA gene (Escherichia coli position 28–513) was used for sequence analysis. The dideoxy chain termination reaction was conducted with a double-stranded DNA template and 27F or 520R (5'-ACCGCGGCTGCTGGC-3') (Lane, 1991) primer using the BigDye Terminator Cycle sequencing kit (Applied Biosystems), and products were analysed on a model ABI PRISM 3100 DNA analyser system (Applied Biosystems). Nucleotide sequences were analysed with the FASTA search and Sequence Match program of the Ribosomal Database Project II (RDP-II) (Cole et al., 2003). All sequences were examined for possible chimeric artifacts by the CHECK CHIMERA program of the RDP-II (Cole et al., 2003).
Sequence data were aligned with the CLUSTAL W package (Thompson et al., 1994) and corrected by manual inspection. Nucleotide substitution rates (Knuc values) were calculated (Kimura, 1980) and phylogenetic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987). An operational taxonomic unit (OTU) has previously been used to describe clusters of clone sequences that differ from known species by about 2 % and are at least 98 % similar to members of their cluster (Suau et al., 1999). Coverage was calculated by Good's method (Good, 1953), according to which the percentage of coverage is calculated with the formula [1–(n/N)]x100, where n is the number of OTUs represented by one clone (single-clone OTUs) and N is the total number of sequences.
T-RFLP analysis.
T-RFLP analysis was performed as described previously (Hayashi et al., 2002b; Sakamoto et al., 2003), using 27F and 1492R primers. Primer 27F was labelled with 6-FAM (6-carboxyfluorescein, Applied Biosystems). PCR conditions were the same as those used for amplification of 16S rRNA gene sequences from faecal samples, except for the extension reaction, which was performed for 30 cycles. PCR products were purified using polyethylene glycol (PEG 6000) (Hiraishi et al., 1995) and redissolved in 20 µl sterile distilled water. Purified PCR products were digested with HhaI or MspI. Each restriction digest was mixed with deionized formamide and DNA fragment length standard (GS-500 ROX and GS-1000 ROX; Applied Biosystems). The lengths of T-RFs were analysed by electrophoresis on a model ABI PRISM 3100 genetic analyser (Applied Biosystems) in GENESCAN mode. Fragment sizes were estimated by the local Southern method in GenScan 3.1 software (Applied Biosystems). T-RFs with peak amplitudes of less than 25 fluorescence units were excluded from the analysis. The major T-RFs were identified by computer simulation, which was performed using 16S rRNA gene sequences registered into the RDP-II and determined by 16S rRNA gene libraries in this study. Predicted T-RFLP patterns of the 16S rRNA genes of OTUs were obtained using GENETYX-MAC version 11.2 computer software (Software Development). The proportions of each T-RF were calculated as the mean percentages of the total T-RF area on the graph after digestion with HhaI and MspI.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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We also analysed the number of common OTUs detected from each compartment of each individual. The number of common OTUs detected in the caecum and recto-sigmoid colon was 11 (13.9 % of total OTUs) in subject A and seven (14.6 % of total OTUs) in subject C. These OTUs belonged to the C. coccoides group, the C. leptum subgroup, lactobaccilli and streptococci. The number of OTUs and T-RFs increased from the ileum to the caecum (Table 1, Fig. 1). The diversity of OTUs is so high in the caecum and recto-sigmoid colon that common OTUs are detected. Two OTUs, belonging to ‘Gammaproteobacteria’ and the Enterococcus group, were detected in all four compartments in subject B. ‘Gammaproteobacteria’ is one of the most predominant groups in the faecal microbiota of elderly humans (Hayashi et al., 2003). This micro-organism is not only the predominant group in the recto-sigmoid colon, but also the jejunum, ileum and caecum. On the other hand, some common OTUs were detected between other compartments.
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No common OTUs were identified in the same compartment of all three individuals. Some common OTUs, belonging to streptococci and the C. coccoides group, were detected in the ileum or caecum in subjects A and B. Eleven OTUs belonging to ‘Deltaproteobacteria', the C. coccoides group, the C. leptum subgroup, the Enterococcus group, ‘Gammaproteobacteria', the Lactobacillus reuteri subgroup and the Sporomusa group were detected in the recto-sigmoid colon of these two subjects. The recto-sigmoid colon microbiota were more complex than those of the jejunum, ileum and caecum, and consisted of various OTUs. Therefore, the number of common OTUs in the recto-sigmoid colon was higher than in the jejunum, ileum and caecum.
Jejunal and ileal microbiota
Jejunal microbiota were characterized in three elderly individuals from 16S rRNA gene libraries and by T-RFLP analysis (Table 1, Fig. 1). Lactobacilli (73.9 %) and streptococci (16.3 %) were detected in subject A from the 16S rRNA gene library (Table 1). The major T-RFs in subject A corresponded to the Lactobacillus mali subgroup (55 % of the total area of T-RFs), the Lactococcus lactis subgroup and streptococci (Fig. 1). Enterobacter cloacae (Enterobacter asburiae subgroup) (66.7 %) was the most predominant group in subject B from the 16S rRNA gene library (Table 1). However, the most dominant T-RF was the Enterobacter asburiae subgroup (about 60 % of the total area of T-RFs) (Fig. 1). The Klebsiella planticola subgroup (93.3 %) was most predominant in subject C from the 16S rRNA gene library (Table 1), and was also the most dominant T-RF (about 70 % of the total area of T-RFs) (Fig. 1).
Lactobacilli (21.3 %), streptococci (36.0 %) and the Actinobacillus actinomycetemcomitans subgroup (22.5 %) were detected in the ileum of subject A from the 16S rRNA gene library (Table 1). The Lactobacillus mali subgroup (about 15 % of the total area of T-RFs), streptococci (about 30 % of the total area of T-RFs) and the A. actinomycetemcomitans subgroup (about 20 % of the total area of T-RFs), were detected by T-RFLP (Fig. 1). Enterococcus faecium (Enterococcus group) (12.8 %) and Enterobacter cloacae (Enterobacter asburiae subgroup) (85.1 %) were detected in the ileum of subject B from the 16S rRNA gene library (Table 1). Two major T-RFs belonging to the Enterococcus group (about 17 % of the total area of T-RFs) and the Enterobacter asburiae subgroup (60 % of the total area of T-RFs) were detected by T-RFLP. The Enterococcus group (32.6 %), the Escherichia subgroup (15.7 %) and the K. planticola subgroup (21.3 %) were detected in the ileum of subject C from the 16S rRNA gene library (Table 1). However, the most dominant T-RFs were the Enterococcus group and the Escherichia subgroup (accounting for approximately 24.5 % and 37 % of the total area of T-RFs, respectively) (Fig. 1).
According to culture-dependent analyses, enterococci, Escherichia coli, klebsiella, lactobacilli, staphylococci and streptococci are present in the jejunum and ileum (Corrodi et al., 1978; Thadepalli et al., 1979). We too detected the Enterococcus group, lactobacilli, streptococci and ‘Gammaproteobacteria’ (the A. actinomycetemcomitans subgroup and Klebsiella subgroup) from the 16S rRNA gene libraries and by T-RFLP in the jejunum and ileum. Most of the bacteria detected in the jejunum and ileum were facultative anaerobes and aerobes. Lactobacilli and streptococci were particularly detected in subject A, while the Enterococcus group and ‘Gammaproteobacteria’ were detected at high frequencies in subjects B and C. However, they were seldom detected in the caecum and recto-sigmoid colon of these two subjects. There was a marked change in bacterial community from the ileum to caecum. The predominant species that inhabited the small-intestinal tract were facultative anaerobes and aerobes belonging to the Enterococcus group, lactobacilli, streptococci and ‘Gammaproteobacteria'. On the other hand, the large-intestinal microbiota consisted of larger numbers and a greater variety of anaerobes.
Wang et al. (2003) reported that clones belonging to the Bacteroides fragilis subgroup (36.6 %) and the Clostridium lituseburense group (33.8 %) were detected in the ileum. Facultative anaerobes and aerobes were hardly detected in that report, in contrast to the present study. Wang et al. (2003) used mucosa obtained by biopsy, whereas we used gut contents, and it is possible that the ileal mucosal microbiota differs from that of the ileal lumen content. In addition, these investigators studied a much younger subject (35 years old), which could explain the difference in the composition of microbiota. In this regard, we have previously reported the existence of notable differences in the composition of faecal microbiota between adult and elderly individuals (Hayashi et al., 2003).
Caecal microbiota
The C. coccoides group (50.0 %) and the C. leptum subgroup (25.6 %) were detected in subject A from the 16S rRNA gene library (Table 1). The major T-RFs also corresponded to the C. coccoides group (about 39 % of the total area of T-RFs) and the C. leptum subgroup (about 16 % of the total area of T-RFs) (Fig. 1). The Enterobacter asburiae subgroup (36.3 %) and the Enterococcus group (Enterococcus faecium, 35.2 %) were detected in subject B using the 16S rRNA gene library (Table 1). The major T-RFs also corresponded to the Enterobacter asburiae subgroup (about 34 % of the total area of T-RFs) and the Enterococcus group (about 23.5 % of the total area of T-RFs) (Fig. 1). Streptococci (57.6 %) and the C. coccoides group (16.3 %) were detected in subject C from the 16S rRNA gene library (Table 1). The major T-RFs also corresponded to streptococci (about 50 % of the total area of T-RFs) and the C. coccoides group (about 11 % of the total area of T-RFs) (Fig. 1).
Caecal microbiota were more complex than jejunal and ileal microbiota. The C. leptum subgroup, the C. coccoides group and the Bacteroides group, which are the major constituents of faecal microbiota, were also detected in the caecum. Marteau et al. (2001) analysed caecal and faecal microbiota by dot-blot hybridization with six 16S rRNA-targeted oligonucleotides, and found that the numbers of the Bacteroides group, the C. coccoides group and the C. leptum subgroup were lower in the caecum than in faeces, representing only 13 % of caecal bacterial rRNA. We also detected these groups in all three subjects (A, 80.0 %; B, 25.3 %; C, 22.8 %). On the other hand, the Lactobacillus–Enterococcus group and Escherichia coli were more prevalent in the caecum (50 % of the caecal bacterial rRNA) (Marteau et al., 2001). We also detected these groups in all three subjects (A, 1.1 %; B, 72.2 %; C, 18.4 %). In addition, we detected streptococci at a high frequency from the 16S rRNA gene library (57.6 %) and by T-RFLP (about 50 %) in subject B. Although obligate anaerobes belonging to the Bacteroides group, C. coccoides group and C. leptum subgroup were also present among the caecal microbiota, facultative aerobes were the predominant species. We also detected the Enterobacter asburiae subgroup, and the Streptococcus salivarius and Streptococcus pneumoniae subgroups. Furthermore, OTUs and T-RFs that belonged to these subgroups were detected together in the small intestine and caecum.
Recto-sigmoidal colonic microbiota
The C. coccoides group (32.2 %) and the C. leptum subgroup (30.0 %) were detected in subject A from the 16S rRNA gene library (Table 1). The major T-RFs also corresponded to these groups (approximately 32 % and 16 % of the total area of T-RFs, respectively). The Enterococcus group (18.9 %) and the Enterobacter asburiae subgroup (22.2 %) were detected in subject B from the 16S rRNA gene library and formed the major T-RFs (approximately 14 % and 30 % of the total area of T-RFs, respectively). The Bifidobacterium subgroup, the C. coccoides group and lactobacilli were detected in subject C from the 16S rRNA gene library (Table 1) and formed the major T-RFs (approximately 11 %, 10 % and 28 % of the total area of T-RFs, respectively) (Fig. 1).
The recto-sigmoidal colonic microbiota consisted of complex microbial communities that consisted of more than 26 OTUs and 16 major T-RFs. They differed completely from the caecal microbiota. The predominant recto-sigmoidal colonic microbial community consisted of strict anaerobic bacteria belonging to the C. coccoides group, the C. leptum subgroup and the Bacteroides group. Other predominant species varied between individuals (Hayashi et al., 2002a, 2003; Zoetendal et al., 1998). We previously reported inter-individual differences in the composition of faecal microbiota of healthy and elderly individuals (Hayashi et al., 2002a, 2003). Inter-individual differences were also observed in the jejunum, ileum and caecum.
`Gammaproteobacteria’ was detected at a high frequency in the recto-sigmoid colon in subjects A and B from the 16S rRNA gene libraries and by T-RFLP. Recently, we detected ‘Gammaproteobacteria’ in the elderly faecal microbiota (the highest percentage was 52.4 % of the total clones) (Hayashi et al., 2003). These organisms have also been detected in healthy elderly individuals by culture-based methods (Benno et al., 1989). Species belonging to ‘Gammaproteobacteria’ are also detected in the jejunum, ileum and caecum (Corrodi et al., 1978; Thadepalli et al., 1979; MacFarlane & Macfarlane, 2004), but are barely detectable in faecal material of healthy individuals (Hayashi et al., 2002a; Suau et al., 1999). The environment of the large bowel changes with age and ‘Gammaproteobacteria’ may inhabit the large bowel. When antibiotics are administered to humans, ‘Gammaproteobacteria’ increase in faeces (Högenauer et al., 1998; Young & Schmidt, 2004). Taken together, increased predominance of ‘Gammaproteobacteria’ may be an important marker of an altered large bowel environment.
Conclusions
In the present study, we characterized the jejunal, ileal, caecal and recto-sigmoidal colonic microbiota of three elderly individuals using both 16S rRNA gene libraries and T-RFLP. We demonstrated similarities and differences in microbiota composition among four different compartments of the human gut. The number of OTUs increased from the jejunum to recto-sigmoid colon. Approximately 10 OTUs were common to the caecum and recto-sigmoid colon. One common OTU (belonging to the facultative anaerobes) was seen in all four compartments. In addition, several common OTUs were found in the same compartment of all three individuals. Facultative anaerobes were the predominant species in jejunal and ileal microbiota. Although facultative anaerobes were also the predominant species in caecal microbiota, obligate aerobes belonging to the Bacteroides group, the C. coccoides group and the C. leptum subgroup were also present. On the other hand, obligate anaerobes of the Bacteroides group, the C. coccoides and the C. leptum subgroup were the predominant species among the recto-sigmoidal colonic microbiota. Major inter-individual differences in the composition of jejunal, ileal and caecal microbiota were evident as determined by molecular biological techniques.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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