Abstract

The bacterial species diversity of three colonic tissue samples from elderly people was investigated by sequence analysis of randomly cloned eubacterial 16S rDNA. The majority of sequences (87%) clustered within three bacterial groups: (1) Bacteroides; (2) low G+C content Gram-positives related to Clostridium coccoides (cluster XIVa); (3) Gram-positives related to Clostridium leptum (cluster IV). These groups have been shown to dominate the human faecal flora. Only 25% of sequences were closely related (>97%) to current species type strains, and 28% were less than 97% related to any database entry. 19% of sequences were most closely related to recently isolated butyrate-producing bacteria belonging to clusters XIVa and IV, with a further 18% of the sequences most closely related to Ruminococcus obeum and Ruminococcus torques (members of cluster XIVa). These results provide the first molecular information on the microbial diversity present in human colonic samples.

Introduction

The human colon harbours a highly diverse microbial ecosystem [1]. Microbial metabolic activity in the gut has important consequences for health through the supply of nutrients including vitamins and short chain fatty acids to the host tissues (reviewed in [2–4)]. Additionally, the commensal microflora provides a natural defense mechanism against invading pathogens [5,6] and interacts at several levels with the intestinal epithelium and immune system [7–9]. However, our understanding of this complex bacterial community and its interactions with the host is still far from complete.

Molecular techniques provide the most powerful tools available for revealing phylogenetic diversity of microorganisms within complex ecosystems independent of cultural bias [10,14]. Recent investigations of 16S rDNA sequences have produced important insights into the diversity of the human faecal flora, showing that Bacteroides species and bacteria belonging to the Clostridium coccoides/cluster XIVa and Clostridium leptum/cluster IV groups are major constituents of the human faecal microflora [15–18]. This work has also shown that as many as 76% of randomly cloned sequences share less than 97% identity with known cultured bacterial strains [16,17,19].

Almost all of the current information concerning microbial diversity in the human intestinal tract, however, is based on faecal samples. One previous study identified the predominant culturable bacteria associated in vivo with human colonic wall tissue as Bacteroides and Fusobacterium spp. [20]. However, it was noted that diversity revealed through culturing was less than that observed through microscopic analysis.

The work reported here is the first attempt to identify the main types of bacteria associated with human colon tissue by direct retrieval and analysis of SSU (small subunit) rDNA sequences.

Materials and methods

DNA extraction and PCR amplification of colonic tissue samples

Three specimens of normal colonic tissue were analysed, one from a sudden death victim (male aged 70) and two from live subjects who had undergone colon resections (male aged 79 – sigmoid colon, female aged 82 – right colon). Samples were received 10 min after surgical removal and were snap frozen in liquid nitrogen prior to placing at −80°C. A small (1 g) section of each tissue was thawed on ice and thoroughly washed in sterile distilled water prior to analysis to remove luminal contents, therefore it was assumed that all bacteria remaining were associated either with the colonic tissue or with the mucus. For DNA extractions, colonic samples were resuspended in 1 ml of sterile distilled water with the addition of sterile zirconium beads (0.1 mm diameter, Sigma, Dorset, UK). Samples were then beaten for 30 s using a minibead beater (Biospec Corp., Stratech Scientific, UK). DNA suitable for PCR amplification was extracted by a modification of the method of Stahl et al. [21] (as described previously by Pryde et al. [22]). 16S rDNA was amplified using the universal eubacterial primers fD1 5′-AGAGTTTGATCCTGGCTCAG-3′ (Escherichia coli positions 8–27) and rP2 5′-ACGGCTACCTTGTTACGACTT-3′ (E. coli positions 1494–1513) [23], yielding a product of approximately 1500 bp. PCR amplifications were performed using the following conditions: initial denaturation of template DNA at 94°C for 5 min; then 20 cycles consisting of denaturation (2 min at 94°C), annealing (30 s at 57°C), extension (2 min at 72°C), and a final extension at 72°C for 10 min. PCR amplification was limited to 20 cycles in order to minimise possible bias. PCR amplicons were purified using the Wizard PCR product purification kit (Promega, Southampton, UK) and then cloned into a pGEM-T vector plasmid (Promega, Southampton, UK). Insert DNA was sequenced in both directions using universal 16S rRNA primers [24] on a 377 automated DNA sequencer.

Phylogenetic analysis

Sequences were analysed by Blast [25] against 16S rDNA sequences from GenBank [26] and the ribosomal database project [27]. Phylogenetic trees were generated by the neighbour-joining method [28], via the PHYLIP package [29] using DNADIST for distance analysis [30]. Bootstrap resampling (data resampled 100 times) used the SEQBOOT program and consensus trees were generated by the CONSENSE program [31].

Nucleotide sequence accession numbers

The 16S rDNA sequences were deposited in the EMBL data library under accession numbers AJ408957–AJ409009 and AJ315481–AJ315487.

Results

16S rDNA sequence diversity of human colonic tissue samples

A total of 110 clones from the three colonic tissue samples were analysed, 34 from colonic sample 1 (HuCA), 39 clones from colonic sample 2 (HuCB) and 37 clones from colonic sample 3 (HuCC). All clones from colonic samples 1 and 2 were subjected to full-length 16S rDNA sequencing with no chimeric clones found using the program CHECK-CHIMERA [32]. However, clones from colonic sample 3 were initially partially sequenced in order to infer phylogenetic affiliation, with full-length sequencing performed on clones which showed closest 16S rDNA sequence similarity to butyrate-producing bacteria [33] or <97% 16S rDNA sequence relatedness to database entries. Therefore, full-length 16S rDNA sequences were obtained for eight clones from colonic sample 3, namely HuCC2, 13, 15, 28, 30, 33, 34, and 43 (Table 1).

1

Percentage sequence identity between random 16S rDNA clones generated from colonic tissue sample sequences present in GenBank

Clone ID Most closely related type strain sequence (GenBank accession no.) Sequence identity (%) Most closely related database sequence (GenBank accession no.) Sequence identity (%) No. of identical clones 
Colon sample 1 
Gram-positive (cluster XIVa)a 
HuCA2 X85101 Ruminococcus obeum 94 AF132243 uncultured bacterium adhufec171 99 
HuCA5 X85101 Ruminococcus obeum 95 b – 
HuCA8 AF202259 Eubacterium oxidoreducens 95 BBA270474 butyrate-producing bacterium L1-83 99 
HuCA13 AF202259 Eubacterium oxidoreducens 94 AB034123 uncultured rumen bacterium 4C28d-8 99 
HuCA15 L34621 Eubacterium halii 97 BBA270490 butyrate-producing bacterium L2-7 99 
HuCA17 Y18184 Clostridium indolis 94 AF052421 uncultured bacterium AZ54 97 
HuCA19 X94966 Ruminococcus productus 92 AF132267 uncultured bacterium adhufec382 95 
HuCA20 Y18184 Clostridium indolis 94 AF132254 uncultured bacterium adhufec25 97 
HuCA22 X85101 Ruminococcus obeum 95 – – 
HuCA23 AF202259 Eubacterium oxidoreducens 94 AF132248 uncultured bacterium adhufec225 97 
HuCA26 X85101 Ruminococcus obeum 94 – – 
HuCA27 M59089 Clostridium clostridiiformes 93 AF052421 uncultured bacterium AZ54 95 
HuCA28 AF202259 Eubacterium oxidoreducens 94 AB034123 uncultured rumen bacterium 4C28d-8 99 
HuCA29 AJ011522 Eubacterium ramulus 99 AF132260 uncultured bacterium adhufec310 99 
HuCA40 L34420 Eubacterium eligens 98 – – 
Low G+C Gram-positive (cluster IV) 
HuCA1 L76596 Ruminococcus callidus 94 – – 
HuCA10 X85022 Fusobacterium prausnitzii 98 UBU270469 butyrate-producing bacterium A2-165 99 
HuCA11 X85022 Fusobacterium prausnitzii 97 UBU270469 butyrate-producing bacterium A2-165 97 
HuCA24 X85099 Ruminococcus bromii 89 AF018544 unidentified rumen bacterium 92 
HuCA25 X85022 Fusobacterium prausnitzii 97 UBU270469 butyrate-producing bacterium A2-165 97 
Bacteroides CFB 
HuCA7 L16497 Bacteroides putredenis 97 AF132279 uncultured bacterium adhufec73 99 
HuCA9 M58762 Bacteroides vulgatus 96 AF132256 uncultured bacterium adhufec27 97 
HuCA21 L16489 Bacteroides thetaiotaomicron 94 BS16SRNAR Bacteroides species 99 
HuCA33 M11656 Bacteroides fragilis 99 – – 
HuCA34 L16484 Bacteroides ovatus 95 AF139525 Bacteroides species 96 
HuCA36 M58762 Bacteroides vulgatus 96 – – 
Low G+C Gram-positive (cluster XVIII) 
HuCA3 Y10164 Dehalobacter restrictus 93 AB034023 uncultured rumen bacterium 4C0d-10 94 
HuCA6 Y10164 Dehalobacter restrictus 93 AB034023 uncultured rumen bacterium 4C0d-10 94 
β-Proteobacteria 
HuCA4 AF244133 Burkholderia cepacia 90 AF232922 uncultured bacterium MS8 91 
γ-Proteobacteria 
HuCA37 AE000474 Escherichia coli 99 – – 
Verrucomicrobiales 
HuCA18 X90515 Verrucomicrobium spinosum 92 UBA400275 uncultured bacterium L10-6 99 
Colon sample 2 
Low G+C Gram-positive (cluster XIVa) 
HuCB1 AF202259 Eubacterium oxidoreducens 94 BBA270479 butyrate-producing bacterium L1-952 99 
HuCB10 L76604 Ruminococcus torques 99 – – 
HuCB12 X85101 Ruminococcus obeum 96 BBA270483 butyrate-producing bacterium T2-132 98 
HuCB14 L34627 Eubacterium rectale 97 BBA270475 butyrate-producing bacterium A1-86 99 
HuCB21 L34619 Eubacterium formicigenerans 97 – – 
HuCB25 X85101 Ruminococcus obeum 94 RSPBIE16 Ruminococcus species 94 
HuCB26 L34621 Eubacterium halii 97 BBA270490 butyrate-producing bacterium L2-7 97 
HuCB37 X94967 Ruminococcus gnavus 93 BBA270473 butyrate-producing bacterium A2-194 99 
HuCB40 X71855 Clostridium xylanolyticum 94 AF132269 uncultured bacterium adhufec406 99 
HuCB56 L34628 Eubacterium xylanophilum 94 – – 
Gram-positive (cluster IV) 
HuCB2 X85099 Ruminococcus bromii 96 – – 
HuCB5 AF030446 Ruminococcus flavefaciens 91 AF052408 uncultured bacterium AZ03 97 
HuCB7 AF167711 Papillibacter cinnaminovorans 92 AB034125 uncultured rumen bacterium 4C28d-4 95 
HuCB24 Y18187 Clostridium orbiscindens 95 AF157051 bacterium ASF500 95 
HuCB29 X85022 Fusobacterium prausnitzii 98 UBU270469 butyrate-producing bacterium A2-165 98 
Bacteroides CFB 
HuCB3 M58762 Bacteroides vulgatus 97 – – 
HuCB6 M58762 Bacteroides vulgatus 98 AF132256 uncultured bacterium adhufec27 99 
HuCB23 L16497 Bacteroides putredenis 94 AF132279 uncultured bacterium adhufec73 96 
Low G+C Gram-positive (cluster I) 
HuCB15 Y18176 Clostridium disporicum 97 – – 
Low G+C Gram-positive (cluster IX) 
HuCB85 AF283705 Megasphaera elsdenii 96 – – 
Low G+C Gram-positive (cluster XI) 
HuCB31 X76750 Clostridium glycolicum 98 UBA404682 unidentified bacterium ZF5 98 
γ-Proteobacteria 
HuCB27 AE000474 Escherichia coli 99 – – 
Colon sample 3 
Low G+C Gram-positive (cluster XIVa)a 
HuCC4 X85101 Ruminococcus obeum 96 AF153854 uncultured bacterium adhufec30.25 97 
HuCC8 L34619 Eubacterium formicigenerans 97 – – 
HuCC9 L76604 Ruminococcus torques 100 – – 
HuCC15 L34621 Eubacterium halii 95 BBA270490 butyrate-producing bacterium L2-7 98 
HuCC18 L76604 Ruminococcus torques 97 AF376447 uncultured bacterium ckncm214-F4M.1G5 98 
HuCC19 X85101 Ruminococcus obeum 94 AF153854 uncultured bacterium adhufec30.25 97 
HuCC21 X85101 Ruminococcus obeum 94 AF253375 uncultured bacterium L127dB 98 
HuCC22 M59089 Clostridium clostridiiformes 99 – – 
HuCC23 M59112 Clostridium symbiosum 98 – – 
HuCC27 L76604 Ruminococcus torques 99 – – 
HuCC34 M59089 Clostridium clostridiiformes 95 CS16SDR6A Clostridium sp. strain DR6A 96 
HuCC43 X94967 Ruminococcus gnavus 94 BBA270484 butyrate-producing bacterium A2-231 94 
Gram-positive (cluster IV) 
HuCC10 L34618 Eubacterium desmolans 96 AF132258 uncultured bacterium adhufec296 99 
HuCC26 Y18187 Clostridium orbiscindens 99 – – 
HuCC32 X85022 Fusobacterium prausnitzii 93 AF132237 uncultured bacterium adhufec13 99 
Bacteroides CFB 
HuCC1 AB050110 Bacteroides uniformis 99 – – 
HuCC2 M58762 Bacteroides vulgatus 93 – – 
HuCC3 M86695 Bacteroides distasonis 97 AF376376 uncultured bacterium ckncm143-F1M.1E3 98 
HuCC11 M58762 Bacteroides vulgatus 98 – – 
HuCC12 X83951 Bacteroides caccae 98 AF132273 uncultured bacterium adhufec51 99 
HuCC17 X83951 Bacteroides caccae 95 AF132273 uncultured bacterium adhufec51 96 
HuCC20 L16484 Bacteroides ovatus 90 AF153865 uncultured bacterium adhufec77.25 98 
HuCC28 AJ005635 Prevotella enoeca 89 AB009238 unidentified rumen bacterium RFN91 89 
HuCC30 L16489 Bacteroides thetaiotaomicron 96 AF139525 Bacteroides sp. AR29 96 
HuCC35 X83951 Bacteroides caccae 97 AF132273 uncultured bacterium adhufec51 98 
β-Proteobacteria 
HuCC33 AF244133 Burkholderia cepacia 89 AF236011 β-proteobacterium A0823 90 
Verrucomicrobiales 
HuCC13 X90515 Verrucomicrobrium spinosum 92 UBA400275 uncultured bacterium L10-6 99 
HuCC16 AF217461 Candidatus Xiphinematobacter rivesi 88 UBA400275 uncultured bacterium L10-6 99 
Clone ID Most closely related type strain sequence (GenBank accession no.) Sequence identity (%) Most closely related database sequence (GenBank accession no.) Sequence identity (%) No. of identical clones 
Colon sample 1 
Gram-positive (cluster XIVa)a 
HuCA2 X85101 Ruminococcus obeum 94 AF132243 uncultured bacterium adhufec171 99 
HuCA5 X85101 Ruminococcus obeum 95 b – 
HuCA8 AF202259 Eubacterium oxidoreducens 95 BBA270474 butyrate-producing bacterium L1-83 99 
HuCA13 AF202259 Eubacterium oxidoreducens 94 AB034123 uncultured rumen bacterium 4C28d-8 99 
HuCA15 L34621 Eubacterium halii 97 BBA270490 butyrate-producing bacterium L2-7 99 
HuCA17 Y18184 Clostridium indolis 94 AF052421 uncultured bacterium AZ54 97 
HuCA19 X94966 Ruminococcus productus 92 AF132267 uncultured bacterium adhufec382 95 
HuCA20 Y18184 Clostridium indolis 94 AF132254 uncultured bacterium adhufec25 97 
HuCA22 X85101 Ruminococcus obeum 95 – – 
HuCA23 AF202259 Eubacterium oxidoreducens 94 AF132248 uncultured bacterium adhufec225 97 
HuCA26 X85101 Ruminococcus obeum 94 – – 
HuCA27 M59089 Clostridium clostridiiformes 93 AF052421 uncultured bacterium AZ54 95 
HuCA28 AF202259 Eubacterium oxidoreducens 94 AB034123 uncultured rumen bacterium 4C28d-8 99 
HuCA29 AJ011522 Eubacterium ramulus 99 AF132260 uncultured bacterium adhufec310 99 
HuCA40 L34420 Eubacterium eligens 98 – – 
Low G+C Gram-positive (cluster IV) 
HuCA1 L76596 Ruminococcus callidus 94 – – 
HuCA10 X85022 Fusobacterium prausnitzii 98 UBU270469 butyrate-producing bacterium A2-165 99 
HuCA11 X85022 Fusobacterium prausnitzii 97 UBU270469 butyrate-producing bacterium A2-165 97 
HuCA24 X85099 Ruminococcus bromii 89 AF018544 unidentified rumen bacterium 92 
HuCA25 X85022 Fusobacterium prausnitzii 97 UBU270469 butyrate-producing bacterium A2-165 97 
Bacteroides CFB 
HuCA7 L16497 Bacteroides putredenis 97 AF132279 uncultured bacterium adhufec73 99 
HuCA9 M58762 Bacteroides vulgatus 96 AF132256 uncultured bacterium adhufec27 97 
HuCA21 L16489 Bacteroides thetaiotaomicron 94 BS16SRNAR Bacteroides species 99 
HuCA33 M11656 Bacteroides fragilis 99 – – 
HuCA34 L16484 Bacteroides ovatus 95 AF139525 Bacteroides species 96 
HuCA36 M58762 Bacteroides vulgatus 96 – – 
Low G+C Gram-positive (cluster XVIII) 
HuCA3 Y10164 Dehalobacter restrictus 93 AB034023 uncultured rumen bacterium 4C0d-10 94 
HuCA6 Y10164 Dehalobacter restrictus 93 AB034023 uncultured rumen bacterium 4C0d-10 94 
β-Proteobacteria 
HuCA4 AF244133 Burkholderia cepacia 90 AF232922 uncultured bacterium MS8 91 
γ-Proteobacteria 
HuCA37 AE000474 Escherichia coli 99 – – 
Verrucomicrobiales 
HuCA18 X90515 Verrucomicrobium spinosum 92 UBA400275 uncultured bacterium L10-6 99 
Colon sample 2 
Low G+C Gram-positive (cluster XIVa) 
HuCB1 AF202259 Eubacterium oxidoreducens 94 BBA270479 butyrate-producing bacterium L1-952 99 
HuCB10 L76604 Ruminococcus torques 99 – – 
HuCB12 X85101 Ruminococcus obeum 96 BBA270483 butyrate-producing bacterium T2-132 98 
HuCB14 L34627 Eubacterium rectale 97 BBA270475 butyrate-producing bacterium A1-86 99 
HuCB21 L34619 Eubacterium formicigenerans 97 – – 
HuCB25 X85101 Ruminococcus obeum 94 RSPBIE16 Ruminococcus species 94 
HuCB26 L34621 Eubacterium halii 97 BBA270490 butyrate-producing bacterium L2-7 97 
HuCB37 X94967 Ruminococcus gnavus 93 BBA270473 butyrate-producing bacterium A2-194 99 
HuCB40 X71855 Clostridium xylanolyticum 94 AF132269 uncultured bacterium adhufec406 99 
HuCB56 L34628 Eubacterium xylanophilum 94 – – 
Gram-positive (cluster IV) 
HuCB2 X85099 Ruminococcus bromii 96 – – 
HuCB5 AF030446 Ruminococcus flavefaciens 91 AF052408 uncultured bacterium AZ03 97 
HuCB7 AF167711 Papillibacter cinnaminovorans 92 AB034125 uncultured rumen bacterium 4C28d-4 95 
HuCB24 Y18187 Clostridium orbiscindens 95 AF157051 bacterium ASF500 95 
HuCB29 X85022 Fusobacterium prausnitzii 98 UBU270469 butyrate-producing bacterium A2-165 98 
Bacteroides CFB 
HuCB3 M58762 Bacteroides vulgatus 97 – – 
HuCB6 M58762 Bacteroides vulgatus 98 AF132256 uncultured bacterium adhufec27 99 
HuCB23 L16497 Bacteroides putredenis 94 AF132279 uncultured bacterium adhufec73 96 
Low G+C Gram-positive (cluster I) 
HuCB15 Y18176 Clostridium disporicum 97 – – 
Low G+C Gram-positive (cluster IX) 
HuCB85 AF283705 Megasphaera elsdenii 96 – – 
Low G+C Gram-positive (cluster XI) 
HuCB31 X76750 Clostridium glycolicum 98 UBA404682 unidentified bacterium ZF5 98 
γ-Proteobacteria 
HuCB27 AE000474 Escherichia coli 99 – – 
Colon sample 3 
Low G+C Gram-positive (cluster XIVa)a 
HuCC4 X85101 Ruminococcus obeum 96 AF153854 uncultured bacterium adhufec30.25 97 
HuCC8 L34619 Eubacterium formicigenerans 97 – – 
HuCC9 L76604 Ruminococcus torques 100 – – 
HuCC15 L34621 Eubacterium halii 95 BBA270490 butyrate-producing bacterium L2-7 98 
HuCC18 L76604 Ruminococcus torques 97 AF376447 uncultured bacterium ckncm214-F4M.1G5 98 
HuCC19 X85101 Ruminococcus obeum 94 AF153854 uncultured bacterium adhufec30.25 97 
HuCC21 X85101 Ruminococcus obeum 94 AF253375 uncultured bacterium L127dB 98 
HuCC22 M59089 Clostridium clostridiiformes 99 – – 
HuCC23 M59112 Clostridium symbiosum 98 – – 
HuCC27 L76604 Ruminococcus torques 99 – – 
HuCC34 M59089 Clostridium clostridiiformes 95 CS16SDR6A Clostridium sp. strain DR6A 96 
HuCC43 X94967 Ruminococcus gnavus 94 BBA270484 butyrate-producing bacterium A2-231 94 
Gram-positive (cluster IV) 
HuCC10 L34618 Eubacterium desmolans 96 AF132258 uncultured bacterium adhufec296 99 
HuCC26 Y18187 Clostridium orbiscindens 99 – – 
HuCC32 X85022 Fusobacterium prausnitzii 93 AF132237 uncultured bacterium adhufec13 99 
Bacteroides CFB 
HuCC1 AB050110 Bacteroides uniformis 99 – – 
HuCC2 M58762 Bacteroides vulgatus 93 – – 
HuCC3 M86695 Bacteroides distasonis 97 AF376376 uncultured bacterium ckncm143-F1M.1E3 98 
HuCC11 M58762 Bacteroides vulgatus 98 – – 
HuCC12 X83951 Bacteroides caccae 98 AF132273 uncultured bacterium adhufec51 99 
HuCC17 X83951 Bacteroides caccae 95 AF132273 uncultured bacterium adhufec51 96 
HuCC20 L16484 Bacteroides ovatus 90 AF153865 uncultured bacterium adhufec77.25 98 
HuCC28 AJ005635 Prevotella enoeca 89 AB009238 unidentified rumen bacterium RFN91 89 
HuCC30 L16489 Bacteroides thetaiotaomicron 96 AF139525 Bacteroides sp. AR29 96 
HuCC35 X83951 Bacteroides caccae 97 AF132273 uncultured bacterium adhufec51 98 
β-Proteobacteria 
HuCC33 AF244133 Burkholderia cepacia 89 AF236011 β-proteobacterium A0823 90 
Verrucomicrobiales 
HuCC13 X90515 Verrucomicrobrium spinosum 92 UBA400275 uncultured bacterium L10-6 99 
HuCC16 AF217461 Candidatus Xiphinematobacter rivesi 88 UBA400275 uncultured bacterium L10-6 99 
a

a Roman numerals indicate phylogenetic cluster of Clostridaceae as defined in [38].

b

b Indicates type strain 16S rDNA sequence was the closest known relative.

Fifty-one of the 110 16S rRNA gene sequences (46%) fell within the C. coccoides group (Gram-positive cluster XIVa) (Table 1; Fig. 1). When compared between colonic samples, cluster XIVa sequences accounted for between 43 and 49% of the total sequence diversity (Table 2). Only 16 of the 51 sequences within this cluster showed closest resemblance to current species type strains, although in four cases sequence identity was below 97%. On the other hand, 16 sequences showed their closest relationships (97–99%) to butyrate-producing isolates from human faeces such as A2-194, A1-86 and L1-82 (loosely related to Eubacterium ramulus, Eubacterium rectale and Roseburia cecicola) and L2-7 (related to Eubacterium halii) [33] (Fig. 1). The remaining 19 sequences most closely resembled faecal bacterial 16S rDNA clones (prefixed ‘hufec’ or ‘AZ’ respectively) reported by Suau et al. [16] or by Zoetendal et al. [18], or in a few cases cloned sequences of ruminal origin, rather than cultured bacteria.

1

Phylogenetic tree showing the relationships of 16S rDNA sequences (random clones) isolated from three human colonic tissues (prefixed – HuCA, HuCB and HuCC) defined as low G+C Gram-positive bacteria and located within clusters IV, XI, XIVa and XVIII (as defined by Collins et al. [38]). Figures in brackets represent the number of clones with identical sequence data. The scale bar represents genetic distance (10 substitutions per 100 nucleotides). The tree was constructed using the neighbour-joining analysis of a distance matrix obtained from a multiple-sequence alignment. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points: values of 97% or more were considered significant. Sequences derived from the database are shown in italics (e.g. E. rectale). E. coli was used as the outgroup sequence.

1

Phylogenetic tree showing the relationships of 16S rDNA sequences (random clones) isolated from three human colonic tissues (prefixed – HuCA, HuCB and HuCC) defined as low G+C Gram-positive bacteria and located within clusters IV, XI, XIVa and XVIII (as defined by Collins et al. [38]). Figures in brackets represent the number of clones with identical sequence data. The scale bar represents genetic distance (10 substitutions per 100 nucleotides). The tree was constructed using the neighbour-joining analysis of a distance matrix obtained from a multiple-sequence alignment. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points: values of 97% or more were considered significant. Sequences derived from the database are shown in italics (e.g. E. rectale). E. coli was used as the outgroup sequence.

2

Comparison of the percentage of clones attributed to the various phylogenetic affiliations within the three colonic tissue samples

Phylogenetic affiliation Colonic tissue sample 
 
XIVaa 47 48.7 43.3 
IV 14.7 17.9 10.8 
CFB 23.5 20.5 35.1 
Other Clostridium clusters 5.8 10.4 
Other sequences 2.5 10.8 
Phylogenetic affiliation Colonic tissue sample 
 
XIVaa 47 48.7 43.3 
IV 14.7 17.9 10.8 
CFB 23.5 20.5 35.1 
Other Clostridium clusters 5.8 10.4 
Other sequences 2.5 10.8 
a

a Roman numerals indicate phylogenetic cluster of Clostridium as defined in [38].

14.5% (16/110) of the sequences were located within the C. leptum group (cluster IV; Fig. 1) although when compared as individual clone libraries, this figure ranged between 11 and 18% (Table 2). Six sequences were related to type strains of Ruminococcus, while four showed their strongest sequence similarity to the butyrate-producing bacterial isolate A2-165 [33] which is related to Fusobacterium prausnitzii (Fig. 1; Table 1). Of the remaining six sequences, five branched deeply with sequences from random cloning studies and one (HuCC26) showed strong 16S rDNA sequence similarity (>99%) to Clostridium orbiscindens (Table 1).

26% (29/110) of the sequences clustered within the Bacteroides group (Table 1) with values ranging between 20 and 35% within the individual sample sets (Table 2). The majority of sequences within this group were closely related to known type strains including Bacteroides vulgatus and Bacteroides uniformis, however one clone from colonic sample 3 (HuCC28) showed closest 16S rDNA sequence similarity (although <90%) to Prevotella enoeca. Many of the clones within the Cytophaga/Flavobacter/Bacteroides (CFB) phylum also showed strong sequence similarity with cloned ‘hufec’ sequences (Table 1).

The remaining 16S rRNA sequences comprised members of other groups of low G+C bacteria (clusters I, IX, XI and XVIII – six sequences), four Verrucomicrobiales-related sequences, two β-proteobacteria (<90% sequence similarity), and two γ-proteobacteria (related to E. coli) (Table 1; Table 2).

Discussion

The complex microbial ecosystem of the human colon plays a key role in human nutrition and health, and a clearer understanding of the physiology, abundance and location of the dominant colonic bacteria is essential. Overall between 85 and 89% (depending on the colonic sample) of the 16S rDNA sequences obtained here fell within three major phylogenetic groups (Bacteroides, C. coccoides/cluster XIVa and C. leptum/cluster IV) that have been shown to dominate the faecal flora [16]. A qualified comparison can be made between the data obtained here from colonic tissue of three elderly subjects with that of Suau et al. from faecal material from a 40-year-old subject [16]. The percentages of clones present in cluster XIVa are 43–49% (colonic-present study) and 44% (faecal study [16]), for cluster IV 11–18% (colonic) and 20% (faecal), and for the CFB group 20–35% (colonic) and 31% (faecal). Recent work by Sghir et al. [34] has indicated that the CFB bacterial component of the faecal flora can vary from 20 to 52% between individuals. Therefore, our results do not suggest any major discrepancy between the bacterial composition of colonic and faecal samples in terms of the major bacterial groups. Bifidobacterial sequences were not detected here in colonic samples, or previously in faecal samples [16] although detectable by fluorescent in situ hybridisation at around 3% of the total bacterial population [15]. We cannot exclude the possibility of PCR bias against this group.

Gut epithelial cells turn over rapidly and there is a continual passage of mucus and sloughed-off cell debris down the digestive tract together with the digesta. Since amplifiable DNA can be retained in dead or quiescent bacterial cells, it is quite likely that most of the diversity present in the large intestinal microflora will be present in freshly voided faeces. On the other hand, it does not follow that the microbial DNA present in faeces will accurately reflect the relative proportions of bacteria present at any given site in the colon.

Our results do not rule out differences in the actual species present within the major groups. For example, more of the sequences from the current study clustered with Ruminococcus obeum and Ruminococcus torques compared with previous data from faecal samples. We cannot say whether this reflects a systematic difference between faecal material and colonic tissue, or differences between the subjects sampled. It is worth noting, however, that R. torques is reported to be a prominent mucin-degrading species [35].

Despite recent efforts to define the microbial diversity of the gastro-intestinal tract through random cloning and sequencing of 16S rRNA genes, the present study still revealed that 28% of sequences recovered were less than 97% related to any database entry. This indicates that our knowledge of bacterial diversity in the colon is still far from being exhaustive. Perhaps one of the most significant observations made here is the close relationship (>97% sequence identity) of 21 out of the 110 of the colonic 16S rDNA sequences with butyrate-producing strains isolated recently from human faeces [33]. Previous cultural studies have often reported an apparent deficit of butyrate-producing isolates, which have been suggested to represent as little as 1% of the cultivable flora [36,37]. The present findings support the view that 16S rRNA gene sequences closely related to those identified as butyrate producers by Barcenilla et al. [33] are abundant in the human colonic microflora, but may often be underestimated by cultural approaches, although it is not known whether the sequences detected here represent butyrate-producing bacteria or non-butyrate-producing relatives. This work emphasises the potential importance within the colon of Gram-positive bacteria belonging to clostridial clusters IV and XIVa and the need for more detailed research on the diversity and physiology of cultured strains.

Acknowledgements

We thank Prof. Charles Campbell and Prof. George MacFarlane for kindly providing the colonic tissue samples and Moira Johnston and Pauline Young for automated DNA sequencing. This work was supported by SEERAD (Scottish Executive Environment Rural Affairs Department).

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Author notes

Present address:Food Standards Agency, St Magnus House, Guild Street, Aberdeen AB11 6NJ, UK.