A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community

Abstract

Background: Butyrate, which is produced by the human microbiome, is essential for a well-functioning colon. Bacteria that produce butyrate are phylogenetically diverse, which hinders their accurate detection based on conventional phylogenetic markers. As a result, reliable information on this important bacterial group is often lacking in microbiome research.

Results: In this study we describe a gene-targeted approach for 454 pyrotag sequencing and quantitative polymerase chain reaction for the final genes in the two primary bacterial butyrate synthesis pathways, butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). We monitored the establishment and early succession of butyrate-producing communities in four patients with ulcerative colitis who underwent a colectomy with ileal pouch anal anastomosis and compared it with three control samples from healthy colons. All patients established an abundant butyrate-producing community (approximately 5% to 26% of the total community) in the pouch within the 2-month study, but patterns were distinctive among individuals. Only one patient harbored a community profile similar to the healthy controls, in which there was a predominance of but genes that are similar to reference genes from Acidaminococcus sp., Eubacterium sp., Faecalibacterium prausnitzii and Roseburia sp., and an almost complete absence of buk genes. Two patients were greatly enriched in buk genes similar to those of Clostridium butyricum and C. perfringens, whereas a fourth patient displayed abundant communities containing both genes. Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community.

Conclusions: The presented approach provides quantitative and genotypic insights into butyrate-producing communities and facilitates a more specific functional characterization of the intestinal microbiome. Furthermore, our analysis refines but and buk reference annotations found in central databases.

Figure 1

Analysis of obtained butyryl-CoA:acetate CoA-transferase (but) sequences. (A) Neighbor joining tree of all but reference sequences (closest hit from FrameBot) matching our amplicon data. Amount of amplicon sequences per closest match and reference sequence GI number (GenBank) are provided. Sequences marked with * are derived from bacteria with known but activity [5,11]. Pie charts illustrate the origin of amplicon sequences (red: patient 200, green: patient 206, blue: patient 207, yellow: patient 210 and grey: healthy controls). Note: relative abundance was investigated and the proportion of each color in the pie charts does not correspond to actual abundance of genes in samples. Relative community patterns per individual sample are presented in Additional file 1: Figure S5. Bootstrap values are indicated. (B) Conservation analysis of reference sequences from Panel A (dashed line) and of obtained amplicon sequences (thick grey line). The displayed sequence on top corresponds to the consensus sequence of all reference sequences. Bold amino acids demonstrate conserved sites (>95;%) in both reference and amplicon sequences. Blue amino acids on both the sequence ends illustrate primer binding sites.

Figure 2

Analysis of obtained butyrate kinase (buk) sequences. (A) Neighbor joining tree of all buk reference sequences (closest hit from FrameBot) matching our amplicon data. Amount of amplicon sequences per closest match and reference sequence GI number (GenBank) are provided. Sequences marked with * or + highlight known butyrate producers [5,29]. Pie charts illustrate the origin of amplicon sequences (red: patient 200, green: patient 206, blue: patient 207, yellow: patient 210 and grey: healthy controls). Note: relative abundance was investigated and the proportion of each color in the pie charts does not correspond to actual abundance of genes in samples. Relative community patterns per individual sample are presented in Additional file 1: Figure S6. Bootstrap values are indicated. (B) Conservation analysis of reference sequences from Panel A (dashed line) and of obtained amplicon sequences (thick grey line). The displayed sequence on top corresponds to the consensus sequence of all reference sequences. Bold amino acids demonstrate conserved sites (>95%) in both reference and amplicon sequences. Blue amino acids on both sequence ends illustrate primer binding sites.

Figure 3

Nonmetric multidimensional scaling analysis of the total butyrate-producing community - butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk) genes together - based on individual patients. For explanation see text. Ellipses represent the 95% confidence interval on standard errors of means. Shepard plot for clustering is shown in Additional file 1: Figure S7.

Figure 4

Diversity analysis based on the Shannon index. The dotted line illustrates the average value of the three healthy control samples.

Figure 5

Quantitative PCR analysis of butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk) genes.But of Acidaminococcus sp. (light green bars), F. prausnitzii (purple bars) and Roseburia sp. /E. rectale (orange bars) as well as buk linked to C. butyricum, C. acetobutylicum and C. perfringens (white coarse bars) were targeted. Percentage was calculated based on total 16S rRNA gene qPCR data. Results were multiplied by five to account for multiple 16S rRNA gene copy numbers of intestinal bacteria. The error bars represent the range on duplicate measurements.

Figure 6

Exploring the butyrate-producing bacterial community based on 16S rRNA gene analysis. (A) Candidates were split into butyryl-CoA:acetate CoA-transferase (but; grey bars) and butyrate kinase (buk; coarse white bars) containing groups. (B) Individual composition of but (Acidaminococcus sp. - olive, Anaerostipes sp. - dark green, Coprococcus sp. - dark red, Eubacterium sp. - black, Faecalibacterium sp. - dark purple, Megasphaera sp. - light purple, Peptoniphilus sp. - blue, Oscillibacter sp. - grey and Roseburia sp. - orange) and buk (Anaerotruncus - coarse grey, C. beijerinckii - coarse white, C. butyricum - coarse light blue, C. perfringens - coarse light yellow, Enterococcus sp. - light green and Subdoligranulum sp. - coarse light red) communities are given. (C) Quantitative PCR data targeting the 16S genes of Faecalibacterium sp. (purple bar), Roseburia sp./E. rectale (orange bar) and C. butyricum (white coarse bar). Note: Coprococcus sp. is considered to contain both but and buk genes. The error bars represent the range on duplicate measurements. All results are corrected for multiple 16S rRNA copy numbers of individual bacteria (see Methods). ? - Butyrate production was shown for one strain of Subdoligranulum sp. and it is unclear whether all members of this genus have the ability to synthesize butyrate.