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

Vital M, Penton CR, Wang Q, Young VB, Antonopoulos DA, Sogin ML, Morrison HG, Raffals L, Chang EB, Huffnagle GB, Schmidt TM, Cole JR, Tiedje JM - Microbiome (2013)

Bottom Line: As a result, reliable information on this important bacterial group is often lacking in microbiome research.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.Furthermore, our analysis refines but and buk reference annotations found in central databases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA. colej@msu.edu.

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.

No MeSH data available.


Related in: MedlinePlus

Quantitative PCR analysis of butyryl-CoA:acetate CoA-transferase(but) and butyrate kinase(buk) genes.But ofAcidaminococcus sp. (light green bars), F. prausnitzii(purple bars) and Roseburia sp. /E. rectale (orange bars) aswell as buk linked to C. butyricum, C. acetobutylicum andC. perfringens (white coarse bars) were targeted. Percentage wascalculated based on total 16S rRNA gene qPCR data. Results weremultiplied by five to account for multiple 16S rRNA gene copy numbersof intestinal bacteria. The error bars represent the range on duplicatemeasurements.
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Figure 5: Quantitative PCR analysis of butyryl-CoA:acetate CoA-transferase(but) and butyrate kinase(buk) genes.But ofAcidaminococcus sp. (light green bars), F. prausnitzii(purple bars) and Roseburia sp. /E. rectale (orange bars) aswell as buk linked to C. butyricum, C. acetobutylicum andC. perfringens (white coarse bars) were targeted. Percentage wascalculated based on total 16S rRNA gene qPCR data. Results weremultiplied by five to account for multiple 16S rRNA gene copy numbersof intestinal bacteria. The error bars represent the range on duplicatemeasurements.

Mentions: Functional gene pyro-sequencing only allows for relative abundance measurements ineach sample. Hence, we developed a complementary qPCR approach. Clear patternsemerged from qPCR of the buk and but genes. Whereas no target genescould be detected in the luminal aspirate before ileostomy takedown (visit 1; nosample was available for patient 200), abundant butyrate-producing communities becameestablished in all patients over time (Figure 5). Theobserved community profiles were distinct between patients. Patients 206 and 207 wereenriched in buk genes (up to 19.9% of the total community), whereasbut was almost absent. Patient 210 was unique in the development of acommunity similar to the healthy controls, harboring but genes most closelyrelated to both F. prausnitzii and Roseburia sp./E.rectale. Additionally, this patient exhibited abundant but-carryingAcidaminococcus sp. communities, which were absent in the healthy controlsamples. At visits three and four, 15.5% and 26% of patient 210’s totalmicrobial community exhibited but genes, which was within the broad rangefor the control samples with 4.4%, 2.6% and 74.1%, respectively. In patient 200, weinitially detected only buk genes, but a considerable but-genecommunity linked to Roseburia sp./E. rectale was established overtime as well.


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

Vital M, Penton CR, Wang Q, Young VB, Antonopoulos DA, Sogin ML, Morrison HG, Raffals L, Chang EB, Huffnagle GB, Schmidt TM, Cole JR, Tiedje JM - Microbiome (2013)

Quantitative PCR analysis of butyryl-CoA:acetate CoA-transferase(but) and butyrate kinase(buk) genes.But ofAcidaminococcus sp. (light green bars), F. prausnitzii(purple bars) and Roseburia sp. /E. rectale (orange bars) aswell as buk linked to C. butyricum, C. acetobutylicum andC. perfringens (white coarse bars) were targeted. Percentage wascalculated based on total 16S rRNA gene qPCR data. Results weremultiplied by five to account for multiple 16S rRNA gene copy numbersof intestinal bacteria. The error bars represent the range on duplicatemeasurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126176&req=5

Figure 5: Quantitative PCR analysis of butyryl-CoA:acetate CoA-transferase(but) and butyrate kinase(buk) genes.But ofAcidaminococcus sp. (light green bars), F. prausnitzii(purple bars) and Roseburia sp. /E. rectale (orange bars) aswell as buk linked to C. butyricum, C. acetobutylicum andC. perfringens (white coarse bars) were targeted. Percentage wascalculated based on total 16S rRNA gene qPCR data. Results weremultiplied by five to account for multiple 16S rRNA gene copy numbersof intestinal bacteria. The error bars represent the range on duplicatemeasurements.
Mentions: Functional gene pyro-sequencing only allows for relative abundance measurements ineach sample. Hence, we developed a complementary qPCR approach. Clear patternsemerged from qPCR of the buk and but genes. Whereas no target genescould be detected in the luminal aspirate before ileostomy takedown (visit 1; nosample was available for patient 200), abundant butyrate-producing communities becameestablished in all patients over time (Figure 5). Theobserved community profiles were distinct between patients. Patients 206 and 207 wereenriched in buk genes (up to 19.9% of the total community), whereasbut was almost absent. Patient 210 was unique in the development of acommunity similar to the healthy controls, harboring but genes most closelyrelated to both F. prausnitzii and Roseburia sp./E.rectale. Additionally, this patient exhibited abundant but-carryingAcidaminococcus sp. communities, which were absent in the healthy controlsamples. At visits three and four, 15.5% and 26% of patient 210’s totalmicrobial community exhibited but genes, which was within the broad rangefor the control samples with 4.4%, 2.6% and 74.1%, respectively. In patient 200, weinitially detected only buk genes, but a considerable but-genecommunity linked to Roseburia sp./E. rectale was established overtime as well.

Bottom Line: As a result, reliable information on this important bacterial group is often lacking in microbiome research.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.Furthermore, our analysis refines but and buk reference annotations found in central databases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA. colej@msu.edu.

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.

No MeSH data available.


Related in: MedlinePlus