Butyrate is formed in the gut during bacterial fermentation of dietary fiber and is attributed numerous beneficial effects on the host metabolism. We aimed to develop a method for the assessment of functional capacity of gut microbiota butyrate synthesis based on the qPCR quantification of bacterial gene coding butyryl-CoA:acetate CoA-transferase, the key enzyme of butyrate synthesis. In silico, we identified bacteria possessing but gene among human gut microbiota by searching but coding sequences in available databases. We designed and validated six sets of degenerate primers covering all selected bacteria, based on their phylogenetic nearness and sequence similarity, and developed a method for gene abundance normalization in human fecal DNA. We determined but gene abundance in fecal DNA of subjects with opposing dietary patterns and metabolic phenotypes-lean vegans (VG) and healthy obese omnivores (OB) with known fecal microbiota and metabolome composition. We found higher but gene copy number in VG compared with OB, in line with higher fecal butyrate content in VG group. We further found a positive correlation between the relative abundance of target bacterial genera identified by next-generation sequencing and groups of but gene-containing bacteria determined by specific primers. In conclusion, this approach represents a simple and feasible tool for estimation of microbial functional capacity.

1st Faculty of Medicine Charles University Katerinska 1660 32 121 08 Prague Czech Republic

Department of Analytical Chemistry Faculty of Science Palacky University Olomouc 17 Listopadu 1192 12 779 00 Olomouc Czech Republic

Department of Internal Medicine Kralovske Vinohrady University Hospital and 3rd Faculty of Medicine Charles University Srobarova 1150 100 34 Prague 10 Czech Republic

Institute for Clinical and Experimental Medicine Videnska 1958 140 21 Prague 4 Czech Republic

Institute of Microbiology AS CR Videnska 1083 142 20 Prague 4 Czech Republic

RECETOX Faculty of Science Masaryk University Kamenice 753 625 00 Brno Czech Republic

Zobrazit více v PubMed

Tilg H., Moschen A.R. Microbiota and diabetes: An evolving relationship. Gut. 2014;63:1513–1521. doi: 10.1136/gutjnl-2014-306928. PubMed DOI

Karlsson F.H., Tremaroli V., Nookaew I., Bergström G., Behre C.J., Fagerberg B., Nielsen J., Bäckhed F. Gut metagenome in European women with normal, impaired and diabetic glucose control. Nature. 2013;498:99–103. doi: 10.1038/nature12198. PubMed DOI

Qin J., Li Y., Cai Z., Li S., Zhu J., Zhang F., Liang S., Zhang W., Guan Y., Shen D., et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature. 2012;490:55–60. doi: 10.1038/nature11450. PubMed DOI

Thorburn A.N., Macia L., Mackay C.R. Diet, Metabolites, and “Western-Lifestyle” Inflammatory Diseases. Immunity. 2014;40:833–842. doi: 10.1016/j.immuni.2014.05.014. PubMed DOI

Barko P.C., McMichael M.A., Swanson K.S., Williams D.A. The Gastrointestinal Microbiome: A Review. J. Vet. Intern. Med. 2018;32:9–25. doi: 10.1111/jvim.14875. PubMed DOI PMC

Hamer H.M., Jonkers D., Venema K., Vanhoutvin S., Troost F.J., Brummer R.J. Review article: The role of butyrate on colonic function. Aliment. Pharmacol. Ther. 2008;27:104–119. doi: 10.1111/j.1365-2036.2007.03562.x. PubMed DOI

Louis P., Hold G.L., Flint H.J. The gut microbiota, bacterial metabolites and colorectal cancer. Nat. Rev. Microbiol. 2014;12:661–672. doi: 10.1038/nrmicro3344. PubMed DOI

Verbeke K.A., Boobis A.R., Chiodini A., Edwards C.A., Franck A., Kleerebezem M., Nauta A., Raes J., Van Tol E.A.F., Tuohy K.M. Towards microbial fermentation metabolites as markers for health benefits of prebiotics. Nutr. Res. Rev. 2015;28:42–66. doi: 10.1017/S0954422415000037. PubMed DOI PMC

Morrison D.J., Preston T. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes. 2016;7:189–200. doi: 10.1080/19490976.2015.1134082. PubMed DOI PMC

Hernández M.A.G., Canfora E.E., Jocken J.W.E., Blaak E.E. The short-chain fatty acid acetate in body weight control and insulin sensitivity. Nutrients. 2019;11:1943. doi: 10.3390/nu11081943. PubMed DOI PMC

Yu Y., Raka F., Adeli K. The Role of the Gut Microbiota in Lipid and Lipoprotein Metabolism. J. Clin. Med. 2019;8:2227. doi: 10.3390/jcm8122227. PubMed DOI PMC

Koh A., De Vadder F., Kovatcheva-Datchary P., Bäckhed F. From dietary fiber to host physiology: Short-chain fatty acids as key bacterial metabolites. Cell. 2016;165:1332–1345. doi: 10.1016/j.cell.2016.05.041. PubMed DOI

Lupton J.R. Diet Induced Changes in the Colonic Environment and Colorectal Cancer. J. Nutr. 2004;134:479–482. doi: 10.1093/jn/134.2.479. PubMed DOI

Guilloteau P., Martin L., Eeckhaut V., Ducatelle R., Zabielski R., Van Immerseel F. From the gut to the peripheral tissues: The multiple effects of butyrate. Nutr. Res. Rev. 2010;23:366–384. doi: 10.1017/S0954422410000247. PubMed DOI

Singh N., Gurav A., Sivaprakasam S., Brady E., Padia R., Shi H., Thangaraju M., Prasad P.D., Manicassamy S., Munn D.H., et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity. 2014;40:128–139. doi: 10.1016/j.immuni.2013.12.007. PubMed DOI PMC

Richards J.L., Yap Y.A., McLeod K.H., MacKay C.R., Marinõ E. Dietary metabolites and the gut microbiota: An alternative approach to control inflammatory and autoimmune diseases. Clin. Transl. Immunol. 2016;5:e82. doi: 10.1038/cti.2016.29. PubMed DOI PMC

Tappenden K.A., McBurney M.I. Systemic short-chain fatty acids rapidly alter gastrointestinal structure, function, and expression of early response genes. Dig. Dis. Sci. 1998;43:1526–1536. doi: 10.1023/A:1018819032620. PubMed DOI

Burger-van Paassen N., Vincent A., Puiman P.J., van der Sluis M., Bouma J., Boehm G., van Goudoever J.B., Van Seuningen I., Renes I.B. The regulation of intestinal mucin MUC2 expression by short-chain fatty acids: Implications for epithelial protection. Biochem. J. 2009;420:211–219. doi: 10.1042/BJ20082222. PubMed DOI

Blaak E.E., Canfora E.E., Theis S., Frost G., Groen A.K., Mithieux G., Nauta A., Scott K., Stahl B., van Harsselaar J., et al. Short chain fatty acids in human gut and metabolic health. Benef. Microbes. 2020;11:411–455. doi: 10.3920/BM2020.0057. PubMed DOI

Lampe J.W., Navarro S.L., Hullar M.A.J., Shojaie A. Inter-individual differences in response to dietary intervention: Integrating omics platforms towards personalised dietary recommendations. Proc. Nutr. Soc. 2013;72:207–218. doi: 10.1017/S0029665113000025. PubMed DOI PMC

Makki K., Deehan E.C., Walter J., Bäckhed F. The Impact of Dietary Fiber on Gut Microbiota in Host Health and Disease. Cell Host Microbe. 2018;23:705–715. doi: 10.1016/j.chom.2018.05.012. PubMed DOI

Laudadio I., Fulci V., Palone F., Stronati L., Cucchiara S., Carissimi C. Quantitative Assessment of Shotgun Metagenomics and 16S rDNA Amplicon Sequencing in the Study of Human Gut Microbiome. Omics J. Integr. Biol. 2018;22:248–254. doi: 10.1089/omi.2018.0013. PubMed DOI

Sanschagrin S., Yergeau E. Next-generation sequencing of 16S ribosomal RNA gene amplicons. J. Vis. Exp. 2014;90:3–8. doi: 10.3791/51709. PubMed DOI PMC

Fiorini N., Lipman D.J., Lu Z. Towards PubMed 2.0. Elife. 2017;6:4–7. doi: 10.7554/eLife.28801. PubMed DOI PMC

Vital M., Penton C.R., Wang Q., Young V.B., Antonopoulos D.A., Sogin M.L., Morrison H.G., Raffals L., Chang E.B., Huffnagle G.B., et al. A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community. Microbiome. 2013;1:1–14. doi: 10.1186/2049-2618-1-8. PubMed DOI PMC

Anand S., Kaur H., Mande S.S. Comparative in silico analysis of butyrate production pathways in gut commensals and pathogens. Front. Microbiol. 2016;7:1–12. doi: 10.3389/fmicb.2016.01945. PubMed DOI PMC

Louis P., Duncan S.H., McCrae S.I., Millar J., Jackson M.S., Flint H.J. Restricted Distribution of the Butyrate Kinase Pathway among Butyrate-Producing Bacteria from the Human Colon. J. Bacteriol. 2004;186:2099–2106. doi: 10.1128/JB.186.7.2099-2106.2004. PubMed DOI PMC

Lissemore J.L., Lackner L.L., Fedoriw G.D., De Stasio E.A. Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc-93 gene using PCR. Biochem. Mol. Biol. Educ. 2005;33:219–226. doi: 10.1002/bmb.2005.494033032452. PubMed DOI

Untergasser A., Cutcutache I., Koressaar T., Ye J., Faircloth B.C., Remm M., Rozen S.G. Primer3-new capabilities and interfaces. Nucleic Acids Res. 2012;40:1–12. doi: 10.1093/nar/gks596. PubMed DOI PMC

McGinnis S., Madden T.L. BLAST: At the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res. 2004;32:20–25. doi: 10.1093/nar/gkh435. PubMed DOI PMC

Apprill A., Mcnally S., Parsons R., Weber L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat. Microb. Ecol. 2015;75:129–137. doi: 10.3354/ame01753. DOI

Budinska E., Gojda J., Heczkova M., Bratova M., Dankova H., Wohl P., Bastova H., Lanska V., Kostovcik M., Dastych M., et al. Microbiome and Metabolome Profiles Associated With Different Types of Short Bowel Syndrome: Implications for Treatment. J. Parenter. Enter. Nutr. 2020;44:105–118. doi: 10.1002/jpen.1595. PubMed DOI

Callahan B.J., McMurdie P.J., Rosen M.J., Han A.W., Johnson A.J.A., Holmes S.P. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods. 2016;13:581–583. doi: 10.1038/nmeth.3869. PubMed DOI PMC

Caporaso J.G., Kuczynski J., Stombaugh J., Bittinger K., Bushman F.D., Costello E.K., Fierer N., Peña A.G., Goodrich J.K., Gordon J.I., et al. Correspondence QIIME allows analysis of high- throughput community sequencing data Intensity normalization improves color calling in SOLiD sequencing. Nat. Publ. Gr. 2010;7:335–336. doi: 10.1038/nmeth0510-335. PubMed DOI PMC

Pelantová H., Bugáňová M., Anýž J., Železná B., Maletínská L., Novák D., Haluzík M., Kuzma M. Strategy for NMR metabolomic analysis of urine in mouse models of obesity- from sample collection to interpretation of acquired data. J. Pharm. Biomed. Anal. 2015;115:225–235. doi: 10.1016/j.jpba.2015.06.036. PubMed DOI

R core Team A language and environment for statistical computing. Found. Stat. Comput. 2013;2:1–12.

Aitchison J. The Statistical Analysis of Compositional Data. Stat. Anal. Compos. Data. 1986;44:139–177. doi: 10.1007/978-94-009-4109-0. DOI

Fish J.A., Chai B., Wang Q., Sun Y., Brown C.T., Tiedje J.M., Cole J.R. FunGene: The functional gene pipeline and repository. Front. Microbiol. 2013;4:1–14. doi: 10.3389/fmicb.2013.00291. PubMed DOI PMC

Louis P., Flint H.J. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol. Lett. 2009;294:1–8. doi: 10.1111/j.1574-6968.2009.01514.x. PubMed DOI

Louis P., Flint H.J. Formation of propionate and butyrate by the human colonic microbiota. Environ. Microbiol. 2017;19:29–41. doi: 10.1111/1462-2920.13589. PubMed DOI

Louis P., McCrae S.I., Charrier C., Flint H.J. Organization of butyrate synthetic genes in human colonic bacteria: Phylogenetic conservation and horizontal gene transfer. FEMS Microbiol. Lett. 2007;269:240–247. doi: 10.1111/j.1574-6968.2006.00629.x. PubMed DOI

Thompson J.D., Gibson T.J., Higgins D.G. Multiple Sequence Alignment Using ClustalW and ClustalX. Curr. Protoc. Bioinform. 2003:1–22. doi: 10.1002/0471250953.bi0203s00. PubMed DOI

Dereeper A., Guignon V., Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J.F., Guindon S., Lefort V., Lescot M., et al. Phylogeny.fr: Robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008;36:465–469. doi: 10.1093/nar/gkn180. PubMed DOI PMC

Lane C.E., Hulgan D., O’Quinn K., Benton M.G. CEMAsuite: Open source degenerate PCR primer design. Bioinformatics. 2015;31:3688–3690. doi: 10.1093/bioinformatics/btv420. PubMed DOI

Ye J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S., Madden T.L. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 2012;13:134. doi: 10.1186/1471-2105-13-134. PubMed DOI PMC

Costea P.I., Zeller G., Sunagawa S., Pelletier E., Alberti A., Levenez F., Tramontano M., Driessen M., Hercog R., Jung F.E., et al. Towards standards for human fecal sample processing in metagenomic studies. Nat. Biotechnol. 2017;35:1069–1076. doi: 10.1038/nbt.3960. PubMed DOI

Leigh Greathouse K., Sinha R., Vogtmann E. DNA extraction for human microbiome studies: The issue of standardization. BMC Microbiol. 2018;18:212. doi: 10.1186/s13059-019-1843-8. PubMed DOI PMC

Jenkins S.V., Vang K.B., Gies A., Griffin R.J., Jun S., Nookaew I., Dings R.P.M. Sample storage conditions induce post- collection biases in microbiome profiles. BMC Microbiol. 2018;18:227. doi: 10.1186/s12866-018-1359-5. PubMed DOI PMC

Větrovský T., Baldrian P. The Variability of the 16S rRNA Gene in Bacterial Genomes and Its Consequences for Bacterial Community Analyses. PLoS ONE. 2013;8:e57923. doi: 10.1371/journal.pone.0057923. PubMed DOI PMC

Dinu M., Abbate R., Gensini G.F., Casini A., Sofi F. Vegetarian, vegan diets and multiple health outcomes: A systematic review with meta-analysis of observational studies. Crit. Rev. Food Sci. Nutr. 2017;57:3640–3649. doi: 10.1080/10408398.2016.1138447. PubMed DOI

Jia W., Zhen J., Liu A., Yuan J., Wu X., Zhao P., Zhao L., Li X., Liu Q., Huang G., et al. Long-Term Vegan Meditation Improved Human Gut Microbiota. Evid. Based Complement. Altern. Med. 2020;2020:9517897. doi: 10.1155/2020/9517897. PubMed DOI PMC

Wu G.D., Compher C., Chen E.Z., Smith S.A., Shah R.D., Bittinger K., Chehoud C., Albenberg L.G., Nessel L., Gilroy E., et al. Comparative metabolomics in vegans and omnivores reveal constraints on diet-dependent gut microbiota metabolite production. Gut. 2016;65:63–72. doi: 10.1136/gutjnl-2014-308209. PubMed DOI PMC

Vegan Diet Is Associated With Favorable Effects on the Metabolic Performance of Intestinal Microbiota: A Cross-Sectional Multi-Omics Study