Abstract
The group of butyrate-producing bacteria within the human gut microbiome may be associated with positive effects on memory improvement, according to previous studies on dementia-associated diseases. Here, fecal samples of four elderly Japanese diagnosed with Alzheimer’s disease (AD) were used to isolate butyrate-producing bacteria. 226 isolates were randomly picked, their 16S rRNA genes were sequenced, and assigned into sixty OTUs (operational taxonomic units) based on BLASTn results. Four isolates with less than 97% homology to known sequences were considered as unique OTUs of potentially butyrate-producing bacteria. In addition, 12 potential butyrate-producing isolates were selected from the remaining 56 OTUs based on scan-searching against the PubMed and the ScienceDirect databases. Those belonged to the phylum Bacteroidetes and to the clostridial clusters I, IV, XI, XV, XIVa within the phylum Firmicutes. 15 out of the 16 isolates were indeed able to produce butyrate in culture as determined by high-performance liquid chromatography with UV detection. Furthermore, encoding genes for butyrate formation in these bacteria were identified by sequencing of degenerately primed PCR products and included the genes for butyrate kinase (buk), butyryl-CoA: acetate CoAtransferase (but), CoA-transferase-related, and propionate CoA-transferase. The results showed that eight isolates possessed buk, while five isolates possessed but. The CoA-transfer-related gene was identified as butyryl-CoA:4-hydroxybutyrate CoA transferase (4-hbt) in four strains. No strains contained the propionate CoA-transferase gene. The biochemical and butyrate-producing pathways analyses of butyrate producers presented in this study may help to characterize the butyrate-producing bacterial community in the gut of AD patients.
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Altschu, F.S., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Atarashi, K., Tanoue, T., Oshima, K., Suda, W., Nagano, Y., Nishikawa, H., Fukuda, S., Saito, T., Narushima, S., Hase, K., et al. 2013. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 500, 232–236.
Barcenilla, A., Pryde, S.E., Martin, J.C., Duncan, S.H., Stewart, C.S., Henderson, C., and Flint, H.J. 2000. Phylogenetic relationships of butyrate-producing bacteria from the human gut. Appl. Environ. Microbiol. 66, 1654–1661.
Bourassa, M.W., Alim, I., Bultman, S.J., and Ratan, R.R. 2016. Butyrate, neuroepigenetics and the gut microbiome: Can a high fiber diet improve brain health? Neurosci. Lett. 625, 56–63.
Browne, H.P., Forster, S.C., Anonye, B.O., Kumar, N., Neville, B.A., Stares, M.D., Goulding, D., and Lawley, T.D. 2016. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533, 543–546.
Bui, T.P.N., Shetty, S.A., Lagkouvardos, I., Ritari, J., Chamlagain, B., Douillard, F.P., Paulin, L., Piironen, V., Clavel, T., Plugge, C.M., et al. 2016. Comparative genomics and physiology of the butyrate-producing bacterium Intestinimonas butyriciproducens. Environ. Microbiol. Rep. 8, 1024–1037.
Carlier, J.P., Bedora-Faure, M., K’Ouas, G., Alauzet, C., and Mory, F. 2010. Proposal to unify Clostridium orbiscindens Winter et al. 1991 and Eubacterium plautii (Seguin 1928) Hofstad and Aasjord 1982, with description of Flavonifractor plautii gen. nov., comb. nov., and reassignment of Bacteroides capillosus to Pseudoflavonifractor capillosus gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 60, 585–590.
Charrier, C., Duncan, G.J., Reid, M.D., Rucklidge, G.J., Henderson, D., Young, P., Russell, V.J., Aminov, R.I., Flint, H.J., and Louis, P. 2006. A novel class of CoA-transferase involved in short-chain fatty acid metabolism in butyrate-producing human colonic bacteria. Microbiology 152, 179–185.
Collins, M.D., Lawson, P.A., Willems, A., Cordoba, J.J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H., and Farrow, J.A. 1994. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int. J. Syst. Bacteriol. 44, 812–826.
Duncan, S.H., Stewart, C.S., Hold, G.L., and Flint, H.J. 2002. Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 52, 2141–2146.
Eeckhaut, V., Van Immerseel, F., Croubels, S., De Baere, S., Haesebrouck, F., Ducatelle, R., Louis, P., and Vandamme, P. 2011. Butyrate production in phylogenetically diverse Firmicutes isolated from the chicken caecum. Microb. Biotechnol. 4, 503–512.
Feehily, C. and Karatzas, K.A. 2013. Role of glutamate metabolism in bacterial responses towards acid and other stresses. J. Appl. Microbiol. 114, 11–24.
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783–791.
García-Villalba, R., Giménez-Bastida, J.A., García-Conesa, M.T., Tomás-Barberán, F.A., Espín, J.C., and Larrosa, M. 2012. Alternative method for gas chromatography-mass spectrometry analysis of short-chain fatty acids in faecal samples. J. Sep. Sci. 35, 1906–1913.
Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D., and Bairoch, A. 2003. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31, 3784–3788.
Geirnaert, A., Calatayud, M., Grootaert, C., Laukens, D., Devriese, S., Smagghe, G., De Vos, M., Boon, N., and Van de Wiele, T. 2017. Butyrate-producing bacteria supplemented in vitro to Crohn’s disease patient microbiota increased butyrate production and enhanced intestinal epithelial barrier integrity. Sci. Rep. 7, 11450.
Govindarajan, N., Agis-Balboa, R.C., Walter, J., Sananbenesi, F., and Fischer, A. 2011. Sodium butyrate improves memory function in an Alzheimer’s disease mouse model when administered at an advanced stage of disease progression. J. Alzheimers. Dis. 26, 187–197.
Hamer, H.M., Jonkers, D., Venema, K., Vanhoutvin, S., Troost, F.J., and Brummer, R.J. 2008. The role of butyrate on colonic function. Aliment. Pharmacol. Ther. 27, 104–119.
Holdeman, L.V. and Moore, W.E.C. 1974. New genus, Coprococcus, twelve new species, and emended descriptions of four previously described species of bacteria from human feces. Int. J. Syst. Bacteriol. 24, 260–277.
Kumar, S., Stecher, G., and Tamura, K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874.
Li, X., Højberg, O., Canibe, N., and Jensen, B.B. 2016a. Phylogenetic diversity of cultivable butyrate-producing bacteria from pig gut content and feces. J. Anim. Sci. 94, 377–381.
Li, Y., Sun, H., Chen, Z., Xu, H., Bu, G., and Zheng, H. 2016b. Implications of GABAergic neurotransmission in Alzheimer’s disease. Front. Aging Neurosci. 8, 31.
Li, Z., Yi, C., Katiraei, S., Kooijman, S., Zhou, E., Chung, K.C., Gao, Y., Heuvel, J.K., Meijer, O.C., Berbée, J.F.P., et al. 2018. Butyrate reduces appetite and activates brown adipose tissue via the gutbrain neural circuit. Gut 67, 1269–1279.
Liu, J., Sun, J., Wang, F., Yu, X., Ling, Z., Li, H., Zhang, H., Jin, J., Chen, W., Pang, M., et al. 2015. Neuroprotective effects of Clostridium butyricum against vascular dementia in mice via metabolic butyrate. Biomed. Res. Int. 2015, 412946.
Louis, P., Duncan, S.H., McCrae, S.I., Millar, J., Jackson, M.S., and Flint, H.J. 2004. Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon. J. Bacteriol. 186, 2099–2106.
Louis, P. and Flint, H.J. 2007. Development of a semiquantitative degenerate real-time PCR-based assay for estimation of numbers of butyryl-coenzyme A (CoA) CoA transferase genes in complex bacterial samples. Appl. Environ. Microbiol. 73, 2009–2012.
Louis, P. and Flint, H.J. 2009. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol. Lett. 294, 1–8.
Louis, P. and Flint, H.J. 2017. Formation of propionate and butyrate by the human colonic microbiota. Environ. Microbiol. 19, 29–41.
Louis, P., Scott, K.P., Duncan, S.H., and Flint, H.J. 2007. Understanding the effects of diet on bacterial metabolism in the large intestine. J. Appl. Microbiol. 102, 1197–1208.
Mandell, A.M. and Green, C.R. 2011. Alzheimer’s disease. In Budson, A.E. and Kowall, N.W. (eds.), The handbook of Alzheimer’s disease and other dementias, 1st ed. Wiley-Blackwell, UK.
Morita, H., Kuwahara, T., Ohshima, K., Sasamoto, H., Itoh, K., Hattori, M., Hayashi, T., and Takami, H. 2007. An improved DNA isolation method for metagenomic analysis of the microbial flora of the human intestine. Microbes Environ. 22, 214–222.
Nei, M. and Kumar, S. 2000. Molecular evolution and phylogenetics. Oxford University Press, New York, USA.
Nishijima, S., Suda, W., Oshima, K., Kim, S.W., Hirose, Y., Morita, H., and Hattori, M. 2016. The gut microbiome of healthy Japanese and its microbial and functional uniqueness. DNA Res. 23, 125–133.
Ott, S.J., Musfeldt, M., Wenderoth, D.F., Hampe, J., Brant, O., Folsch, U.R., Timmis, K.N., and Schreiber, S. 2004. Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease. Gut 53, 685–693.
Pruesse, E., Peplies, J., and Glockner, F.O. 2012. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28, 1823–1829.
Pryde, S.E., Duncan, S.H., Hold, G.L., Stewart, C.S., and Flint, H.J. 2002. The microbiology of butyrate formation in the human colon. FEMS Microbiol. Lett. 217, 133–139.
Rainey, F.A. 2009. Class II. Clostridia class. nov. In De Vos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K., and Whitman, W.B. (eds.), Bergey’s manual of systematic bacteriology, 2nd ed. Springer, Spring Street, New York, NY, USA.
Rivière, A., Selak, M., Lantin, D., Leroy, F., and De Vuyst, L. 2016. Bifidobacteria and butyrate-producing colon bacteria: Importance and strategies for their stimulation in the human gut. Front. Microbiol. 7, 979.
Saitou, N. and Nei, M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
Sakamoto, M., Tanaka, Y., Benno, Y., and Ohkuma, M. 2014. Butyricimonas faecihominis sp. nov. and Butyricimonas paravirosa sp. nov., isolated from human faeces, and emended description of the genus Butyricimonas. Int. J. Syst. Evol. Microbiol. 64, 2992–2997.
Sakurazawa, T. and Ohkusa, T. 2005. Cytotoxicity of organic acids produced by anaerobic intestinal bacteria on cultured epithelial cells. J. Gastroenterol. 40, 600–609.
Schulze-Schweifing, K., Banerjee, A., and Wade, W.G. 2014. Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and pyrosequencing for the characterization of the dentine caries-associated microbiome. Front. Cell. Infect. Microbiol. 4, 164.
Schwiertz, A., Hold, G.L., Duncan, S.H., Gruhl, B., Collins, M.D., Lawson, P.A., Flint, H.J., and Blaut, M. 2002. Anaerostipes caccae gen. nov., sp. nov., a new saccharolytic, acetate-utilising, butyrateproducing bacterium from human faeces. Syst. Appl. Microbiol. 25, 46–51.
Sommer, F. and Backhed, F. 2013. The gut microbiota-masters of host development and physiology. Nat. Rev. Microbiol. 11, 227–238.
Vital, M., Howe, A.C., and Tiedje, J.M. 2014. Revealing the bacterial butyrate synthesis pathways by analyzing (meta)genomic data. MBio 5, e00889.
Vital, M., Karch, A., and Pieper, D.H. 2017. Colonic butyrate-producing communities in humans: An overview using omics data. mSystems 2, e00130-00117.
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. 2013. A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community. Microbiome 1, 8.
Wallace, T.C., Guarner, F., Madsen, K., Cabana, M.D., Gibson, G., Hentges, E., and Sanders, M.E. 2011. Human gut microbiota and its relationship to health and disease. Nutr. Rev. 69, 392–403.
Yoon, S.H., Ha, S.M., Kwon, S., Lim, J., Kim, Y., Seo, H., and Chun, J. 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613–1617.
Yutin, N. and Galperin, M.Y. 2013. A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environ. Microbiol. 15, 2631–2641.
Zuckerkandl, E. and Pauling, L. 1965. Evolutionary divergence and convergence in proteins, pp. 97−166. In Bryson, V. and Vogel, H.J. (eds.), Evolving genes and proteins. Academic Press, New York, USA.
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Nguyen, T.T.T., Fujimura, Y., Mimura, I. et al. Cultivable butyrate-producing bacteria of elderly Japanese diagnosed with Alzheimer’s disease. J Microbiol. 56, 760–771 (2018). https://doi.org/10.1007/s12275-018-8297-7
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DOI: https://doi.org/10.1007/s12275-018-8297-7