Abstract
One of the fascinating functions of mammalian intestinal microbiota is fermentation of plant cell wall components. Eight-week continuous culture enrichments of pig feces with cellulose and xylan/pectin were used to isolate bacteria from this community. A total of 575 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented in the bacterial isolates: Firmicutes (242), Bacteroidetes (185), Proteobacteria (65), Fusobacteria (55), Actinobacteria (23), and Synergistetes (5). The majority of the bacterial isolates had ≥97 % similarity to cultured bacteria with sequences in the RDP, but 179 isolates represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families of Lachnospiraceae, Enterococcaceae, Staphylococcaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, Bacteroides ovatus, and B. xylanisolvens. Many of the Firmicutes and Bacteroidetes isolates were identified as species that possess enzymes that ferment plant cell wall components, and the rest likely support these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 30 % of the isolates not represented in culture, there are new opportunities to study genomic and metabolic capacities of these members of the complex intestinal microbiota.
Similar content being viewed by others
References
Allison MJ, Robinson IM, Bucklin JA, Booth GD (1979) Comparison of bacterial populations of the pig cecum and colon based upon enumeration with specific energy sources. Appl Environ Microbiol 37:1142–1151
Brulc JM, Antonopoulos DA, Miller ME, Wilson MK, Yannarell AC, Dinsdale EA, Edwards RE, Frank ED, Emerson JB, Wacklin P, Coutinho PM, Henrissat B, Nelson KE, White BA (2009) Gene-centric metagemomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases. Proc Natl Acad Sci USA 106:1948–1953
Castillo M, Skene G, Roca M, Anguita M, Badiola I, Duncan SH, Flint HJ, Martín-Orúe SM (2007) Application of 16S rRNA gene-targeted fluorescence in situ hybridization and restriction fragment length polymorphism to study porcine microbiota along the gastrointestinal tract in response to different sources of dietary fiber. FEMS Microbiol Ecol 59:138–146
Cole JR, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrel DM, Garrity GM, Tiedje JM (2005) The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33:D294–D296
Collins MD, Lawson PA, Willems A, Cordoba JJ, Frenandez-Garayzabal J, Garcia P, Cai J, Hippe H, Farrow JAE (1994) The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44:812–826
Cotta MA, Whitehead TR, Zeltwanger RL (2003) Isolation, characterization and comparison of bacteria from swine faeces and manure storage pits. Environ Microbiol 5:737–745
Crittenden R, Karppinen S, Ojanen S, Tenkanen M, Fagerström R, Mättö J, Saarele M, Mattila-Sandholm T, Poutanen K (2002) In vitro fermentation of cereal dietary fiber carbohydrates by probiotic and intestinal bacteria. J Sci Food Agric 82:781–789
Dai Z-L, Zhang J, Wu G, Zhu W-Y (2010) Utilization of amino acids by bacteria from the pig small intestine. Amino Acids 39:1201–1215
Durmic Z, Pethick DW, Pluske JR, Hampson DJ (1998) Changes in bacterial populations in the colon of pigs fed different sources of dietary fibre, and the development of swine diysentery after experimental infection. J Appl Microbiol 85:574–582
Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Santos VA, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov MM, Timis KN, Golyshin PN (2005) Novel hydrolase diversity retrieved from a metagemone library of bovine rumen microflora. Environ Microbiol 7:1996–2010
Furet J-P, Firmesse O, Gourmelon M, Bridonneau C, Tap J, Mondot S, Doré J, Corthier G (2009) Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR. FEMS Microbiol Ecol 68:351–352
Gill SR, Pop M, DeBoy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, Gordon JI, Relman DA, Fraser-Ligget CM, Nelson KE (2006) Metagenomic analysis of the human distal gut microbiome. Science 312:1355–1359
Högberg A, Lindberg JE, Leser T, Wallgren P (2004) Influence of cereal non-starch polysaccharides on ileo-caecal and rectal microbial populations in growing pigs. Acta Vet Scand 45:87–98
Karlsson FH, Ussery DW, Nielsen J, Nookaew I (2011) A closer look at Bacteroides: phylogenetic relationship and genomic implications of a life in the human gut. Microb Ecol 61:473–485
Kass ML, Van Soest PJ, Pond WG (1980) Utilization of dietary fiber from alfalfa by growing swine. II. Volatile fatty acid concentrations in and disappearance from the gastrointestinal tract. J Anim Sci 50:192–197
Konstantinov SR, Zhu W-Y, Williams BA, Tamminga S, de Vos WM, Akkermans ADL (2003) Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA. FEMS Microbiol Ecol 43:225–235
Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, Takami H, Morita H, Sharma VK, Srivastava TP, Taylor TD, Noguchi H, Mori H, Ogura Y, Ehrlich DS, Itoh K, Takagi T, Sakaki Y, Hayashi T, Hattori M (2007) Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes. DNA Res 14:169–181
Lamendella R, Santo Domingo SW, Ghosh S, Martinson J, Oerther DB (2011) Comparative fecal metagenomics unveils unique functional capacity of the swine gut. BMC Microbiol 11:103 (http//www.biomedcentral.com/1471-2180/11/103)
Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp 115–163
Lederberg J, Lederberg EM (1952) Replicate plating and indirect selection of bacterial mutants. J Bacteriol 63:399–406
Leedle JAZ, Hespell RB (1980) Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations. Appl Environ Microbiol 39:709–719
Leser TD, Amenuvor JZ, Jensen TK, Lindecrona R, Boye M, Møller K (2002) Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol 68:673–690
Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI (2008) Worlds with in worlds: evolution of the vertebrate gut microbiota. Nat Rev Microbiol 6:776–788
Ley RE, Peterson DA, Gordon JI (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124:837–848
Lineback DR (1999) The chemistry of complex carbohydrates. In: Cho SS, Prosky L, Dreher M (eds) Complex carbohydrates in foods. Marcel Dekker, New York, pp 115–129
Macfarlane GT, Allison C, Gibson SAW, Cummings JH (1988) Contribution of the microflora to proteolysis in the human large intestine. J Appl Bacteriol 64:37–46
Maki M, Leung KT, Qin W (2009) The prospects of cellulose-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci 5:500–516
Marchandin H, Damay A, Roudière L, Teyssier C, Zorgniotti I, Dechaud H, Jean-Pierre H, Jumas-Bilak E (2010) Phylogeny, diversity and host specialization in the phylum Synergistetes with emphasis on strains and clones of human origin. Res Microbiol 161:91–100
Martens EC, Low EC, Chiang H, Pudlo NA, Wu M, McNulty NP, Abbott DW, Henrissat B, Gilbert HJ, Bolam DN, Gordon JI (2011) Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts. PLoS Biol 9:e1001221. doi:10.1371/journal.pbio.1001221
Metzler-Zebeli BU, Hooda S, Pieper R, Zijlstra RT, van Kessel AG, Mosenthin R, Gänzle MG (2010) Nonstarch polysaccharides modulate bacterial microbiota, pathways for butyrate production, and abundance of pathogenic Escherichia coli in the pig gastrointestinal tract. Appl Microbiol Biotechnol 76:3692–3701
Mirande C, Mosoni P, Béra-Maillet C, Bernalier-Donadille A, Forano E (2010) Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A. Appl Microbiol Biotechnol 87:2097–2105
Pond WG (1987) Thoughts on fiber utilization in swine. J Anim Sci 65:497–499
Palop MLL, Valles S, Piñaga F, Flors A (1989) Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov. Int J Syst Bacteriol 39:68–71
Qu A, Brulc JM, Wilson MK, Law BF, Theoret JR, Joens LA, Konkel ME, Angly F, Dinsdale EA, Edwards RA, Nelson KE, White BA (2008) Comparative metagenomics reveals host specific metavirulomes and horizontal gene transfer elements in the chicken cecum microbiome. PLoS One 3:e2945. doi:10.1371/journal/.pone.0002945
Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, Frederick WJ Jr, Hallett JP, Leak DJ, Liotta CL, Mielenz JR, Murphy R, Templer R, Tschaplinski T (2006) The path forward for biofuels and biomaterials. Sci 311:484–489
Reŕat A, Fiszlewicz M, Giusi A, Vaugelade P (1987) Influence of meal frequency on postprandial variations in the production and absorption of volatile fatty acids in the digestive tract of conscious pigs. J Anim Sci 64:448–456
Roberfroid M, Gibson GR, Hoyles L, McCartney AL, Rastall R, Rowland I, Wolvers D, Watzl B, Szajewska H, Stahl B, Guarner F, Respondek F, Whelan K, Coxam V, Davicco M-J, Léotoing L, Wittrant Y, Delzenne NM, Cani PD, Neyrinck AM, Meheust A (2010) Prebiotic effects: metabolic and health benefits. Br J Nutr 104(Suppl 2):S1–S63
Salyers AA (1979) Energy sources of major intestinal fermentative anaerobes. Am J Clin Nutr 32:158–163
Salyers AA, Vercellotti JR, West SHE, Wilkens TD (1977) Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl Environ Microbiol 33:319–322
Sankar M, Delgado O, Mattiasson B (2003) Isolation and characterization of solvenogenic, cellulase-free xylanolytic Clostridia from cow rumen. Water Sci Technol 48:185–188
Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbio 71:1501–1506
Schwarz WH (2001) the cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 56:634–649
Schwarz WH, Zverlov VV, Bahl H (2004) Extracellular glycosyl hydrolases from Clostridia. Adv Appl Microbiol 56:215–261
Shah HN, Olsen I, Bernard K, Finegold SM, Gharbia S, Gupta RS (2009) Approaches to the study of the systematic of anaerobic, Gram-negative, non-sporeforming rods: current status and perspectives. Anaerobe 15:179–194
Slade AP, Wyatt GM, Bayliss CE, Waites WM (1987) Comparison of populations of faecal bacteria before and after in vitro incubation with plant cell wall substrates. J Appl Bacteriol 62:231–240
Van Laere KMJ, Hartemink R, Bosveld M, Schols HA, Voragen AGJ (2000) Fermentation of plant cell wall derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria. J Agric Food Chem 48:1644–1652
Zoetendal EG, Collier CT, Koike S, Mackie RI, Gaskins HR (2004) Molecular ecological analysis of the gastrointestinal microbiota: a review. J Nutr 134:465–472
Acknowledgments
This research was supported by a grant from Defense Advanced Research Projects Agency as part of its Intestinal Fortitude Program to C.J. Ziemer. The author would like to thank Todd Atherly, Kerrie Franzen, and John Tenhundfeld for technical analyses.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ziemer, C.J. Broad Diversity and Newly Cultured Bacterial Isolates from Enrichment of Pig Feces on Complex Polysaccharides. Microb Ecol 66, 448–461 (2013). https://doi.org/10.1007/s00248-013-0185-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00248-013-0185-4