Profiling of the intestinal community of Clostridia: taxonomy and evolutionary analysis

Abstract

Aim:Clostridia are relevant commensals of the human gut due to their major presence and correlations to the host. In this study, we investigated intestinal Clostridia of 51 healthy subjects and reconstructed their taxonomy and phylogeny. The relatively small number of intestinal Clostridia allowed a systematic whole genome approach based on average amino acid identity (AAI) and core genome with the aim of revising the current classification into genera and determining evolutionary relationships.

Methods: 51 healthy subjects’ metagenomes were retrieved from public databases. After the dataset’s validation through comparison with Human Microbiome Project (HMP) samples, the metagenomes were profiled using MetaPhlAn3 to identify the population ascribed to the class Clostridia. Intestinal Clostridia genomes were retrieved and subjected to AAI analysis and core genome identification. Phylogeny investigation was conducted with RAxML and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithms, and SplitsTree for split decomposition.

Results: 225 out of 406 bacterial taxonomic units were ascribed to Bacillota [Firmicutes], among which 124 were assigned to the class Clostridia. 77 out of the 124 taxonomic units were referred to a species, altogether covering 87.7% of Clostridia abundance. According to the lowest AAI genus boundary set at 55%, 15 putative genera encompassing more than one species (G1 to G15) were identified, while 19 species did not cluster with any other one and each appeared to belong to a diverse genus. Phylogenetic investigations highlighted that most of the species clustered into three main evolutive clades.

Conclusion: This study shed light on the species of Clostridia colonizing the gut of healthy adults and pinpointed several gaps in knowledge regarding the taxonomy and the phylogeny of Clostridia.

Keywords

Clostridia / gut microbiome / metagenomics / average amino acid identity (AAI) / taxonomy / phylogenomics

References

[1] Prazmowski A. Untersuchungen über die entwickelungsgeschichte und fermentwirkung einiger bacterien-arten. Available from: https://gdz.sub.uni-goettingen.de/id/PPN1030662479 [Last accessed on 17 Apr 2023]

[2] Rainey FA,Small AM.Clostridium. In: Whitman WB, Rainey F, Kämpfer P, Trujillo M, Chun J, Devos P, Hedlund B, Dedysh S, editors. Bergey’s manual of systematics of archaea and bacteria. Wiley; 2015. pp. 1-122.

[3] Parte AC,Meier-Kolthoff JP,Göker M.List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ.Int J Syst Evol Microbiol2020;70:5607-12 PMCID:PMC7723251

[4] Dworkin M. The prokaryotes: a handbook on the biology of bacteria. Available from: https://link.springer.com/book/9780387254999 [Last accessed on 17 Apr 2023]

[5] Béchon N.Gut biofilms: Bacteroides as model symbionts to study biofilm formation by intestinal anaerobes.FEMS Microbiol Rev2022;46:fuab054

[6] Louis P.Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine.FEMS Microbiol Lett2009;294:1-8

[7] Guo P,Ma X.Clostridium species as probiotics: potentials and challenges.J Anim Sci Biotechnol2020;11:24 PMCID:PMC7031906

[8] Lopetuso LR,Petito V.Commensal Clostridia: leading players in the maintenance of gut homeostasis.Gut Pathog2013;5:23 PMCID:PMC3751348

[9] Fernandes J,Rahat-Rozenbloom S,Comelli EM.Adiposity, gut microbiota and faecal short chain fatty acids are linked in adult humans.Nutr Diabetes2014;4:e121 PMCID:PMC4079931

[10] Amaretti A,Simone M.Profiling of protein degraders in cultures of human gut microbiota.Front Microbiol2019;10:2614 PMCID:PMC6874058

[11] Raimondi S,Candeliere F.Multivariate analysis in microbiome description: correlation of human gut protein degraders, metabolites, and predicted metabolic functions.Front Microbiol2021;12:723479 PMCID:PMC8484906

[12] Kamada N,Chen GY.Role of the gut microbiota in immunity and inflammatory disease.Nat Rev Immunol2013;13:321-35

[13] Tanoue T,Honda K.Development and maintenance of intestinal regulatory T cells.Nat Rev Immunol2016;16:295-309

[14] Nagano Y,Honda K.The induction of Treg cells by gut-indigenous Clostridium.Curr Opin Immunol2012;24:392-7

[15] Johnson JL.Taxonomy of the Clostridia: ribosomal ribonucleic acid homologies among the species.J Gen Microbiol1975;88:229-44

[16] Collins MD,Willems A.The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations.Int J Syst Bacteriol1994;44:812-26

[17] Yutin N.A genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia.Environ Microbiol2013;15:2631-41 PMCID:PMC4056668

[18] Lawson PA. The taxonomy of the genus Clostridium: current status and future perspectives. Available from: https://wswxtb.ijournals.cn/html/wswxtbcn/2016/5/tb16051070.htm [Last accessed on 17 Apr 2023]

[19] Woese CR.Phylogenetic structure of the prokaryotic domain: the primary kingdoms.Proc Natl Acad Sci USA1977;74:5088-90 PMCID:PMC432104

[20] Lawson PA.Proposal to restrict the genus Clostridium Prazmowski to Clostridium butyricum and related species.Int J Syst Evol Microbiol2016;66:1009-16

[21] Rossi-Tamisier M,Raoult D.Cautionary tale of using 16S rRNA gene sequence similarity values in identification of human-associated bacterial species.Int J Syst Evol Microbiol2015;65:1929-34

[22] Richter M.Shifting the genomic gold standard for the prokaryotic species definition.Proc Natl Acad Sci USA2009;106:19126-31 PMCID:PMC2776425

[23] Chun J.Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea.Int J Syst Evol Microbiol2014;64:316-24

[24] Rodriguez-R LM. Bypassing cultivation to identify bacterial species. Available from: https://www.researchgate.net/profile/Luis-Rodriguez-R/publication/304587401_Bypassing_Cultivation_To_Identify_Bacterial_Species_Culture-independent_genomic_approaches_identify_credibly_distinct_clusters_avoid_cultivation_bias_and_provide_true_insights_into_microbial_species/links/58c18324aca272e36dcc8314/Bypassing-Cultivation-To-Identify-Bacterial-Species-Culture-independent-genomic-approaches-identify-credibly-distinct-clusters-avoid-cultivation-bias-and-provide-true-insights-into-microbial-species.pdf [Last accessed on 17 Apr 2023]

[25] Pérez-Cataluña A,Diéguez AL.Revisiting the taxonomy of the genus arcobacter: getting order from the chaos.Front Microbiol2018;9:2077

[26] Orata FD,Sauvageau D.Phylogenomic analysis of the gammaproteobacterial methanotrophs (order methylococcales) calls for the reclassification of members at the genus and species levels.Front Microbiol2018;9:3162 PMCID:PMC6315193

[27] Stott CM.Impact of homologous recombination on core genome phylogenies.BMC Genomics2020;21:829 PMCID:PMC7691112

[28] Gophna U,Nielsen HB.Oscillospira and related bacteria - from metagenomic species to metabolic features.Environ Microbiol2017;19:835-41

[29] Cruz-Morales P,Moutafis G.Revisiting the evolution and taxonomy of clostridia, a phylogenomic update.Genome Biol Evol2019;11:2035-44

[30] Mukherjee A,Ross RP.Gut microbes from the phylogenetically diverse genus Eubacterium and their various contributions to gut health.Gut Microbes2020;12:1802866 PMCID:PMC7524325

[31] Waters JL.The human gut bacteria Christensenellaceae are widespread, heritable, and associated with health.BMC Biol2019;17:83 PMCID:PMC6819567

[32] Meehan CJ.A phylogenomic view of ecological specialization in the Lachnospiraceae, a family of digestive tract-associated bacteria.Genome Biol Evol2014;6:703-13 PMCID:PMC3971600

[33] Sorbara MT,Fontana E.Functional and genomic variation between human-derived isolates of lachnospiraceae reveals inter- and intra-species diversity.Cell Host Microbe2020;28:134-146.e4 PMCID:PMC7351604

[34] Galperin MY,Tolstoy I.Phylogenomic analysis of the family Peptostreptococcaceae (Clostridium cluster XI) and proposal for reclassification of Clostridium litorale (Fendrich et al. 1991) and Eubacterium acidaminophilum (Zindel et al. 1989) as Peptoclostridium litorale gen. nov. comb. nov. and Peptoclostridium acidaminophilum comb. nov.Int J Syst Evol Microbiol2016;66:5506-13 PMCID:PMC5244501

[35] Biddle A,Blanchard J.Untangling the genetic basis of fibrolytic specialization by Lachnospiraceae and Ruminococcaceae in diverse gut communities.Diversity2013;5:627-40

[36] Whelan S.Inferring trees. In: Keith JM, editor. Bioinformatics. New York: Springer; 2017. pp. 349-77.

[37] Turnbaugh PJ,Hamady M,Knight R.The human microbiome project.Nature2007;449:804-10 PMCID:PMC3709439

[38] Lloyd-Price J,Rahnavard G.Strains, functions and dynamics in the expanded Human Microbiome Project.Nature2017;550:61-6 PMCID:PMC5831082

[39] Truong DT,Tickle TL.MetaPhlAn2 for enhanced metagenomic taxonomic profiling.Nat Methods2015;12:902-3

[40] Beghini F,Blanco-Míguez A.Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3.Elife2021;10

[41] Bolyen E,Dillon MR.Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2.Nat Biotechnol2019;37:852-7

[42] Kabeerdoss J,Pugazhendhi S.Clostridium leptum group bacteria abundance and diversity in the fecal microbiota of patients with inflammatory bowel disease: a case-control study in India.BMC Gastroenterol2013;13:20 PMCID:PMC3565871

[43] Raimondi S,Candeliere F,Rossi M.Identification of mucin degraders of the human gut microbiota.Sci Rep2021;11:11094 PMCID:PMC8159939

[44] Rodriguez-R LM.The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes.PeerJ Preprints2016;4, e1900v1

[45] Seemann T.Prokka: rapid prokaryotic genome annotation.Bioinformatics2014;30:2068-9

[46] Page AJ,Hunt M.Roary: rapid large-scale prokaryote pan genome analysis.Bioinformatics2015;31:3691-3

[47] Stamatakis A.RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies.Bioinformatics2014;30:1312-3 PMCID:PMC3998144

[48] Garcia-Vallvé S,Romeu A.Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis.Mol Biol Evol1999;16:1125-34

[49] Letunic I.Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation.Nucleic Acids Res2021;49:W293-6 PMCID:PMC8265157

[50] Huson DH.Application of phylogenetic networks in evolutionary studies.Mol Biol Evol2006;23:254-67

[51] Bandelt HJ.Split decomposition: a new and useful approach to phylogenetic analysis of distance data.Mol Phylogenet Evol1992;1:242-52

[52] Sender R,Milo R.Revised estimates for the number of human and bacteria cells in the body.PLoS Biol2016;14:e1002533 PMCID:PMC4991899

[53] Konstantinidis KT,Amann R.Uncultivated microbes in need of their own taxonomy.ISME J2017;11:2399-406 PMCID:PMC5649169

[54] Vandeputte D,D'hoe K.Quantitative microbiome profiling links gut community variation to microbial load.Nature2017;551:507-11

[55] Candeliere F,Ranieri R.β-glucuronidase pattern predicted from gut metagenomes indicates potentially diversified pharmacomicrobiomics.Front Microbiol2022;13:826994 PMCID:PMC8928169

[56] Cassir N,La Scola B.Clostridium butyricum: from beneficial to a new emerging pathogen.Clin Microbiol Infect2016;22:37-45

[57] Wood DE,Langmead B.Improved metagenomic analysis with Kraken 2.Genome Biol2019;20:257 PMCID:PMC6883579

[58] Sokol H,Watterlot L.Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients.Proc Natl Acad Sci USA2008;105:16731-6

[59] Sang J,Zhang T,Yu J.Convergent and divergent age patterning of gut microbiota diversity in humans and nonhuman primates.mSystems2022;7:e0151221

[60] Zeevi D,Godneva A.Structural variation in the gut microbiome associates with host health.Nature2019;568:43-8

[61] Zhou P,Sun D.Gut microbiome: new biomarkers in early screening of colorectal cancer.J Clin Lab Anal2022;36:e24359 PMCID:PMC9102648

[62] Karcher N,Asnicar F.Analysis of 1321 Eubacterium rectale genomes from metagenomes uncovers complex phylogeographic population structure and subspecies functional adaptations.Genome Biol2020;21:138 PMCID:PMC7278147

[63] Molino S,Jiménez-Hernández N,Francino MP.Evaluation of the Effects of a Short Supplementation With Tannins on the Gut Microbiota of Healthy Subjects.Front Microbiol2022;13:848611 PMCID:PMC9093706

[64] Sánchez-Tapia M,Pichardo-Ontiveros E.Consumption of cooked black beans stimulates a cluster of some clostridia class bacteria decreasing inflammatory response and improving insulin sensitivity.Nutrients2020;12:1182 PMCID:PMC7230233

[65] Cann I,Mackie RI.Cellulose degradation in the human gut: ruminococcus champanellensis expands the cellulosome paradigm.Environ Microbiol2016;18:307-10

[66] Alfa MJ,Davi M,Van Caeseele P.An outbreak of necrotizing enterocolitis associated with a novel clostridium species in a neonatal intensive care unit.Clin Infect Dis2002;35:S101-5

[67] Bouvet P,Dauphin B,Butel MJ.16S rRNA gene sequencing, multilocus sequence analysis, and mass spectrometry identification of the proposed new species “Clostridium neonatale”.J Clin Microbiol2014;52:4129-36 PMCID:PMC4313276

[68] Groussin M,Alm EJ.Co-evolution and co-speciation of host-gut bacteria systems.Cell Host Microbe2020;28:12-22