Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank

Abstract

Background: In gut microbiome studies, the cultured gut microbial resource plays essential roles, such as helping to unravel gut microbial functions and host-microbe interactions. Although several major studies have been performed to elucidate the cultured human gut microbiota, up to 70% of the Unified Human Gastrointestinal Genome species have not been cultured to date. Large-scale gut microbial isolation and identification as well as availability to the public are imperative for gut microbial studies and further characterizing human gut microbial functions.

Results: In this study, we constructed a human Gut Microbial Biobank (hGMB; homepage: hgmb.nmdc.cn ) through the cultivation of 10,558 isolates from 31 sample mixtures of 239 fresh fecal samples from healthy Chinese volunteers, and deposited 1170 strains representing 400 different species in culture collections of the International Depository Authority for long-term preservation and public access worldwide. Following the rules of the International Code of Nomenclature of Prokaryotes, 102 new species were characterized and denominated, while 28 new genera and 3 new families were proposed. hGMB represented over 80% of the common and dominant human gut microbial genera and species characterized from global human gut 16S rRNA gene amplicon data (n = 11,647) and cultured 24 "most-wanted" and "medium priority" taxa proposed by the Human Microbiome Project. We in total sequenced 115 genomes representing 102 novel taxa and 13 previously known species. Further in silico analysis revealed that the newly sequenced hGMB genomes represented 22 previously uncultured species in the Unified Human Gastrointestinal Genome (UHGG) and contributed 24 representatives of potentially "dark taxa" that had not been discovered by UHGG. The nonredundant gene catalogs generated from the hGMB genomes covered over 50% of the functionally known genes (KEGG orthologs) in the largest global human gut gene catalogs and approximately 10% of the "most wanted" functionally unknown proteins in the FUnkFams database.

Conclusions: A publicly accessible human Gut Microbial Biobank (hGMB) was established that contained 1170 strains and represents 400 human gut microbial species. hGMB expands the gut microbial resources and genomic repository by adding 102 novel species, 28 new genera, 3 new families, and 115 new genomes of human gut microbes. Video abstract.

Keywords: Biobank; Cultivation; Human gut microbiomes; Novel taxa; hGMB.

Fig. 1

The taxonomic diversity and specificity of hGMB. a The taxonomic cladogram displaying the taxonomic diversity of hGMB. The nodes of 102 newly characterized species, 28 novel genus and 3 novel family are indicated in red. The background is color-coded according to 6 phyla, yellow: Firmicutes, green: Bacteroides, red: Proteobacteria, blue: Actinobacteria, gray: Fusobacteria, purple: Verrucomicrobia. The outer ring (the coral red pointers) shows the unique 138 species that are solely covered by hGMB. b The taxonomic diversity of gut microbes from different gut microbial collections. hGMB (this study): a human gut microbial culture collection constructed in this study contains 400 species with 102 novel taxa; SPORE [23]: a human gut microbial culture collection constructed in 2016 comprises 121 species including 68 novel-taxon candidates; BIO-ML [21]: a human gut microbial culture collection constructed in 2019 comprises 106 species with 20 novel-taxon candidates; Culturomics [25]: the culturomics study of human gut microbes in 2016 reveal the discovery of 1056 species including 247 novel taxa, of which 117 were still novel-taxon candidates by the time of manuscript preparation; CGR [20]: a human gut microbial culture collection constructed in 2019 comprises 319 species determined based on the 16S rRNA gene sequence clustering at identity of 98.7%, of which 141 taxa are novel-taxon candidates; HBC [24]: a human gut microbial culture collection constructed in 2019 contains 236 species with 100 novel-taxon candidates. c The Venn diagram displaying the unique and shared taxa in each study. The numbers of taxa uniquely in one collection or shared by different studies are labeled in the panel. d Summary of novel taxa claimed by 1 or more than 1 study. Numbers in the bar represent the number of novel taxa. Note: The taxonomic diversity of each previously published study compared in panel b and c was re-mined and summarized as described in “Methods,” and by the time of this writing, all the mentioned “novel-taxon candidates” are never described

Fig. 2

The cultured recovery of major composition of human gut microbiota by hGMB at genus and species levels. a and b The rarefaction curves displaying the increase trend of the numbers of assigned genera (a) and species (b) as 1 to 26 16S rRNA gene amplicon datasets (Table S8) were sampled for combined analysis. c The coverage of human gut common (bar chart) and dominant (box-and-whiskers plot) genera by hGMB. All the genera covered by hGMB were colored in blue while the genera absent in hGMB were color in olive brown. d The coverage of human gut common (bar chart), dominant (box and whiskers plot) species by hGMB. All the genera covered by hGMB were colored in purple while the genera absent in hGMB were color in olive brown. Common genera/species: genera/species with equally weighted average frequency of occurrence (FO) > 30% (definition: FO = 100% is defined when a taxon presents in all samples, while FO = 0 is defined when a taxon presents in none of the samples; The equally weighted average FO is the mean value of the average FOs of the 26 analyzed studies); Dominant genera/species: genera/species with equally weighted average relative abundance (RA) > 0.1% (log 10 (RA(%)) > − 1) (definition: The equally weighted average RA is the mean value of the average RAs of the 26 analyzed studies). The light-pink background in panel c and d highlighted the core genera/species shared by both dominant and common genera/species, while the light-blue background marked out the taxa presenting uniquely in either dominant or common genera/species. The bar chart in panel c and d shows the mean values of the 26 FO averages (%), while the box-and-whiskers plot shows the Log 10 of average Ras (%) of each taxon in each study, center line: median, bounds of box: quartile, whiskers: Tukey extreme

Fig. 3

The prevalence of novel taxa in hGMB among global health human gut metagenomes (n = 1129). The bar charts demonstrated the frequency of occurrence (FO) of each novel taxa among 1129 analyzed health human gut metagenomes (Table S9) (definition: FO = 100% is defined when a taxon presents in all samples, while FO = 0 is defined when a taxon presents in none of the samples); The box-and-whiskers plot displayed the relative abundance (RA) of each novel taxa among all samples in Log 10 format. center line: median, bounds of box: quartile, whiskers: Tukey extreme

Fig. 4

The functional coverage of global human gut gene catalogs by new hGMB genomes. a The coverage of IGC [49], UHGP [8] and FUnkFams [50] by hGMB.catalog. The hGMB.catalog was constructed by extraction of 341,876 nonredundant genes from 115 newly sequenced genomes in hGMB and was BLAST analyzed against subject gene catalog IGC (pink bars), UHGP (yellow bars) and FUnkFams (green bars) with cutoff sequence identities of 40% and 60%, respectively. The y-axis names indicated the names of subject gene catalog and the sequence identities used for BLAST (in bracket). The coverage rates were listed in panel on the right side of each bar. b and c The rarefaction curves displaying the accumulatively increased coverage of the KOs (purple), GOs (blue) and unannotated genes (gray) in IGC (b) and UHGP (c) catalogs. The sampling was repeated for 50 times at each x-axis point; Light purple dot: the coverage rates of KO functions of IGC or UHGP gene catalogs when specified numbers of genomes were randomly sampled from 115 hGMB genomes; dark purple line: the mean coverage rate of KO functions; light blue dot: the coverage rates of GO functions of IGC or UHGP gene catalogs; dark blue line: the mean coverage rate of GO functions; gray dot: the coverage rates of unannotated genes of IGC or UHGP; black line: the mean coverage rate of unannotated genes of IGC or UHGP. d The rarefaction curves displaying the accumulatively increased coverage of conserved functionally unknown proteins in FUnkFams. The sampling was repeated for 50 times at each x-axis point; light green dot: the coverage rates of FUnkFams proteins when sampled randomly; dark green line: the mean value of the coverage rates