title: Capturing the diversity of the human gut microbiota through culture-enriched molecular profiling.
abstract: The human gut microbiota has been implicated in most aspects of health and disease; however, most of the bacteria in this community are considered unculturable, so studies have relied on molecular-based methods. These methods generally do not permit the isolation of organisms, which is required to fully explore the functional roles of bacteria for definitive association with host phenotypes. Using a combination of culture and 16S rRNA gene sequencing, referred to as culture-enriched molecular profiling, we show that the majority of the bacteria identified by 16S sequencing of the human gut microbiota can be cultured.
  
  
  
  Five fresh, anaerobic fecal samples were cultured using 33 media and incubation of plates anaerobically and aerobically resulted in 66 culture conditions for culture-enriched molecular profiling. The cultivable portion of the fecal microbiota was determined by comparing the operational taxonomic units (OTUs) recovered by 16S sequencing of the culture plates to OTUs from culture-independent sequencing of the fecal sample. Targeted isolation of Lachnospiraceae strains using conditions defined by culture-enriched molecular profiling was carried out on two fresh stool samples.
  
  
  
  We show that culture-enriched molecular profiling, utilizing 66 culture conditions combined with 16S rRNA gene sequencing, allowed for the culturing of an average of 95 % of the OTUs present at greater than 0.1 % abundance in fecal samples. Uncultured OTUs were low abundance in stool. Importantly, comparing culture-enrichment to culture-independent sequencing revealed that the majority of OTUs were detected only by culture, highlighting the advantage of culture for studying the diversity of the gut microbiota. Applying culture-enriched molecular profiling to target Lachnospiraceae strains resulted in the recovery of 79 isolates, 12 of which are on the Human Microbiome Project's "Most Wanted" list.
  
  
  
  We show that, through culture-enriched molecular profiling, the majority of the bacteria in the human gut microbiota can be cultured and this method revealed greater bacterial diversity compared to culture-independent sequencing. Additionally, this method could be applied for the targeted recovery of a specific bacterial group. This approach allows for the isolation of bacteria of interest from the gut microbiota, providing new opportunities to explore mechanisms of microbiota-host interactions and the diversity of the human microbiota.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, L8S 4K1, Canada.
authors.forename: Jennifer T
authors.initials: JT
authors.lastname: Lau
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, L8S 4K1, Canada.
authors.forename: Fiona J
authors.initials: FJ
authors.lastname: Whelan
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, L8S 4K1, Canada.
authors.forename: Isiri
authors.initials: I
authors.lastname: Herath
authors.affiliation: Department of Medicine, Division of Infectious Diseases, McMaster University, Hamilton, ON, L8S 4K1, Canada.|Hamilton Regional Laboratory Medicine Program, Hamilton, ON, L8N 4A6, Canada.
authors.forename: Christine H
authors.initials: CH
authors.lastname: Lee
authors.affiliation: Department of Medicine, Farncombe Family Digestive Health Research Institute, McMaster University, 1280 Main St W, HSC 3N-9, Hamilton, ON, L8S 4K1, Canada.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Collins
authors.affiliation: Department of Medicine, Farncombe Family Digestive Health Research Institute, McMaster University, 1280 Main St W, HSC 3N-9, Hamilton, ON, L8S 4K1, Canada.
authors.forename: Premysl
authors.initials: P
authors.lastname: Bercik
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, L8S 4K1, Canada. surette@mcmaster.ca.|Department of Medicine, Farncombe Family Digestive Health Research Institute, McMaster University, 1280 Main St W, HSC 3N-9, Hamilton, ON, L8S 4K1, Canada. surette@mcmaster.ca.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Surette
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D044822:Biodiversity; D018929:Cell Culture Techniques; D000068016:Eubacteriales / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D000069196:Gastrointestinal Microbiome / Q000235:genetics*; D041981:Gastrointestinal Tract / Q000382:microbiology; D005798:Genes, Bacterial; D064368:Healthy Volunteers; D006801:Humans; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D012336:RNA, Ribosomal, 16S