Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and well-being1,2. Intestinal colonisation begins at birth and climaxes with the acquisition of two dominant groups of strict anaerobic bacteria belonging to the Firmicutes and Bacteroidetes phyla2. Culture independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases1. However, due to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown3. Here we describe a novel workflow based on targeted phenotypic culturing linked to large-scale whole genome sequencing, phylogenetic analysis and computational modeling that demonstrates that a substantial proportion of the intestinal bacteria are culturable. Applying this approach to healthy individuals, we isolated 137 bacterial species from characterised and candidate novel families, genera and species that were archived as pure cultures. Whole genome and metagenomic sequencing, combined with computational and phenotypic analysis, suggests that at least 50-60% of the bacterial genera from the intestinal microbiota of a healthy individual produce resilient spores, specialised for host-to-host transmission. Our approach unlocks the human intestinal microbiota for phenotypic analysis and reveals how a significant proportion of oxygen-sensitive intestinal bacteria can transmit between individuals, impacting microbiota heritability.

Immediately after birth, newborn babies experience rapid colonisation by microorganisms from their mothers and the surrounding environment 1. Diseases in childhood and later in life are potentially mediated through perturbation of the infant gut microbiota colonisations 2. However, the impact of modern clinical practices, such as caesarean section delivery and antibiotic usage, on the earliest stages of gut microbiota acquisition and development during the neonatal period (≤1 month) remains controversial 3,4. Here we report disrupted maternal transmission of Bacteroides strains and high-level colonisation by healthcare-associated opportunistic pathogens, including Enterococcus, Enterobacter and Klebsiella species, in babies delivered by caesarean section (C-section), and to a lesser extent, in those delivered vaginally with maternal antibiotic prophylaxis or not breastfed during the neonatal period. Applying longitudinal sampling and whole-genome shotgun metagenomic analysis on 1,679 gut microbiotas of 772 full term, UK-hospital born babies and mothers, we demonstrate that the mode of delivery is a significant factor impacting gut microbiota composition during the neonatal period that persists into infancy (1 month-1 year). Matched large-scale culturing and whole-genome sequencing (WGS) of over 800 bacterial strains cultured from these babies identified virulence factors and clinically relevant antimicrobial resistance (AMR) in opportunistic pathogens that may predispose to opportunistic infections. Our findings highlight the critical early roles of the local environment (i.e. mother and hospital) in establishing the gut microbiota in very early life, and identifies colonisation with AMR carrying, healthcare-associated opportunistic pathogens as a previously unappreciated risk factor.

Understanding gut microbiome functions requires cultivated bacteria for experimental validation and reference bacterial genome sequences to interpret metagenome datasets and guide functional analyses. We present the Human Gastrointestinal Bacteria Culture Collection (HBC), a comprehensive set of 737 whole-genome-sequenced bacterial isolates, representing 273 species (105 novel species) from 31 families found in the human gastrointestinal microbiota. The HBC increases the number of bacterial genomes derived from human gastrointestinal microbiota by 37%. The resulting global Human Gastrointestinal Bacteria Genome Collection (HGG) classifies 83% of genera by abundance across 13,490 shotgun-sequenced metagenomic samples, improves taxonomic classification by 61% compared to the Human Microbiome Project (HMP) genome collection and achieves subspecies-level classification for almost 50% of sequences. The improved resource of gastrointestinal bacterial reference sequences circumvents dependence on de novo assembly of metagenomes and enables accurate and cost-effective shotgun metagenomic analyses of human gastrointestinal microbiota.

Metagenomic analyses have indicated that the female bladder harbors an indigenous microbiota. However, there are few cultured reference strains with sequenced genomes available for functional and experimental analyses. Here we isolate and genome-sequence 149 bacterial strains from catheterized urine of 77 women. This culture collection spans 78 species, representing approximately two thirds of the bacterial diversity within the sampled bladders, including Proteobacteria, Actinobacteria, and Firmicutes. Detailed genomic and functional comparison of the bladder microbiota to the gastrointestinal and vaginal microbiotas demonstrates similar vaginal and bladder microbiota, with functional capacities that are distinct from those observed in the gastrointestinal microbiota. Whole-genome phylogenetic analysis of bacterial strains isolated from the vagina and bladder in the same women identifies highly similar Escherichia coli, Streptococcus anginosus, Lactobacillus iners, and Lactobacillus crispatus, suggesting an interlinked female urogenital microbiota that is not only limited to pathogens but is also characteristic of health-associated commensals.

We identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.