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A novel approach is used to cultivate a substantial proportion of the human gut microbiota, representing an important step forward in characterizing the role of these bacteria in health and disease. Metagenomic analysis of human gut microbiota The human intestinal microbiota comprises perhaps hundreds of bacterial species. Most of them are regarded as 'unculturable' and have never been isolated in the laboratory. Trevor Lawley and colleagues have used a novel approach based on targeted phenotypic culturing linked to large-scale whole-genome sequencing, phylogenetic analysis and computational modelling, to cultivate a substantial proportion of the human gut microbiota. Applying this workflow to faecal samples from healthy individuals, the authors isolated 137 distinct bacterial species as pure cultures, including 45 candidate novel species. More than half of the bacterial genera identified produce resilient spores, specialized for host-to-host transmission. This work demonstrates the value of phenotypic-based studies in determining the role of the gut microbiota in health and disease. Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and wellbeing 1 , 2 . Intestinal colonization begins at birth and climaxes with the acquisition of two dominant groups of strict anaerobic bacteria belonging to the Firmicutes and Bacteroidetes phyla 2 . Culture-independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases 1 . However, owing to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown 3 . Here we describe a novel workflow based on targeted phenotypic culturing linked to large-scale whole-genome sequencing, phylogenetic analysis and computational modelling that demonstrates that a substantial proportion of the intestinal bacteria are culturable. Applying this approach to healthy individuals, we isolated 137 bacterial species from characterized 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, specialized for host-to-host transmission. Our approach unlocks the human intestinal microbiota for phenotypic analysis and reveals how a marked proportion of oxygen-sensitive intestinal bacteria can be transmitted between individuals, affecting microbiota heritability.

Key Points The human intestinal microbiota is dominated by anaerobic health-associated bacteria that are established and maintained in individuals through host-to-host transmission. The transmission process incorporates excretion from a host in faecal matter, survival and persistence in the external environment, and concludes with ingestion and subsequent colonization of a new host. Studies of the routes of transmission of intestinal pathogens provide a useful framework to better understand intestinal commensal transmission. Both share some common transmission features, such as the use of the faecal–oral transmission route and similar survival mechanisms to persist in the external environment. Environmental survival mechanisms that are used by the intestinal microbiota once expelled by a host include sporulation, aerotolerance and entering a viable but non-culturable (VBNC) dormancy state. For anaerobic bacteria, these mechanisms protect against harmful oxygen, and in the case of sporulation and VBNC states, can provide varying resistance against other environmental conditions, such as desiccation and a lack of nutrients. Reservoirs are a source or a sink for bacteria during transmission. Other people in the community are the principal reservoirs of intestinal bacteria, but food, water, animals and the built environment may also facilitate transmission. Transmission of commensal bacteria may be disrupted by human sanitation practices, through the use of antibiotics or a long-term change in diet, which can eliminate species within an individual thereby preventing their onward transmission. Direct interventions to restore a depleted microbiota, such as faecal microbiota transplantation (FMT), are effective in some cases. A greater general awareness of the transmission of the commensal microbiota is facilitated by technological advances in different disciplines, including microbiology, bioinformatics and genomics. Fostering the transmission of commensal bacteria between people through the maintenance of a healthy lifestyle and the discerning use of antibiotics and sanitation processes may promote human health. The transmission of commensal intestinal bacteria between humans could promote health by establishing, maintaining and replenishing microbial diversity in the microbiota of an individual. In this Review, Browne and colleagues discuss the mechanisms and factors that influence host-to-host transmission of the intestinal microbiota. Transmission of commensal intestinal bacteria between humans could promote health by establishing, maintaining and replenishing microbial diversity in the microbiota of an individual. Unlike pathogens, the routes of transmission for commensal bacteria remain unappreciated and poorly understood, despite the likely commonalities between both. Consequently, broad infection control measures that are designed to prevent pathogen transmission and infection, such as oversanitation and the overuse of antibiotics, may inadvertently affect human health by altering normal commensal transmission. In this Review, we discuss the mechanisms and factors that influence host-to-host transmission of the intestinal microbiota and examine how a better understanding of these processes will identify new approaches to nurture and restore transmission routes that are used by beneficial bacteria.

Background Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112–5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq. Results Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the S taphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset. Conclusion The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.

Mobile genetic elements (MGEs) carrying antibiotic resistance genes (ARGs) disseminate ARGs when they mobilise into new bacterial hosts. The nature of such horizontal gene transfer (HGT) events between human gut commensals and pathogens remain poorly characterised. Here, we compare 1354 cultured commensal strains (540 species) to 45,403 pathogen strains (12 species) and find 64,188 MGE-mediated ARG transfer events between the two groups using established methods. Among the 5931 MGEs, we find 15 broad host range elements predicted to have crossed different bacterial phyla while also occurring in animal and environmental microbiomes. We experimentally demonstrate that predicted broad host range MGEs can mobilise from commensals Dorea longicatena and Hungatella hathewayi to pathogen Klebsiella oxytoca , crossing phyla simultaneously. Our work establishes the MGE-mediated ARG dissemination network between human gut commensals and pathogens and highlights broad host range MGEs as targets for future ARG dissemination management. Here, Forster et al. compare 1354 cultured commensal strains (540 species) to 45,403 pathogen strains (12 species), identifying 64,188 MGE-mediated antibiotic resistance gene transfer events between the two groups, and show that 15 broad host range MGEs are able to transfer between phyla.

Background Human-to-human transmission of symbiotic, anaerobic bacteria is a fundamental evolutionary adaptation essential for membership of the human gut microbiota. However, despite its importance, the genomic and biological adaptations underpinning symbiont transmission remain poorly understood. The Firmicutes are a dominant phylum within the intestinal microbiota that are capable of producing resistant endospores that maintain viability within the environment and germinate within the intestine to facilitate transmission. However, the impact of host transmission on the evolutionary and adaptive processes within the intestinal microbiota remains unknown. Results We analyze 1358 genomes of Firmicutes bacteria derived from host and environment-associated habitats. Characterization of genomes as spore-forming based on the presence of sporulation-predictive genes reveals multiple losses of sporulation in many distinct lineages. Loss of sporulation in gut Firmicutes is associated with features of host-adaptation such as genome reduction and specialized metabolic capabilities. Consistent with these data, analysis of 9966 gut metagenomes from adults around the world demonstrates that bacteria now incapable of sporulation are more abundant within individuals but less prevalent in the human population compared to spore-forming bacteria. Conclusions Our results suggest host adaptation in gut Firmicutes is an evolutionary trade-off between transmission range and colonization abundance. We reveal host transmission as an underappreciated process that shapes the evolution, assembly, and functions of gut Firmicutes.

Bacterial speciation is a fundamental evolutionary process characterized by diverging genotypic and phenotypic properties. However, the selective forces that affect genetic adaptations and how they relate to the biological changes that underpin the formation of a new bacterial species remain poorly understood. Here, we show that the spore-forming, healthcare-associated enteropathogen Clostridium difficile is actively undergoing speciation. Through large-scale genomic analysis of 906 strains, we demonstrate that the ongoing speciation process is linked to positive selection on core genes in the newly forming species that are involved in sporulation and the metabolism of simple dietary sugars. Functional validation shows that the new C. difficile produces spores that are more resistant and have increased sporulation and host colonization capacity when glucose or fructose is available for metabolism. Thus, we report the formation of an emerging C. difficil e species, selected for metabolizing simple dietary sugars and producing high levels of resistant spores, that is adapted for healthcare-mediated transmission. Genomic analysis of 906 Clostridium difficile strains shows that this enteropathogen is undergoing speciation, which is linked to the selection of genes involved in sporulation and in the metabolism of simple dietary sugars.

Gut bacterial infections involve three-way interactions between virulence factors, the host immune responses, and the microbiome. While the microbiome erects colonization resistance barriers, pathogens employ virulence factors to overcome them. The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium , a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing and succumbing to the infection, respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in relocalization of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumens of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon that we term antibiotic-induced bacterial commensalization (AIBC). AIBC C. rodentium was well tolerated by the host, which showed few signs of inflammation; passaged AIBC C. rodentium robustly infected naive C3H/HeN mice, suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalization in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus , which inhibited the growth of C. rodentium in vitro in a contact-dependent manner and the luminal growth of AIBC C. rodentium in vivo . Overall, our data suggest that commensal species can confer colonization resistance to closely related pathogenic species. IMPORTANCE Gut bacterial infections involve three-way interactions between virulence factors, the host immune responses, and the microbiome. While the microbiome erects colonization resistance barriers, pathogens employ virulence factors to overcome them. Treating mice infected with kanamycin-resistant Citrobacter rodentium with kanamycin caused displacement of the pathogen from the colonic mucosa to the cecal lumen. Following withdrawal of the kanamycin treatment, 65% of the mice were persistently colonized by C. rodentium , which seemed to downregulate virulence factor expression. In this model of luminal gut colonization, 35% of mice were refractory to stable C. rodentium colonization, suggesting that their microbiotas were able to confer colonization resistance. We identify a commensal bacterium of the Citrobacter genus, C. amalonaticus , which inhibits C. rodentium growth in vitro and in vivo . These results show that the line separating commensal and pathogenic lifestyles is thin and multifactorial and that commensals may play a major role in combating enteric infection.

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.A large bacterial strain collection and genome sequences will boost gut microbiome research.

Studies of the microbes living on and in our bodies are conducted mainly in a few rich countries, squandering opportunities to improve the health of people globally. Studies of the microbes living on and in our bodies are conducted mainly in a few rich countries, squandering opportunities to improve the health of people globally. Two young researchers at icddr,b Genome Centre are seen in the lab processing microbiome samples

Clostridium difficile is an anaerobic, Gram-positive bacterium that can reside as a commensal within the intestinal microbiota of healthy individuals or cause life-threatening antibiotic-associated diarrhea in immunocompromised hosts. C. difficile can also form highly resistant spores that are excreted facilitating host-to-host transmission. The C. difficile spo0A gene encodes a highly conserved transcriptional regulator of sporulation that is required for relapsing disease and transmission in mice. Here we describe a genome-wide approach using a combined transcriptomic and proteomic analysis to identify Spo0A regulated genes. Our results validate Spo0A as a positive regulator of putative and novel sporulation genes as well as components of the mature spore proteome. We also show that Spo0A regulates a number of virulence-associated factors such as flagella and metabolic pathways including glucose fermentation leading to butyrate production. The C. difficile spo0A gene is a global transcriptional regulator that controls diverse sporulation, virulence and metabolic phenotypes coordinating pathogen adaptation to a wide range of host interactions. Additionally, the rich breadth of functional data allowed us to significantly update the annotation of the C. difficile 630 reference genome which will facilitate basic and applied research on this emerging pathogen.