Bacteriotherapy for the treatment of intestinal dysbiosis caused by Clostridium difficile infection

Faecal microbiota transplantation (FMT) has been used for more than five decades to treat a variety of intestinal diseases associated with pathological imbalances within the resident microbiota, termed dysbiosis. FMT has been particularly effective for treating patients with recurrent Clostridium difficile infection who are left with few clinical options other than continued antibiotic therapy. Our increasing knowledge of the structure and function of the human intestinal microbiota and C. difficile pathogenesis has led to the understanding that FMT promotes intestinal ecological restoration and highlights the microbiota as a viable therapeutic target. However, the use of undefined faecal samples creates a barrier for widespread clinical use because of safety and aesthetic issues. An emerging concept of bacteriotherapy, the therapeutic use of a defined mixture of harmless, health-associated bacteria, holds promise for the treatment of patients with severe C. difficile infection, and possibly represents a paradigm shift for the treatment of diseases linked to intestinal dysbiosis

Emerging insights on intestinal dysbiosis during bacterial infections

Infection of the gastrointestinal tract is commonly linked to pathological imbalances of the resident microbiota, termed dysbiosis. In recent years, advanced high-throughput genomic approaches have allowed us to examine the microbiota in an unprecedented manner, revealing novel biological insights about infection-associated dysbiosis at the community and individual species levels. A dysbiotic microbiota is typically reduced in taxonomic diversity and metabolic function, and can harbour pathobionts that exacerbate intestinal inflammation or manifest systemic disease. Dysbiosis can also promote pathogen genome evolution, while allowing the pathogens to persist at high density and transmit to new hosts. A deeper understanding of bacterial pathogenicity in the context of the intestinal microbiota should unveil new approaches for developing diagnostics and therapies for enteropathogens

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

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 Staphylococcus 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

Should we modulate the neonatal microbiome and what should be the goal?

Peer reviewe

Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation

Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and wellbeing. 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. Culture-independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases. However, owing to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown. 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

Polysaccharide utilization loci and nutritional specialization in a dominant group of butyrate-producing human colonic Firmicutes

Acknowledgements The Rowett Institute of Nutrition and Health (University of Aberdeen) receives financial support from the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS). POS is a PhD student supported by the Scottish Government (RESAS) and the Science Foundation Ireland, through a centre award to the APC Microbiome Institute, Cork, Ireland. Data Summary The high-quality draft genomes generated in this work were deposited at the European Nucleotide Archive under the following accession numbers: 1. Eubacterium rectale T1-815; CVRQ01000001–CVRQ0100 0090: http://www.ebi.ac.uk/ena/data/view/PRJEB9320 2. Roseburia faecis M72/1; CVRR01000001–CVRR010001 01: http://www.ebi.ac.uk/ena/data/view/PRJEB9321 3. Roseburia inulinivorans L1-83; CVRS01000001–CVRS0 100 0151: http://www.ebi.ac.uk/ena/data/view/PRJEB9322Peer reviewedPublisher PD

Genome-Wide Screen for Salmonella Genes Required for Long-Term Systemic Infection of the Mouse

A microarray-based negative selection screen was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium) genes that contribute to long-term systemic infection in 129X1/SvJ (Nramp1(r)) mice. A high-complexity transposon-mutagenized library was used to infect mice intraperitoneally, and the selective disappearance of mutants was monitored after 7, 14, 21, and 28 d postinfection. One hundred and eighteen genes were identified to contribute to serovar Typhimurium infection of the spleens of mice by 28 d postinfection. The negatively selected mutants represent many known aspects of Salmonella physiology and pathogenesis, although the majority of the identified genes are of putative or unknown function. Approximately 30% of the negatively selected genes correspond to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1–5), prophages (Gifsy-1 and −2 and remnant), and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. Competitive index experiments demonstrated that the secreted SPI2 effectors SseK2 and SseJ as well as the SPI4 locus are attenuated relative to wild-type bacteria during systemic infection. Interestingly, several SPI1-encoded type III secretion system effectors/translocases are required by serovar Typhimurium to establish and, unexpectedly, to persist systemically, challenging the present description of Salmonella pathogenesis. Moreover, we observed a progressive selection against serovar Typhimurium mutants based upon the duration of the infection, suggesting that different classes of genes may be required at distinct stages of infection. Overall, these data indicate that Salmonella long-term systemic infection in the mouse requires a diverse repertoire of virulence factors. This diversity of genes presumably reflects the fact that bacteria sequentially encounter a variety of host environments and that Salmonella has evolved to respond to these selective forces in a way that permits both the bacteria and the host to survive

The association between early-life gut microbiota and childhood respiratory diseases: a systematic review

Data from animal models suggest a role of early-life gut microbiota in lung immune development, and in establishing susceptibility to respiratory infections and asthma in humans. This systematic review summarises the association between infant (ages 0-12 months) gut microbiota composition measured by genomic sequencing, and childhood (ages 0-18 years) respiratory diseases (ie, respiratory infections, wheezing, or asthma). Overall, there was evidence that low α-diversity and relative abundance of particular gut-commensal bacteria genera (Bifidobacterium, Faecalibacterium, Ruminococcus, and Roseburia) are associated with childhood respiratory diseases. However, results were inconsistent and studies had important limitations, including insufficient characterisation of bacterial taxa to species level, heterogeneous outcome definitions, residual confounding, and small sample sizes. Large longitudinal studies with stool sampling during the first month of life and shotgun metagenomic approaches to improve bacterial and fungal taxa resolution are needed. Standardising follow-up times and respiratory disease definitions and optimising causal statistical approaches might identify targets for primary prevention of childhood respiratory diseases

Stunted microbiota and opportunistic pathogen colonization in caesarean-section birth

Immediately after birth, newborn babies experience rapid colonization by microorganisms from their mothers and the surrounding environment1. Diseases in childhood and later in life are potentially mediated by the perturbation of the colonization of the infant gut microbiota2. However, the effects of delivery via caesarean section on the earliest stages of the acquisition and development of the gut microbiota, during the neonatal period (≤1 month), remain controversial3,4. Here we report the disrupted transmission of maternal Bacteroides strains, and high-level colonization by opportunistic pathogens associated with the hospital environment (including Enterococcus, Enterobacter and Klebsiella species), in babies delivered by caesarean section. These effects were also seen, to a lesser extent, in vaginally delivered babies whose mothers underwent antibiotic prophylaxis and in babies who were not breastfed during the neonatal period. We applied longitudinal sampling and whole-genome shotgun metagenomic analysis to 1,679 gut microbiota samples (taken at several time points during the neonatal period, and in infancy) from 596 full-term babies born in UK hospitals; for a subset of these babies, we collected additional matched samples from mothers (175 mothers paired with 178 babies). This analysis demonstrates that the mode of delivery is a significant factor that affects the composition of the gut microbiota throughout the neonatal period, and into infancy. Matched large-scale culturing and whole-genome sequencing of over 800 bacterial strains from these babies identified virulence factors and clinically relevant antimicrobial resistance in opportunistic pathogens that may predispose individuals to opportunistic infections. Our findings highlight the critical role of the local environment in establishing the gut microbiota in very early life, and identify colonization with antimicrobial-resistance-containing opportunistic pathogens as a previously underappreciated risk factor in hospital births