Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans

Peer reviewedPublisher PD

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

Genome sequences and comparative genomics of two Lactobacillus ruminis strains from the bovine and human intestinal tracts

peer-reviewedBackground: The genus Lactobacillus is characterized by an extraordinary degree of phenotypic and genotypic diversity, which recent genomic analyses have further highlighted. However, the choice of species for sequencing has been non-random and unequal in distribution, with only a single representative genome from the L. salivarius clade available to date. Furthermore, there is no data to facilitate a functional genomic analysis of motility in the lactobacilli, a trait that is restricted to the L. salivarius clade. Results: The 2.06 Mb genome of the bovine isolate Lactobacillus ruminis ATCC 27782 comprises a single circular chromosome, and has a G+C content of 44.4%. In silico analysis identified 1901 coding sequences, including genes for a pediocin-like bacteriocin, a single large exopolysaccharide-related cluster, two sortase enzymes, two CRISPR loci and numerous IS elements and pseudogenes. A cluster of genes related to a putative pilin was identified, and shown to be transcribed in vitro. A high quality draft assembly of the genome of a second L. ruminis strain, ATCC 25644 isolated from humans, suggested a slightly larger genome of 2.138 Mb, that exhibited a high degree of synteny with the ATCC 27782 genome. In contrast, comparative analysis of L. ruminis and L. salivarius identified a lack of long-range synteny between these closely related species. Comparison of the L. salivarius clade core proteins with those of nine other Lactobacillus species distributed across 4 major phylogenetic groups identified the set of shared proteins, and proteins unique to each group. Conclusions: The genome of L. ruminis provides a comparative tool for directing functional analyses of other members of the L. salivarius clade, and it increases understanding of the divergence of this distinct Lactobacillus lineage from other commensal lactobacilli. The genome sequence provides a definitive resource to facilitate investigation of the genetics, biochemistry and host interactions of these motile intestinal lactobacilli

Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota

Lactobacilli are Gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on Gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent

The MITRE trial protocol: a study to evaluate the microbiome as a biomarker of efficacy and toxicity in cancer patients receiving immune checkpoint inhibitor therapy.

BACKGROUND: The gut microbiome is implicated as a marker of response to  immune checkpoint inhibitors (ICI) based on preclinical mouse models and preliminary observations in limited patient series. Furthermore, early studies suggest faecal microbial transfer may have therapeutic potential, converting ICI non-responders into responders. So far, identification of specific responsible bacterial taxa has been inconsistent, which limits future application. The MITRE study will explore and validate a microbiome signature in a larger scale prospective study across several different cancer types. METHODS: Melanoma, renal cancer and non-small cell lung cancer patients who are planned to receive standard immune checkpoint inhibitors are being recruited to the MITRE study. Longitudinal stool samples are collected prior to treatment, then at 6 weeks, 3, 6 and 12 months during treatment, or at disease progression/recurrence (whichever is sooner), as well as after a severe (≥grade 3 CTCAE v5.0) immune-related adverse event. Additionally, whole blood, plasma, buffy coat, RNA and peripheral blood mononuclear cells (PBMCs) is collected at similar time points and will be used for exploratory analyses. Archival tumour tissue, tumour biopsies at progression/relapse, as well as any biopsies from body organs collected after a severe toxicity are collected. The primary outcome measure is the ability of the microbiome signature to predict 1 year progression-free survival (PFS) in patients with advanced disease. Secondary outcomes include microbiome correlations with toxicity and other efficacy end-points. Biosamples will be used to explore immunological and genomic correlates. A sub-study will evaluate both COVID-19 antigen and antibody associations with the microbiome. DISCUSSION: There is an urgent need to identify biomarkers that are predictive of treatment response, resistance and toxicity to immunotherapy. The data generated from this study will both help inform patient selection for these drugs and provide information that may allow therapeutic manipulation of the microbiome to improve future patient outcomes. TRIAL REGISTRATION: NCT04107168 , ClinicalTrials.gov, registered 09/27/2019. Protocol V3.2 (16/04/2021)

A human gut bacterial genome and culture collection for improved metagenomic analyses

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

Effects of Using Alternative Extreme Pressure (EP) and Anti-Wear (AW) Additives with Oxy-Nitrided Samples

Oxy-nitriding is a widely used industrial process aiming to improve the tribological properties and performance of components. Previous studies have shown the effectiveness of the treatment with friction and wear performance, but very few have focussed on optimising this behaviour. The lubrication properties of several EP and AW additives were examined to investigate their effectiveness in improving the tribological properties of the layers formed after treatment. Previous studies showed the presence of an oxide layer on the sample could improve the effectiveness of the sulphurised olefin (SO) and tricresyl phosphate (TCP) additives. The friction and wear behaviour of oxy-nitrided samples were analysed using a tribometer and surface profiler. Scanning electron microscope, energy-dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy were employed to identify the morphologies and chemical compositions of the treated surface before and after testing. No real effect on friction was observed when using the SO or TCP additives, mostly due to lack of interaction with the less reactive iron nitride layer and their roles as anti-wear additives. However, when the zinc dialkyldithiophosphate-containing lubricant was used, a higher friction coefficient was observed. Greater improvements in anti-wear properties with the presence of additives in comparison with only using base oil were reported, with the TCP additive producing the lowest wear rates. The study effectively demonstrated that the additive package type used could impact the tribological and tribochemical properties of oxy-nitrided surfaces

Using Remote Sensing to Map the Risk of Human Monkeypox Virus in the Congo Basin

Although the incidence of human monkeypox has greatly increased in Central Africa over the last decade, resources for surveillance remain extremely limited. We conducted a geospatial analysis using existing data to better inform future surveillance efforts. Using active surveillance data collected between 2005 and 2007, we identified locations in Sankuru district, Democratic Republic of Congo (DRC) where there have been one or more cases of human monkeypox. To assess what taxa constitute the main reservoirs of monkeypox, we tested whether human cases were associated with (i) rope squirrels (Funisciurus sp.), which were implicated in monkeypox outbreaks elsewhere in the DRC in the 1980s, or (ii) terrestrial rodents in the genera Cricetomys and Graphiurus, which are believed to be monkeypox reservoirs in West Africa. Results suggest that the best predictors of human monkeypox cases are proximity to dense forests and associated habitat preferred by rope squirrels. The risk of contracting monkeypox is significantly greater near sites predicted to be habitable for squirrels (OR = 1.32; 95% CI 1.08–1.63). We recommend that semi-deciduous rainforests with oil-palm, the rope squirrel’s main food source, be prioritized for monitoring

New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes

Identification and functional characterization of G6PC2 coding variants influencing glycemic traits define an effector transcript at the G6PC2-ABCB11 locus.

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights