Networks of gut bacteria relate to cardiovascular disease in a multi-ethnic population: the HELIUS study.

AIMS: Gut microbiota have been linked to blood lipid levels and cardiovascular diseases (CVDs). The composition and abundance of gut microbiota trophic networks differ between ethnicities. We aim to evaluate the relationship between gut microbiotal trophic networks and CVD phenotypes. METHODS AND RESULTS: We included cross-sectional data from 3860 individuals without CVD history from 6 ethnicities living in the Amsterdam region participating in the prospective Healthy Life in Urban Setting (HELIUS) study. Genetic variants were genotyped, faecal gut microbiota were profiled, and blood and anthropometric parameters were measured. A machine learning approach was used to assess the relationship between CVD risk (Framingham score) and gut microbiota stratified by ethnicity. Potential causal relationships between gut microbiota composition and CVD were inferred by performing two-sample Mendelian randomization with hard CVD events from the Pan-UK Biobank and microbiome genome-wide association studies summary data from a subset of the HELIUS cohort (n = 4117). Microbial taxa identified to be associated with CVD by machine learning and Mendelian randomization were often ethnic-specific, but some concordance across ethnicities was found. The microbes Akkermansia muciniphila and Ruminococcaceae UCG-002 were protective against ischaemic heart disease in African-Surinamese and Moroccans, respectively. We identified a strong inverse association between blood lipids, CVD risk, and the combined abundance of the correlated microbes Christensenellaceae-Methanobrevibacter-Ruminococcaceae (CMR). The CMR cluster was also identified in two independent cohorts and the association with triglycerides was replicated. CONCLUSION: Certain gut microbes can have a potentially causal relationship with CVD events, with possible ethnic-specific effects. We identified a trophic network centred around Christensenellaceae, Methanobrevibacter, and various Ruminococcaceae, frequently lacking in South-Asian Surinamese, to be protective against CVD risk and associated with low triglyceride levels.

Placental Streptococcus agalactiae DNA is associated with neonatal unit admission and foetal pro-inflammatory cytokines in term infants.

Streptococcus agalactiae (Group B Streptococcus; GBS) is a common cause of sepsis in neonates. Previous work detected GBS DNA in the placenta in ~5% of women before the onset of labour, but the clinical significance of this finding is unknown. Here we re-analysed this dataset as a case control study of neonatal unit (NNU) admission. Of 436 infants born at term (≥37 weeks of gestation), 7/30 with placental GBS and 34/406 without placental GBS were admitted to the NNU (odds ratio (OR) 3.3, 95% confidence interval (CI) 1.3-7.8). We then performed a validation study using non-overlapping subjects from the same cohort. This included a further 239 cases of term NNU admission and 686 term controls: 16/36 with placental GBS and 223/889 without GBS were admitted to the NNU (OR 2.4, 95% CI 1.2-4.6). Of the 36 infants with placental GBS, 10 were admitted to the NNU with evidence of probable but culture-negative sepsis (OR 4.8, 95% CI 2.2-10.3), 2 were admitted with proven GBS sepsis (OR 66.6, 95% CI 7.3-963.7), 6 were admitted and had chorioamnionitis (inflammation of the foetal membranes) (OR 5.3, 95% CI 2.0-13.4), and 5 were admitted and had funisitis (inflammation of the umbilical cord) (OR 6.7, 95% CI 12.5-17.7). Foetal cytokine storm (two or more pro-inflammatory cytokines >10 times median control levels in umbilical cord blood) was present in 36% of infants with placental GBS DNA and 4% of cases where the placenta was negative (OR 14.2, 95% CI 3.6-60.8). Overall, ~1 in 200 term births had GBS detected in the placenta, which was associated with infant NNU admission and morbidity.

Optimization of high-throughput 16S rRNA gene amplicon sequencing: an assessment of PCR pooling, mastermix use and contamination.

16S rRNA gene sequencing is widely used to characterize human and environmental microbiomes. Sequencing at scale facilitates better powered studies but is limited by cost and time. We identified two areas in our 16S rRNA gene library preparation protocol where modifications could provide efficiency gains, including (1) pooling of multiple PCR amplifications per sample to reduce PCR drift and (2) manual preparation of mastermix to reduce liquid handling. Using nasal samples from healthy human participants and a serially diluted mock microbial community, we compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. One hundred and fifty-eight 16S rRNA gene sequencing libraries were prepared, including a replicate experiment. Comparing PCR pooling strategies, we found no significant difference in high-quality read counts and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate on principal coordinate analysis (PCoA) and non-metric dimensional scaling (NMDS) analysis. Choice of mastermix had no significant impact on high-quality read and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate in PCoA and NMDS analysis. Importantly, we observed contamination and variability of rare species (<0.01 %) across replicate experiments; the majority of contaminants were accounted for by removal of species present at <0.1 %, or were linked to reagents (including a primer stock). We demonstrate no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol.

Gut microbiome transitions across generations in different ethnicities in an urban setting-the HELIUS study.

BACKGROUND: During the course of history, various important lifestyle changes have caused profound transitions of the gut microbiome. These include the introduction of agriculture and animal husbandry, a shift from a nomadic to a more sedentary lifestyle, and recently increased levels of urbanization and a transition towards a more Western lifestyle. The latter is linked with shifts in the gut microbiome that have a reduced fermentative capability and which are commonly associated with diseases of affluence. In this study, in which 5193 subjects are included, we investigated the direction of microbiome shifts that occur in various ethnicities living in Amsterdam by comparing 1st and 2nd generation participants. We furthermore validated part of these findings with a cohort of subjects that moved from rural Thailand to the USA. RESULTS: The abundance of the Prevotella cluster, which includes P. copri and the P. stercorea trophic network, diminished in the 2nd generation Moroccans and Turks but also in younger Dutch, whilst the Western-associated Bacteroides/Blautia/Bifidobacterium (BBB) cluster, which has an inverse correlation with α-diversity, increased. At the same time, the Christensenellaceae/Methanobrevibacter/Oscillibacter trophic network, which is positively associated with α-diversity and a healthy BMI, decreased in younger Turks and Dutch. Large compositional shifts were not observed in South-Asian and African Surinamese, in whom the BBB cluster is already dominant in the 1st generation, but ASV-level shifts towards certain species, associated amongst others with obesity, were observed. CONCLUSION: The Moroccan and Turkish populations, but also the Dutch population are transitioning towards a less complex and fermentative less capable configuration of the gut microbiota, which includes a higher abundance of the Western-associated BBB cluster. The Surinamese, whom have the highest prevalence of diabetes and other diseases of affluence, are already dominated by the BBB cluster. Given the continuous increase in diseases of affluence, this devolution towards low-diversity and fermentatively less capable gut microbiome compositions in urban environments is a worrying development. Video Abstract.

Identifying Gut Microbiota associated with Gastrointestinal Symptoms upon Roux-en-Y Gastric Bypass.

PURPOSE: Roux-en-Y gastric bypasses (RYGB) are frequently accompanied by long-term gastrointestinal (GI) symptoms. Direct mechanistic insight into the causation of these symptoms is lacking, but changes in the intestinal microbiome have been proposed to play a role. With this study, we aimed to investigate whether a microbial predisposition exists before RYGB which is associated with GI symptoms during follow-up and to evaluate which microbial groups are involved. MATERIALS AND METHODS: In total, 67 RYGB patients were included. Shotgun metagenomic sequencing was performed on fecal samples obtained just before and 1 year after surgery. To assess GI symptoms, patients filled out Gastrointestinal Quality of Life Index (GIQLI) questionnaires and were divided into groups based on their total GIQLI score and change in score (postsurgery versus baseline). Extremely randomized tree predictor models were used to identify the most distinctive microbial species associated with postoperative GI symptoms. RESULTS: Beta diversity differed significantly between baseline and 1-year post-surgery samples, with the post-surgery microbiome resembling a more dysbiotic profile. The most predictive species regarding total GIQLI (AUC 0.77) or delta GIQLI score (AUC 0.83) were identified. Many of these species are known butyrate producers or species known to support them and/or species with anti-inflammatory properties, including Coprococcus eutactus, Faecalibacterium prausnitzii, and Ruminococcus callidus. CONCLUSION: Beneficial commensal gut microbiota related to a high GI score were associated to adequate intestinal fermentative capacity, suggesting these species might have protective properties against postoperative GI malfunctioning.

Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Gut Microbiota in Nutrition and Health with a Special Focus on Specific Bacterial Clusters.

Health is influenced by how the gut microbiome develops as a result of external and internal factors, such as nutrition, the environment, medication use, age, sex, and genetics. Alpha and beta diversity metrics and (enterotype) clustering methods are commonly employed to perform population studies and to analyse the effects of various treatments, yet, with the continuous development of (new) sequencing technologies, and as various omics fields as a result become more accessible for investigation, increasingly sophisticated methodologies are needed and indeed being developed in order to disentangle the complex ways in which the gut microbiome and health are intertwined. Diseases of affluence, such as type 2 diabetes (T2D) and cardiovascular diseases (CVD), are commonly linked to species associated with the Bacteroides enterotype(s) and a decline of various (beneficial) complex microbial trophic networks, which are in turn linked to the aforementioned factors. In this review, we (1) explore the effects that some of the most common internal and external factors have on the gut microbiome composition and how these in turn relate to T2D and CVD, and (2) discuss research opportunities enabled by and the limitations of some of the latest technical developments in the microbiome sector, including the use of artificial intelligence (AI), strain tracking, and peak to trough ratios.

Recent insights into the role of microbiome in the pathogenesis of obesity.

Obesity is a risk factor for many chronic diseases and its rising prevalence the last couple of decades is a healthcare concern in many countries. Obesity is a multifactorial problem that is not only limited in its causation by diet and lack of exercise. Genetics but also environmental factors such as the gut microbiome should similarly be taken into account. A plethora of articles have been published, that from various different angles, attempt to disentangle the complex interaction between gut microbiota and obesity. Examples range from the effect of the gut microbiota on the host immune system to the pathophysiological pathways in which microbial-derived metabolites affect obesity. Various discordant gut microbiota findings are a result of this complexity. In this review, in addition to summarizing the classical role of the gut microbiome in the pathogenesis of obesity, we attempt to view both the healthy and obesogenic effects of the gut microbiota as a consequence of the presence or absence of collective guilds/trophic networks. Lastly, we propose avenues and strategies for the future of gut microbiome research concerning obesity.

Patients With Inflammatory Bowel Disease Show IgG Immune Responses Towards Specific Intestinal Bacterial Genera.

Introduction: Inflammatory bowel disease (IBD) is characterized by a disturbed gut microbiota composition. Patients with IBD have both elevated mucosal and serum levels of IgG-antibodies directed against bacterial antigens, including flagellins. In this study, we aimed to determine to which intestinal bacteria the humoral immune response is directed to in patients with IBD. Methods: Fecal and serum samples were collected from patients with IBD (n=55) and age- and sex-matched healthy controls (n=55). Fecal samples were incubated with autologous serum and IgG-coated fractions were isolated by magnetic-activated cell sorting (MACS) and its efficiency was assessed by flow cytometry. The bacterial composition of both untreated and IgG-coated fecal samples was determined by 16S rRNA-gene Illumina sequencing. Results: IgG-coated fecal samples were characterized by significantly lower microbial diversity compared to the fecal microbiome. Both in patients with IBD and controls, serum IgG responses were primarily directed to Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Veillonella and Enterobacteriaceae, as well as against specific Lachnospiraceae bacteria, including Coprococcus and Dorea (all P<0.001), and to Ruminococcus gnavus-like bacteria (P<0.05). In contrast, serological IgG responses against typical commensal, anaerobic and colonic microbial species were rather low, e.g. to the Lachnospiraceae members Roseburia and Blautia, to Faecalibacterium, as well as to Bacteroides. Patients with IBD showed more IgG-coating of Streptococcus, Lactobacillus, and Lactococcus bacteria compared to healthy controls (all P<0.05). No differences in IgG-coated bacterial fractions were observed between Crohn's disease and ulcerative colitis, between active or non-active disease, nor between different disease locations. Conclusion: The IgG immune response is specifically targeted at distinct intestinal bacterial genera that are typically associated with the small intestinal microbiota, whereas responses against more colonic-type commensals are lower, which was particularly the case for patients with IBD. These findings may be indicative of a strong immunological exposure to potentially pathogenic intestinal bacteria in concordance with relative immune tolerance against commensal bacteria.

Gut microbiomes from Gambian infants reveal the development of a non-industrialized Prevotella-based trophic network.

Distinct bacterial trophic networks exist in the gut microbiota of individuals in industrialized and non-industrialized countries. In particular, non-industrialized gut microbiomes tend to be enriched with Prevotella species. To study the development of these Prevotella-rich compositions, we investigated the gut microbiota of children aged between 7 and 37 months living in rural Gambia (616 children, 1,389 stool samples, stratified by 3-month age groups). These infants, who typically eat a high-fibre, low-protein diet, were part of a double-blind, randomized iron intervention trial (NCT02941081) and here we report the secondary outcome. We found that child age was the largest discriminating factor between samples and that anthropometric indices (collection time points, season, geographic collection site, and iron supplementation) did not significantly influence the gut microbiome. Prevotella copri, Faecalibacterium prausnitzii and Prevotella stercorea were, on average, the most abundant species in these 1,389 samples (35%, 11% and 7%, respectively). Distinct bacterial trophic network clusters were identified, centred around either P. stercorea or F. prausnitzii and were found to develop steadily with age, whereas P. copri, independently of other species, rapidly became dominant after weaning. This dataset, set within a critical gut microbial developmental time frame, provides insights into the development of Prevotella-rich gut microbiomes, which are typically understudied and are underrepresented in western populations.

Effects of fecal microbiota transplant on DNA methylation in subjects with metabolic syndrome.

Accumulating evidence shows that microbes with their theater of activity residing within the human intestinal tract (i.e., the gut microbiome) influence host metabolism. Some of the strongest results come from recent fecal microbial transplant (FMT) studies that relate changes in intestinal microbiota to various markers of metabolism as well as the pathophysiology of insulin resistance. Despite these developments, there is still a limited understanding of the multitude of effects associated with FMT on the general physiology of the host, beyond changes in gut microbiome composition. We examined the effect of either allogenic (lean donor) or autologous FMTs on the gut microbiome, plasma metabolome, and epigenomic (DNA methylation) reprogramming in peripheral blood mononuclear cells in individuals with metabolic syndrome measured at baseline (pre-FMT) and after 6 weeks (post-FMT). Insulin sensitivity was determined with a stable isotope-based 2 step hyperinsulinemic clamp and multivariate machine learning methodology was used to uncover discriminative microbes, metabolites, and DNA methylation loci. A larger gut microbiota shift was associated with an allogenic than with autologous FMT. Furthemore, the data results of the the allogenic FMT group data indicates that the introduction of new species can potentially modulate the plasma metabolome and (as a result) the epigenome. Most notably, the introduction of Prevotella ASVs directly correlated with methylation of AFAP1, a gene involved in mitochondrial function, insulin sensitivity, and peripheral insulin resistance (Rd, rate of glucose disappearance). FMT was found to have notable effects on the gut microbiome but also on the host plasma metabolome and the epigenome of immune cells providing new avenues of inquiry in the context of metabolic syndrome treatment for the manipulation of host physiology to achieve improved insulin sensitivity.

Weight shapes the intestinal microbiome in preterm infants: results of a prospective observational study.

BACKGROUND: The intestinal microbiome in preterm infants differs markedly from term infants. It is unclear whether the microbiome develops over time according to infant specific factors. METHODS: We analysed (clinical) metadata - to identify the main factors influencing the microbiome composition development - and the first meconium and faecal samples til the 4th week via 16 S rRNA amplican sequencing. RESULTS: We included 41 infants (gestational age 25-30 weeks; birth weight 430-990 g. Birth via Caesarean section (CS) was associated with placental insufficiency during pregnancy and lower BW. In meconium samples and in samples from weeks 2 and 3 the abundance of Escherichia and Bacteroides (maternal faecal representatives) were associated with vaginal delivery while Staphylococcus (skin microbiome representative) was associated with CS. Secondly, irrespective of the week of sampling or the mode of birth, a transition was observed as children children gradually increased in weight from a microbiome dominated by Staphylococcus (Bacilli) towards a microbiome dominated by Enterobacteriaceae (Gammaproteobacteria). CONCLUSIONS: Our data show that the mode of delivery affects the meconium microbiome composition. They also suggest that the weight of the infant at the time of sampling is a better predictor for the stage of progression of the intestinal microbiome development/maturation than postconceptional age as it less confounded by various infant-specific factors.

Batch effects account for the main findings of an in utero human intestinal bacterial colonization study.

A recent study by Rackaityte et al. reported evidence for a low level of bacterial colonization, specifically of Micrococcus luteus, in the intestine of second trimester human fetuses. We have re-analyzed their sequence data and identified a batch effect which violates the underlying assumptions of the bioinformatic method used for contamination removal. This batch effect resulted in Micrococcus not being identified as a contaminant in the original work and being falsely assigned to the fetal samples. We further provide evidence that the micrographs presented by Rackaityte et al. are unlikely to show Micrococci or other bacteria as the size of the particles shown exceeds that of related bacterial cells. Finally, phylogenetic analysis showed that the microbes cultured from the fetal samples differed significantly from those detected by sequencing. Overall, our findings show that the presence of Micrococcus in the fetal gut is not supported by the primary sequence data. Our findings underline important aspects of the nature of contamination for both sequencing and culture approaches in microbiome studies and the appropriate use of automated contamination identification tools.

STROBE-metagenomics: a STROBE extension statement to guide the reporting of metagenomics studies.

The term metagenomics refers to the use of sequencing methods to simultaneously identify genomic material from all organisms present in a sample, with the advantage of greater taxonomic resolution than culture or other methods. Applications include pathogen detection and discovery, species characterisation, antimicrobial resistance detection, virulence profiling, and study of the microbiome and microecological factors affecting health. However, metagenomics involves complex and multistep processes and there are important technical and methodological challenges that require careful consideration to support valid inference. We co-ordinated a multidisciplinary, international expert group to establish reporting guidelines that address specimen processing, nucleic acid extraction, sequencing platforms, bioinformatics considerations, quality assurance, limits of detection, power and sample size, confirmatory testing, causality criteria, cost, and ethical issues. The guidance recognises that metagenomics research requires pragmatism and caution in interpretation, and that this field is rapidly evolving.

Increasing incidence of group B streptococcus neonatal infections in the Netherlands is associated with clonal expansion of CC17 and CC23.

Group B streptococcus (GBS) is the leading cause of neonatal invasive disease worldwide. In the Netherlands incidence of the disease increased despite implementation of preventive guidelines. We describe a genomic analysis of 1345 GBS isolates from neonatal (age 0-89 days) invasive infections in the Netherlands reported between 1987 and 2016. Most isolates clustered into one of five major lineages: CC17 (39%), CC19 (25%), CC23 (18%), CC10 (9%) and CC1 (7%). There was a significant rise in the number of infections due to isolates from CC17 and CC23. Phylogenetic clustering analysis revealed that this was caused by expansion of specific sub-lineages, designated CC17-A1, CC17-A2 and CC23-A1. Dating of phylogenetic trees estimated that these clones diverged in the 1960s/1970s, representing historical rather than recently emerged clones. For CC17-A1 the expansion correlated with acquisition of a new phage, carrying gene encoding a putative cell-surface protein. Representatives of CC17-A1, CC17-A2 and CC23-A1 clones were identified in datasets from other countries demonstrating their global distribution.

Fetal inheritance of chromosomally integrated human herpesvirus 6 predisposes the mother to pre-eclampsia.

Pre-eclampsia (typically characterized by new-onset hypertension and proteinuria in the second half of pregnancy) represents a major determinant of the global burden of disease1,2. Its pathophysiology involves placental dysfunction, but the mechanism is unclear. Viral infection can cause organ dysfunction, but its role in placentally related disorders of human pregnancy is unknown3. We addressed this using RNA sequencing metagenomics4-6 of placental samples from normal and complicated pregnancies. Here, we show that human herpesvirus 6 (HHV-6, A or B) RNA was detected in 6.1% of cases of pre-eclampsia and 2.2% of other pregnancies. Fetal genotyping demonstrated that 70% of samples with HHV-6 RNA in the placenta exhibited inherited, chromosomally integrated HHV-6 (iciHHV-6). We genotyped 467 pre-eclampsia cases and 3,854 controls and found an excess of iciHHV-6 in the cases (odds ratio of 2.8, 95% confidence intervals of 1.4-5.6, P = 0.008). We validated this finding by comparing iciHHV-6 in a further 740 cases with controls from large-scale population studies (odds ratio of 2.5, 95% confidence intervals of 1.4-4.4, P = 0.0013). We conclude that iciHHV-6 results in the transcription of viral RNA in the human placenta and predisposes the mother to pre-eclampsia.

Maturation of Gut Microbiota and Circulating Regulatory T Cells and Development of IgE Sensitization in Early Life.

Recent studies suggest that the cross-talk between the gut microbiota and human immune system during the first year of life is an important regulator of the later development of atopic diseases. We explored the changes in the gut microbiota, blood regulatory T cells, and atopic sensitization in a birth-cohort of Estonian and Finnish children followed from 3 to 36 months of age. We describe here an infant Treg phenotype characterized by high Treg frequency, the maturation of Treg population characterized by a decrease in their frequency accompanied with an increase in the highly activated Treg cells. These changes in Treg population associated first with the relative abundance of Bifidobacterium longum followed by increasing colonization with butyrate producing bacteria. High bifidobacterial abundance in the neonatal microbiota appeared to be protective, while colonization with Bacteroides and E. coli was associated with later risk of allergy. Estonian children with lower risk of IgE mediated allergic diseases than Finnish children showed an earlier maturation of the gut microbiota, detected as earlier switch to an increasing abundance of butyrate-producing bacteria, combined with an earlier maturation of Treg cell phenotype and total IgE production. The children with established allergic diseases by age 3 showed a decreased abundance of butyrate producing Faecalibacterium. These results suggest that as well as the maintenance of a bifidobacterial dominated gut microbiota is important during the first weeks of life, the overtake by butyrate producing bacteria seems to be a beneficial shift, which should not be postponed.

Author Correction: Human placenta has no microbiome but can contain potential pathogens.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human placenta has no microbiome but can contain potential pathogens.

We sought to determine whether pre-eclampsia, spontaneous preterm birth or the delivery of infants who are small for gestational age were associated with the presence of bacterial DNA in the human placenta. Here we show that there was no evidence for the presence of bacteria in the large majority of placental samples, from both complicated and uncomplicated pregnancies. Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA. The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour. We conclude that bacterial infection of the placenta is not a common cause of adverse pregnancy outcome and that the human placenta does not have a microbiome, but it does represent a potential site of perinatal acquisition of S. agalactiae, a major cause of neonatal sepsis.