Conducting a microbiome study.

Human microbiome research is an actively developing area of inquiry, with ramifications for our lifestyles, our interactions with microbes, and how we treat disease. Advances depend on carefully executed, controlled, and reproducible studies. Here, we provide a Primer for researchers from diverse disciplines interested in conducting microbiome research. We discuss factors to be considered in the design, execution, and data analysis of microbiome studies. These recommendations should help researchers to enter and contribute to this rapidly developing field.

Human genetics shape the gut microbiome.

Host genetics and the gut microbiome can both influence metabolic phenotypes. However, whether host genetic variation shapes the gut microbiome and interacts with it to affect host phenotype is unclear. Here, we compared microbiotas across >1,000 fecal samples obtained from the TwinsUK population, including 416 twin pairs. We identified many microbial taxa whose abundances were influenced by host genetics. The most heritable taxon, the family Christensenellaceae, formed a co-occurrence network with other heritable Bacteria and with methanogenic Archaea. Furthermore, Christensenellaceae and its partners were enriched in individuals with low body mass index (BMI). An obese-associated microbiome was amended with Christensenella minuta, a cultured member of the Christensenellaceae, and transplanted to germ-free mice. C. minuta amendment reduced weight gain and altered the microbiome of recipient mice. Our findings indicate that host genetics influence the composition of the human gut microbiome and can do so in ways that impact host metabolism.

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.

Salmonella manipulates the host to drive pathogenicity via induction of interleukin 1ß.

Acute gastrointestinal infection with intracellular pathogens like Salmonella Typhimurium triggers the release of the proinflammatory cytokine interleukin 1ß (IL-1ß). However, the role of IL-1ß in intestinal defense against Salmonella remains unclear. Here, we show that IL-1ß production is detrimental during Salmonella infection. Mice lacking IL-1ß (IL-1ß -/-) failed to recruit neutrophils to the gut during infection, which reduced tissue damage and prevented depletion of short-chain fatty acid (SCFA)-producing commensals. Changes in epithelial cell metabolism that typically support pathogen expansion, such as switching energy production from fatty acid oxidation to fermentation, were absent in infected IL-1ß -/- mice which inhibited Salmonella expansion. Additionally, we found that IL-1ß induces expression of complement anaphylatoxins and suppresses the complement-inactivator carboxypeptidase N (CPN1). Disrupting this process via IL-1ß loss prevented mortality in Salmonella-infected IL-1ß -/- mice. Finally, we found that IL-1ß expression correlates with expression of the complement receptor in patients suffering from sepsis, but not uninfected patients and healthy individuals. Thus, Salmonella exploits IL-1ß signaling to outcompete commensal microbes and establish gut colonization. Moreover, our findings identify the intersection of IL-1ß signaling and the complement system as key host factors involved in controlling mortality during invasive Salmonellosis.

Host remodeling of the gut microbiome and metabolic changes during pregnancy.

Many of the immune and metabolic changes occurring during normal pregnancy also describe metabolic syndrome. Gut microbiota can cause symptoms of metabolic syndrome in nonpregnant hosts. Here, to explore their role in pregnancy, we characterized fecal bacteria of 91 pregnant women of varying prepregnancy BMIs and gestational diabetes status and their infants. Similarities between infant-mother microbiotas increased with children's age, and the infant microbiota was unaffected by mother's health status. Gut microbiota changed dramatically from first (T1) to third (T3) trimesters, with vast expansion of diversity between mothers, an overall increase in Proteobacteria and Actinobacteria, and reduced richness. T3 stool showed strongest signs of inflammation and energy loss; however, microbiome gene repertoires were constant between trimesters. When transferred to germ-free mice, T3 microbiota induced greater adiposity and insulin insensitivity compared to T1. Our findings indicate that host-microbial interactions that impact host metabolism can occur and may be beneficial in pregnancy.

Ontogeny of the B Cell Receptor Repertoire and Microbiome in Mice.

The immune system matures throughout childhood to achieve full functionality in protecting our bodies against threats. The immune system has a strong reciprocal symbiosis with the host bacterial population and the two systems co-develop, shaping each other. Despite their fundamental role in health physiology, the ontogeny of these systems is poorly characterized. In this study, we investigated the development of the BCR repertoire by analyzing high-throughput sequencing of their receptors in several time points of young C57BL/6J mice. In parallel, we explored the development of the gut microbiome. We discovered that the gut IgA repertoires change from birth to adolescence, including an increase in CDR3 lengths and somatic hypermutation levels. This contrasts with the spleen IgM repertoires that remain stable and distinct from the IgA repertoires in the gut. We also discovered that large clones that germinate in the gut are initially confined to a specific gut compartment, then expand to nearby compartments and later on expand also to the spleen and remain there. Finally, we explored the associations between diversity indices of the B cell repertoires and the microbiome, as well as associations between bacterial and BCR clusters. Our results shed light on the ontogeny of the adaptive immune system and the microbiome, providing a baseline for future research.

Gestational diabetes is driven by microbiota-induced inflammation months before diagnosis.

OBJECTIVE: Gestational diabetes mellitus (GDM) is a condition in which women without diabetes are diagnosed with glucose intolerance during pregnancy, typically in the second or third trimester. Early diagnosis, along with a better understanding of its pathophysiology during the first trimester of pregnancy, may be effective in reducing incidence and associated short-term and long-term morbidities. DESIGN: We comprehensively profiled the gut microbiome, metabolome, inflammatory cytokines, nutrition and clinical records of 394 women during the first trimester of pregnancy, before GDM diagnosis. We then built a model that can predict GDM onset weeks before it is typically diagnosed. Further, we demonstrated the role of the microbiome in disease using faecal microbiota transplant (FMT) of first trimester samples from pregnant women across three unique cohorts. RESULTS: We found elevated levels of proinflammatory cytokines in women who later developed GDM, decreased faecal short-chain fatty acids and altered microbiome. We next confirmed that differences in GDM-associated microbial composition during the first trimester drove inflammation and insulin resistance more than 10 weeks prior to GDM diagnosis using FMT experiments. Following these observations, we used a machine learning approach to predict GDM based on first trimester clinical, microbial and inflammatory markers with high accuracy. CONCLUSION: GDM onset can be identified in the first trimester of pregnancy, earlier than currently accepted. Furthermore, the gut microbiome appears to play a role in inflammation-induced GDM pathogenesis, with interleukin-6 as a potential contributor to pathogenesis. Potential GDM markers, including microbiota, can serve as targets for early diagnostics and therapeutic intervention leading to prevention.

Effects of Diet-Modulated Autologous Fecal Microbiota Transplantation on Weight Regain.

BACKGROUND & AIMS: We evaluated the efficacy and safety of diet-modulated autologous fecal microbiota transplantation (aFMT) for treatment of weight regain after the weight-loss phase. METHODS: In the DIRECT PLUS (Dietary Intervention Randomized Controlled Trial Polyphenols-Unprocessed) weight-loss trial (May 2017 through July 2018), abdominally obese or dyslipidemic participants in Israel were randomly assigned to healthy dietary guidelines, Mediterranean diet, and green-Mediterranean diet weight-loss groups. All groups received free gym membership and physical activity guidelines. Both isocaloric Mediterranean groups consumed 28 g/d walnuts (+440 mg/d polyphenols provided). The green-Mediterranean dieters also consumed green tea (3-4 cups/d) and a Wolffia globosa (Mankai strain, 100 g/d) green shake (+800 mg/d polyphenols provided). After 6 months (weight-loss phase), 90 eligible participants (mean age, 52 years; mean weight loss, 8.3 kg) provided a fecal sample that was processed into aFMT by frozen, opaque, and odorless capsules. The participants were then randomly assigned to groups that received 100 capsules containing their own fecal microbiota or placebo until month 14. The primary outcome was regain of the lost weight over the expected weight-regain phase (months 6-14). Secondary outcomes were gastrointestinal symptoms, waist circumference, glycemic status, and changes in the gut microbiome, as measured by metagenomic sequencing and 16s ribosomal RNA. We validated the results in a parallel in vivo study of mice specifically fed with Mankai compared with control chow diet. RESULTS: Of the 90 participants in the aFMT trial, 96% ingested at least 80 of 100 oral aFMT or placebo frozen capsules during the transplantation period. No aFMT-related adverse events or symptoms were observed. For the primary outcome, although no significant differences in weight regain were observed among the participants in the different lifestyle interventions during months 6-14 (aFMT, 30.4% vs placebo, 40.6%; P = .28), aFMT significantly attenuated weight regain in the green-Mediterranean group (aFMT, 17.1%, vs placebo, 50%; P = .02), but not in the dietary guidelines (P = .57) or Mediterranean diet (P = .64) groups (P for the interaction = .03). Accordingly, aFMT attenuated waist circumference gain (aFMT, 1.89 cm vs placebo, 5.05 cm; P = .01) and insulin rebound (aFMT, -1.46 ± 3.6 µIU/mL vs placebo, 1.64 ± 4.7 µIU/mL; P = .04) in the green-Mediterranean group but not in the dietary guidelines or Mediterranean diet (P for the interaction = .04 and .03, respectively). The green-Mediterranean diet was the only intervention to induce a significant change in microbiome composition during the weight-loss phase, and to prompt preservation of weight-loss-associated specific bacteria and microbial metabolic pathways (mainly microbial sugar transport) after the aFMT. In mice, Mankai-modulated aFMT in the weight-loss phase compared with control diet aFMT, significantly prevented weight regain and resulted in better glucose tolerance during a high-fat diet-induced regain phase (all, P < .05). CONCLUSIONS: Autologous FMT, collected during the weight-loss phase and administrated in the regain phase, might preserve weight loss and glycemic control, and is associated with specific microbiome signatures. A high-polyphenols, green plant-based or Mankai diet better optimizes the microbiome for an aFMT procedure. ClinicalTrials.gov number, NCT03020186.

The microbial diversity following antibiotic treatment of Clostridioides difficile infection.

BACKGROUND: Clostridioides difficile (C. difficile) is a major nosocomial pathogen that infects the human gut and can cause diarrheal disease. A dominant risk factor is antibiotic treatment that disrupts the normal gut microbiota. The aim of the study was to examine the correlation between antibiotic treatment received prior to C. difficile infection (CDI) onset and patient gut microbiota. METHODS: Stool samples were collected from patients with CDI, presenting at the Baruch Padeh Medical Center Poriya, Israel. Demographic and clinical information, including previous antibiotic treatments, was collected from patient charts, and CDI severity score was calculated. Bacteria were isolated from stool samples, and gut microbiome was analyzed by sequencing the 16S rRNA gene using the Illumina MiSeq platform and QIIME2. RESULTS: In total, 84 patients with CDI were enrolled in the study; all had received antibiotics prior to disease onset. Due to comorbidities, 46 patients (55%) had received more than one class of antibiotics. The most common class of antibiotics used was cephalosporins (n = 44 cases). The intestinal microbiota of the patients was not uniform and was mainly dominated by Proteobacteria. Differences in intestinal microbiome were influenced by the different combinations of antibiotics that the patients had received (p = 0.022) CONCLUSIONS: The number of different antibiotics administered has a major impact on the CDI patients gut microbiome, mainly on bacterial richness.

Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome.

The intestinal tract is inhabited by a large and diverse community of microbes collectively referred to as the gut microbiota. While the gut microbiota provides important benefits to its host, especially in metabolism and immune development, disturbance of the microbiota-host relationship is associated with numerous chronic inflammatory diseases, including inflammatory bowel disease and the group of obesity-associated diseases collectively referred to as metabolic syndrome. A primary means by which the intestine is protected from its microbiota is via multi-layered mucus structures that cover the intestinal surface, thereby allowing the vast majority of gut bacteria to be kept at a safe distance from epithelial cells that line the intestine. Thus, agents that disrupt mucus-bacterial interactions might have the potential to promote diseases associated with gut inflammation. Consequently, it has been hypothesized that emulsifiers, detergent-like molecules that are a ubiquitous component of processed foods and that can increase bacterial translocation across epithelia in vitro, might be promoting the increase in inflammatory bowel disease observed since the mid-twentieth century. Here we report that, in mice, relatively low concentrations of two commonly used emulsifiers, namely carboxymethylcellulose and polysorbate-80, induced low-grade inflammation and obesity/metabolic syndrome in wild-type hosts and promoted robust colitis in mice predisposed to this disorder. Emulsifier-induced metabolic syndrome was associated with microbiota encroachment, altered species composition and increased pro-inflammatory potential. Use of germ-free mice and faecal transplants indicated that such changes in microbiota were necessary and sufficient for both low-grade inflammation and metabolic syndrome. These results support the emerging concept that perturbed host-microbiota interactions resulting in low-grade inflammation can promote adiposity and its associated metabolic effects. Moreover, they suggest that the broad use of emulsifying agents might be contributing to an increased societal incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.

Large-scale replicated field study of maize rhizosphere identifies heritable microbes.

Soil microbes that colonize plant roots and are responsive to differences in plant genotype remain to be ascertained for agronomically important crops. From a very large-scale longitudinal field study of 27 maize inbred lines planted in three fields, with partial replication 5 y later, we identify root-associated microbiota exhibiting reproducible associations with plant genotype. Analysis of 4,866 samples identified 143 operational taxonomic units (OTUs) whose variation in relative abundances across the samples was significantly regulated by plant genotype, and included five of seven core OTUs present in all samples. Plant genetic effects were significant amid the large effects of plant age on the rhizosphere microbiome, regardless of the specific community of each field, and despite microbiome responses to climate events. Seasonal patterns showed that the plant root microbiome is locally seeded, changes with plant growth, and responds to weather events. However, against this background of variation, specific taxa responded to differences in host genotype. If shown to have beneficial functions, microbes may be considered candidate traits for selective breeding.

Modulation of cytokine patterns and microbiome during pregnancy in IBD.

OBJECTIVE: Pregnancy may affect the disease course of IBD. Both pregnancy and IBD are associated with altered immunology and intestinal microbiology. However, to what extent immunological and microbial profiles are affected by pregnancy in patients with IBD remains unclear. DESIGN: Faecal and serum samples were collected from 46 IBD patients (31 Crohn's disease (CD) and 15 UC) and 179 healthy controls during first, second and third trimester of pregnancy, and prepregnancy and postpartum for patients with IBD. Peripheral blood cytokine profiles were determined by ELISA, and microbiome analysis was performed by sequencing the V4 region of the bacterial 16S rRNA gene. RESULTS: Proinflammatory serum cytokine levels in patients with IBD decrease significantly on conception. Reduced interleukin (IL)-10 and IL-5 levels but increased IL-8 and interferon (IFN)γ levels compared with healthy controls were seen throughout pregnancy, but cytokine patterns remained stable during gestation. Microbial diversity in pregnant patients with IBD was reduced compared with that in healthy women, and significant differences existed between patients with UC and CD in early pregnancy. However, these microbial differences were no longer present during middle and late pregnancy. Dynamic modelling showed considerable interaction between cytokine and microbial composition. CONCLUSION: Serum proinflammatory cytokine levels markedly improve on conception in pregnant patients with IBD, and intestinal microbiome diversity of patients with IBD normalises during middle and late pregnancy. We thus conclude that pregnancy is safe and even potentially beneficial for patients with IBD.

The intestinal microbiome, weight, and metabolic changes in women treated by adjuvant chemotherapy for breast and gynecological malignancies.

BACKGROUND: Adjuvant chemotherapy induces weight gain, glucose intolerance, and hypertension in about a third of women. The mechanisms underlying these events have not been defined. This study assessed the association between the microbiome and weight gain in patients treated with adjuvant chemotherapy for breast and gynecological cancers. METHODS: Patients were recruited before starting adjuvant therapy. Weight and height were measured before treatment and 4-6 weeks after treatment completion. Weight gain was defined as an increase of 3% or more in body weight. A stool sample was collected before treatment, and 16S rRNA gene sequencing was performed. Data regarding oncological therapy, menopausal status, and antibiotic use was prospectively collected. Patients were excluded if they were treated by antibiotics during the study. Fecal transplant experiments from patients were conducted using Swiss Webster germ-free mice. RESULTS: Thirty-three patients were recruited; of them, 9 gained 3.5-10.6% of baseline weight. The pretreatment microbiome of women who gained weight following treatment was significantly different in diversity and taxonomy from that of control women. Fecal microbiota transplantation from pretreatment samples of patients that gained weight induced metabolic changes in germ-free mice compared to mice transplanted with pretreatment fecal samples from the control women. CONCLUSION: The microbiome composition is predictive of weight gain following adjuvant chemotherapy and induces adverse metabolic changes in germ-free mice, suggesting it contributes to adverse metabolic changes seen in patients. Confirmation of these results in a larger patient cohort is warranted.

Prenatal low-dose penicillin results in long-term sex-specific changes to murine behaviour, immune regulation, and gut microbiota.

Growing evidence suggests that environmental disruptors of maternal microbes may have significant detrimental consequences for the developing fetus. Antibiotic exposure during early life can have long-term effects on neurodevelopment in mice and humans. Here we explore whether exposure to low-dose penicillin during only the last week of gestation in mice has long-term effects on offspring behaviour, brain, immune function, and gut microbiota. We found that this treatment had sex-specific effects in the adult mouse offspring. Female, but not male, mice demonstrated decreased anxiety-like behaviours, while male, but not female, mice had abnormal social behaviours which correlated with altered brain expression of AVPR1A, AVPR1B, and OXTR, and decreases in the balance of splenic FOXP3+ regulatory T cells. Prenatal penicillin exposure also led to distinct microbiota compositions that clustered differently by sex. These data suggest that exposure of pregnant mice to even a low dose of penicillin through only the last week before birth is nonetheless sufficient to induce long-term sex-specific developmental changes in both male and female offspring.

Attributes of Physical Activity and Gut Microbiome in Adults: A Systematic Review.

Growing evidence shows the contribution of physical activity interventions to the gut microbiome. However, specific physical activity characteristics that can modify the gut microbiome are unknown. This review's aim was to explore the contribution of physical activity intervention characteristics on human gut microbiome composition, in terms of diversity, specific bacterial groups, and associated gut microbiome metabolites. A literature search in PubMed; Cochrane Library; CINAHL-EBSCO; SCOPUS; Web of Science; ClinicalTrials.gov; PROSPERO; and ProQuest. Five studies met the inclusion criteria of a physical activity intervention duration of at least five weeks, with any description of the type or dose used. All included studies reported an endurance training; two studies used endurance and an additional muscle-strengthening training regimen. All studies reported using a dietary intervention control. Reported gut microbiome α-diversity changes were non-significant, ß-diversity changes were mixed (three studies reported an increase, two reported non-significant changes). All studies reported significant changes in the abundances of specific bacterial/archaea groups and bacteria-related metabolites following interventions. In conclusion, physical activity (regardless of specific characteristics) has significant contribution to gut microbiome composition and associated metabolites. There are no current recommendations for physical activity to promote gut microbiome composition. Future studies should focus on the contribution of current recommended physical activity dose to gut microbiome composition.

The effects of antipsychotic medications on microbiome and weight gain in children and adolescents.

BACKGROUND: Atypical antipsychotics, also known as second-generation antipsychotics, are commonly prescribed as treatment for psychotic disorders in adults, as well as in children and adolescents with behavioral problems. However, in many cases, second-generation antipsychotics have unwanted side effects, such as weight gain, potentially further increasing risk for morbidities including obesity, diabetes, and cardiovascular disease. While various mechanisms for this weight gain have been proposed, including effects on metabolic hormone signaling, recent evidence points to the importance of the gut microbiome in this process. The microbial communities residing within the gut are affected by second-generation antipsychotics and can confer weight gain. MAIN TEXT: This review summarizes recent findings and presents data linking second-generation antipsychotics, gut microbiota alterations and weight gain. The review focuses on children and adolescent populations, which have not previously received much attention, but are of great interest because they may be most vulnerable to gut microbiome changes and may carry long-term metabolic effects into adulthood. CONCLUSIONS: We present correlations between second-generation antipsychotics, gut microbiota alterations and weight gain, and suggest some mechanisms that may link them. A better understanding of the underlying mechanisms may lead to the design of improved treatments for psychotic disorders with fewer harmful side effects.

Radiation induces proinflammatory dysbiosis: transmission of inflammatory susceptibility by host cytokine induction.

OBJECTIVE: Radiation proctitis (RP) is a complication of pelvic radiotherapy which affects both the host and microbiota. Herein we assessed the radiation effect on microbiota and its relationship to tissue damage using a rectal radiation mouse model. DESIGN: We evaluated luminal and mucosa-associated dysbiosis in irradiated and control mice at two postradiation time points and correlated it with clinical and immunological parameters. Epithelial cytokine response was evaluated using bacterial-epithelial co-cultures. Subsequently, germ-free (GF) mice were colonised with postradiation microbiota and controls and exposed to radiation, or dextran sulfate-sodium (DSS). Interleukin (IL)-1ß correlated with tissue damage and was induced by dysbiosis. Therefore, we tested its direct role in radiation-induced damage by IL-1 receptor antagonist administration to irradiated mice. RESULTS: A postradiation shift in microbiota was observed. A unique microbial signature correlated with histopathology. Increased colonic tumor necrosis factor (TNF)α, IL-1ß and IL-6 expression was observed at two different time points. Adherent microbiota from RP differed from those in uninvolved segments and was associated with tissue damage. Using bacterial-epithelial co-cultures, postradiation microbiota enhanced IL-1ß and TNFα expression compared with naïve microbiota. GF mice colonisation by irradiated microbiota versus controls predisposed mice to both radiation injury and DSS-induced colitis. IL-1 receptor antagonist administration ameliorated intestinal radiation injury. CONCLUSIONS: The results demonstrate that rectal radiation induces dysbiosis, which transmits radiation and inflammatory susceptibility and provide evidence that microbial-induced radiation tissue damage is at least in part mediated by IL-1ß. Environmental factors may affect the host via modifications of the microbiome and potentially allow for novel interventional approaches via its manipulation.