Microbiome composition recovery after liver transplantation correlates with initial liver disease severity and antibiotics treatment.

Liver transplantation (LT) is crucial for end-stage liver disease, but it is linked to infection risks. Pathobionts, microorganisms potentially harmful under specific conditions, can cause complications posttransplant. Monitoring such pathogens in fecal samples can be challenging and therefore remains underexplored post-LT. This study aimed to analyze the gut microbiome before and after LT, tracking pathobionts and correlating clinical data. The study involved 17 liver transplant recipients, 17 healthy relatives (spouses), and 13 donors. Gut samples collected pretranplantation and posttransplantation underwent bacterial and fungal profiling through DNA sequencing. Quantitative polymerase chain reaction was used to assess microbial load. Statistical analyses included alpha and beta diversity measures, differential abundance analysis, and correlation tests between microbiome and clinical parameters. Microbiome analysis revealed dynamic changes in diversity posttransplant. Notably, high-severity patients showed persistent and greater dysbiosis during the first months post-LT compared with low-severity patients, partly due to an antibiotic treatment pre-LT. The analysis identified a higher proportion of pathogens such as Escherichia coli/Shigella flexneri in high-severity cases posttransplant. Furthermore, butyrate producers including Roseburia intestinalis, Anaerostipes hadrus, and Eubacterium coprostanoligenes were positively correlated with levels of albumin. This study offers valuable insights into post-LT microbiome changes, shedding light on the need for tailored prophylactic treatment post-LT.

Colonic bacterial diversity and dysbiosis in active microscopic colitis as compared to chronic diarrhoea and healthy controls: effect of polyethylene glycol after bowel lavage for colonoscopy.

BACKGROUND: Most microbiota studies in microscopic colitis patients are performed after diagnostic colonoscopy without considering the potential effect of colonic lavage. Patients may achieve clinical remission after colonoscopy and it is unknown whether lavage-induced changes play a role. AIM: To assess the effect of polyethylene glycol (PEG) colonic lavage on clinical remission rate, microbial diversity, microbial dysbiosis index and specific microbial changes in patients with active microscopic colitis as compared to other diarrhoeal diseases and healthy controls. METHODS: Fifty-five consecutive patients presenting chronic watery diarrhoea and 12 healthy controls were included. Faecal samples were collected three days before and 30 days after PEG in patients and controls for microbiome analysis. RESULTS: Clinical remission was observed in 53% of microscopic colitis patients, and in 32% of non-microscopic colitis patients (p = 0.16). Considering patients with persisting diarrhoea after colonoscopy, 71% of non-microscopic colitis patients had bile acid diarrhoea. Baseline Shannon Index was lower in diarrhoea groups than in healthy controls (p = 0.0025); there were no differences between microscopic colitis, bile-acid diarrhoea and functional diarrhoea. The microbial dysbiosis index was significantly higher in microscopic colitis than in bile acid diarrhoea plus functional diarrhoea (p = 0.0095), but no bacterial species showed a significantly different relative abundance among the diarrheal groups. CONCLUSIONS: Dysbiosis is a feature in active microscopic colitis, but loss of microbial diversity was similar in all diarrheal groups, suggesting that faecal microbial changes are not due to microscopic colitis itself but associated with stool form. A considerable number of microscopic colitis patients achieved clinical remission after colonoscopy, but we were unable to demonstrate related PEG-induced changes in faecal microbiome.

Cervical pessary and cerclage placement for preterm birth prevention and cervicovaginal microbiome changes.

INTRODUCTION: Our objective was to compare the vaginal microbiome in low-risk and high-risk pregnant women and to explore a potential association between vaginal microbiome and preterm birth. MATERIAL AND METHODS: A pilot, consecutive, longitudinal, multicenter study was conducted in pregnant women at 18-22 weeks of gestation. Participants were assigned to one of three groups: control (normal cervix), pessary (cervical length ≤25 mm) and cerclage (cervical length ≤25 mm or history of preterm birth). Analysis and comparison of vaginal microbiota as a primary outcome was performed at inclusion and at 30 weeks of gestation, along with a follow-up of pregnancy and perinatal outcomes. We assessed the vaginal microbiome of pregnant women presenting a short cervix with that of pregnant women having a normal cervix, and compared the vaginal microbiome of women with a short cervix before and after placement of a cervical pessary or a cervical cerclage. RESULTS: The microbiome of our control cohort was dominated by Lactobacillus crispatus and inners. Five community state types were identified and microbiome diversity did not change significantly over 10 weeks in controls. On the other hand, a short cervix was associated with a lower microbial load and higher microbial richness, and was not correlated with Lactobacillus relative abundance. After intervention, the cerclage group (n = 19) had a significant increase in microbial richness and a shift towards community state types driven by various bacterial species, including Lactobacillus mulieris, unidentified Bifidobacterium or Enterococcus. These changes were not significantly observed in the pessary (n = 26) and control (n = 35) groups. The cerclage group had more threatened preterm labor episodes and poorer outcomes than the control and pessary groups. CONCLUSIONS: These findings indicate that a short cervix is associated with an altered vaginal microbiome community structure. The use of a cerclage for preterm birth prevention, as compared with a pessary, was associated with a microbial community harboring a relatively low abundance of Lactobacillus, with more threatened preterm labor episodes, and with poorer clinical outcomes.

Intercontinental Gut Microbiome Variances in IBD.

The development of biomarkers for inflammatory bowel disease (IBD) diagnosis would be relevant in a generalized context. However, intercontinental investigation on these microbial biomarkers remains scarce. We examined taxonomic microbiome variations in IBD using published DNA shotgun metagenomic data. For this purpose, we used sequenced data from our previous Spanish Crohn's disease (CD) and ulcerative colitis (UC) cohort, downloaded sequence data from a Chinese CD cohort, and downloaded taxonomic and functional profiling tables from a USA CD and UC cohort. At the global level, geographical location and disease phenotype were the main explanatory covariates of microbiome variations. In healthy controls (HC) and UC, geography turned out to be the most important factor, while disease intestinal location was the most important one in CD. Disease severity correlated with lower alpha-diversity in UC but not in CD. Across geography, alpha-diversity was significantly different independently of health status, except for CD. Despite recruitment from different countries and with different disease severity scores, CD patients may harbor a very similar microbial taxonomic profile. Our study pointed out that geographic location, disease activity status, and other environmental factors are important contributing factors in microbiota changes in IBD. We therefore strongly recommend taking these factors into consideration for future IBD studies to obtain globally valid and reproducible biomarkers.

Dysbiosis: An Indicator of COVID-19 Severity in Critically Ill Patients.

Here, we examined the dynamics of the gut and respiratory microbiomes in severe COVID-19 patients in need of mechanical ventilation in the intensive care unit (ICU). We recruited 85 critically ill patients (53 with COVID-19 and 32 without COVID-19) and 17 healthy controls (HCs) and monitored them for up to 4 weeks. We analyzed the bacterial and fungal taxonomic profiles and loads of 232 gut and respiratory samples and we measured the blood levels of Interleukin 6, IgG, and IgM in COVID-19 patients. Upon ICU admission, the bacterial composition and load in the gut and respiratory samples were altered in critically ill patients compared with HCs. During their ICU stay, the patients experienced increased bacterial and fungal loads, drastic decreased bacterial richness, and progressive changes in bacterial and fungal taxonomic profiles. In the gut samples, six bacterial taxa could discriminate ICU-COV(+) from ICU-COV(-) cases upon ICU admission and the bacterial taxa were associated according to age, PaO2/FiO2, and CRP levels. In the respiratory samples of the ICU-COV(+) patients, bacterial signatures including Pseudomonas and Streptococcus were found to be correlated with the length of ICU stay. Our findings demonstrated that the gut and respiratory microbiome dysbiosis and bacterial signatures associated with critical illness emerged as biomarkers of COVID-19 severity and could be a potential predictor of ICU length of stay. We propose using a high-throughput sequencing approach as an alternative to traditional isolation techniques to monitor ICU patient infection.

Effects of Prebiotics vs a Diet Low in FODMAPs in Patients With Functional Gut Disorders.

Prebiotics and diets low in fermentable oligo-, di-, mono-saccharides and polyols (low-FODMAP diet) might reduce symptoms in patients with functional gastrointestinal disorders, despite reports that some nonabsorbable, fermentable meal products (prebiotics) provide substrates for colonic bacteria and thereby increase gas production. We performed a randomized, parallel, double-blind study of patients with functional gastrointestinal disorders with flatulence. We compared the effects of a prebiotic supplement (2.8 g/d Bimuno containing 1.37 g beta-galactooligosaccharide) plus a placebo (Mediterranean-type diet (prebiotic group, n = 19) vs a placebo supplement (2.8 g xylose) plus a diet low in FODMAP (low-FODMAP group, n = 21) for 4 weeks; patients were then followed for 2 weeks. The primary outcome was effects on composition of the fecal microbiota, analyzed by 16S sequencing. Secondary outcomes were intestinal gas production and digestive sensations. After 4 weeks, we observed opposite effects on microbiota in each group, particularly in relation to the abundance of Bifidobacterium sequences (increase in the prebiotic group and decrease in the low-FODMAP group; P = .042), and Bilophila wadsworthia (decrease in the prebiotic group and increase in the low-FODMAP group; P = .050). After 4 weeks, both groups had statistically significant reductions in all symptom scores, except reductions in flatulence and borborygmi were not significant in the prebiotic group. Although the decrease in symptoms persisted for 2 weeks after patients discontinued prebiotic supplementation, symptoms reappeared immediately after patients discontinued the low-FODMAP diet. Intermittent prebiotic administration might therefore be an alternative to dietary restrictions for patients with functional gut symptoms. ClinicalTrials.gov no.: NCT02210572.

Restoration of a healthy intestinal microbiota normalizes portal hypertension in a rat model of nonalcoholic steatohepatitis.

Portal hypertension (PH) drives most of the clinical complications in chronic liver diseases. However, its progression in nonalcoholic steatohepatitis (NASH) and its association with the intestinal microbiota (IM) have been scarcely studied. Our aim was to investigate the role of the IM in the mechanisms leading to PH in early NASH. The experimental design was divided in two stages. In stage 1, Sprague-Dawley rats were fed for 8 weeks a high-fat, high-glucose/fructose diet (HFGFD) or a control diet/water (CD). Representative rats were selected as IM donors for stage 2. In stage 2, additional HFGFD and CD rats underwent intestinal decontamination, followed by IM transplantation with feces from opposite-diet donors (heterologous transplant) or autologous fecal transplant (as controls), generating four groups: CD-autotransplanted, CD-transplanted, HFGFD-autotransplanted, HFGFD-transplanted. After IM transplantation, the original diet was maintained for 12-14 days until death. HFGFD rats developed obesity, insulin resistance, NASH without fibrosis but with PH, intrahepatic endothelial dysfunction, and IM dysbiosis. In HFGFD rats, transplantation with feces from CD donors caused a significant reduction of PH to levels comparable to CD without significant changes in NASH histology. The reduction in PH was due to a 31% decrease of intrahepatic vascular resistance compared to the HFGFD-autotransplanted group (P < 0.05). This effect occurs through restoration of the sensitivity to insulin of the hepatic protein kinase B-dependent endothelial nitric oxide synthase signaling pathway. CONCLUSION: The IM exerts a direct influence in the development of PH in rats with diet-induced NASH and dysbiosis; PH, insulin resistance, and endothelial dysfunction revert when a healthy IM is restored. (Hepatology 2018;67:1485-1498).

Norfloxacin is more effective than Rifaximin in avoiding bacterial translocation in an animal model of cirrhosis.

BACKGROUND & AIMS: Norfloxacin administration is useful in preventing bacterial infections in cirrhosis but associated to the generation of resistant species. Rifaximin is known to reach high concentrations in the intestinal lumen without generating relevant resistance in the intestinal flora. Our aim was to compare the effect of Norfloxacin and Rifaximin on intestinal flora composition, bacterial translocation and survival in cirrhotic rats. METHODS: Cirrhosis was induced in rats by oral administration of CCl4 . Animals were divided into three groups: only CCl4 (group I, n = 10); CCl4 + Norfloxacin (group II, n = 17) and CCl4 + Rifaximin (group III, n = 14). Gut bacterial composition, bacterial translocation and cytokine levels were measured. RESULTS: Forty-one rats were finally included. The incidence of viable and non-viable bacterial translocation was significantly reduced in animals receiving Norfloxacin; Rifaximin also decreased the incidence of viable and non-viable bacterial translocation, but did not reach statistical significance. Serum TNF-α levels were significantly lower in antibiotic groups. Norfloxacin modified intestinal microbiota, depleting significantly more pathobionts than Rifaximin. CONCLUSION: Norfloxacin is more effective than Rifaximin in preventing bacterial translocation in rats with cirrhosis probably because of its capacity to reduce pathobionts from intestinal microbiota.

A microbial signature for Crohn's disease.

OBJECTIVE: A decade of microbiome studies has linked IBD to an alteration in the gut microbial community of genetically predisposed subjects. However, existing profiles of gut microbiome dysbiosis in adult IBD patients are inconsistent among published studies, and did not allow the identification of microbial signatures for CD and UC. Here, we aimed to compare the faecal microbiome of CD with patients having UC and with non-IBD subjects in a longitudinal study. DESIGN: We analysed a cohort of 2045 non-IBD and IBD faecal samples from four countries (Spain, Belgium, the UK and Germany), applied a 16S rRNA sequencing approach and analysed a total dataset of 115 million sequences. RESULTS: In the Spanish cohort, dysbiosis was found significantly greater in patients with CD than with UC, as shown by a more reduced diversity, a less stable microbial community and eight microbial groups were proposed as a specific microbial signature for CD. Tested against the whole cohort, the signature achieved an overall sensitivity of 80% and a specificity of 94%, 94%, 89% and 91% for the detection of CD versus healthy controls, patients with anorexia, IBS and UC, respectively. CONCLUSIONS: Although UC and CD share many epidemiologic, immunologic, therapeutic and clinical features, our results showed that they are two distinct subtypes of IBD at the microbiome level. For the first time, we are proposing microbiomarkers to discriminate between CD and non-CD independently of geographical regions.

The microbiome in respiratory medicine: current challenges and future perspectives.

The healthy lung has previously been considered to be a sterile organ because standard microbiological culture techniques consistently yield negative results. However, culture-independent techniques report that large numbers of microorganisms coexist in the lung. There are many unknown aspects in the field, but available reports show that the lower respiratory tract microbiota: 1) is similar in healthy subjects to the oropharyngeal microbiota and dominated by members of the Firmicutes, Bacteroidetes and Proteobacteria phyla; 2) shows changes in smokers and well-defined differences in chronic respiratory diseases, although the temporal and spatial kinetics of these changes are only partially known; and 3) shows relatively abundant non-cultivable bacteria in chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, cystic fibrosis and bronchiectasis, with specific patterns for each disease. In all of these diseases, a loss of diversity, paralleled by an over-representation of Proteobacteria (dysbiosis), has been related to disease severity and exacerbations. However, it is unknown whether dysbiosis is a cause or a consequence of the damage to bronchoalveolar surfaces.Finally, little is known about bacterial functionality and the interactions between viruses, fungi and bacteria. It is expected that future research in bacterial gene expressions, metagenomics longitudinal analysis and host-microbiome animal models will help to move towards targeted microbiome interventions in respiratory diseases.

Effect of Chicory-derived Inulin on Abdominal Sensations and Bowel Motor Function.

GOAL: To determine the effect of a prebiotic chicory-derived inulin-type fructan on the tolerance of intestinal gas. BACKGROUND: Subjects with gas-related complaints exhibit impaired handling of intestinal gas loads and we hypothesized that inulin would have a beneficial effect. STUDY: Placebo-controlled, parallel, randomized, double-blind trial. Subjects with abdominal symptoms and reduced tolerance of intestinal gas (selected by a pretest) received either inulin (8 g/d, n=18) or maltodextrin as a placebo (8 g/d, n=18) for 4 weeks. A gas challenge test (4 h jejunal gas infusion at 12 mL/min while measuring abdominal symptoms and gas retention for 3 h) was performed before and at the end of the intervention phase. Gastrointestinal symptoms and bowel habits (using daily questionnaires for 1 wk) and fecal bifidobacteria counts were measured before and at the end of the intervention. RESULTS: Inulin decreased gas retention during the gas challenge test (by 22%; P=0.035 vs. baseline), while the placebo did not, but the intergroup difference was not statistically significant (P=0.343). Inulin and placebo reduced the perception of abdominal sensations in the gas challenge test to a similar extent (by 52% and 43%, respectively). Participants reported moderate gastrointestinal symptoms and normal bowel habits during baseline examination, and these findings remained unchanged in both groups during the intervention. Inulin led to a higher relative abundance of bifidobacteria counts (P=0.01 vs. placebo). CONCLUSIONS: A daily dose of inulin that promotes bifidobacteria growth and may improve gut function, is well tolerated by subjects with gastrointestinal complaints.

Enterotypes of the human gut microbiome.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

A human gut microbial gene catalogue established by metagenomic sequencing.

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage.

By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.

Intestinal microbiota and diet in IBS: causes, consequences, or epiphenomena?

Irritable bowel syndrome (IBS) is a heterogeneous functional disorder with a multifactorial etiology that involves the interplay of both host and environmental factors. Among environmental factors relevant for IBS etiology, the diet stands out given that the majority of IBS patients report their symptoms to be triggered by meals or specific foods. The diet provides substrates for microbial fermentation, and, as the composition of the intestinal microbiota is disturbed in IBS patients, the link between diet, microbiota composition, and microbial fermentation products might have an essential role in IBS etiology. In this review, we summarize current evidence regarding the impact of diet and the intestinal microbiota on IBS symptoms, as well as the reported interactions between diet and the microbiota composition. On the basis of the existing data, we suggest pathways (mechanisms) by which diet components, via the microbial fermentation, could trigger IBS symptoms. Finally, this review provides recommendations for future studies that would enable elucidation of the role of diet and microbiota and how these factors may be (inter)related in the pathophysiology of IBS.

Topoisomerase II regulates yeast genes with singular chromatin architectures.

Eukaryotic topoisomerase II (topo II) is the essential decatenase of newly replicated chromosomes and the main relaxase of nucleosomal DNA. Apart from these general tasks, topo II participates in more specialized functions. In mammals, topo IIα interacts with specific RNA polymerases and chromatin-remodeling complexes, whereas topo IIß regulates developmental genes in conjunction with chromatin remodeling and heterochromatin transitions. Here we show that in budding yeast, topo II regulates the expression of specific gene subsets. To uncover this, we carried out a genomic transcription run-on shortly after the thermal inactivation of topo II. We identified a modest number of genes not involved in the general stress response but strictly dependent on topo II. These genes present distinctive functional and structural traits in comparison with the genome average. Yeast topo II is a positive regulator of genes with well-defined promoter architecture that associates to chromatin remodeling complexes; it is a negative regulator of genes extremely hypo-acetylated with complex promoters and undefined nucleosome positioning, many of which are involved in polyamine transport. These findings indicate that yeast topo II operates on singular chromatin architectures to activate or repress DNA transcription and that this activity produces functional responses to ensure chromatin stability.

Anal gas evacuation and colonic microbiota in patients with flatulence: effect of diet.

OBJECTIVE: To characterise the influence of diet on abdominal symptoms, anal gas evacuation, intestinal gas distribution and colonic microbiota in patients complaining of flatulence. DESIGN: Patients complaining of flatulence (n=30) and healthy subjects (n=20) were instructed to follow their usual diet for 3 days (basal phase) and to consume a high-flatulogenic diet for another 3 days (challenge phase). RESULTS: During basal phase, patients recorded more abdominal symptoms than healthy subjects in daily questionnaires (5.8±0.3 vs 0.4±0.2 mean discomfort/pain score, respectively; p=<0.0001) and more gas evacuations by an event marker (21.9±2.8 vs 7.4±1.0 daytime evacuations, respectively; p=0.0001), without differences in the volume of gas evacuated after a standard meal (262±22 and 265±25 mL, respectively). On flatulogenic diet, both groups recorded more abdominal symptoms (7.9±0.3 and 2.8±0.4 discomfort/pain, respectively), number of gas evacuations (44.4±5.3 and 21.7±2.9 daytime evacuations, respectively) and had more gas production (656±52 and 673±78 mL, respectively; p<0.05 vs basal diet for all). When challenged with flatulogenic diet, patients' microbiota developed instability in composition, exhibiting variations in the main phyla and reduction of microbial diversity, whereas healthy subjects' microbiota were stable. Taxa from Bacteroides fragilis or Bilophila wadsworthia correlated with number of gas evacuations or volume of gas evacuated, respectively. CONCLUSIONS: Patients complaining of flatulence have a poor tolerance of intestinal gas, which is associated with instability of the microbial ecosystem.

Processing faecal samples: a step forward for standards in microbial community analysis.

BACKGROUND: The microbial community analysis of stools requires optimised and standardised protocols for their collection, homogenisation, microbial disruption and nucleic acid extraction. Here we examined whether different layers of the stool are equally representative of the microbiome. We also studied the effect of stool water content, which typically increases in diarrhoeic samples, and of a microbial disruption method on DNA integrity and, therefore, on providing an unbiased microbial composition analysis. RESULTS: We collected faecal samples from healthy subjects and performed microbial composition analysis by pyrosequencing the V4 region of the 16S rRNA gene. To examine the effect of stool structure, we compared the inner and outer layers of the samples (N = 8). Both layers presented minor differences in microbial composition and abundance at the species level. These differences did not significantly bias the microbial community specific to an individual. To evaluate the effect of stool water content and bead-beating, we used various volumes of a water-based salt solution and beads of distinct weights before nucleic acid extraction (N = 4). The different proportions of water did not affect the UniFrac-based clustering of samples from the same subject However, the use or omission of a bead-beating step produced different proportions of Gram-positive and Gram-negative bacteria and significant changes in the UniFrac-based clustering of the samples. CONCLUSION: The degree of hydration and homogenisation of faecal samples do not significantly alter their microbial community composition. However, the use of bead-beating is critical for the proper detection of Gram-positive bacteria such as Blautia and Bifidobacterium.

GSR-DB: a manually curated and optimized taxonomical database for 16S rRNA amplicon analysis.

Amplicon-based 16S ribosomal RNA sequencing remains a widely used method to profile microbial communities, especially in low biomass samples, due to its cost-effectiveness and low-complexity approach. Reference databases are a mainstay for taxonomic assignments, which typically rely on popular databases such as SILVA, Greengenes, Genome Taxonomy Database (GTDB), or Ribosomal Database Project (RDP). However, the inconsistency of the nomenclature across databases and the presence of shortcomings in the annotation of these databases are limiting the resolution of the analysis. To overcome these limitations, we created the GSR database (Greengenes, SILVA, and RDP database), an integrated and manually curated database for bacterial and archaeal 16S amplicon taxonomy analysis. Unlike previous integration approaches, this database creation pipeline includes a taxonomy unification step to ensure consistency in taxonomical annotations. The database was validated with three mock communities, two real data sets, and a 10-fold cross-validation method and compared with existing 16S databases such as Greengenes, Greengenes 2, GTDB, ITGDB, SILVA, RDP, and MetaSquare. Results showed that the GSR database enhances taxonomical annotations of 16S sequences, outperforming current 16S databases at the species level, based on the evaluation of the mock communities. This was confirmed by the 10-fold cross-validation, except for Greengenes 2. The GSR database is available for full-length 16S sequences and the most commonly used hypervariable regions: V4, V1-V3, V3-V4, and V3-V5.IMPORTANCETaxonomic assignments of microorganisms have long been hindered by inconsistent nomenclature and annotation issues in existing databases like SILVA, Greengenes, Greengenes2, Genome Taxonomy Database, or Ribosomal Database Project. To overcome these issues, we created Greengenes-SILVA-RDP database (GSR-DB), accurate and comprehensive taxonomic annotations of 16S amplicon data. Unlike previous approaches, our innovative pipeline includes a unique taxonomy unification step, ensuring consistent and reliable annotations. Our evaluation analyses showed that GSR-DB outperforms existing databases in providing species-level resolution, especially based on mock-community analysis, making it a game-changer for microbiome studies. Moreover, GSR-DB is designed to be accessible to researchers with limited computational resources, making it a powerful tool for scientists across the board. Available for full-length 16S sequences and commonly used hypervariable regions, including V4, V1-V3, V3-V4, and V3-V5, GSR-DB is a go-to database for robust and accurate microbial taxonomy analysis.

Reshaping the gut microbiome with bacterial transplantation and antibiotic intake.

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.