Microbiota In Samples Research Articles

Genomic and Phenotypic Characteristics in Geographically Separated Clinical Campylobacter jejuni ST353CC Isolates.

Campylobacter jejuni fecal isolates of eight international travelers, 5 of which had traveled to Ecuador and 3 to Bangladesh, were characterized, and the possible relationship between bacterial traits and clinical symptoms was further analyzed. All eight isolates belonged to the same Multi-Locus Sequence Type clonal complex (ST353CC). The three isolates from Bangladesh were all of the same sequence type (ST-9438), and when compared to isolates of various other sequence types, they had a larger quantity of unique genetic content, higher expression levels of some putative virulence genes involved in adhesion and invasion (flpA, ciaB and iamA), and showed higher adhesion levels to human HT-29 colon cancer cells in an in vitro infection model. However, in contrast to the seemingly higher pathogenic potential of these bacterial isolates, travelers infected with the ST-9438 isolates had no or only very mild symptoms, whereas the other individuals, whose bacterial isolates seemed to have less pathogenic potential, generally reported severe symptoms. When studying the 16S rRNA gene-based fecal microbiota in samples collected prior to travel, there was an individual variation in the relative abundance of the three major bacterial phyla Actinobacteria, Bacteroidetes and Firmicutes, but there were no associations between composition and diversity of microbiota and development of severe symptoms from the infection. It remains to be confirmed by larger studies whether an individual’s characteristics such as gut microbiota, might be related to the severity of symptoms in Campylobacter infections.

1017. Gut Microbiota Diversity and Beneficial Metabolite Production is Reduced in Liver Transplant Recipients and Associated with Post Operative Infection

BackgroundLiver transplant (LT) recipients have abnormal microbiota before and after transplantation. (1,2) Associations between fecal microbiota, microbial metabolites, and clinical outcomes in liver transplantation are not well established. We correlated fecal microbiota composition and metabolite concentrations with early LT outcomes, including infection.MethodsIn a prospective observational study, we collected peri-transplant fecal samples and determined microbiota composition by 16S ribosomal RNA gene sequencing in LT recipients. Fecal short chain fatty acid (SCFA) and bile acid concentrations were measured by targeted GC- and LC-MS analyses, respectively. Inverse Simpson index was used to determine microbiota alpha-diversity in subjects and healthy controls. Clinical outcomes including length of stay, ICU admission, liver function, antibiotic use, immunosuppressive requirement and post-operative infection were correlated with microbiota composition.Results69 patients were enrolled, 70 liver transplants were performed and 307 peri-transplant fecal samples were collected and analyzed. Compared to healthy controls, the fecal microbiota of LT recipients had reduced alpha-diversity (p< 0.001). [Fig1] Bacteroidetes, Ruminococcaceae, and Lachnospiraceae, three taxa associated with a health-promoting microbiota, and their metabolites, SCFA and secondary bile acids, were markedly diminished 55% of LT patients.(3) Intestinal domination ( >30% frequency) by Enterococcus or Proteobacteria species was common and occurred in 36% of LT recipients. 76 post-operative infections occurred in 40 LT recipients, with Enterococci causing 52% and Proteobacteria 41% of bacterial infections. In subjects with fecal samples collected within 5 days of infection, 9/17 infections were caused by the organism dominating the microbiota. [Fig2]Microbiota Composition and Metabolite Production 16s gene sequencing color coded by taxonomy. Each bar represents one stool sample nearest to LT compared to healthy controls. Alpha diversity measured by inverse simpson index. Absolute values of microbial metabolites and ratio of primary to secondary bile acids.Comparison of Microbiota Composition and Post Operative Infection All bacterial infections captured with a microbiota sample within 5 days of infection.ConclusionMicrobiota diversity and microbially derived metabolites are markedly reduced in >50% of LT recipients. Intestinal domination and post-operative infections caused by antibiotic-resistant Enterococcus and Proteobacteria correlate with loss of Bacteroidetes, Ruminococcaceae, and Lachnospiraceae species, suggesting a potential role for microbiota reconstitution therapy in LT patients.Disclosures Eric G. Pamer, MD;FIDSA, Nothing to disclose

Longitudinal Changes in Early Nasal Microbiota and the Risk of Childhood Asthma

To explore the association between longitudinal changes in early nasal microbiota and the risk of developing asthma.In the study, researchers included 704 children (aged 0 to 7 years old) born in the Hospital District of Southwest Finland between January 2008 and April 2010.This was a prospective, population-based birth cohort study. Children with a qualified baseline nasal microbiota sample at 2 months old and at least 1 follow-up sample were included in the analysis. Samples were collected by using nasal and nasopharyngeal swabs. The nasal microbiota were tested by using 16S ribosomal RNA gene sequencing. An unsupervised machine learning approach was used to identify mutually exclusive, longitudinal nasal microbiota profiles. Changes in profiles between the ages of 2 to 13 months and 2 to 24 months were examined. The profile with persistent Moraxella dominance was used as the reference group because this was the largest group, a finding also supported by the literature. The primary outcome was the risk of physician-diagnosed asthma at the ages of 6.5 to 7.5 years old. The secondary outcome was the number of acute respiratory infections (ARIs) during 13 to 24 months of age. The association of the microbiota longitudinal profiles with the risk of developing asthma and rate of ARIs was measured by using multivariable logistic regression and negative binomial models.Of the 704 children in the analytic cohort, 57 (8%) developed asthma. Across all samples, the nasal airway microbiota was dominated by Moraxella, which generally increased with age. During age 2 to 13 months, 4 distinct longitudinal nasal microbiota profiles were identified: (1) persistent Moraxelladominance profile (reference group), (2) Streptococcus to Moraxella transition profile, (3) persistent Dolosigranulum and Corynebacteriaceae dominance profile, and (4) persistent Moraxella sparsity profile. During 2 to 24 months of age, an additional distinct longitudinal nasal microbiota profiles was identified: (5) a mixed longitudinal pattern profile. Overall, children with a persistent Moraxella sparsity profile were at a significantly higher risk of developing asthma (adjusted odds ratio: 2.74; 95% confidence interval: 1.20 to 6.27 for those in this profile at 2 to 13 months of age, which persisted in the 2 to 24 month profile) and had significantly higher incidence rates of ARIs (incidence rate ratio: 1.16; 95% confidence interval: 1.01 to 1.32, from 13 to 24 months of age).In this study, researchers found that, compared with the reference group (Moraxella dominant profile), children with an altered longitudinal nasal microbiota pattern (Moraxella sparsity profile) during early childhood had a higher risk of developing asthma and ARIs.This is the first study to reveal an association between longitudinal changes in early airway microbiota and later development of childhood asthma. Further efforts to develop primary prevention strategies aimed at modifying the microbiota may help to lower the incidence of childhood asthma and ARIs.

Gingival Crevicular Fluid as Biomarker’s Source for Alzheimer’s Disease

Periodontitis is a low-grade inflammatory disease caused by a subgingival dysbiotic microbiota. Multiple studies have determined the higher prevalence of tooth loss and poor oral hygiene in patients with Alzheimer’s disease (AD). However, the periodontal diagnosis, periodontal bacteria or mediators has not been measured to date. Aim: To determine the periodontal status, the pro-inflammatory mediators, Porphyromonas gingivalis load, and Apoliporpotein E (ApoE) in patients with AD. A complete dental examination was performed on 30 patients, and cognitive status was determined by the Montreal Cognitive Assessment (MoCA). Subgingival microbiota and GCF samples were then taken from all patients from the deepest sites. Total DNA was isolated from the microbiota samples for the quantification of the 16S ribosomal subunit. Pro-inflammatory mediators and ApoE were quantified from the gingival crevicular fluid (GCF). Patients with AD had periodontitis stage III-IV in 80%, a higher concentration of pro-inflammatory and ApoE mediators, and a higher P. gingivalis load compared to healthy subjects. The pro-inflammatory mediators, P. gingivalis load had a negative correlation with the MoCA test scores. Finally, a ROC curve was performed to assess the specificity and sensitivity of ApoE levels, detecting an area of 84.9%. In AD patients, we found a more severe periodontitis, a higher levels of pro-inflammatory mediators, and higher bacterial load. In addition, there is an increase in ApoE that allows to clearly determine patients with health, periodontitis and periodontitis and AD.

Current Insights: The Impact of Gut Microbiota on Postoperative Complications in Visceral Surgery-A Narrative Review.

Postoperative complications are a major problem occurring in up to 50% of patients undergoing major abdominal surgery. Occurrence of postoperative complications is associated with a significantly higher morbidity and mortality in affected patients. The most common postoperative complications are caused by an infectious genesis and include anastomotic leakage in case of gastrointestinal anastomosis and surgical site infections. Recent research highlighted the importance of gut microbiota in health and disease. It is plausible that the gut microbiota also plays a pivotal role in the development of postoperative complications. This narrative review critically summarizes results of recent research in this particular field. The review evaluates the role of gut microbiota alteration in postoperative complications, including postoperative ileus, anastomotic leakage, and surgical site infections in visceral surgery. We tried to put a special focus on a potential diagnostic value of pre- and post-operative gut microbiota sampling showing that recent data are inhomogeneous to identify a high-risk microbial profile for development of postoperative complications.

Charting host-microbe co-metabolism in skin aging and application to metagenomics data.

During aging of human skin, a number of intrinsic and extrinsic factors cause the alteration of the skin's structure, function and cutaneous physiology. Many studies have investigated the influence of the skin microbiome on these alterations, but the molecular mechanisms that dictate the interplay between these factors and the skin microbiome are still not fully understood. To obtain more insight into the connection between the skin microbiome and the human physiological processes involved in skin aging, we performed a systematic study on interconnected pathways of human and bacterial metabolic processes that are known to play a role in skin aging. The bacterial genes in these pathways were subsequently used to create Hidden Markov Models (HMMs), which were applied to screen for presence of defined functionalities in both genomic and metagenomic datasets of skin-associated bacteria. These models were further applied on 16S rRNA gene sequencing data from skin microbiota samples derived from female volunteers of two different age groups (25-28 years ('young') and 59-68 years ('old')). The results show that the main bacterial pathways associated with aging skin are those involved in the production of pigmentation intermediates, fatty acids and ceramides. This study furthermore provides evidence for a relation between skin aging and bacterial enzymes involved in protein glycation. Taken together, the results and insights described in this paper provide new leads for intervening with bacterial processes that are associated with aging of human skin.

The Relationship Between Mucosal Microbiota, Colitis, and Systemic Inflammation in Chronic Granulomatous Disorder.

Chronic granulomatous disorder (CGD) is a primary immunodeficiency which is frequently complicated by inflammatory colitis and is associated with systemic inflammation. Herein, we aimed to investigate the role of the microbiome in the pathogenesis of colitis and systemic inflammation. We performed 16S rDNA sequencing on mucosal biopsy samples from each segment of 10 CGD patients' colons and conducted compositional and functional pathway prediction analyses. The microbiota in samples from colitis patients demonstrated reduced taxonomic alpha-diversity compared to unaffected patients, even in apparently normal bowel segments. Functional pathway richness was similar between the colitic and non-colitic mucosa, although metabolic pathways involved in butyrate biosynthesis or utilization were enriched in patients with colitis and correlated positively with fecal calprotectin levels. One patient with very severe colitis was dominated by Enterococcus spp., while among other patients Bacteroides spp. abundance correlated with colitis severity measured by fecal calprotectin and an endoscopic severity score. In contrast, Blautia abundance is associated with low severity scores and mucosal health. Several taxa and functional pathways correlated with concentrations of inflammatory cytokines in blood but not with colitis severity. Notably, dividing patients into "high" and "low" systemic inflammation groups demonstrated clearer separation than on the basis of colitis status in beta-diversity analyses. The microbiome is abnormal in CGD-associated colitis and altered functional characteristics probably contribute to pathogenesis. Furthermore, the relationship between the mucosal microbiome and systemic inflammation, independent of colitis status, implies that the microbiome in CGD can influence the inflammatory phenotype of the condition.

Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro-inflammatory host-microbe interactions in the colon.

The colonic epithelial barrier is vital to preserve gut and host health by maintaining the immune homeostasis between host and microbes. The mechanisms underlying beneficial or harmful host-microbe interactions are poorly understood and impossible to study in vivo given the limited accessibility and ethical constraints. Moreover, existing in vitro models lack the required cellular complexity for the routine, yet profound, analysis of the intricate interplay between different types of host and microbial cells. We developed and characterized a broadly applicable, easy-to-handle in vitro triple coculture model that combines chemically-induced macrophage-like, goblet and epithelial cells covered by a mucus layer, which can be coincubated with complex human-derived gut microbiota samples for 16h. Comparison with a standard epithelial monolayer model revealed that triple cocultures produce thicker mucus layers, morphologically organize in a network and upon exposure to human-derived gut microbiota samples, respond via pro-inflammatory cytokine production. Both model systems, however, were not suffering from cytotoxic stress or different microbial loads, indicating that the obtained endpoints were caused by the imposed conditions. Addition of the probiotic Lactobacillus rhamnosus GG to assess its immunomodulating capacity in the triple coculture slightly suppressed pro-inflammatory cytokine responses, based on transcriptomic microarray analyses. TNF conditioning of the models prior to microbial exposure did not cause shifts in cytokines, suggesting a strong epithelial barrier in which TNF did not reach the basolateral side. To conclude, the triple coculture model is tolerable towards manipulations and allows to address mechanistic host-microbe research questions in a stable in vitro environment.

The Role of Intestinal Microbiota in Metastatic Colorectal Cancer Patients Treated With Capecitabine

BackgroundPrevious pre-clinical research has indicated that the intestinal microbiota can potentiate anti-tumour efficacy of capecitabine and that capecitabine treatment impacts intestinal microbiota composition and diversity. Using a longitudinal design, this study explores the associations between the intestinal microbiota and treatment response in patients with metastatic colorectal cancer (mCRC) during capecitabine treatment. Patients and MethodsPatients with mCRC treated with capecitabine were prospectively enrolled in a multicentre cohort study. Patients collected a faecal sample and completed a questionnaire before, during, and after three cycles of capecitabine. Several clinical characteristics, including tumour response, toxicity and antibiotic use were recorded. Intestinal microbiota were analysed by amplicon sequencing of the 16S rRNA V4 gene-region. ResultsThirty-three patients were included. After three cycles of capecitabine, six patients (18%) achieved a partial response, 25 (76%) showed stable disease, and one (3%) experienced progressive disease. Of the 90 faecal samples were collected. Microbial diversity (α-diversity), community structure (β-diversity), and bacterial abundance on phylum and genus level were not significantly different between responders and non-responders and were not significantly affected by three cycles of capecitabine. ConclusionThis is the first clinical study with longitudinal intestinal microbiota sampling in mCRC patients that explores the role of the intestinal microbiota during treatment with capecitabine. Intestinal microbiota composition and diversity before, during, and after three cycles of capecitabine were not associated with response in this study population. Capecitabine did not induce significant changes in the microbiota composition and diversity during the treatment period. Individual effects of antibiotics during capecitabine treatment were observed.

Recovery of strain-resolved genomes from human microbiome through an integration framework of single-cell genomics and metagenomics

BackgroundObtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once.ResultsOur sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis).ConclusionsSMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker.3Xqp2i4EXkShdVYdkpPFdnVideo abstract

Black-and-White Ruffed Lemur (Varecia variegata) in Captivity: Analysis of the Oral Microbiota in a One Health Perspective.

Simple SummaryThe emergence of multidrug-resistant bacteria is a serious public health problem. Wild animals are known to be sources of multidrug-resistant bacteria as well as infectious diseases, some of them transmissible to humans. Due to many factors, such as the destruction of natural habitats or climate change, contact between wild animal species and humans is increasing. Thus, it is particularly important that studies be carried out in wildlife species to assess the possible existence of multidrug-resistant bacteria. In this case, the chosen species was the black-and-white ruffed lemur belonging to a zoo. Through a sample of the oral cavity, it was possible to know that half of the bacteria isolated in this group of animals were resistant to, at least, one antibiotic and one of them is resistant to an antibiotic for exclusive use in a hospital environment. Captive wild mammals can be a source of multidrug-resistant bacteria and studies such as this could contribute to the development of strategies to prevent the spread of this public health program.This study aimed to characterize the susceptibility profile to antibiotics and biofilm formation of Gram-negative bacterial isolates obtained from the oral cavity of the black-and-white ruffed lemur (Varecia variegata). From eight individuals from a zoo located in Portugal, samples of the oral microbiota were collected with sterile swabs and then placed in closed tubes with a transport medium. Culture was carried out for media of Gram-negative bacteria. Twenty-two isolates were obtained and subjected to susceptibility tests to twenty-five antimicrobial agents belonging to seven different classes. All tested isolates demonstrated resistance to, at least, one antibiotic, and it was possible to observe multidrug resistance in 11 of the 22 isolates (50%). It should be noted that an isolate showed phenotypic resistance to imipenem, an antibiotic for exclusive use in a hospital environment. All the isolates showed an increasing ability of biofilm formation over time. The obtained results show that wild mammals in captivity could be reservoirs and potential sources of multi-resistant pathogens. In view of this fact and considering the One Health concept, it will be advisable to establish local monitoring programs worldwide that benefit and protect human, animal and environmental health.

138 Expression of epidermal differentiation markers is modulated by AD-microbiota

Atopic Dermatitis (AD) is the most common chronic inflammatory skin disease, which is caused by an exaggerated T helper 2 cell-mediated immune responses and impairment of the skin barrier function. Single loss of function (LOF) mutations of filaggrin (FLG) are the strongest known genetic risk factor for AD. In Europeans an allele frequency of approximately 10% for a single LOF mutation of FLG can be found, but does not necessarily lead to AD-manifestation. FLG is essential for the skin barrier function. Mutations can thus lead to a disrupted skin barrier. It is known that atopic and healthy skin differ in bacterial diversity and that AD-skin is associated with dysbiosis. It is also known that healthy individuals with a single LOF mutation of FLG have a skin microbiota which is in part similar to that of AD patients. However, the mechanism through which the microbiota contributes in the pathogenesis of AD need further investigation and it is still unclear to what extent the AD microbiota is a driver of the disease. To investigate this question further, we stimulated 3D skin equivalents (3DSE) with collected microbiota of localization-, gender- and age-matched probands (AD- and healthy). Defined skin areas were rinsed with a saline/detergent solution to harvest the microbiota. Subsequently, the rinsing solution containing the microbiota was sequentially centrifuged to remove host mediators/cellular debris. The microbiota samples were then applied to the surface of 3DSE and the expression of skin differentiation markers like FLG were analyzed by real-time PCR. Our findings indicate that the treatment of the 3DSE with the AD-derived microbiota induce a decreased expression of FLG compared to 3DSE treated with the control-microbiota. These results support the hypothesis, that the AD-microbiota maintain the disrupted skin barrier and provide further evidence that the dysbiosis of the AD microbiota is linked to the manifestation/ development and course of this skin disease.

P62.01 PREVALUNG: Evaluation of Lung Cancer Prevalence in Patients With Smocking-associated Atherosclerotic Cardiovascular Diseases

Lung cancer is the leading cause of cancer related death in France and Worldwide. Lung cancer screening based on low dose thoracic computed tomography (low dose CT) in patients selected on age and lifelong tobacco consumption has proven to improve lung cancer related mortality. However, the definition and identification of patients eligible for lung cancer screening is a challenge that may limit lung cancer screening program implementation. Epidemiological data shows that 30 to 40% of patients with lung cancer present a history of cardiovascular disease, mainly atheromatous diseases such as peripheral arterial diseases and coronary artery stenoses. We hypothesized that patients with history of smoking who developed atheromatous disease could represent a population at high risk to develop lung cancer. Our main objective was to evaluate the prevalence of lung cancer among patients with atherosclerotic disease and history of tobacco consumption. Our second objective was to evaluate the lung cancer screening program implemented to measure the prevalence. We implemented a monocentric prospective epidemiological study to evaluate the prevalence of lung cancer among 500 patients with inclusion criteria i.e age 45-75 years old, history of at least 10 years of daily tobacco consumption preceding the onset of an atherosclerotic disease. Principal exclusion criteria were history of active carcinoma < 5 years, symptoms of lung cancer, follow-up for lung nodules and grade IV dyspnea. Patients with inclusion criteria are referred for an inclusion visit (V0) with a thoracic surgeon by adult cardiologists, vascular surgeons and adult cardiac surgeons in charge of the patient. After information and inclusion, a low dose CT scan is scheduled within 7 months and a smoking cessation visit is proposed in case of active smoking. Blood and gut microbiota samples are harvested the day of the low dose CT. Positive low-dose CT are discussed weekly at the multidisciplinary thoracic oncologic staff meeting and managed accordingly to the current European and national recommendations. A clinical follow-up is scheduled by phone at 3, 6 and 12 months after the low-dose CT to evaluate oncologic and cardiovascular events. Between November 2019 and April 2021 we included 487 of the 500 patients scheduled, 330 low dose CT were completed, 7 localized primary lung cancer were resected by minimally invasive lobectomy, 1 patient was treated by radiation alone without pathological proof for a lung nodule, 1 patient was related with systemic therapy for a stage IV primary lung cancer and 2 patients are waiting for surgical resection for clinically localized lung cancers. The implementation of a lung cancer screening program dedicated to patients followed-up for tobacco related atherosclerotic diseases is feasible and will allow to evaluate both lung cancer prevalence and short-term benefits of lung cancer screening in this population.

Elevated colonic microbiota-associated paucimannosidic and truncated N-glycans in pediatric ulcerative colitis

Pediatric ulcerative colitis (UC) is a distinct type of inflammatory bowel disease with severe disease activity and rapid progression, which can lead to detrimental life-long consequences. The pathogenesis of pediatric UC remains unclear, although dysbiosis of the gut microbiota has been considered an important factor. In this study, we collected intestinal mucosal-luminal interface microbiota samples from a cohort of treatment-naïve pediatric UC or control patients and used two different mass spectrometry-based glycomic approaches to examine the N-glycans that were associated with the microbiota. We observed abundant small N-glycans that were associated with the microbiota and found that the pediatric UC microbiota samples contained significantly higher levels of these atypical N-glycans compared to those of controls. Four paucimannosidic or other truncated N-glycans were identified to successfully segregate UC from control patients with an area under the ROC curve of ≥0.9. This study indicates that the aberrant metabolism of glycans in the intestinal by gut microbiota may be involved in the pathogenesis of UC and intestinal N-glycans, including small glycans, can act as novel biomarker candidates for pediatric UC. SignificanceThere is no cure for pediatric ulcerative colitis (UC) due to its unclear pathogenesis and the diagnosis of UC in children still largely depends on invasive colonoscopic examination. Recent evidence suggests that the dysbiosis of intestinal microbiota is associated with the onset and development of UC, however how the microbiota interact with the host remains unclear. This study used two different mass spectrometry-based glycomic approaches to quantitatively examine N-glycans that are associated with colonic mucosal-luminal interface microbiota of pediatric UC or control patients. To the best of our knowledge, this is the first comprehensive glycomic study of intestinal microbiota samples in UC, which demonstrated that intestinal microbiota was associated with abundant atypical small N-glycans with elevated levels in UC than controls. This study also identified four intestinal paucimannosidic or other truncated N-glycans as promising biomarker candidates for pediatric UC. These findings shed light on the mechanism study of host-microbiome interactions in UC and indicate that atypical glycans present in the gut can be a source for UC biomarker discovery.

Environment-Dependent Variation in Gut Microbiota of an Oviparous Lizard (Calotes versicolor).

Simple SummaryThe different gut sections potentially provide different habitats for gut microbiota. We found that Bacteroidetes, Firmicutes, and Proteobacteria were the three primary phyla in gut microbiota of C. versicolor. The relative abundance of dominant phyla Bacteroidetes and Firmicutes exhibited an increasing trend from the small intestine to the large intestine, and there was a higher abundance of genus Bacteroides (Class: Bacteroidia), Coprobacillus and Eubacterium (Class: Erysipelotrichia), Parabacteroides (Family: Porphyromonadaceae) and Ruminococcus (Family: Lachnospiraceae), and Family Odoribacteraceae and Rikenellaceae in the hindgut, and some metabolic pathways were higher in the hindgut. Our results reveal the variations of gut microbiota composition and metabolic pathways in different parts of the lizards’ intestine.Vertebrates maintain complex symbiotic relationships with microbiota living within their gastrointestinal tracts which reflects the ecological and evolutionary relationship between hosts and their gut microbiota. However, this understanding is limited in lizards and the spatial heterogeneity and co-occurrence patterns of gut microbiota inside the gastrointestinal tracts of a host and variations of microbial community among samples remain poorly understood. To address this issue and provide a guide for gut microbiota sampling from lizards, we investigated the bacteria in three gut locations of the oriental garden lizard (Calotes versicolor) and the data were analyzed for bacterial composition by 16S ribosomal RNA (16S rRNA) gene amplicon sequencing. We found the relative abundance of the dominant phyla exhibited an increasing trend from the small intestine to the large intestine, and phyla Firmicutes, Bacteroidetes and Proteobacteria were the three primary phyla in the gut microbiota of C. versicolor. There were a higher abundance of genus Bacteroides (Class: Bacteroidia), Coprobacillus and Eubacterium (Class: Erysipelotrichia), Parabacteroides (Family: Porphyromonadaceae) and Ruminococcus (Family: Lachnospiraceae), and Family Odoribacteraceae and Rikenellaceae in the sample from the hindgut. The secondary bile acid biosynthesis, glycosaminoglycan degradation, sphingolipid metabolism and lysosome were significantly higher in the hindgut than that in the small intestine. Taken together our results indicate variations of gut microbiota composition and metabolic pathway in different parts of the oriental garden lizard.

Ecological Adaptation and Succession of Human Fecal Microbial Communities in an Automated In Vitro Fermentation System.

ABSTRACTLongitudinal studies of gut microbiota following specific interventions are vital for understanding how they influence host health. However, robust longitudinal sampling of gut microbiota is a major challenge, which can be addressed using in vitro fermentors hosting complex microbial communities. Here, by employing 16S rRNA gene amplicon sequencing, we investigated the adaptation and succession of human fecal microbial communities in an automated multistage fermentor. We performed two independent experiments using different human donor fecal samples, one configured with two units of three colon compartments each studied for 22 days and another with one unit of two colon compartments studied for 31 days. The fermentor maintained a trend of increasing microbial alpha diversity along colon compartments. Within each experiment, microbial compositions followed compartment-specific trajectories and reached independent stable configurations. While compositions were highly similar between replicate units, they were clearly separated between different experiments, showing that they maintained the individuality of fecal inoculum rather than converging on a fermentor-specific composition. While some fecal amplicon sequence variants (ASVs) were undetected in the fermentor, many ASVs undetected in the fecal samples flourished in vitro. These bloomer ASVs accounted for significant proportions of the population and included prominent health-associated microbes such as Bacteroides fragilis and Akkermansia muciniphila. Turnover in community compositions is likely explained by feed composition and pH, suggesting that these communities can be easily modulated. Our results suggest that in vitro fermentors are promising tools to study complex microbial communities harboring important members of human gut microbiota.IMPORTANCE In vitro fermentors that can host complex gut microbial communities are promising tools to investigate the dynamics of human gut microbiota. In this work, using an automated in vitro gut fermentor consisting of different colon compartments, we investigated the adaptation dynamics of two different human fecal microbial communities over 22 and 31 days. By observing the temporal trends of different community members, we found that many dominant members of the fecal microbiota failed to maintain their dominance in vitro, and some of the low-abundance microbes undetected in the fecal microbiota successfully grew in the in vitro communities. Microbiome compositional changes and blooming could largely be explained by feed composition and pH, suggesting that these communities can be modulated to desired compositions via optimizing culture conditions. Thus, our results open up the possibility of modulating in vitro microbial communities to predefined compositions by optimizing feed composition and culture conditions.

Measuring and mitigating PCR bias in microbiota datasets.

PCR amplification plays an integral role in the measurement of mixed microbial communities via high-throughput DNA sequencing of the 16S ribosomal RNA (rRNA) gene. Yet PCR is also known to introduce multiple forms of bias in 16S rRNA studies. Here we present a paired modeling and experimental approach to characterize and mitigate PCR NPM-bias (PCR bias from non-primer-mismatch sources) in microbiota surveys. We use experimental data from mock bacterial communities to validate our approach and human gut microbiota samples to characterize PCR NPM-bias under real-world conditions. Our results suggest that PCR NPM-bias can skew estimates of microbial relative abundances by a factor of 4 or more, but that this bias can be mitigated using log-ratio linear models.

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

Patient-specific establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering.

Dysbiosis, a loss of balance in the microbiota, is a potential factor of peri-implantitis. However, compositional change of the peri-implant microbiota soon after implant uncovering is still unknown. In this study, bacterial composition in the peri-implant sulcus was examined to understand the establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering. Microbiota samples were collected at weeks 1, 2, 4, and 6 after stage-two surgery. Bacterial DNA was isolated from the samples, and a 16S rRNA gene library was constructed. Sequence reads were obtained using a high-throughput sequencing platform and were taxonomically assigned at the phylum and genus levels. Alpha diversity indices, which did not include taxonomic information, were at similar levels throughout the four time points. At 1 and 2weeks, the bacterial composition was similar among patients with the predominance of Firmicutes and Proteobacteria. However, the composition was diverse at 4 and 6weeks and significantly dissimilar to the composition at 1week. At 1week, the peri-implant microbiota was already formed with alpha diversity as high as that at the later time points. However, the bacterial composition was not highly dissimilar among patients at 1week. The composition changed over the passage of several weeks and was specific for each patient.

Experimental validation of small mammal gut microbiota sampling from faeces and from the caecum after death.

Data on the gut microbiota (GM) of wild animals are key to studies on evolutionary biology (host-GM interactions under natural selection), ecology and conservation biology (GM as a fitness component closely connected to the environment). Wildlife GM sampling often requires non-invasive techniques or sampling from dead animals. In a controlled experiment profiling microbial 16S rRNA in 52 house mice (Mus musculus) from eight families and four genetic backgrounds, we studied the effects of live- and snap-trapping on small mammal GM and evaluated the suitability of microbiota from non-fresh faeces as a proxy for caecal GM. We compared CM from individuals sampled 16-18 h after death with those in live traps and caged controls, and caecal and faecal GM collected from mice in live-traps. Sampling delay did not affect GM composition, validating data from fresh cadavers or snap-trapped animals. Animals trapped overnight displayed a slight but significant difference in GM composition to the caged controls, though the change only had negligible effect on GM diversity, composition and inter-individual divergence. Hence, the trapping process appears not to bias GM profiling. Despite their significant difference, caecal and faecal microbiota were correlated in composition and, to a lesser extent, diversity. Both showed congruent patterns of inter-individual divergence following the natural structure of the dataset. Thus, the faecal microbiome represents a good non-invasive proxy of the caecal microbiome, making it suitable for detecting biologically relevant patterns. However, care should be taken when analysing mixed datasets containing both faecal and caecal samples.