Ribosomal DNA Amplicon Sequencing Research Articles

Association of anti-TNF-α treatment with gut microbiota of patients with ankylosing spondylitis.

ObjectiveGut dysbiosis contributes to multiple autoimmune diseases, including ankylosing spondylitis, which is commonly treated with tumor necrosis factor (TNF)-α inhibitors (TNFis). Because host TNF-α levels are considered to interact with gut microbiota, we aimed to systematically investigate the microbiota profile of ankylosing spondylitis patients with anti-TNF-α-based treatment and identify potential key bacteria.MethodsFecal samples were collected from 11 healthy controls and 24 ankylosing spondylitis patients before/after anti-TNF-α treatment, the microbiota profiles of which were evaluated by 16S ribosomal DNA amplicon sequencing and subsequent bioinformatic analysis.ResultsSignificantly different microbial compositions were observed in samples from ankylosing spondylitis patients compared with healthy controls, characterized by a lower abundance of short-chain fatty acid (SCFA)-producing bacteria. All patients exhibited a positive response after anti-TNF-α treatment, accompanied by a trend of restoration in the microbiota compositions and functional profile of ankylosing spondylitis patients to healthy controls. In particular, the abundance of SCFA-producing bacteria (e.g. Megamonsa and Lachnoclostridium) was not only significantly lower in ankylosing spondylitis patients than in healthy controls and restored after anti-TNF-α treatment but also negatively correlated with disease severity (e.g. cor = -0.52, P = 8 × 10−5 for Megamonsa). In contrast, Bacilli and Haemophilus may contribute to ankylosing spondylitis onset and severity.ConclusionsMicrobiota dysbiosis in ankylosing spondylitis patients can be restored after anti-TNF-α treatment, possibly by impacting SCFA-producing bacteria.

Differences in the Oral Microbiome in Patients With Early Rheumatoid Arthritis and Individuals at Risk of Rheumatoid Arthritis Compared to Healthy Individuals.

ObjectiveIt has been suggested that rheumatoid arthritis (RA) may originate at the oral mucosa. The aim of the present study was to assess the oral microbiome and periodontal condition in patients with early RA and individuals at risk of developing RA compared to healthy controls.MethodsThree groups were recruited (n = 50 participants per group): 1) patients with early RA (meeting the American College of Rheumatology/European Alliance of Associations for Rheumatology 2010 classification criteria), 2) individuals at risk of developing RA (those with arthralgia who were positive for RA‐associated autoantibodies), and 3) healthy controls. A periodontal examination was conducted to assess the presence of bleeding on probing (BOP), pocket probing depth (PPD), and periodontal inflamed surface area (PISA). The microbial composition of subgingival dental plaque, saliva, and tongue coating was assessed using 16S ribosomal DNA amplicon sequencing, and findings were compared between groups with permutational multivariate analysis of variance (PERMANOVA).ResultsThere were no significant differences in any of the 3 periodontal variables between patients with early RA, at‐risk individuals, and healthy controls (P = 0.70 for BOP, P = 0.30 for PPD, and P = 0.57 for PISA, by Kruskal‐Wallis test). PERMANOVA analyses comparing microbial composition between the groups showed significant differences in the microbial composition of saliva (F = 2.08, P = 0.0002) and tongue coating (F = 2.04, P = 0.008), but not subgingival dental plaque (F = 0.948, P = 0.51). However, in post hoc tests, no significant differences in microbial composition of the saliva or tongue coating were observed between the early RA group and the at‐risk group (F = 1.12, P = 0.28 for saliva; F = 0.834, P = 0.59 for tongue coating). In assessing microbial diversity based on the number of zero‐radius operational taxonomic units per sample, Prevotella in the saliva and Veillonella in the saliva and tongue coating were each found at a higher relative abundance in samples from patients with early RA and at‐risk individuals compared to healthy controls.ConclusionThe results show similarities in the oral microbiome between patients with early RA and at‐risk individuals, since in both groups, the oral microbiome was characterized by an increased relative abundance of potentially proinflammatory species when compared to that in healthy controls. These findings suggest a possible association between the oral microbiome and the onset of RA.

Transcriptomics Provides a Genetic Signature of Vineyard Site and Offers Insight into Vintage-Independent Inoculated Fermentation Outcomes.

ABSTRACTRibosomal DNA amplicon sequencing of grape musts has demonstrated that microorganisms occur nonrandomly and are associated with the vineyard of origin, suggesting a role for the vineyard, grape, and wine microbiome in shaping wine fermentation outcomes. Here, ribosomal DNA amplicon sequencing from grape musts and RNA sequencing of eukaryotic transcripts from primary fermentations inoculated with the wine yeast Saccharomyces cerevisiae RC212 were used to profile fermentations from 15 vineyards in California and Oregon across two vintages. These data demonstrate that the relative abundance of fungal organisms detected by ribosomal DNA amplicon sequencing correlated with neither transcript abundance from those same organisms within the RNA sequencing data nor gene expression of the inoculated RC212 yeast strain. These data suggest that the majority of the fungi detected in must by ribosomal DNA amplicon sequencing were not active during the primary stage of these inoculated fermentations and were not a major factor in determining RC212 gene expression. However, unique genetic signatures were detected within the ribosomal DNA amplicon and eukaryotic transcriptomic sequencing that were predictive of vineyard site and region. These signatures included S. cerevisiae gene expression patterns linked to nitrogen, sulfur, and thiamine metabolism. These genetic signatures of site offer insight into specific environmental factors to consider with respect to fermentation outcomes and vineyard site and regional wine characteristics.IMPORTANCE The wine industry generates billions of dollars of revenue annually, and economic productivity is in part associated with regional distinctiveness of wine sensory attributes. Microorganisms associated with grapes and wineries are influenced by region of origin, and given that some microorganisms play a role in fermentation, it is thought that microbes may contribute to the regional distinctiveness of wine. In this work, as in previous studies, it is demonstrated that specific bacteria and fungi are associated with individual wine regions and vineyard sites. However, this work further shows that their presence is not associated with detectable fungal gene expression during the primary fermentation or the expression of specific genes by the inoculate Saccharomyces cerevisiae strain RC212. The detected RC212 gene expression signatures associated with region and vineyard site also allowed the identification of flavor-associated metabolic processes and environmental factors that could impact primary fermentation outcomes. These data offer novel insights into the complexities and subtleties of vineyard-specific inoculated wine fermentation and starting points for future investigations into factors that contribute to regional wine distinctiveness.

Preliminary microbiological and chemical analysis of two historical stock ales from Victorian and Edwardian brewing

Historical beers are a valuable source of information on past brewing microbiology providing opportunity for analysis and isolation. Although rarely found intact and suffering variable degrees of deterioration they can contain living microbial cells and residues of chemical components indicating the character of the beers and their production processes. This report summarises preliminary analysis of two beers from Victorian and Edwardian times and provides an indication of their chemistry and microbiology. One beer, recovered from the 1895 Scottish shipwreck Wallachia, was a 7.5% ABV stout, the other an 11% ABV celebration King's Ale, a barley wine, brewed by Bass in 1902 for the visit of King Edward VII. Live yeast was isolated from both beers: Brettanomyces and Debaryomyces from the Wallachia stout and Saccharomyces from the Bass Ale. Ribosomal DNA amplicon sequencing indicated the presence of a wide range of microorganisms in both beers including lactobacilli and pediococci in the Wallachia beer and staphylococci in the Bass Ale. Both beers are likely to be in the category of stock ales of the time and would have been matured for a period before bottling. The presence of Brettanomyces bruxellensis in both beers confirms the understanding of this species as an important contributor to these beers but it is also interesting that Debaryomyces species are consistently present as a major component and that Saccharomyces are less prevalent. © 2021 The Institute of Brewing & Distilling

Schistosomiasis Vector Snails and Their Microbiota Display a Phylosymbiosis Pattern.

Planorbidae snails are the intermediate host for the trematode parasite of the Schistosoma genus, which is responsible for schistosomiasis, a disease that affects both humans and cattle. The microbiota for Schistosoma has already been described as having an effect on host/parasite interactions, specifically through immunological interactions. Here, we sought to characterize the microbiota composition of seven Planorbidae species and strains. Individual snail microbiota was determined using 16S ribosomal DNA amplicon sequencing. The bacterial composition was highly specific to the host strain with limited interindividual variation. In addition, it displayed complete congruence with host phylogeny, revealing a phylosymbiosis pattern. These results were confirmed in a common garden, suggesting that the host highly constrains microbial composition. This study presents the first comparison of bacterial communities between several intermediate snail hosts of Schistosoma parasites, paving the way for further studies on the understanding of this tripartite interaction.

2628. Nasopharyngeal Microbiome in the First Weeks of Life Distinguishes Infants Who Subsequently Develop Lower Respiratory Tract Infections

BackgroundColonization of the nasopharynx (NP) is the initial event in the pathogenesis of lower respiratory tract infections (LRTI). Evidence is accumulating that the NP microbiome influences host immune responses and whether colonization progresses to disease or not. We hypothesized that infants who experience LRTI early in life display distinct NP microbiome characteristics prior to infection, and potentially as early as the newborn period.MethodsAs part of the “Southern Africa Mother Infant Pertussis Study” in Zambia, NP samples were prospectively collected approximately every 2 weeks beginning at birth, through 3 months of age, in conjunction with clinical data. Samples were also collected when an infant experienced respiratory symptoms. We identified infants from the cohort with LRTI and matched with asymptomatic controls. We performed 16S ribosomal DNA amplicon sequencing on DNA extracted from the NP samples using Illumina MiSeq, and analyzed the data using Qiime2 and PathoScope2. We described the NP microbiome characteristics of asymptomatic infants and infants with LRTI and their changes over time and compared between the two populations at each 2-week interval using the R package DESeq2.ResultsTen infants who had LRTI during the study period were matched with 17 healthy asymptomatic controls, together contributing 183 samples with high-quality reads. In asymptomatic infants, Dolosigranulum, Haemophilus and Moraxella’s relative abundance increased over the first 3 months of life, while Corynebacterium and Staphylococcus relative abundance decreased in the NP microbiome (Figure 1). In contrast, infants who developed LRTI had increased abundance of Staphylococcus, Anaerobacillus, and Bacillus, and decreased relative abundance of Dolosigranulum and Moraxella compared with asymptomatic controls (Figure 2). These differences were already present at the time of first sample collection (age 1 week).ConclusionInfants who develop LRTI early in life demonstrate altered NP microbial composition as early as the first week of life. These differences could potentially lead to early identification of at-risk infants. If confirmed, interventions to prevent LRTI in infancy could be evaluated to reduce respiratory mortality and morbidity. Disclosures All authors: No reported disclosures.

Changes in the gut microbiota during and after commercial helium–oxygen saturation diving in China

ObjectivesThe influence of commercial helium–oxygen saturation diving on divers’ gut microbiotas was assessed to provide dietary suggestion.MethodsFaecal samples of 47 divers working offshore were collected before (T1), during (T2) and...

Differential composition of gut microbiota among healthy volunteers, morbidly obese patients and post‑bariatric surgery patients

The modulation of the gut microbiota was recently deemed one of the mechanisms responsible for the excellent outcomes of bariatric surgery. However, to date, only few studies have assessed this, and they have high heterogeneity. In the present study, next-generation 16S ribosomal DNA amplicon sequencing was used to characterize the gut microbiota of healthy volunteers, as well as patients prior to and after sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB). Significant differences in α diversity, β diversity and species were identified between the different groups/time-points. The results demonstrated excellent outcomes of SG and RYGB. The β diversity was lower in healthy volunteers compared with that in morbidly obese patients with or without type 2 diabetes mellitus. At 3 months after SG, the α diversity was increased and the β diversity was decreased. The abundance of certain species changed significantly after SG and RYGB. It was also revealed that the abundance of certain microbes was significantly correlated with the body mass index, fasting blood glucose and glycosylated haemoglobin. It may be concluded that bariatric surgery may cause obvious alterations in the gut microbiota and compared with healthy volunteers and obese patients without bariatric surgery, the microbiota composition of post-bariatric surgery has unique characteristics. However, studies with a larger cohort and longer follow-up may be required to confirm these results.

Increased Tryptophan Metabolism Is Associated With Activity of Inflammatory Bowel Diseases

Administration of tryptophan and some of its metabolites reduces the severity of colitis in mice, whereas removing tryptophan from the diet increases susceptibility to colitis. Transfer of the intestinal microbiome transfers the colitogenic phenotype from tryptophan starved animals to normally nourished mice. We aimed to systematically evaluate serum levels of tryptophan and its metabolites in patients with inflammatory bowel diseases (IBD), and study their association with clinical and serologic features. We studied 535 consecutive patients with IBD (211 with ulcerative colitis [UC], 234 with Crohn's disease [CD]; 236 male), enrolled in Germany from August 2013 through April 2014 and followed until July 2016. Serum samples were collected from patients and 291 matched individuals without IBD (controls); levels of tryptophan were measured using high-performance liquid chromatography. Metabolites of tryptophan were measured in serum from 148 patients and 100 controls by mass spectrometry. We measured levels of interleukin 22 in serum from 28 patients by enzyme-linked immunosorbent assay. Paired stool and serum samples were collected from a subset of patients with active UC (n= 10) or CD (n= 8) to investigate associations between serum levels of tryptophan and composition of the fecal microbiota, analyzed by 16S ribosomal DNA amplicon sequencing. We used real-time polymerase chain reaction to measure levels of messenger RNAs in colonic biopsies from 60 patients with UC, 50 with CD, and 30 controls. We collected information on patients' disease activity scores, medications, laboratory assessments, and clinical examinations during recruitment and follow-up visits. Serum levels of tryptophan were significantly lower in patients with IBD than in controls (P= 5.3× 10-6) with a stronger reduction in patients with CD (vs control; P= 1.1× 10-10) than UC (vscontrol; P= 2.8× 10-3). We found a negative correlation between serum levels of tryptophan and disease activity or levels of C-reactive protein. Levels of messenger RNAs encoding tryptophan 2,3-dioxygenase-2 and solute carrier family 6member 19 (also called B0AT1) were significantly decreased in colonic biopsies from patients with IBD compared with controls, whereas level of messenger RNA encoding indoleamine 2,3-dioxygenase-1 was significantly increased. The composition of the fecal microbiota associated with serum levels of tryptophan. Analysis of tryptophan metabolites revealed activation of the kynurenine pathway, based on high levels of quinolinic acid, in patients with IBD compared with controls. Serum concentration of interleukin 22 associated with disease activity in patients with IBD; there was an inverse association between levels of interleukin 22 and serum levels of tryptophan. In an analysis of serum samples from more than 500 patients with IBD, we observed a negative correlation between serumlevels of tryptophan and disease activity. Increased levels of tryptophan metabolites-especially of quinolinic acid-indicated a high activity of tryptophan degradation in patients with active IBD. Tryptophan deficiency could contribute to development of IBD or aggravate disease activity. Interventional clinical studies areneeded to determine whether modification of intestinal tryptophan pathways affects the severity of IBD.

Correction: Factors That Affect Large Subunit Ribosomal DNA Amplicon Sequencing Studies of Fungal Communities: Classification Method, Primer Choice, and Error

Correction: Factors That Affect Large Subunit Ribosomal DNA Amplicon Sequencing Studies of Fungal Communities: Classification Method, Primer Choice, and Error

Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error.

Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project naïve Bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50–100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys.