AB1232 ORAL DYSBIOSIS REFLECTS THE IMMUNOLOGICAL ALTERATION OF RA REGARDING TO ACPA AND HLA DRB1*SE: NAGASAKI ISLAND STUDY

Background:Anti-citrullinated protein antibody (ACPA) production is observed in several organs even prior to the onset of rheumatoid arthritis (RA), and oral mucosa is considered to be one of the important tissues. The presence of HLA-DRB1*SE closely associates with ACPA production. Saliva is considered to reflect the oral microbiota including periodontal disease. Alteration of oral microbiota of RA becomes to be normalized by DMARDs treatment, however, the interaction of HLA-DRB1*SE, ACPA and oral microbiota of RA patients remains to be elucidated.Objectives:The Nagasaki Island Study, which had started in 2014 collaborating with Goto City, is intended for research of the preclinical stage of RA, including ACPA/HLA genotype screening and ultrasound and magnetic resonance imaging examinations in high-risk subjects. Using the samples accumulated in this cohort, we have tried to investigate the difference of oral microbiota among RA patients and healthy subjects regarding to ACPA and HLA-DRB1*SE.Methods:Blood and salivary samples were obtained from 1422 subjects out of 4276 who have participated in the Nagasaki Island Study from 2016 to 2018. ACPA positivity was 1.7 % in total. Some of RA patients resided in Goto City participated in the Nagasaki Island Study. At this point, we selected 291 subjects, who were ACPA positive non-RA healthy subjects (n=22) and patients with RA (n=33, 11 subjects were ACPA positive and 22 ACPA negative respectively) as the case, age and gender matched ACPA negative non-RA healthy subjects (n=236) as the control. ACPA was measured by an enzyme-linked immunosorbent assay, and HLA genotyping was quantified by next-generation sequencing (Ref.1). The operational taxonomic unit (OUT) analysis using 16S rRNA gene sequencing were performed. The richness of microbial diversity within-subject (alpha diversity) was scaled via Shannon entropy. The dissimilarity between microbial community composition was calculated using Bray-Curtis distance as a scale, and differences between groups (beta diversity) were tested by permutational multivariate analysis of variance (PERMANOVA). In addition, UniFrac distance calculated in consideration of the distance on the phylogenetic tree were performed.Results:Median age 70 y.o., % Female 58.8 %. Among RA and non-RA subjects, not alpha diversity but beta diversity was statistically significance (p=0.022, small in RA). In RA subjects, both alpha and beta diversity is small (p<0.0001), especially significant in ACPA positive RA (Figure 1). Amongt RA subjects, presence of HLA-DRB1*SE did not show the difference but the tendency of being small of alpha diversity (p=0.29).Conclusion:Our study has suggested for the first time the association of oral microbiota alteration with the presence of ACPA and HLA-DRB1*SE. Oral dysbiosis may reflect the immunological status of patients with RA.References:[1]Kawaguchi S, et al. Methods Mol Biol 2018;1802: 22Disclosure of Interests:None declared

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Evaluation of the gut microbiome in association with biological signatures of inflammation in murine polytrauma and shock

Scientific Reports ◽

10.1038/s41598-021-85897-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sandra A. Appiah ◽

Christine L. Foxx ◽

Dominik Langgartner ◽

Annette Palmer ◽

Cristian A. Zambrano ◽

...

Keyword(s):

Gut Microbiota ◽

Community Composition ◽

Gut Microbiome ◽

Beta Diversity ◽

Microbial Community Composition ◽

Alpha Diversity ◽

Global Changes ◽

Rrna Gene ◽

Alpha And Beta Diversity ◽

Increased Risk

AbstractSevere injuries are frequently accompanied by hemorrhagic shock and harbor an increased risk for complications. Local or systemic inflammation after trauma/hemorrhage may lead to a leaky intestinal epithelial barrier and subsequent translocation of gut microbiota, potentially worsening outcomes. To evaluate the extent with which trauma affects the gut microbiota composition, we performed a post hoc analysis of a murine model of polytrauma and hemorrhage. Four hours after injury, organs and plasma samples were collected, and the diversity and composition of the cecal microbiome were evaluated using 16S rRNA gene sequencing. Although cecal microbial alpha diversity and microbial community composition were not found to be different between experimental groups, norepinephrine support in shock animals resulted in increased alpha diversity, as indicated by higher numbers of distinct microbial features. We observed that the concentrations of proinflammatory mediators in plasma and intestinal tissue were associated with measures of microbial alpha and beta diversity and the presence of specific microbial drivers of inflammation, suggesting that the composition of the gut microbiome at the time of trauma, or shortly after trauma exposure, may play an important role in determining physiological outcomes. In conclusion, we found associations between measures of gut microbial alpha and beta diversity and the severity of systemic and local gut inflammation. Furthermore, our data suggest that four hours following injury is too early for development of global changes in the alpha diversity or community composition of the intestinal microbiome. Future investigations with increased temporal-spatial resolution are needed in order to fully elucidate the effects of trauma and shock on the gut microbiome, biological signatures of inflammation, and proximal and distal outcomes.

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Effect of Bovine MFGM and LF in Infant Formula on Gut Microbiome and Metabolome at Four Months of Age

Current Developments in Nutrition ◽

10.1093/cdn/nzab027 ◽

2021 ◽

Author(s):

Maciej Chichlowski ◽

Nicholas Bokulich ◽

Cheryl L Harris ◽

Jennifer L Wampler ◽

Fei Li ◽

...

Keyword(s):

Infant Formula ◽

Beta Diversity ◽

Milk Fat ◽

Alpha Diversity ◽

Illumina Miseq ◽

Rrna Gene ◽

Term Infants ◽

Linear Discriminant ◽

Alpha And Beta Diversity ◽

Metabolite Profiles

Abstract Background Milk fat globule membrane (MFGM) and lactoferrin (LF) are human milk bioactive components demonstrated to support gastrointestinal (GI) and immune development. Significantly fewer diarrhea and respiratory-associated adverse events through 18 months of age were previously reported in healthy term infants fed a cow's milk-based infant formula with added source of bovine MFGM and bovine LF through 12 months of age. Objectives To compare microbiota and metabolite profiles in a subset of study participants. Methods Stool samples were collected at Baseline (10–14 days of age) and Day 120 (MFGM + LF: 26, Control: 33). Bacterial community profiling was performed via16S rRNA gene sequencing (Illumina MiSeq) and alpha and beta diversity were analyzed (QIIME 2). Differentially abundant taxa were determined using Linear discriminant analysis effect size (LefSE) and visualized (Metacoder). Untargeted stool metabolites were analyzed (HPLC/mass spectroscopy) and expressed as the fold-change between group means (Control: MFGM + LF ratio). Results Alpha diversity increased significantly in both groups from baseline to 4 months. Subtle group differences in beta diversity were demonstrated at 4 months (Jaccard distance; R2 = 0.01, P = 0.042). Specifically, Bacteroides uniformis and Bacteroides plebeius were more abundant in the MFGM + LF group at 4 months. Metabolite profile differences for MFGM + LF vs Control included: lower fecal medium chain fatty acids, deoxycarnitine, and glycochenodeoxycholate, and some higher fecal carbohydrates and steroids (P < 0.05). After applying multiple test correction, the differences in stool metabolomics were not significant. Conclusions Addition of bovine MFGM and LF in infant formula was associated with subtle differences in stool microbiome and metabolome by four months of age, including increased prevalence of Bacteroides species. Stool metabolite profiles may be consistent with altered microbial metabolism. Trial registration: https://clinicaltrials.gov/ct2/show/NCT02274883).

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Gut microbiota composition is associated with narcolepsy type 1

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000000896 ◽

2020 ◽

Vol 7 (6) ◽

pp. e896

Author(s):

Alexandre Lecomte ◽

Lucie Barateau ◽

Pedro Pereira ◽

Lars Paulin ◽

Petri Auvinen ◽

...

Keyword(s):

Community Structure ◽

Gut Microbiota ◽

Bacterial Communities ◽

Beta Diversity ◽

Alpha Diversity ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Differential Abundance ◽

Alpha And Beta Diversity

ObjectiveTo test the hypothesis that narcolepsy type 1 (NT1) is related to the gut microbiota, we compared the microbiota bacterial communities of patients with NT1 and control subjects.MethodsThirty-five patients with NT1 (51.43% women, mean age 38.29 ± 19.98 years) and 41 controls (57.14% women, mean age 36.14 ± 12.68 years) were included. Stool samples were collected, and the fecal microbiota bacterial communities were compared between patients and controls using the well-standardized 16S rRNA gene amplicon sequencing approach. We studied alpha and beta diversity and differential abundance analysis between patients and controls, and between subgroups of patients with NT1.ResultsWe found no between-group differences for alpha diversity, but we discovered in NT1 a link with NT1 disease duration. We highlighted differences in the global bacterial community structure as assessed by beta diversity metrics even after adjustments for potential confounders as body mass index (BMI), often increased in NT1. Our results revealed differential abundance of several operational taxonomic units within Bacteroidetes, Bacteroides, and Flavonifractor between patients and controls, but not after adjusting for BMI.ConclusionWe provide evidence of gut microbial community structure alterations in NT1. However, further larger and longitudinal multiomics studies are required to replicate and elucidate the relationship between the gut microbiota, immunity dysregulation and NT1.

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Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals

Frontiers in Microbiology ◽

10.3389/fmicb.2021.740932 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hannah E. Epstein ◽

Alejandra Hernandez-Agreda ◽

Samuel Starko ◽

Julia K. Baum ◽

Rebecca Vega Thurber

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Beta Diversity ◽

Sequence Data ◽

Alpha Diversity ◽

Gene Profiling ◽

Rrna Gene ◽

Sequencing Platform ◽

Alpha And Beta Diversity ◽

Diversity Metrics

16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata, we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.

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Resident microbes of lactation rooms and daycares

PeerJ ◽

10.7717/peerj.8168 ◽

2019 ◽

Vol 7 ◽

pp. e8168

Author(s):

Diana H. Taft ◽

Samir Akre ◽

Nicolas Madrid ◽

Andre Knoesen ◽

David A. Mills ◽

...

Keyword(s):

Beta Diversity ◽

Alpha Diversity ◽

Illumina Miseq ◽

Shannon Index ◽

Rrna Gene ◽

Sample Collection ◽

Unifrac Distance ◽

Modern Development ◽

Temperature And Humidity ◽

The Impact

Dedicated lactation rooms are a modern development as mothers return to work while still providing breastmilk to their absent infants. This study describes the built environment microbiome of lactation rooms and daycares, and explores the influence of temperature and humidity on the microbiome of lactation rooms. Sterile swabs were used to collect samples from five different sites in lactation rooms at University of California, Davis and from five different sites in daycares located in Davis, California. DNA from the swabs was extracted and the V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Temperature and relative humidity data were collected on a subset of the lactation rooms. Sampled lactation rooms could be either dedicated lactation rooms or could also serve other functions (e.g., combined lactation room and restroom lounge). The majority of sequence reads were identified as belonging to family Moraxellaceae, with 73% of all reads included in analysis identified as an unknown species of Acinetobacter. Alpha diversity was analyzed using the Shannon index, while beta diversity was analyzed using unweighted and weighted UniFrac distance. The Jaccard distance was used to measure amount of change at sampling locations between time points for analysis of the impact of temperature and humidity on the microbiome. There were significant differences in the beta diversity of the microbiome of lactation rooms by room type. There were also significant differences in the beta diversity of the microbiome by sample collection location. There were no significant differences in either alpha or beta diversity associated with room temperature or humidity. Additional studies are needed to understand if the differences in lactation room type may result in differences in the breastmilk microbiome of milk collected in those rooms, and to what extent any such differences may influence the infant microbiome.

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The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota

10.21203/rs.3.rs-17416/v2 ◽

2020 ◽

Author(s):

Kristina Feye ◽

Christina L. Swaggerty ◽

Michael H. Kogut ◽

Steven C. Ricke ◽

Andrea Piva ◽

...

Keyword(s):

Organic Acids ◽

Beta Diversity ◽

Biological Effects ◽

Alpha Diversity ◽

Distance Matrix ◽

Microbial Consortia ◽

Poultry Industry ◽

Dissimilarity Index ◽

Unifrac Distance ◽

Alpha And Beta Diversity

Abstract Background: Microencapsulated organic acids and botanicals have the potential to develop into important tools for the poultry industry. A blend of organic acids and botanicals (AviPlus®P) has previously shown to reduce Salmonella and Campylobacter in chickens; however, changes to the microbiota of the jejunum and ileum have not been evaluated. Microbiota diversity is linked to, but not correlated with, the efficacy of natural products; therefore, understanding the effects on the microbiota is necessary for evaluating their potential as an antibiotic alternative. Results: Ileal and jejunal segments from control and supplement-fed chickens (300 and 500g/metric ton [MT]) were subjected to alpha diversity analysis including Shannon’s diversity and Pielou’s Evenness. In both analytics, the diversity in the ileum was significantly decreased compared to the jejunum irrespective of treatment. Similarly, beta diversity metrics including Bray-Curtis dissimilarity index and Weighted Unifrac Distance Matrix, were significant (Q<0.05) for both tissue and treatments comparisons. Alpha and beta diversity analytics indicated compartmentalization effects between the ileum and jejunum. Additionally, analysis of communities in the microbiota (ANCOM) analysis showed Lactobacilliaceae predominated the total operational taxonomic units (OTU), with a stepwise increase from 53% in the no treatment control (NTC) to 56% in the 300g/MT and 67% in the 500g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500g/MT groups. Enterobacteriaceae decreased in the 500g/MT (31%) and increased in the 300g/MT (37%) compared to the NTC (35%). Aerococcaceae was 0% for both doses and 7% in NTC. Ruminococcaceae were 0% in NTC and 2% and 1% in the 300 and 500g/MT. These changes in the microbial consortia were statistically (Q<0.05) associated with treatment groups in the jejunum that were not observed in the ileum. Least discriminant analysis effect size (LEfSE) indicated different changes directly corresponding to treatment. Enterobacteriaceae demonstrated a stepwise decrease (from NTC onward) while Clostridiaceae, were significantly increased in the 500g/MT compared to NTC and 300g/MT (P<0.05).Conclusion: The bioactive site for the microencapsulated blend of organic acids and botanicals was the jejunum, and dietary inclusion enhanced the GIT microbiota and may be a viable antibiotic alternative for the poultry industry.

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Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating the Illumina platform

10.1101/010058 ◽

2014 ◽

Cited By ~ 2

Author(s):

Lucas Sinclair ◽

Omneya Ahmed Osman ◽

Stefan Bertilsson ◽

Alexander Eiler

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Beta Diversity ◽

Sequence Data ◽

Soda Lakes ◽

Microbial Community Composition ◽

Rrna Gene ◽

Clustering Methods ◽

Illumina Platform ◽

Alpha And Beta Diversity

As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner -- with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50\% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition. (version 1.1 resubmitted to PLOS one 2014-Sept-08)

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Alpha and Beta-diversity of Microbial Communities Associated to Plant Disease Suppressive Functions of On-farm Green Composts

Agriculture ◽

10.3390/agriculture10040113 ◽

2020 ◽

Vol 10 (4) ◽

pp. 113 ◽

Cited By ~ 3

Author(s):

Catello Pane ◽

Roberto Sorrentino ◽

Riccardo Scotti ◽

Marcella Molisso ◽

Antonio Di Matteo ◽

...

Keyword(s):

Microbial Communities ◽

Beta Diversity ◽

Alpha Diversity ◽

Ecological Niches ◽

Rrna Gene ◽

Alpha And Beta Diversity ◽

Length Polymorphisms ◽

Differential Pattern ◽

Curtis Dissimilarity ◽

Underlying Mechanisms

Green waste composts are obtained from agricultural production chains; their suppressive properties are increasingly being developed as a promising biological control option in the management of soil-borne phytopathogens. The wide variety of microbes harbored in the compost ecological niches may regulate suppressive functions through not yet fully known underlying mechanisms. This study investigates alpha- and beta-diversity of the compost microbial communities, as indicators of the biological features. Our green composts displayed a differential pattern of suppressiveness over the two assayed pathosystems. Fungal and bacterial densities, as well as catabolic and enzyme functionalities did not correlate with the compost control efficacy on cress disease. Differences in the suppressive potential of composts can be better predicted by the variations in the community levels of physiological profiles indicating that functional alpha-diversity is more predictive than that which is calculated on terminal restriction fragments length polymorphisms (T-RFLPs) targeting the 16S rRNA gene. However, beta-diversity described by nMDS analysis of the Bray–Curtis dissimilarity allowed for separating compost samples into distinct functionally meaningful clusters and indicated that suppressiveness could be regulated by selected groups of microorganisms as major deterministic mechanisms. This study contributes to individuating new suitable characterization procedures applicable to the suppressive green compost chain.

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Feline conjunctival microbiota in a shelter: effects of time, upper respiratory disease and famciclovir administration

Journal of Feline Medicine and Surgery ◽

10.1177/1098612x20949038 ◽

2020 ◽

pp. 1098612X2094903

Author(s):

Danica R Lucyshyn ◽

David J Maggs ◽

Ann E Cooper ◽

Joyce D Rousseau ◽

J Scott Weese

Keyword(s):

Respiratory Disease ◽

Relative Abundance ◽

Ocular Surface ◽

Beta Diversity ◽

Alpha Diversity ◽

Taxonomic Diversity ◽

Rrna Gene ◽

Alpha And Beta Diversity ◽

Treatment Groups ◽

Upper Respiratory

Objectives The aim of this study was to evaluate changes in the conjunctival microbiota of shelter-housed cats with time, upper respiratory disease (URD) and famciclovir administration. Methods Cats were assigned to treatment groups on shelter entry. Healthy cats or cats with URD received ~30 mg/kg or ~90 mg/kg of famciclovir or placebo PO q12h for 7 days, or were untreated. Swabs were collected from ventral conjunctival fornices prior to (day 1) and immediately after (day 8) the treatment period. Microbiota analysis was conducted on 124 randomly selected swabs from healthy (56 swabs) or URD-affected (68 swabs) cats. Following DNA extraction and amplification of the V4 region of the 16S rRNA gene, sequences were assembled into operational taxonomic units (OTUs). Over-represented OTUs (as determined by linear discriminate analysis effect size), alpha and beta diversity, and median relative abundance of known feline ocular surface pathogens were assessed for the entire population and in 10 clinically relevant subpopulations of cats. Results Bacteria from 33 phyla and 70 genera were identified. Considering all cats, median relative abundance of Mycoplasma increased from day 1 to day 8, while Proteobacteria decreased. Community membership and structure (beta diversity) differed between days 1 and 8 for all famciclovir-treated cats (regardless of health status or dose) and healthy or URD-affected cats (regardless of famciclovir dose). Differences in taxonomic diversity within a sample (alpha diversity) between day 1 and day 8 were not detected in any subpopulations. Conclusions and relevance Within 1 week of shelter entry, there were significant changes in community structure and membership of the feline conjunctival microbiota, with a shift towards over-representation of feline ocular surface pathogens. Although famciclovir may impact beta diversity of the feline conjunctival microbiota, absence of change in alpha diversity suggests minimal shift in individual cats.

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Choice of 16S ribosomal RNA primers affects the microbiome analysis in chicken ceca

Scientific Reports ◽

10.1038/s41598-021-91387-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nadia Darwish ◽

Jonathan Shao ◽

Lori L. Schreier ◽

Monika Proszkowiec-Weglarz

Keyword(s):

16S Rrna ◽

Dna Sequences ◽

Bacterial Communities ◽

Beta Diversity ◽

Taxonomic Composition ◽

Rrna Gene ◽

Unifrac Distance ◽

Variable Regions ◽

Alpha And Beta Diversity ◽

Predicted Function

AbstractWe evaluated the effect of applying different sets of 16S rRNA primers on bacterial composition, diversity, and predicted function in chicken ceca. Cecal contents from Ross 708 birds at 1, 3, and 5 weeks of age were collected for DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA gene were employed. DNA sequences were analyzed using open-source platform QIIME2 and the Greengenes database. PICRUSt2 was used to determine the predicted function of bacterial communities. Changes in bacterial relative abundance due to 16S primers were determined by GLMs. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4–V6). Alpha- and beta-diversity, taxonomic composition, and predicted functions were significantly affected by the primer choice. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of microbiota with four different clusters of bacterial communities. Based on the alpha- and beta-diversity and taxonomic composition, variable regions V1–V3(1) and (2), and V3–V4 and V3–V5 were in most consensus. Our data strongly suggest that selection of particular sets of the 16S rRNA primers can impact microbiota analysis and interpretation of results in chicken as was shown previously for humans and other animal species.

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