Ribosomal Ribonucleic Acid Gene Sequencing Research Articles

Comparative evaluation of 16S rRNA primer pairs in identifying nitrifying guilds in soils under long-term organic fertilization and water management

Compared with 454 sequencing technology, short-read sequencing (e.g., Illumina) technology generates sequences of high accuracy, but limited length (<500 bp). Such a limitation can prove that studying a target gene using a large amplicon (>500 bp) is challenging. The ammonia monooxygenase subunit A (amoA) gene of ammonia-oxidizing archaea (AOA), which plays a crucial part in the nitrification process, is such a gene. By providing a full overview of the community of a functional microbial guild, 16S ribosomal ribonucleic acid (rRNA) gene sequencing could overcome this problem. However, it remains unclear how 16S rRNA primer selection influences the quantification of relative abundance and the identification of community composition of nitrifiers, especially AOA. In the present study, a comparison was made between the performance of primer pairs 338F-806R, 515F-806R, and 515F-907R to a shotgun metagenome approach. The structure of nitrifier communities subjected to different long-term organic matter amendment and water management protocols was assessed. Overall, we observed higher Chao1 richness diversity of soil total bacteria by using 515F-806R compared to 338F-806R and 515F-907R, while higher Pielou’s evenness diversity was observed by using 515F-806R and 515F-907R compared to 338F-806R. The studied primer pairs revealed different performances on the relative abundance of Thaumarchaeota, AOB, and NOB. The Thaumarchaeota 16S rRNA sequence was rarely detected using 338F-806R, while the relative abundances of Thaumarchaeota detected using 515F-806R were higher than those detected by using 515F-907R. AOB showed higher proportions in the 338F-806R and 515F-907R data, than in 515F-806R data. Different primers pairs showed significant change in relative proportion of NOB. Nonetheless, we found consistent patterns of the phylotype distribution of nitrifiers in different treatments. Nitrosopumilales (NP) and Nitrososphaerales (NS) clades were the dominant members of the AOA community in soils subject to controlled irrigation, whereas Ca. Nitrosotaleales (NT) and NS clades dominated the AOA community in soils subject to flooding irrigation. Nitrospira lineage II was the dominant NOB phylotype in all samples. Overall, ideal 16S rRNA primer pairs were identified for the analysis of nitrifier communities. Moreover, NP and NT clades of AOA might have distinct environmental adaptation strategies under different irrigation treatments.

Unveiling the gut microbiota and metabolite profiles in guinea pigs with form deprivation myopia through 16S rRNA gene sequencing and untargeted metabolomics

AimThe aim of this study was to confirm the presence of the form deprivation myopia (FDM) guinea pig eye-gut axis and investigate the relationship between serum vasoactive intestinal peptide (VIP), lipopolysaccharides (LPS), specific gut microbiota and their metabolites. Method20 specific-pathogen-free (SPF) guinea pigs were divided into the FDM and the control(Con) group. Following model induction, serum levels of VIP and LPS were quantified. A combination of 16S ribosomal ribosomal Ribonucleic Acid (rRNA) gene sequencing, non-targeted metabolomics and bioinformatics analysis were employed to identify disparities in gut microbiota and metabolites between the two groups of guinea pigs. ResultCompared to the control group, FDM guinea pigs exhibited a significant trend towards myopia, along with significantly elevated concentrations of LPS and VIP (p < 0.0001). Furthermore, Ruminococcus_albus emerged as the predominant bacterial community enriched in FDM (p < 0.05), and demonstrated positive correlations with 10 metabolites, including l-Glutamic acid, Additionally, Ruminococcus_albus exhibited positive correlations with VIP and LPS levels (p < 0.05). ConclusionThe findings suggest that the Ruminococcus_Albus and glutamate metabolic pathways play a significant role in myopia development, leading to concurrent alterations in serum VIP and LPS levels in FDM guinea pigs. This underscores the potential of specific gut microbiota and their metabolites as pivotal biomarkers involved in the pathogenesis of myopia.

Temporal gut microbiota variability and association with dietary patterns: From the one-year observational Diet, Cancer, and Health - Next Generations MAX study

BackgroundKnowledge about the variability of gut microbiota within an individual over time is important to allow meaningful investigations of the gut microbiota in relation to diet and health outcomes in observational studies. Plant-based dietary patterns have been associated with a lower risk of morbidity and mortality and may alter gut microbiota in a favorable direction. ObjectivesTo assess the gut microbiota variability during one year and investigate the association between adherence to diet indexes and the gut microbiota in a Danish population. MethodsFour hundred forty-four participants were included in the Diet, Cancer, and Health - Next Generations MAX study (DCH-NG MAX). Stool samples collected up to three times during a year were analyzed by 16S ribosomal ribonucleic acid gene sequencing. Diet was obtained by 24-hour dietary recalls. Intraclass correlation coefficient (ICC) was calculated to assess temporal microbial variability based on 214 individuals. Diet indexes (Nordic, Mediterranean, and plant-based diets) and food groups thereof were associated with gut microbiota using linear regression analyses. ResultsWe found that 91 out of 234 genera had an ICC >0.5. We identified three subgroups dominated by Bacteroides, Prevotella 9, and Ruminococcaceae and adherence to diet indexes differed between subgroups. Higher adherence to diet indexes was associated with the relative abundance of 22 genera. Across diet indexes, higher intakes of fruit, vegetables, whole grains/cereals, and nuts were most frequently associated with these genera. ConclusionsIn the DCH-NG MAX study, 39% of the genera had an ICC >0.5 over one year, suggesting that these genera could be studied with health outcomes in prospective analyses with acceptable precision. Adherence to the Nordic, Mediterranean, and plant-based diets differed between bacterial subgroups and was associated with a higher abundance of genera with fiber-degrading properties. Fruits, vegetables, whole grains/cereals, and nuts were frequently associated with these genera.

Short term antibiotic effects on gut microbiome in Indian preschoolers: A 16S rRNA analysis

Objectives: Antibiotic use is associated with dysbiosis of the gut microbiome. The objective of this study is to investigate the effect of antibiotics on gut composition in children aged 3–5 years receiving antibiotics compared to children who did not receive antibiotics. Materials and Methods: A total of 54 participants aged 3–5 years were included in this multi-centric cohort study. Participants were divided into two equal groups, that is the treatment-experienced group (Group 1, n = 27, antibiotic) and the treatment-not-experienced group (Group 2, n = 27, non-antibiotic). Stool samples of study participants were collected on days 0 and 5 (± 1 day) and analyzed using 16Svedberg ribosomal ribonucleic acid (16S rRNA) gene sequencing. Statistical Analysis: The Kruskal-Wallis H-test and Benjamini-Hochberg FDR correction were applied to determine the differentially abundant pathways across the zones using Statistical Analysis of Metagenomic Profiles (STAMP) (v2.1.3). Results: A non-significant increase in the mean abundance of the Phyla Bacteroidota, Proteobacteria, Actinobacteria, and Verrucomicrobiota was observed in both groups from day 0 to day 5. An alteration in the Firmicutes/Bacteroidetes ratio was observed. A significant (P &lt; 0.05) abundance of genus Enterobacteriaceae, Enterococcaceae, and Lactobacillaceae was observed in the participants with antibiotic treatment. The relative abundance of families Enterococcus, Lactobacillus, Sellimonas, Ruminococcus, Torques, and Eggerthella groups was observed to be significantly higher (P &lt; 0.05) in participants with antibiotic treatment. Beta-diversity indices revealed significant differences at group and subgroup levels regarding the bacterial counts. Conclusions: It was observed that a short-term course of 5 days of antibiotic usage is associated with altered microbial abundance and diversity.

Ocular surface microbiota in patients with varying degrees of dry eye severity.

To elucidate the profiles of commensal bacteria on the ocular surfaces of patients with varying severity of dry eye (DE). The single-center, prospective, case-control, observational study categorized all participants into three distinct groups: 1) control group (n=61), 2) mild DE group (n=56), and 3) moderate-to-severe DE group (n=82). Schirmer's tear secretion strips were used, and the bacterial microbiota was analyzed using 16S ribosomal ribonucleic acid gene sequencing. The three groups had significant differences in alpha diversity: the control group had the highest richness (Chao1, Faith's phylogenetic diversity), the mild DE group showed the highest diversity (Shannon, Simpson), and the moderate-to-severe DE group had the lowest of the above-mentioned indices. DE severity was positively correlated with a reduction in beta diversity of the microbial community, with the moderate-to-severe DE group exhibiting the lowest beta diversity. Linear discriminant analysis effect size presented distinct dominant taxa that significantly differed between each. Furthermore, the exacerbation of DE corresponded with the enrichment of certain pathogenic bacteria, as determined by random forest analysis. As DE severity worsens, microbial community diversity tends to decrease. DE development corresponds with changes in microbial constituents, primarily characterized by reduced microbial diversity and a more homogenous species composition.

Optimizing waste molasses utilization to enhance electron transfer via micromagnetic carriers: Mechanisms and high-nitrate wastewater denitrification performance

The biological denitrification of high-nitrate wastewater (HNW) is primarily hindered by insufficient carbon sources and excessive nitrite accumulation. In this study, micromagnetic carriers with varying micromagnetic field (MMF) strengths (0.0, 0.3, 0.6, 0.9 mT) were employed to enhance the denitrification of HNW using waste molasses (WMs) as a carbon source. The results revealed that 0.6 mT MMF significantly improved the total nitrogen removal (TN) efficiency at 96.3%. A high nitrate (NO3−-N) removal efficiency at 99.3% with a low nitrite (NO2−-N) accumulation at 25.5 mg/L was achieved at 0.6 mT MMF. The application of MMF facilitated the synthesis of adenosine triphosphate (ATP) and stimulated denitrifying enzymes (e.g., nitrate reductase (NAR), nitrite reductase (NIR), and nitric oxide reductase (NOR)), which thereby promoting denitrification. Moreover, the effluent chemical oxygen demand (COD), tryptophan and fulvic-like substances exhibited their lowest levels at 0.6 mT MMF. Analysis through 16S ribosomal ribonucleic acid gene sequencing indicated a significant enrichment of denitrifying bacteria including Castellaniella Klebsiella under the influence of MMF. Besides, the proliferation of Acholeplasma, Klebsiella and Proteiniphilum at 0.6 mT MMF promoted the hydrolysis and acidification of WMs. This study offers new insights into the enhanced utilization of WMs and the denitrification of HNW through the application of MMF.

Antimicrobial mechanism and biocontrol effect of Bacillus cereus XZ30-2 on Aspergillus niger

Aspergillus niger is a major mold-causing spoilage in cereals, fruits and vegetables. Controlling of mold in stored grains is essential for safety of food. Currently, application of microorganisms to control A. niger is a safer and more effective method. In this study, strain XZ30-2 against A. niger was isolated and identified as Bacillus cereus according to morphological and biochemical characteristics as well as 16 Svedberg ribosomal ribonucleic acid (16S rRNA) gene sequence analysis. The investigation of action mechanism showed XZ30-2 culture filtrate caused the mycelia inflated or contract, increasing the membrane permeability, leading to the intracellular leakage and nucleic acids release, disrupting the proton pump, decreasing the ergosterol content, inducing the membrane lipid peroxidation and reactive oxygen species (ROS) accumulation in A. niger. Moreover, B. cereus XZ30-2 culture filtrate could produce hydrolases and lipopeptides, including iturin, surfactin and fengycin. This work also evaluated the control effect of XZ30-2 on A. niger in wheat grains, and indicated that 40 μL/g of culture filtrate significantly controlled the infection of A. niger. Therefore, B. cereus XZ30-2 can be developed as a biological agent for controlling A. niger in stored grains.

Critical review of 16S rRNA gene sequencing workflow in microbiome studies: From primer selection to advanced data analysis.

The multi-batch reanalysis approach of jointly reevaluating gene/genome sequences from different works has gained particular relevance in the literature in recent years. The large amount of 16S ribosomal ribonucleic acid (rRNA) gene sequence data stored in public repositories and information in taxonomic databases of the same gene far exceeds that related to complete genomes. This review is intended to guide researchers new to studying microbiota, particularly the oral microbiota, using 16S rRNA gene sequencing and those who want to expand and update their knowledge to optimise their decision-making and improve their research results. First, we describe the advantages and disadvantages of using the 16S rRNA gene as a phylogenetic marker and the latest findings on the impact of primer pair selection on diversity and taxonomic assignment outcomes in oral microbiome studies. Strategies for primer selection based on these results are introduced. Second, we identified the key factors to consider in selecting the sequencing technology and platform. The process and particularities of the main steps for processing 16S rRNA gene-derived data are described in detail to enable researchers to choose the most appropriate bioinformatics pipeline and analysis methods based on the available evidence. We then produce an overview of the different types of advanced analyses, both the most widely used in the literature and the most recent approaches. Several indices, metrics and software for studying microbial communities are included, highlighting their advantages and disadvantages. Considering the principles of clinical metagenomics, we conclude that future research should focus on rigorous analytical approaches, such as developing predictive models to identify microbiome-based biomarkers to classify health and disease states. Finally, we address the batch effect concept and the microbiome-specific methods for accounting for or correcting them.

Re-evaluation of the systematic position of the order Prostomatida (Protista: Ciliophora), with the establishment of two new genera and two new species

Abstract Prostomatean ciliates inhabit a wide range of aquatic environments and serve as a trophic link in food webs. However, the biodiversity and molecular phylogeny of the Prostomatea, especially the order Prostomatida, is poorly known owing to undersampling and the paucity of molecular data. Here, three prostomatid ciliates, Platina marina gen. nov., sp. nov., Parametacystis pulchra gen. nov., sp. nov. and Apsiktrata gracilis, were studied based on morphological data and small subunit ribosomal ribonucleic acid (SSU rRNA) gene sequences. The two new genera show a close relationship with the class Plagiopylea but a distant relationship with Metacystis, which bears a strong morphological similarity to the two new genera. The simplified oral ciliature can be regarded as a synapomorphy of the two new genera. The SSU rRNA gene of a representative of the family Apsiktratidae was sequenced for the first time. Morphological and phylogenetic analyses suggest that Apsiktratidae might be more closely related to Prorodontida than to Prostomatida, and the oral basket might be more phylogenetically informative than the brosse in distinguishing Prorodontida and Prostomatida. Supplementation with rare taxa reveals a close association between the Prostomatida and Trimyemidae. The putative secondary structure of the SSU rRNA V9 region suggests a sister relationship between the order Prostomatida and the class Plagiopylea.

Bioprospecting the potential of metabolites from a Saharan saline soil strain Nocardiopsis dassonvillei GSBS4.

Saharan soil samples collected in El-Oued province have been investigated for actinobacteria as a valuable source for the production of bioactive metabolites. A total of 273 isolates were obtained and subjected to antagonistic activity tests against human pathogenic germs. A strain with a broad-spectrum antimicrobial activity was selected and identified as Nocardiopsis dassonvillei GSBS4, with high sequence similarities to N. dassonvillei subsp. dassonvilleiT X97886.1 (99%) based on polyphasic taxonomy approach and 16S ribosomal ribonucleic acid gene sequence analysis. The GSBS4 ethyl acetate crude extract showed strong antibacterial activity towards pathogenic bacteria and Candida albicans. It inhibited biofilm formation by Staphylococcus aureus and methicillin-resistant S. aureus with minimum inhibitory concentrations estimated at 0.144 and 1.15 mg·mL-1 , respectively. A 44% biofilm reduction was obtained for S. aureus and 61% for Pseudomonas aeruginosa. Furthermore, phenols composition of the crude extract showed a significant dose-dependent antioxidant activity by α-diphenyl-β-picrylhydrazyl (57.21%) and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (64.29%) radicals scavenging assays. Although no inhibition was obtained on human coronavirus human coronavirus (HCoV) 229E and on model enterovirus (poliovirus 1) infection, a dose-dependent increase in cell viability of HCoV 229E-infected cells was noticed as the viability increased from 21% to 37%. Bioassay-guided fractionation of the crude extract gave a fraction showing antibacterial activity, which was analyzed by liquid chromatography-electrospray mass spectrometric technique, providing structural features on a major purple metabolite.

Heavy-metal tolerant bacterial strains isolated from industrial sites and scrap yards in Kashmir, India.

Heavy metal pollution has posed a severe danger to environmental stability due to its high toxicity and lack of biodegradability. The present study deals with the appraisement of tolerance shown by various bacteria in varied copper and iron concentrations. Among the 20 isolates, four isolates, GN2, SC5, SC8, and SC10, exhibiting more significant iron and copper tolerance, were selected and identified by 16 S ribosomal ribonucleic acid (rRNA) gene sequence analysis as Pantoea agglomerans strain GN2, Pantoea sp. strain SC5, Bacillus sp. strain SC8 and Priestia aryabhattaistrain SC10. The minimum inhibitory concentration of molecularly identified strains revealed that P. agglomerans strain GN2 showed tolerance to iron sulfate and copper sulfate upto 600 and 400 µg/mL, whereas Bacillus sp. SC8 (OQ202165) showed tolerance of 700 and 250 µg/mL were tolerant to iron sulfate and copper sulfate up to 700 and 150 µg/mL, respectively. Pantoea sp. strain SC5 showed significant tolerance to both heavy metals. The isolates were further studied for their ability to grow at varying temperatures and pH ranges. Most of the isolates showed optimal growth at 37°C and pH 7. However, Pantoea sp. SC5 was competent to have prominent growth at 45°C and pH 8.0. Microbial remediation, which is eco-friendly, has proven the most effective method for bioremediation of heavy metal-contaminated environments. Using heavy metal-resistant bacteria for microbial remediation of iron and copper-contaminated environments could be a viable and valuable strategy. These isolates could also be used to decontaminate heavy metal-polluted agricultural soils.

Insect-based diets (house crickets and mulberry silkworm pupae): A comparison of their effects on canine gut microbiota.

The gut microbiome plays an important role in the overall health and well-being of dogs, influencing various physiological processes such as metabolism, nutrient absorption, and immune function. Edible insects are a sustainable and nutritious alternative protein source attracting increasing attention as a potential component of animal feeds, including pet food. However, little is known about the effects of insect-based diets on the gut microbiota of dogs. This study aimed to examine the fecal microbiota of dogs fed a diet that substituted common protein sources (poultry meal) with the house cricket (Acheta domesticus [AD]) or mulberry silkworm pupae (Bombyx mori pupae [BMp]) at different levels. Fifteen healthy adult mixed-breed dogs were systemically randomized and assigned into each block under a completed randomized block design into the following five experimental dietary groups: control diet, 10% AD, 20% AD, 7% BMp, or 14% BMp for 29 days. The amounts fed to the dogs were based on the daily energy requirement. Fecal samples were collected on days 14 and 29 and analyzed for bacterial community structure using 16S ribosomal ribonucleic acid gene sequencing. At the phylum and genus levels, microbiota and their diversity were generally relatively similar among all treatments. The diets containing insects did not significantly alter the major phyla in the gut microbiome of dogs (p > 0.05). A few significant changes were found in the relative abundance of bacterial genera, with the levels of Allobaculum and Turicibacter being reduced in dogs fed a higher level of BMp. In contrast, only a decrease in Turicibacter was found in dogs fed the lower level of AD than the control diet (p < 0.05). Corynebacterium and Lactobacillus levels in the dogs fed 14% BMp were significantly increased compared with those in the control group (p < 0.05). These findings suggest that insect-based diets may slightly alter the gut microbiota of dogs. Further research is needed to fully understand the mechanisms by which insect-based diets influence the gut microbiota of dogs and the long-term potential health implications.

Relative and Quantitative Characterization of the Bovine Bacterial Ocular Surface Microbiome in the Context of Suspected Ocular Squamous Cell Carcinoma.

The ocular surface microbiome is altered in certain disease states. The aim of this study was to characterize the bovine bacterial ocular surface microbiome (BBOSM) in the context of ocular squamous cell carcinoma (OSCC). The conjunctiva of normal (n = 28) and OSCC (n = 10) eyes of cows aged 2 to 13 years from two farms in Louisiana and Wyoming were sampled using individual sterile swabs. DNA extraction followed by 16S ribosomal ribonucleic acid (rRNA) gene sequencing and real-time polymerase chain reaction (RT-PCR) were performed to, respectively, assess the relative and absolute BBOSM. Discriminant analysis (DA) was performed using RT-PCR data, and relative abundance analysis was performed using 16S rRNA gene sequencing data. The 11 most abundant phyla in both normal and OSCC-affected cows were identified using 16S rRNA gene sequencing analysis. The relative abundance of Euryarchaeota was found to be significantly lower (p = 0.0372) in OSCC eyes compared to normal eyes. Relative abundance differences within and between geographic locations were also identified. Quadratic DA categorized samples as OSCC or normal with 100% sensitivity and 83.3-100% specificity. Relative abundance analysis identified relative BBOSM phylum alterations in OSCC. Quadratic DA can be used to accurately categorize BBOSM from normal and OSCC ocular surface samples.

ABC-12: Exploring the microbiome in patients (pts) with advanced biliary tract cancer (BTC) in a first-line study of durvalumab in combination with cisplatin/gemcitabine (cis/gem).

TPS4183 Background: Durvalumab/cis/gem improved overall survival (OS) in pts with advanced BTC versus placebo/cis/gem (Oh et al. NEJM Evid 2022). Disruption of the microbiota may impair tumour response to immunotherapy and chemotherapy and a better understanding of its role in the efficacy of these therapeutics in advanced BTC is required. Methods: This is a multi-centre, single arm trial exploring the microbiome in pts receiving durvalumab 1500 mg intravenously (IV) Q3w, in combination with cis 25 mg/m2, gem 1000 mg/m2 (Days 1 and 8, Q3w) up to 8 cycles, followed by durvalumab 1500 mg as monotherapy Q4w, until progression or intolerable toxicity. Pts with an ECOG performance status of ≤1 and histologically-proven BTC, including cholangiocarcinoma and gallbladder carcinoma, who have had no prior systemic chemotherapy for locally advanced or metastatic disease are eligible. Pts must provide a saliva and stool sample prior to commencement of durvalumab/cis/gem and at 18 weeks, or at progression (if earlier than 18 weeks). Taxonomic profiling via 16S Ribosomal ribonucleic acid gene sequencing will examine the differences in the diversity and composition of the pt gut microbiome. Pts must also have availability of a tumour biopsy. This study plans to recruit 70 pts from 10 UK centres (over 12 months). The primary objective is to determine the difference in baseline alpha diversity between “responders” (partial or complete response) and “non-responders” at 18 weeks (RECIST 1.1) in patients treated with durvalumab/cis/gem. Secondary objectives include investigation of the association between microbiome parameters and objective response rate, tumour control (partial + complete response + stable disease), progression-free and OS, and to investigate the interaction between treatment effect and microbiome parameters on clinical outcomes. The tumour biopsy will be used for research into the tumour microbiome and/or factors that may influence response to chemotherapy/immunotherapy, including, but not limited to tumour mutation burden, programmed cell death 1/programmed death-ligand 1 status, and microsatellite instability status. Clinical trial information: ISRCTN11210442 .

Gut microbiota indole-3-propionic acid mediates neuroprotective effect of probiotic consumption in healthy elderly: A randomized, double-blind, placebo-controlled, multicenter trial and in vitro study

The beneficial effects of probiotic consumption on age-related decline in cerebral function have been previously reported in the literature; however, the mechanistic link between gut and brain interactions has not yet been fully elucidated. Therefore, this study aimed to identify the role of gut microbiota-derived metabolites in gut-brain interactions via blood metabolomic profiling analysis in clinical trials and invitro mechanistic studies. A randomized, double-blind, placebo-controlled, multicenter clinical trial was conducted in 63 healthy elderly individuals (≥65 years of age). Participants were administered either placebo (placebo group, N=31) or probiotic capsules (Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI; probiotics group, N=32) for 12 weeks. Global and targeted metabolomic profiling analyses of their blood samples were then performed using 1H nuclear magnetic resonance and liquid chromatography-mass spectrometry methods, both at baseline and at the end of the trial. Gut microbial analysis was conducted using the 16S ribosomal ribonucleic acid gene sequencing method. Subsequently, microglial BV2 cells were treated invitro with indole-3-propionic acid (IPA) following lipopolysaccharide stimulation, and neuronal SH-SY5Y cells were treated with conditioned media from the BV2 cells. Finally, the levels of pro-inflammatory cytokines in BV2 cells and neurotrophins in SH-SY5Y cells were quantified using a real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The metabolomic profiling analyses showed that probiotic consumption significantly altered the levels of metabolites involved in tryptophan metabolism (P<0.01). Among these metabolites, gut microbiota-produced IPA had a 1.91-fold increase in the probiotics group (P<0.05) and showed a significant relation to gut bacterial profiles (P<0.01). Elevated IPA levels were also positively associated with the level of serum brain-derived neurotropic factor (BDNF) in the probiotics group (r=0.28, P<0.05), showing an inverse trend compared to the placebo group. In addition, invitro treatment with IPA (5μM) significantly reduced the concentration of proinflammatory TNF-α in activated microglia (P<0.05), and neuronal cells cultured with conditioned media from IPA-treated microglia showed a significant increase in BDNF and nerve growth factor production (P<0.05). These results show that gut microbiota-produced IPA plays a role in protecting the microglia from inflammation, thus promoting neuronal function. Therefore, this suggests that IPA is a significant mediator linking the interaction between the gut and the brain in the elderly with probiotic supplementation.

Galacto-Oligosaccharides Increase the Abundance of Beneficial Probiotic Bacteria and Improve Gut Architecture and Goblet Cell Expression in Poorly Performing Piglets, but Not Performance.

Poorly performing piglets receiving commercial milk replacers do not benefit from the naturally occurring probiotic galacto-oligosaccharides otherwise found in sow milk. Study objectives were to investigate the effects of complete milk replacer supplemented with galacto-oligosaccharides on the microbiome, gut architecture and immunomodulatory goblet cell expression of poorly performing piglets that could benefit from milk replacement feeding when separated from sows and housed with fit siblings in environmentally controlled pens. The study is novel in that it is one of the first to investigate the effects of supplementing complete milk replacer with galacto-oligosaccharides in poorly performing piglets. Gastrointestinal tract samples were collected from piglets, and the microbiome composition was assessed by 16s ribosomal ribonucleic acid gene sequencing. Gut architectural features, villus/crypt ratio and enumeration of goblet cells in tissues were assessed by histopathological techniques. The most abundant taxa identified at the genus level were Lactobacillus, Streptococcus, Prevotella, Lactococcus and Leuconostoc. Milk replacer plus galacto-oligosaccharides significantly improved gut architectural features and villus/crypt ratio throughout the gastrointestinal tract, increased the number of goblet cells and revealed a differential abundance of beneficial probiotic bacteria, particularly Lactobacillus and Bifidobacterium. In these respects, galacto-oligosaccharide-supplemented milk replacer may be a useful addition to animal husbandry in poorly performing, non-thriving animals when moved to environmentally controlled pens away from sows and fit siblings, thereby modulating the microbiome and gastrointestinal tract performance.

Abstract 13266: Sex Specific Differences in the Gut Microbiome in Patients With Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) patients exhibit sexual dimorphism in pulmonary vascular remodeling and right ventricular (RV) dysfunction. While female PAH patients have severe pulmonary vascular remodeling, male PAH patients have worse RV function. Inflammation, a key component of PAH pathobiology, has been implicated as a potential mechanism for sexual dimorphism. Gut dysbiosis causes systemic inflammation. However, it is unknown whether gut dysbiosis contributes to sexual dimorphism in PAH. Hypothesis: There are sex-specific differences in the gut microbiome and microbial metabolites of PAH patients. Methods: 16S ribosomal ribonucleic acid gene sequencing was performed on stool samples from PAH patients (n=73). Markers of inflammation and circulating microbial metabolites were measured using plasma samples. Results: There was no significant difference in the Shannon diversity indices or species richness (Chao1 indices) between the gut microbiomes of male and female PAH patients. Moreover, principal component analysis of pairwise Bray-Curtis dissimilarity indices revealed no distinct microbiome compositions dependent on sex. There was no difference in serum claudin-3, a marker of gut permeability, and serum interleukin 6 levels between male and female PAH patients. When examining microbial metabolites, including trimethylamine N-oxide, short chain fatty acids, and secondary bile acids, no sex-specific differences were observed. Linear discriminant analysis of effect size revealed that there were no taxonomic differences based on sex. Conclusions: Male and female PAH patients do not possess distinct differences in their gut microbiomes or metabolites. There are also no sex-specific taxonomic differences. These findings do not support our hypothesis that gut dysbiosis may serve as a possible explanation for the sexual dimorphism observed in PAH.

The Lung Allograft Microbiome Associates with Pepsin, Inflammation, and Primary Graft Dysfunction.

Rationale: Primary graft dysfunction (PGD) is the principal cause of early morbidity and mortality after lung transplantation. The lung microbiome has been implicated in later transplantation outcomes but has not been investigated in PGD. Objectives: To define the peritransplant bacterial lung microbiome and relationship to host response and PGD. Methods: This was a single-center prospective cohort study. Airway lavage samples from donor lungs before organ procurement and recipient allografts immediately after implantation underwent bacterial 16S ribosomal ribonucleic acid gene sequencing. Recipient allograft samples were analyzed for cytokines by multiplex array and pepsin by ELISA. Measurements and Main Results: We enrolled 139 transplant subjects and obtained donor lung (n = 109) and recipient allograft (n = 136) samples. Severe PGD (persistent grade 3) developed in 15 subjects over the first 72 hours, and 40 remained without PGD (persistent grade 0). The microbiome of donor lungs differed from healthy lungs, and recipient allograft microbiomes differed from donor lungs. Development of severe PGD was associated with enrichment in the immediate postimplantation lung of oropharyngeal anaerobic taxa, particularly Prevotella. Elevated pepsin, a gastric biomarker, and a hyperinflammatory cytokine profile were present in recipient allografts in severe PGD and strongly correlated with microbiome composition. Together, immediate postimplantation allograft Prevotella/Streptococcus ratio, pepsin, and indicator cytokines were associated with development of severe PGD during the 72-hour post-transplantation period (area under the curve = 0.81). Conclusions: Lung allografts that develop PGD have a microbiome enriched in anaerobic oropharyngeal taxa, elevated gastric pepsin, and hyperinflammatory phenotype. These findings suggest a possible role for peritransplant aspiration in PGD, a potentially actionable mechanism that warrants further investigation.

Soybean isoflavones modulate gut microbiota to benefit the health weight and metabolism.

Soybean isoflavones (SIs) are widely found in food and herbal medicines. Although the pharmacological activities of SIs have been widely reported, their effects on the intestinal microecology of normal hosts have received little attention. Five-week-old Kunming (KM) mice were administered SIs (10 mg/kg/day) for 15 days. Food intake, body weight, and digestive enzyme activity were measured. Small intestine microbiota, including lumen-associated bacteria (LAB) and mucosa-associated bacteria (MAB), were analyzed using 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing. Short-chain fatty acids (SCFAs) were analyzed using gas chromatography-mass spectrometry (GC-MS). The results showed that the mice that consuming SIs showed a higher food intake but a lower body weight gain rate than that of normal mice. Sucrase, cellulase, and amylase activities reduced, while protease activity increased after SIs intervention. Moreover, SIs increased the intestinal bacterial diversity in both LAB and MAB of normal mice. The composition of LAB was more sensitive to SIs than those of MAB. Lactobacillus, Adlercreutzia, Coprococcus, Ruminococcus, Butyricicoccus, and Desulfovibrio were the differential bacteria among the LAB of mice treated with SIs. In addition, acetic acid, valeric acid, isobutyric acid, isovaleric acid, and caproic acid decreased, while butyric acid and propionic acid increased in the mice treated with SIs. Taken together, SIs are beneficial for weight control, even in short-term interventions. The specific mechanism is related to regulating the gut microbiota, changing digestive enzyme activities, and further affecting carbohydrate absorption and metabolism.

The Impact of Synbiotic Treatment on the Levels of Gut-Derived Uremic Toxins, Inflammation, and Gut Microbiome of Chronic Kidney Disease Patients—A Randomized Trial

Altering dysbiotic gut flora through synbiotic supplementation has recently been recognized as a potential treatment strategy to reduce the levels of gut-derived uremic toxins and decrease inflammation. Assessing its efficacy and safety has been the main goal of our randomized, double-blind, placebo-controlled study. A total of 34 nondialyzed chronic kidney disease patients, aged ≥18years, with an estimated glomerular filtration rate between 15 and 45mL/minute, were randomized either to an intervention group (n=17), receiving synbiotic (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis, 32 billion colony forming units per day plus 3.2g of inulin), or control group (n=17), receiving placebo during 12weeks. The impact of treatment on the dynamic of serum levels of gut-derived uremic toxins, total serum indoxyl sulfate, p-cresyl sulfate, and trimethylamine N-oxide, was defined as the primary outcome of the study. Secondary outcomes included changes in the stool microbiome, serum interleukin-6 levels, high-sensitivity C-reactive protein, estimated glomerular filtration rate, albuminuria, diet, gastrointestinal symptom dynamics, and safety. Serum levels of uremic toxins were determined using ultraperformance liquid chromatography. The stool microbiome analysis was performed using the 16S ribosomal ribonucleic acid gene sequencing approach. Synbiotic treatment significantly modified gut microbiome with Bifidobacteria, Lactobacillus, and Subdoligranulum genera enrichment and consequently reduced serum level of indoxyl sulfate (ΔIS -21.5% vs. 5.3%, P<.001), improved estimated glomerular filtration rate (ΔeGFR 12% vs. 8%, P=.029), and decreased level of high-sensitivity C-reactive protein (-39.5 vs. -8.5%, P<.001) in treated patients. Two patients of the intervention arm complained of increased flatulence. No other safety issues were noted. Synbiotics could be available, safe, and an effective therapeutic strategy we could use in daily practice in order to decrease levels of uremic toxins and microinflammation in chronic kidney disease patients.