V4 Variable Region Research Articles

Diversity and functional roles of the symbiotic microbiome associated to marine sponges off Karah Island, Terengganu, Malaysia

Marine sponges harbor a complex microbial consortium that plays important roles in host metabolisms and the production of chemical compounds. Symbiotic microbial community structures of marine sponges have been studied in recent years especially in the Caribbean and the Mediterranean Sea, whereas there is still a paucity of research in Malaysia. This study investigated the microbial community structures associated with four different marine sponge species (Aaptos suberitoides (Brøndsted, 1934), Neopetrosia exigua (Kirkpatrick, 1900), Theonella swinhoei (Gray, 1868), and Xestospongia testudinaria (Lamarck, 1815)) collected from Karah Island, Peninsular Malaysia. Microbial genomic DNA of four sponges and surrounding seawater were extracted and 16S rRNA gene amplicon sequencing was conducted targeting V3–V4 variable region using Illumina MiSeq platform. Paired-end sequences were analyzed using QIIME2 and the potential microbial functions related to the microbial communities of marine sponges and seawater were annotated using functional annotation of prokaryotic taxa (FAPROTAX). Microbial community structures and compositions were clearly different between sponges and seawater, as well as within different sponge species. Taxonomic analysis showed that Chloroflexi was the predominant phylum in all sponge species. Proteobacteria and Actinobacteriota were composing the core taxa of the microbial communities in N. exigua, T. swinhoei, and X. testudinaria, whereas Cyanobacteria and Proteobacteria were dominant in A. suberitoides. The functional analysis revealed that the abundance of the “predatory or exoparasitic” function was high in all sponges. The functions of aerobic ammonia oxidation and nitrification were dominant in marine sponges, rather than seawater. This study has important implications in understanding the microbial community structures associated with marine sponges in Malaysia, and gives fundamental information of sponge–microbe interactions and functional roles for future investigations.

Can the Gut Microbiota Serve as a Guide to the Diagnosis and Treatment of Childhood Epilepsy?

BackgroundTo investigate the activity of the gut-brain axis in the pathogenesis of childhood epilepsy and to define biomarkers capable of assisting with determining new strategies in that context. MethodsTwenty children with epilepsy of “unknown etiology” and seven healthy controls in the same age group were included in the study. The groups were compared using a questionnaire. Stool samples were stored in tubes containing DNA/RNA Shield (Zymo Research) with a sterile swab. Sequencing was carried out using the MiSeq System (Illumina). The 16S rRNA sequencing of samples using next-generation sequencing involved V4 variable region polymerase chain reaction amplification concluded by 2 × 250-bp paired-end sequencing of amplicons and at least 50,000 reads (>Q30) per sample. DNA sequences were classified at the genus level using the Kraken program. Bioinformatics and statistical analysis were then performed. ResultsIndividuals’ gut microbiota relative abundance values differed between the groups at the genus, order, class, family, and phylum levels. Flavihumibacter, Niabella, Anoxybacillus, Brevundimonas, Devosia, and Delftia were seen only in the control group, whereas Megamonas and Coriobacterium were observed only in the epilepsy group. The linear discriminant analysis effect size method identified 33 taxa as important in differentiating the groups. ConclusionsWe think that bacterial varieties (such as Megamonas and Coriobacterium) that differ between the two groups can be employed as useful biomarkers in the diagnosis and follow-up of epileptic patients. We also predict that, in addition to epilepsy treatment protocols, the restoration of eubiotic microbiota may increase the success of treatment.

Analysis of the Bacterial Community of Metal Scrap Using an Enrichment Culture Approach

Microbiologically influenced corrosion (MIC) of metal alloys is promoted by biofilms formed on metal surfaces. In the marine environment, MIC causes serious metal infrastructure problems, which lead to significant economic losses. In this study, we used an enrichment culture approach to examine the bacterial community that grows on metal surface at levels below the detection limit as a preliminary study for developing guidelines to prevent biofilm formation. An enrichment culture approach was employed to analyze the bacterial community on metal surface without biofilms and corrosion. Genomic DNA was extracted from culture sample after incubation in the enrichment culture with a metal piece, and then the V3–V4 variable regions of the bacterial 16S rRNA gene were amplified using the extracted genomic DNA as the template. Subsequently, using a next-generation sequencing approach, the amplified V3–V4 regions were sequenced, and the bacterial community was analyzed using the QIIME 2 microbiome bioinformatics platform. Using this enrichment culture approach, more than 80 bacterial genera were detected with Sphingomonas bacteria exhibiting the highest relative abundance (44%). These results demonstrated that this method could be useful for bacterial community analysis for bacteria below detection limits, and will serve as a basis for the development of the guidelines.

Bacterial Communities of Lamiacea L. Medicinal Plants: Structural Features and Rhizosphere Effect.

Bacterial communities associated with medicinal plants are an essential part of ecosystems. The rhizosphere effect is rather important in the cultivation process. The purpose of the study was to analyze the rhizosphere effect of oregano (Origanum vulgare L.), peppermint (Mentha piperita L.), thyme (Thymus vulgaris L.), creeping thyme (Thymus serpillum L.) and sage (Salvia officinalis L.). To estimate the quantity of 16S bacteria ribosomal genes, qPCR assays were used. To compare bacterial communities' structure of medicinal plants rhizosphere with bulk soil high-throughput sequencing of the 16S rRNA targeting variable regions V3-V4 of bacteria was carried out. The highest bacterial abundance was associated with T. vulgaris L., M. piperita L. and S. officinalis L., and the lowest was associated with the O. vulgare L. rhizosphere. Phylum Actinobacteriota was predominant in all rhizosphere samples. The maximum bacterial α-diversity was found in S. officinalis L. rhizosphere. According to bacterial β-diversity calculated by the Bray-Curtis metric, T. vulgaris L. root zone significantly differed from bulk soil. The rhizosphere effect was positive to the Myxococcota, Bacteroidota, Verrucomicrobiota, Proteobacteria and Gemmatimonadota.

Microbiota attached to and encapsulated by granulomas dissected from tilapia spleen: A case report

AbstractGranulomatous diseases are caused mainly by bacterial infections that the fish immune system cannot eliminate; consequently, pathogens are encapsulated, forming a complex structure where immune cells participate in containing the damage. The granuloma is covered by a collagenous layer that may be an attractive surface for external bacteria, leading to the potential development of a microbiota associated with the outer side of granulomas. Therefore, this study aimed to describe the microbiota attached to and encapsulated by granulomas dissected from tilapia spleen obtained from a commercial farm. Tissue samples with an advanced granulomatosis stage were analyzed by amplifying and sequencing the V4 variable region of the 16S rRNA gene. More than 20 phyla were detected attached to granulomas; however, only five represented 94% of the relative abundance, and the genus Gordonia was the most abundant with 21.9%. This bacterium has been previously identified in the tilapia gut and has also been reported as the etiologic agent of granulomatous disease in humans and terrestrial animals. This is the first time it has been detected in fish granulomas. Finally, these results provide the first evidence of microbiota attached to granulomatous lesions while demonstrating that these can provide asurface that can harbour highly diverse microbial communities.

Microbial Community Structure of Colostrum in Women with Antibiotic Exposure Immediately After Delivery.

<b><i>Background:</i></b> The microbial community in human milk is associated with many maternal and neonatal factors. This study aimed to investigate the effect of antibiotic exposure on the microbial community structure of colostrum. <b><i>Methods:</i></b> Twenty women with antibiotic treatment immediately after delivery and 10 age-matched control women were enrolled at the Guangdong Women and Children Hospital. Colostrum samples were collected within postpartum 30 hours. The V4 variable region of the bacterial 16S rRNA gene was sequenced to characterize the microbial profile using Illumina MiSeq platform. <b><i>Results:</i></b> Phyla <i>Proteobacteria</i> and <i>Firmicutes</i> were the predominant bacteria in colostrum samples. The core and abundant genera in colostrum included <i>Streptococcus</i>, <i>Staphylococcus</i>, and <i>Pseudomonas</i>. Compared with the control group, principal coordinate analysis based on the Bray-Curtis distance showed a significant difference in milk microbial community in women with antibiotic exposure, accompanied by a significantly lower alpha diversity and a different microbial ecological network. Furthermore, the relative abundances of genera <i>Actinomyces</i>, <i>Anaerobacter</i>, and <i>Clostridium_</i>sensu<i>_stricto</i> significantly decreased after antibiotic treatment. <b><i>Conclusions:</i></b> This study provided evidence of alterations in the colostrum microbial community with antibiotic exposure, improving our understanding of the effects of antibiotic treatment on the milk microbiome.

Developing a Feline Immunodeficiency Virus Subtype B Vaccine Prototype Using a Recombinant MVA Vector.

The feline immunodeficiency virus (FIV) is a retrovirus with global impact and distribution, affecting both domestic and wild cats. This virus can cause severe and progressive immunosuppression culminating in the death of felids. Since the discovery of FIV, only one vaccine has been commercially available. This vaccine has proven efficiency against FIV subtypes A and D, whereas subtype B (FIV-B), found in multiple continents, is not currently preventable by vaccination. We, therefore, developed and evaluated a vaccine prototype against FIV-B using the recombinant viral vector modified vaccinia virus Ankara (MVA) expressing the variable region V1–V3 of the FIV-B envelope protein. We conducted preclinical tests in immunized mice (C57BL/6) using a prime-boost protocol with a 21 day interval and evaluated cellular and humoral responses as well the vaccine viability after lyophilization and storage. The animals immunized with the recombinant MVA/FIV virus developed specific splenocyte proliferation when stimulated with designed peptides. We also detected cellular and humoral immunity activation with IFN-y and antibody production. The data obtained in this study support further development of this immunogen and testing in cats.

Bacterial Community Analysis of Biofilm Formed on Metal Joint

Microbiologically influenced corrosion (MIC) is caused by biofilms formed on metal surfaces, and MIC of metal alloys on marine infrastructure leads to severe accidents and great economic losses. Although bacterial community analyses of the biofilms collected from corroded metal have been studied, the analyses of biofilms collected from uncorroded metal are rarely reported. In this study, a biofilm formed on an uncorroded metal joint attached to a metal dock mooring at Akitsu Port was used as a model for bacterial community analysis. The bacterial community was analyzed by high-throughput sequencing of the V3–V4 variable regions of the 16S rRNA gene. Bacterial species contained in the biofilms were identified at the genus level, and Alkanindiges bacteria were the dominant species, which have been not reported as the dominant species in previous research on MIC. The genome sequences of known Alkanindiges bacteria do not have conserved gene clusters required to cause metal corrosion, which suggests that Alkanindiges bacteria do not corrode metals but act on the formation of biofilms. Those findings indicated that the bacterial community may change significantly during the process from biofilm formation to the occurrence of metal corrosion.

Effect of Routine Gastric Residual Aspiration on the Preterm Infant Fecal Microbiome.

Enteral feeding tubes are used in neonatal intensive care units (NICUs) to assess feeding tolerance by utilizing preprandial gastric residual aspiration. This study evaluates the effect of gastric residual aspiration on the preterm infant fecal microbiome and gastrointestinal inflammation. Fifty-one very low birth weight (VLBW) infants (≤32 weeks' gestational age and ≤1,250 g) enrolled in a larger single-center randomized controlled trial evaluating the effects of routine and nonroutine gastric residual aspiration were selected for further analysis. Of those infants, 30 had microbiome analysis performed on stools collected at 6 weeks by sequencing the bacterial V1 to V3 variable regions of the genes encoding for 16S rRNA. In an additional 21 infants, stool samples collected at 3 and 6 weeks were analyzed for intestinal inflammation using a cytokine multiplex panel. Microbial communities between groups were not distinct from each other and there was no difference in intestinal inflammation between groups. Analyses using gene expression packages DESeq2 and edgeR produced statistically significant differences in several taxa, possibly indicating a more commensal intestinal microbiome in infants not undergoing gastric residual aspiration. Omission of routine gastric residual aspiration was not associated with intestinal dysbiosis or inflammation, providing additional evidence that monitors preprandial gastric residuals is unnecessary. · Omission of routine gastric residual aspiration was not associated with intestinal dysbiosis or inflammation.. · Existing literature indicates preprandial gastric aspiration does not reliably correlate with development of necrotizing enterocolitis but does correlate with delayed enteral nutrition.. · Further study is required but this data that suggest monitoring preprandial gastric residuals are unnecessary..

Endophytic Klebsiella aerogenes HGG15 stimulates mulberry growth in hydro-fluctuation belt and the potential mechanisms as revealed by microbiome and metabolomics.

Growth promotion and stress tolerance induced by endophytes have been observed in various plants, but their effects on mulberry regularly suffering flood in the hydro-fluctuation belt are less understood. In the present study, endophytic Klebsiella aerogenes HGG15 was screened out from 28 plant growth promotion (PGP) bacteria as having superior PGP traits in vitro and in planta as well as biosafety for silkworms. K. aerogenes HGG15 could actively colonize into roots of mulberry and subsequently transferred to stems and leaves. The 16S ribosomal RNA (V3–V4 variable regions) amplicon sequencing revealed that exogenous application of K. aerogenes HGG15 altered the bacterial community structures of mulberry roots and stems. Moreover, the genus of Klebsiella was particularly enriched in inoculated mulberry roots and was positively correlated with mulberry development and soil potassium content. Untargeted metabolic profiles uncovered 201 differentially abundant metabolites (DEMs) between inoculated and control mulberry, with lipids and organo-heterocyclic compounds being particularly abundant DEMs. In addition, a high abundance of abiotic stress response factors and promotion growth stimulators such as glycerolipid, sphingolipid, indole, pyridine, and coumarin were observed in inoculated mulberry. Collectively, the knowledge gained from this study sheds light on potential strategies to enhance mulberry growth in hydro-fluctuation belt, and microbiome and metabolite analyses provide new insights into the growth promotion mechanisms used by plant-associated bacteria.

Salivary Microbiome Profile of Diabetes and Periodontitis in a Chinese Population.

AimThere is a bidirectional association between diabetes and periodontitis. However, the effect of diabetes on the periodontitis salivary microbiota has not been elucidated. The aim of this study was to determine the effect of the presence of diabetes on the microbiota among Chinese patients with periodontitis.Materials and MethodsUnstimulated whole saliva samples were collected from the periodontitis with diabetes group (TC), chronic periodontitis group (CP), and periodontally healthy and systemically healthy group (H) by spitting method. Bacterial genomic DNA was PCR-amplified at the V4 variable region of 16S rRNA gene. The library was constructed according to the obtained sequence results, and biological analysis and statistical analysis were carried out. Functional prediction of three groups of microbial communities was performed by the PICRUSt algorithm.ResultsThere was no significant difference in bacterial diversity between the TC and CP groups. Compared with the H group, the TC group and CP group presented a higher diversity of salivary flora. Firmicutes, Streptococcus, Haemophilus, Veillonella, and Haemophilus parainfluenzae dominated the H group. Corynebacterium, Leptotrichia, Dialister, Comamonas, Capnocytophaga, Catonella, Filifactor, Campylobacter, Treponema, Campylobacter concisus, Prevotella oralis, and Porphyromonas gingivalis were significantly enriched in the TC and CP groups. Among them, Treponema and P. oralis were the most abundant in the TC group. The PICRUSt results showed that many pathways related to cell motility and functional metabolism of the salivary microbial flora changed in the TC group and the CP group.ConclusionsDiabetes was not the main factor causing the altered diversity of salivary microbiota in patients with periodontitis; however, the presence of diabetes altered the abundance of some microbiota in saliva.

Effects of calcium-magnesium carbonate and calcium-magnesium hydroxide as supplemental sources of magnesium on ruminal microbiome.

Our objective was to evaluate the inclusion of calcium-magnesium carbonate [CaMg(CO3)2] and calcium-magnesium hydroxide [CaMg(OH)4] in corn silage-based diets and their impact on ruminal microbiome. Our previous work showed a lower pH and molar proportion of butyrate from diets supplemented with [CaMg(CO3)2] compared to [CaMg(OH)4]; therefore, we hypothesized that ruminal microbiome would be affected by Mg source. Four continuous culture fermenters were arranged in a 4 × 4 Latin square with the following treatments defined by the supplemental source of Mg: 1) Control (100% MgO, plus sodium sesquicarbonate as a buffer); 2) CO3 [100% CaMg(CO3)2]; 3) OH [100% CaMg(OH)4]; and 4) CO3/OH [50% Mg from CaMg(CO3)2, 50% Mg from CaMg(OH)4]. Diet nutrient concentration was held constant across treatments (16% CP, 30% NDF, 1.66 MCal NEl/kg, 0.67% Ca, and 0.25% Mg). We conducted four fermentation periods of 10 d, with the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 16 solid and 16 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R and SAS to determine treatment effects on taxa relative abundance of liquid and solid fractions. Correlation of butyrate molar proportion with taxa relative abundance was also analyzed. Treatments did not affect alpha and beta diversities or relative abundance of phylum, class and order in either liquid or solid fractions. At the family level, relative abundance of Lachnospiraceae in solid fraction was lower for CO3 and CO3/OH compared to OH and Control (P < 0.01). For genera, abundance of Butyrivibrio (P = 0.01) and Lachnospiraceae ND3007 (P < 0.01) (both from Lachnospiraceae family) was lower and unclassified Ruminococcaceae (P = 0.03) was greater in CO3 than Control and OH in solid fraction; while abundance of Pseudobutyrivibrio (P = 0.10) and Lachnospiraceae FD2005 (P = 0.09) (both from Lachnospiraceae family) and Ruminobacter (P = 0.09) tended to decrease in CO3 compared to Control in liquid fraction. Butyrate molar proportion was negatively correlated to Ruminococcaceae (r = –0.55) in solid fraction and positively correlated to Pseudobutyrivibrio (r = 0.61) and Lachnospiraceae FD2005 (r = 0.61) in liquid. Our results indicate that source of Mg has an impact on bacterial taxa associated with ruminal butyrate synthesis, which is important for epithelial health and fatty acid synthesis.

Changes in Gut Microbiota Composition Associated with the Presence of Enteric Protist Blastocystis in Captive Forest Musk Deer (Moschus Berezovskii).

ABSTRACTBlastocystis is a common protistan parasite inhabiting the gastrointestinal tract of a wide range of hosts including humans and domestic and wild animals. Many studies have revealed the associations between Blastocystis and gut microbiome in humans. However, only a few studies have focused on the associations between Blastocystis and gut microbiome of animals, especially in forest musk deer (Moschus berezovskii). We investigated the effects of the Blastocystis colonization on the intestinal bacterial community compositions using amplicon sequencing targeting the V4 variable region of the 16S rRNA. Two subtypes of Blastocystis (ST5 and ST10) and Blastocystis-free (control) were included in this study. We found that compared with the forest musk deer without Blastocystis, ST10-colonized forest musk deer had higher bacterial richness and diversity, while ST5-colonized forest musk deer showed a comparable bacterial diversity. Likewise, beta diversity revealed significant differences in bacterial community structure between ST10-colonized and Blastocystis-free forest musk deer. The proportion of Bacteroidetes were significantly enriched in ST10-colonized forest musk deer. Bacterial community structure between ST5-colonized and Blastocystis-free forest musk deer did not differ significantly. The present study explored the associations between Blastocystis and gut microbial community of forest musk deer for the first time, and revealed ST10 colonization, instead of ST5, is associated with higher bacterial diversity and shifted microbial structure. Our data provides valuable insights into the associations between gut microbiomes and parasites.IMPORTANCE Forest musk deer is listed as an endangered species by International Union for Conservation of Nature Red List, and the Chinese government has introduced captivity breeding measures to curb the rapid decline of the musk deer population since the 1950s. It has been suggested that Blastocystis colonization can modulate the composition of the host's intestinal microbiota, thereby affecting the host health. The present study investigated the effects of the Blastocystis colonization on the gut microbiota in the feces of forest musk deer in Sichuan Province, China. Two subtypes (ST5 and ST10) have differential effects on the bacterial diversity and community composition, suggesting that the study of Blastocystis should be distinguished at the subtype level. Because the pathogenicity of Blastocystis is controversial, pathogenic, or commensal, continuous monitoring of the impact of Blastocystis colonization on the intestinal microbiota is of great significance to assess its health effects on forest musk deer.

Assessing the seasonal dynamics of zooplankton in a recreational marina of the northwest of Portugal through multi-marker DNA metabarcoding

The monitoring of larvae in plankton samples in recreational marinas, ports, or the close vicinities, may provide key information about non-indigenous species (NIS) introduction status or detect their presence at an earlier stage. DNA metabarcoding is a powerful method to assess biodiversity but its efficiency is dependent on the methodologies employed along the analytical chain, namely the targeted genetic markers. Therefore, we aim to analyze the ability of two genetic markers - the mitochondrial cytochrome C oxidase subunit I gene (COI) and the variable region V4 of the nuclear small subunit ribosomal gene (18S) - to assess the seasonal dynamics of zooplankton communities, including NIS, through DNA metabarcoding, and further to compare it with a compiled list of zooplanktonic species identified via morphology that occur in the Lima Estuary. To this end, we sampled zooplankton communities, spanning three consecutive seasons (spring, autumn, and winter 2020/2021) in three sampling points in the recreational marina of Viana do Castelo, located in the Lima Estuary, North of Portugal. Globally, metabarcoding recovered 157 species belonging to 19 phyla, with a dominance of Annelida, Crustacea, and Mollusca that represent 64.3% of all species. Even though our sampling area poorly represents the Lima Estuary, this approach allowed the detection of 6 NIS, which were not yet reported by traditional methods of identification. The composition of the zooplankton communities recovered was greatly affected by the markers employed, and to a lesser extent by the season. Overall 18S detected more species and fewer species were recovered in winter. NIS were predominantly detected during spring and autumn, but only 5.1% of all species were detected in all seasons. This study shows the DNA metabarcoding efficiency in assessing the dynamics of zooplankton communities and highlights the interest in using a multi-marker and seasonal approach to species detection, particularly to target NIS.

Rhizosphere 16S-ITS Metabarcoding Profiles in Banana Crops Are Affected by Nematodes, Cultivation, and Local Climatic Variations.

Agriculture affects soil and root microbial communities. However, detailed knowledge is needed on the effects of cropping on rhizosphere, including biological control agents (BCA) of nematodes. A metabarcoding study was carried out on the microbiota associated with plant parasitic and other nematode functional groups present in banana farms in Tenerife (Canary Islands, Spain). Samples included rhizosphere soil from cv Pequeña Enana or Gruesa and controls collected from adjacent sites, with the same agroecological conditions, without banana roots. To characterize the bacterial communities, the V3 and V4 variable regions of the 16S rRNA ribosomal gene were amplified, whereas the internal transcribed spacer (ITS) region was used for the fungi present in the same samples. Libraries were sequenced with an Illumina MiSeq™ in paired ends with a 300-bp read length. For each sample, plant parasitic nematodes (PPN) and other nematodes were extracted from the soil, counted, and identified. Phytoparasitic nematodes were mostly found in banana rhizosphere. They included Pratylenchus goodeyi, present in northern farms, and Helicotylenchus spp., including H. multicinctus, found in both northern and southern farms. Metabarcoding data showed a direct effect of cropping on microbial communities, and latitude-related factors that separated northern and southern controls from banana rizosphere samples. Several fungal taxa known as nematode BCA were identified, with endophytes, mycorrhizal species, and obligate Rozellomycota endoparasites, almost only present in the banana samples. The dominant bacterial phyla were Proteobacteria, Actinobacteria, Planctomycetes, Bacteroidetes, Chloroflexi, and Acidobacteria. The ITS data showed several operational taxonomic units (OTUs) belonging to Sordariomycetes, including biocontrol agents, such as Beauveria spp., Arthrobotrys spp., Pochonia chlamydosporia, and Metarhizium anisopliae. Other taxa included Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma virens, and Fusarium spp., together with mycoparasites such as Acrostalagmus luteoalbus. However, only one Dactylella spp. showed a correlation with predatory nematodes. Differences among the nematode guilds were found, as phytoparasitic, free-living, and predatory nematode groups were correlated with specific subsets of other bacteria and fungi. Crop cultivation method and soil texture showed differences in taxa representations when considering other farm and soil variables. The data showed changes in the rhizosphere and soil microbiota related to trophic specialization and specific adaptations, affecting decomposers, beneficial endophytes, mycorrhizae, or BCA, and plant pathogens.

Rubus chingii Hu. unripe fruits extract ameliorates carbon tetrachloride-induced liver fibrosis and improves the associated gut microbiota imbalance

BackgroundThe unripe fruits of Rubus chingii Hu. (“Fu-peng-zi” in Chinese) is a well-known herbal tonic in traditional Chinese medicine (TCM) for tonifying liver and kidney. However, little is known regarding its therapeutic efficacy against liver fibrosis and the underlying mechanism.MethodsThe current research aims to explore the potential of Rubus chingii Hu. unripe fruits extract (RF) in the treatment of liver fibrosis and explore the underlying mechanism. RF was administered (450 and 900 mg·kg− 1 of body weight per day) orally to male C57BL/6 mice with CCl4-induced liver fibrosis for 3 weeks. The histopathological changes and fibrosis stage in liver tissue were assessed using hematoxylin and eosin (H&E) and Sirius red staining. The distribution of α-SMA and Col1A1 in the liver was analyzed to determine the hepatic stellate cells (HSCs) activation using immunohistochemistry and immunofluorescent analysis. Various biochemical markers in serum (ALT, AST) and liver (Hyp, IL1-β, IL6, TNF-α and MCP-1) were observed to assess the liver’s injury, fibrosis, and inflammation. In liver tissue, fibrosis-associated proteins including α-SMA, TGF-β1, Smad2/3, p-Smad2/3, and Smad4 were detected through a Western blot assay. Pyrosequencing-based analysis of bacterial 16 S ribosomal RNA from variable regions V3–V4 of fecal samples characterized the gut microbiota. Spearman’s rank correlation analysis was performed for the association between altered bacterial genera by RF and pharmacodynamics parameters.ResultsThree weeks of RF treatment can significantly lower liver inflammatory levels, pathological abnormalities, and collagen fibrous deposition in mice with CCl4-induced liver fibrosis. The expressions of α-SMA and Col1A1 were lowered by RF, while the expression levels of TGF-β/Smads signaling pathway-related proteins, including TGF-β1, p-Smad2/3, and Smad4, were dramatically decreased by RF. The RF treatment significantly increased or reduced 18 different bacterial species, restoring the CCl4-induced gut microbiota imbalance to the normal group’s levels. According to correlation analysis, the bacterial genera Bifidobacterium and Turicibacter were the most significant in restoring CCl4-induced liver fibrosis.ConclusionsRF can reduce liver damage and delay the onset of liver fibrosis through modulating TGF-β/Smads signaling pathway. Furthermore, RF’s anti-liver fibrosis effect was related to balancing the gut microbial community, partly attained by increasing Bifidobacterium and Turicibacter in liver fibrosis.

The impacts of bovine milk, soy beverage, or almond beverage on the growing rat microbiome.

BackgroundMilk, the first food of mammals, helps to establish a baseline gut microbiota. In humans, milk and milk products are consumed beyond infancy, providing comprehensive nutritional value. Non-dairy beverages, produced from plant, are increasingly popular as alternatives to dairy milk. The nutritive value of some plant-based products continues to be debated, whilst investigations into impacts on the microbiome are rare. The aim of this study was to compare the impact of bovine milk, soy and almond beverages on the rat gut microbiome. We previously showed soy and milk supplemented rats had similar bone density whereas the almond supplemented group had compromised bone health. There is an established link between bone health and the microbiota, leading us to hypothesise that the microbiota of groups supplemented with soy and milk would be somewhat similar, whilst almond supplementation would be different.MethodsThree-week-old male Sprague Dawley rats were randomly assigned to five groups (n = 10/group) and fed ad libitum for four weeks. Two control groups were fed either standard diet (AIN-93G food) or AIN-93G amino acids (AA, containing amino acids equivalent to casein but with no intact protein) and with water provided ad libitum. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages as their sole liquid source. At trial end, DNA was extracted from caecum contents, and microbial abundance and diversity assessed using high throughput sequencing of the V3 to V4 variable regions of the 16S ribosomal RNA gene.ResultsAlmost all phyla (91%) differed significantly (FDR < 0.05) in relative abundance according to treatment and there were distinct differences seen in community structure between treatment groups at this level. At family level, forty taxa showed significantly different relative abundance (FDR < 0.05). Bacteroidetes (Bacteroidaceae) and Firmicutes populations (Lactobacillaceae, Clostridiaceae and Peptostreptococcaceae) increased in relative abundance in the AA almond supplemented group. Supplementation with milk resulted in increased abundance of Actinobacteria (Coriobacteriaceae and Bifidobacteriaceae) compared with other groups. Soy supplementation increased abundance of some Firmicutes (Lactobacilliaceae) but not Actinobacteria, as previously reported by others.ConclusionSupplementation with milk or plant-based drinks has broad impacts on the intestinal microbiome of young rats. Changes induced by cow milk were generally in line with previous reports showing increased relative abundance of Bifidobacteriacea, whilst soy and almond beverage did not. Changes induced by soy and almond drink supplementation were in taxa commonly associated with carbohydrate utilisation. This research provides new insight into effects on the microbiome of three commercially available products marketed for similar uses.

Dysbiosis in the Gut Microbiota in Patients with Inflammatory Bowel Disease during Remission.

ABSTRACTInflammatory bowel disease (IBD) is a chronic, relapsing, inflammatory disorder which comprises two main conditions: Crohn’s disease (CD) and ulcerative colitis (UC). Although the etiology of IBD has not been fully elucidated, the gut microbiota is hypothesized to play a vital role in its development. The aim of this cross-sectional study was to characterize the fecal microbiota in CD or UC patients in a state of remission to reveal potential factors sustaining residual levels of inflammation and triggering disease relapses. Ninety-eight IBD patients in a state of clinical remission (66 UC, 32 CD) and 97 controls were recruited, and stool samples, as well as detailed patient data, were collected. After DNA extraction, the variable regions V1 and V2 of the 16S rRNA gene were amplified and sequenced. Patients with IBD had a decrease in alpha diversity compared to that of healthy controls, and the beta diversity indices showed dissimilarity between the cohorts. Healthy controls were associated with the beneficial organisms unclassified Akkermansia species (Akkermansia uncl.), Oscillibacter uncl., and Coprococcus uncl., while flavonoid-degrading bacteria were associated with IBD. Network analysis identified highly central and influential disease markers and a strongly correlated network module of Enterobacteriaceae which was associated with IBD and could act as drivers for residual inflammatory processes sustaining and triggering IBD, even in a state of low disease activity. The microbiota in IBD patients is significantly different from that of healthy controls, even in a state of remission, which implicates the microbiota as an important driver of chronicity in IBD.IMPORTANCE Dysbiosis in inflammatory bowel disease (IBD) has been implicated as a causal or contributory factor to the pathogenesis of the disease. This study, done on patients in remission while accounting for various confounding factors, shows significant community differences and altered community dynamics, even after acute inflammation has subsided. A cluster of Enterobacteriaceae was linked with Crohn’s disease, suggesting that this cluster, which contains members known to disrupt colonization resistance and form biofilms, persists during quiescence and can lead to chronic inflammation. Flavonoid-degrading bacteria were also associated with IBD, raising the possibility that modification of dietary flavonoids might induce and maintain remission in IBD.

113 Effects of Maternal Parity and Prebiotics on the Microbiome of Nursery Pigs

Abstract The objective of this study was to investigate the effect of maternal parity on sow and offspring microbiomes, and the influence of parity on pig fecal microbiome and performance in response to a prebiotic post-weaning. Fecal and vaginal swabs were collected from 22 primiparous and 74 multiparous sows in a commercial farm at weaning. Fecal swabs were also collected from 48 randomly selected piglets (n = 24, from primiparous and multiparous sows respectively) at d 0, 21, and 42 post-weaning. At weaning, piglets were allotted into 3 dietary treatment groups: a standard nursery diet including pharmacological doses of Zn and Cu (CTRL), a group fed a commercial prebiotic only (PRE), and a group fed the prebiotic plus Zn and Cu (PRE+ZnCu). Extracted DNA was sequenced on the MiSeq platform targeting the V4 variable region of the 16S rRNA bacterial gene, and sequence data were analyzed using DADA2 plus various packages within the R statistical software. Although there were no differences in vaginal microbiome composition between primiparous and multiparous sows, fecal microbiome composition was different (PERMANOVA, R2 = 0.02, P = 0.03). The fecal microbiomes of primiparous offspring displayed significantly higher bacterial diversity compared with multiparous offspring at d0 and d21 (Shannon’s H, P &amp;lt; 0.01), with differences in community composition observed at d21 only (PERMANOVA, R2 = 0.03, P = 0.04). When analyzing the effects of maternal parity within each treatment, only the PRE diet showed significant compositional distinctions between primiparous and multiparous microbiomes (d21: PERMANOVA, R2 = 0.13, P = 0.01; d42: PERMANOVA, R2 = 0.19, P = 0.001). Compositional differences in pig fecal microbiomes between treatments, regardless of maternal parity, were observed only at d21 (PERMANOVA, R2 = 0.12, P = 0.001). No significant differences in growth performance between treatment groups were observed. These results indicate that maternal parity influences pig gut microbiomes post-weaning, and may influence the effects of dietary prebiotics on nursery pig microbiomes.

Effects of choline chloride on the ruminal microbiome at 2 dietary neutral detergent fiber concentrations in continuous culture

Our objective was to evaluate the effects of unprotected choline chloride (Cho) on the ruminal microbiome at 2 dietary neutral detergent fiber (NDF) concentrations. We hypothesized that the effects of Cho on ruminal bacterial populations would depend on NDF. Eight dual-flow continuous-culture fermentors were arranged in a duplicated 4 × 4 Latin square as a 2 × 2 factorial with the following treatments: (1) 30% NDF-control (30% NDF diet, no supplemental choline); (2) 30% NDF-Cho (30% NDF diet plus 1.9 g of choline ion per kg of dry matter); (3) 40% NDF-control (40% NDF diet, no supplemental choline); and (4) 40% NDF-Cho (40% NDF diet plus 1.9 g of choline ion per kg of dry matter). We did 4 fermentation periods of 10 d each and used the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 32 solid and 32 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R (R Project for Statistical Computing) and SAS (SAS Institute Inc.) to determine effects of Cho, NDF, and NDF × Cho on taxa relative abundance. The correlation of propionate molar proportion with taxa relative abundance was also analyzed. At the phylum level, relative abundance of Firmicutes in the liquid fraction tended to be greater when Cho was supplemented with a 30% NDF diet. At the order level, Cho increased Coriobacteriales in solid fraction and decreased Fibrobacterales in liquid fraction. Moreover, Cho decreased abundance of Clostridiales and increased Selenomonadales in the solid fraction, only with the 30% NDF diet. For genera, lower abundance of Pseudobutyrivibrio resulted from Cho in solid and liquid fractions. Greater abundance of Succinivibrio in solid and Selenomonas and Selenomonas 1 in liquid resulted from Cho with the 30% NDF diet. Propionate molar proportion was positively correlated with relative abundance of order Selenomonadales in solid and liquid fractions, and with genus Succinivibrio in solid and genera Selenomonas and Selenomonas 1 in liquid. Our results indicate that Cho primarily decreases abundance of bacteria involved in fiber degradation and increases abundance of bacteria mainly involved in nonstructural carbohydrate degradation and synthesis of propionate, particularly when a diet with 30% NDF is provided.