Gut microbiomes from Gambian infants reveal the development of a non-industrialized Prevotella-based trophic network.

Distinct bacterial trophic networks exist in the gut microbiota of individuals in industrialized and non-industrialized countries. In particular, non-industrialized gut microbiomes tend to be enriched with Prevotella species. To study the development of these Prevotella-rich compositions, we investigated the gut microbiota of children aged between 7 and 37 months living in rural Gambia (616 children, 1,389 stool samples, stratified by 3-month age groups). These infants, who typically eat a high-fibre, low-protein diet, were part of a double-blind, randomized iron intervention trial (NCT02941081) and here we report the secondary outcome. We found that child age was the largest discriminating factor between samples and that anthropometric indices (collection time points, season, geographic collection site, and iron supplementation) did not significantly influence the gut microbiome. Prevotella copri, Faecalibacterium prausnitzii and Prevotella stercorea were, on average, the most abundant species in these 1,389 samples (35%, 11% and 7%, respectively). Distinct bacterial trophic network clusters were identified, centred around either P. stercorea or F. prausnitzii and were found to develop steadily with age, whereas P. copri, independently of other species, rapidly became dominant after weaning. This dataset, set within a critical gut microbial developmental time frame, provides insights into the development of Prevotella-rich gut microbiomes, which are typically understudied and are underrepresented in western populations.

Exploring the signature gut and oral microbiome in individuals of specific Ayurveda prakriti.

Diagnosis and treatment of various diseases in Ayurveda, the Indian system of medicine, relies on 'prakriti' phenotyping of individuals into predominantly three constitutions, kapha, pitta and vata. Recent studies propose that microbiome play an integral role in precision medicine. A study of the relationship between prakriti - the basis of personalized medicine in Ayurveda and that of gut microbiome, and possible biomarker of an individual's health, would vastly improve precision therapy. Towards this, we analyzed bacterial metagenomes from buccal (oral microbiome) and fecal (gut microbiome) samples of 272 healthy individuals of various predominant prakritis. Major bacterial genera from gut microbiome included Prevotella, Bacteroides and Dialister while oral microbiome included Streptococcus, Neisseria, Veilonella, Haemophilus, Porphyromonas and Prevotella. Though the core microbiome was shared across all individuals, we found prakriti specific signatures such as preferential presence of Paraprevotella and Christensenellaceae in vata individuals. A comparison of core gut microbiome of each prakriti with a database of 'healthy' microbes identified microbes unique to each prakriti with functional roles similar to the physiological characteristics of various prakritis as described in Ayurveda. Our findings provide evidence to Ayurvedic interventions based on prakriti phenotyping and possible microbial biomarkers that can stratify the heterogenous population and aid in precision therapy.

Prevotella histicola Protects From Arthritis by Expansion of Allobaculum and Augmenting Butyrate Production in Humanized Mice.

Bacterial therapeutics are the emergent alternatives in treating autoimmune diseases such as Rheumatoid Arthritis [RA]. P. histicola MCI 001 is one such therapeutic bacterium that has been proven to treat autoimmune diseases such as RA and multiple sclerosis [MS] in animal models. The present study characterized P. histicola MCI 001 isolated from a human duodenal biopsy, and evaluated its impact on the gut microbial and metabolic profile in a longitudinal study using the collagen-induced arthritis model in HLA-DQ8.AEo transgenic mice. P. histicola MCI 001 though closely related to the type strain of P. histicola, DSM 19854, differed in utilizing glycerol. In culture, P. histicola MCI 001 produced vitamins such as biotin and folate, and was involved in digesting complex carbohydrates and production of acetate. Colonization study showed that duodenum was the predominant niche for the gavaged MCI 001. A longitudinal follow-up of gut microbial profile in arthritic mice treated with MCI 001 suggested that dysbiosis caused due to arthritis was partially restored to the profile of naïve mice after treatment. A taxon-level analysis suggested an expansion of intestinal genus Allobaculum in MCI001 treated arthritic mice. Eubiosis achieved post treatment with P. histicola MCI 001 was also reflected in the increased production of short-chain fatty acids [SCFAs]. Present study suggests that the treatment with P. histicola MCI 001 leads to an expansion of Allobaculum by increasing the availability of simple carbohydrates and acetate. Restoration of microbial profile and metabolites like butyrate induce immune and gut homeostasis.

Effects of dietary supplementation with two alternatives to antibiotics on intestinal microbiota of preweaned calves challenged with Escherichia coli K99.

The aim of this study was to investigate the effects of dietary supplementation with two alternatives to antibiotics (Candida tropicalis and mulberry leaf flavonoids) on intestinal microbiota of preweaned calves challenged with Escherichia coli K99. Sixty Holstein calves were randomly assigned to 5 treatments: fed a basal diet (N-CON); fed a basal diet and challenged with E.coli K99 (P-CON); fed a basal diet supplemented with C.tropicalis (CT), mulberry leaf flavonoids (MLF), and the combination of the two additives (CM), respectively, and challenged with E.coli K99. The MLF and CM groups had significantly higher average daily grain and feed efficiency, and significantly lower fecal scores compared with the P-CON group after E. coli K99 challenge. The supplementation groups increased the relative abundance, at the phylum level, of Bacteroidetes and Proteobacteria, whereas at the genus level, they increased the relative abundance of Prevotella, Lactobacillus, and Enterococcus. Quantitative PCR revealed that the CT, MLF, and CM groups had significantly lower copy numbers of E.coli K99 compared with the P-CON group. The CT, MLF, and CM treatments reduce days of diarrhea, improve intestinal health, and beneficially manipulate the intestinal microbiota in preweaned calves.

Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17- and TLR4-dependent arthritis.

BACKGROUND: Perturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn -/- mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn -/- mice. RESULTS: Using high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn -/- mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn -/- mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn -/- mice. The arthritis phenotype in IL1rn -/- mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn -/- microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis. CONCLUSIONS: These findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.

Effect of urea-supplemented diets on the ruminal bacterial and archaeal community composition of finishing bulls.

In this study, we evaluated the effects of urea-supplemented diets on the ruminal bacterial and archaeal communities of finishing bulls using sequencing technology. Eighteen bulls were fed a total mixed ration based on maize silage and concentrate (40:60) and randomly allocated to one of three experimental diets: a basal diet with no urea (UC, 0%), a basal diet supplemented with low urea levels (UL, 0.8% dry matter (DM) basis), and a basal diet supplemented with high urea levels (UH, 2% DM basis). All treatments were iso-nitrogenous (14% crude protein, DM basis) and iso-metabolic energetic (ME = 11.3 MJ/kg, DM basis). After a 12-week feeding trial, DNA was isolated from ruminal samples and used for 16S rRNA gene amplicon sequencing. For bacteria, the most abundant phyla were Firmicutes (44.47%) and Bacteroidetes (41.83%), and the dominant genera were Prevotella (13.17%), Succiniclasticum (4.24%), Butyrivibrio (2.36%), and Ruminococcus (1.93%). Urea supplementation had no effect on most phyla (P > 0.05), while there was a decreasing tendency in phylum TM7 with increasing urea levels (P = 0.0914). Compared to UC, UH had lower abundance of genera Butyrivibrio and Coprococcus (P = 0.0092 and P = 0.0222, respectively). For archaea, the most abundant phylum was Euryarchaeota (99.81% of the sequence reads), and the most abundant genus was Methanobrevibacter (90.87% of the sequence reads). UH increased the abundance of genus Methanobrevibacter and Methanobacterium (P = 0.0299 and P = 0.0007, respectively) and decreased the abundance of vadinCA11 (P = 0.0151). These findings suggest that urea-supplemented diets were associated with a shift in archaeal biodiversity and changes in the bacterial community in the rumen.

Mining of Ruminant Microbial Phytase (RPHY1) from Metagenomic Data of Mehsani Buffalo Breed: Identification, Gene Cloning, and Characterization.

Phytases have been widely used as animal feed supplements to increase the availability of digestible phosphorus, especially in monogastric animals fed cereal grains. The present study describes the identification of a full-length phytase gene of Prevotella species present in Mehsani buffalo rumen. The gene, designated as RPHY1, consists of 1,251 bp and is expressed into protein with 417 amino acids. A homology search of the deduced amino acid sequence of the RPHY1 phytase gene in a nonredundant protein database showed that it shares 92% similarity with the histidine acid phosphatase domain. Subsequently, the RPHY1 gene was expressed using a pET32a expression vector in Escherichia coli BL21 and purified using a His60 Ni-NTA gravity column. The mass of the purified RPHY1 was estimated to be approximately 63 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal RPHY1 enzyme activity was observed at 55°C (pH 5) and exhibited good stability at 5°C and within the acidic pH range. Significant inhibition of RPHY1 activity was observed for Mg2+ and K+ metal ions, while Ca2+, Mn2+, and Na+ slightly inhibited enzyme activity. The RPHY1 phytase was susceptible to SDS, and it was highly stimulated in the presence of EDTA. Overall, the observed comparatively high enzyme activity levels and characteristics of the RPHY1 gene mined from rumen prove its promising candidature as a feed supplement enzyme in animal farming.

Structural modulation of gut microbiota by chondroitin sulfate and its oligosaccharide.

Chondroitin sulfate (CS) as a dietary supplement and a symptomatic slow acting (SYSA) drug has been used for years. Recently, CS has been demonstrated to be readily degraded and fermented in vitro by specific human gut microbes, hinting that dietary CS may pose a potential effect on gut microbiota composition in vivo. However, until now, little information is available on modulations of gut microbiota by CS. In the present study, modulations of gut microbiota in Kunming mice by CS and its oligosaccharide (CSO) were investigated by high-throughput sequencing. As evidenced by Heatmap and principal component analysis (PCA), the female microbiota were more vulnerable than the male microbiota to CS and CSO treatment. Besides, it is of interest to found that CS and CSO had differing effects on the abundance of Bacteroidales S24-7, Bacteroides, Helicobacter, Odoribacter, Prevotellaceae and Lactobacillus in male mice versus female mice. Collectively, we demonstrated a sex-dependent effect on gut microbiota of CS and CSO. In addition, since gut microbiota exerts a major effect on host physiology, our study highlighted that certain beneficial effects of CS may be associated with modulations of gut microbiota, which merits further investigation.

Gut microbiota richness promotes its stability upon increased dietary fibre intake in healthy adults.

Gut microbiota richness and stability are important parameters in host-microbe symbiosis. Diet modification, notably using dietary fibres, might be a way to restore a high richness and stability in the gut microbiota. In this work, during a 6-week nutritional trial, 19 healthy adults consumed a basal diet supplemented with 10 or 40 g dietary fibre per day for 5 days, followed by 15-day washout periods. Fecal samples were analysed by a combination of 16S rRNA gene pyrosequencing, intestinal cell genotoxicity assay, metatranscriptomics sequencing approach and short-chain fatty analysis. This short-term change in the dietary fibre level did not have the same impact for all individuals but remained significant within each individual gut microbiota at genus level. Higher microbiota richness was associated with higher microbiota stability upon increased dietary fibre intake. Increasing fibre modulated the expression of numerous microbiota metabolic pathways such as glycan metabolism, with genes encoding carbohydrate-active enzymes active on fibre or host glycans. High microbial richness was also associated with high proportions of Prevotella and Coprococcus species and high levels of caproate and valerate. This study provides new insights on the role of gut microbial richness in healthy adults upon dietary changes and host microbes' interaction.

Relationship between the rumen microbiome and residual feed intake-efficiency of Brahman bulls stocked on bermudagrass pastures.

Residual feed intake (RFI) testing has increased selection pressure on biological efficiency in cattle. The objective of this study was to assess the association of the rumen microbiome in inefficient, positive RFI (p-RFI) and efficient, negative RFI (n-RFI) Brahman bulls grazing 'Coastal' bermudagrass [Cynodondactylon (L.) Pers.]under two levels of forage allowance (high and low stocking intensity). Sixteen Brahman bulls were previously fed in confinement for 70 d to determine the RFI phenotype. Bulls were then allotted 60 d stocking on bermudagrass pastures to estimate RFI using the n-alkane technique. At the conclusion of the grazing period, rumen liquid samples were collected from each bull by stomach tube to evaluate the rumen microbiome. Extraction of DNA, amplification of the V4-V6 region of the 16S rRNA gene, and 454 pyrosequencing were performed on each sample. After denoising the sequences, chimera checking, and quality trimming, 4,573 ± 1,287 sequences were generated per sample. Sequences were then assigned taxonomy from the Greengenes database using the RDP classifier. Overall, 67.5 and 22.9% of sequences were classified as Bacteroidetes and Firmicutes, respectively. Within the phylum Bacteroidetes, Prevotella was the most predominant genus and was observed in greater relative abundance in p-RFI bulls compared with n-RFI bulls (P = 0.01). In contrast, an unidentified Bacteroidales family was greater in relative abundance for n-RFI bulls than p-RFI (26.7 vs. 19.1%; P = 0.03). Ruminococcaceae was the third most abundant family in our samples, but it was not affected by RFI phenotype. No effect of stocking intensity was observed for bacterial taxa, but there was a tendency for alpha diversity and operational taxonomic unit richness to increase with lower stocking intensity. Results suggested the rumen microbiome of p-RFI Brahman bulls has greater levels of Prevotella, but the bacterial community composition was unaffected by stocking intensity.

Establishment of ruminal bacterial community in dairy calves from birth to weaning is sequential.

AIM: Establishment of ruminal bacterial community in dairy calves. METHODS AND RESULTS: Rumen bacterial community was analysed on 6 calves bred according to commercial practices from day one to weaning at day 83 of age, using 454 16S rRNA-based pyrosequencing. Samples taken at day 1 did not produce amplicons. Analysis of data revealed a three-stage implantation process with a progressive but important shift of composition. At day 2, the bacterial community was mainly composed of Proteobacteria (70%) and Bacteroidetes (14%), and Pasteurellaceae was the dominant family (58%). The bacterial community abruptly changed between days 2 and 3, and until day 12, dominant genera were Bacteroides (21%), Prevotella (11%), Fusobacterium (5%) and Streptococcus (4%). From 15 to 83 days, when solid food intake rapidly increased, Prevotella became dominant (42%) and many genera strongly decreased or were no longer detected. A limited number of bacteria genera correlated with feed intake, rumen volatile fatty acids and enzymatic activities. CONCLUSION: The ruminal bacterial community is established before intake of solid food, but solid food arrival in turn shapes this community. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insight into the establishment of calves' rumen bacterial community and suggests a strong effect of diet.

Quantification of cultivable bacteria and endotoxin in post-treatment apical periodontitis before and after chemo-mechanical preparation.

This clinical study was conducted to quantify cultivable bacteria and endotoxin in root canals with post-treatment apical periodontitis by correlating their levels with clinical features and to evaluate the effect of chemo-mechanical preparation (CMP) with 2 % chlorhexidine gel + 17 % EDTA on bacterial and endotoxin removal/elimination. Moreover, target strict Gram-negative anaerobic bacteria were investigated by polymerase chain reaction (PCR). Fifteen teeth with post-treatment apical periodontitis were sampled before (s1) and after (s2) CMP. Culture techniques determined the number of colony-forming units (CFU). PCR (16S rDNA) and limulus amebocyte lysate (LAL) assay were used for bacterial and endotoxin detection, respectively. Prevotella nigrescens (4/15), Prevotella intermedia (2/15), and Tannerella forsythia (2/15) were the most frequently detected species. Endotoxin was recovered in 100 % of the samples. At s1, bacteria and endotoxin were detected at a median value of 5.14 × 10(3) CFU/mL and 3.96 EU/mL, respectively. Higher levels of endotoxin were related to a larger size of radiolucent area (>5 mm) (p < 0.05). CMP was more effective in reducing bacteria (99.61 %) than endotoxin (60.6 %) (both p < 0.05). Our findings indicated that the levels of endotoxin found in infected root canals were related to a larger size of radiolucent area in the periapical region. Moreover, CMP was effective in reducing both bacterial and endotoxin contents in post-treatment apical periodontitis.

Rumen bacterial diversity dynamics associated with changing from bermudagrass hay to grazed winter wheat diets.

Rumen bacterial communities in forage-fed and grazing cattle continually adapt to a wide range of changing dietary composition, nutrient density, and environmental conditions. We hypothesized that very distinct community assemblages would develop between the fiber and liquid fractions of rumen contents in animals transitioned from bermudagrass hay diet to a grazed wheat diet. To address this hypothesis, we designed an experiment utilizing a 16S-based bTEFAP pyrosequencing technique to characterize and elucidate changes in bacterial diversity among the fiber and liquid rumen fractions and whole rumen contents of 14 (Angus x Hereford) ruminally cannulated steers sequentially fed bermudagrass hay (Cynodon dactylon; 34 days) and grazing wheat forage (28 days). Bermudagrass hay was a conserved C4 perennial grass lower in protein and higher in fiber (11% and 67%, respectively) content than grazed winter wheat (Triticum aestivum), a C3 annual grass with higher protein (20%) and a large (66%) soluble fraction.Significant differences in the OTU estimates (Chao1, Ace,and Rarefaction) were detected between fractions of both diets, with bermudagrass hay supporting greater diversity than wheat forage. Sequences were compared with a 16S database using BLASTn and assigned sequences to respective genera and genera-like units based on the similarity value to known sequences in the database. Predominant genera were Prevotella (up to 33%) and Rikenella-like (upto 28%) genera on the bermudagrass diet and Prevotella (upto 56%) genus on the wheat diet irrespective of the fractions. Principle component analyses accounted for over 95% of variation in 16S estimated bacterial community composition in all three fractions and clearly differentiated communities associated with each diet. Overall, bermudagrass hay diets clustered more clearly than wheat diets.These data are the first to explore bacterial diversity dynamics in a common population of animals in response to contrasting grass forage diets.

Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen.

AIMS: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. METHODS AND RESULTS: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60:40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. CONCLUSIONS: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.

Phototargeting oral black-pigmented bacteria.

We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

Nucleic acid-based diagnosis of bacterial vaginosis and improved management using probiotic lactobacilli.

Bacterial vaginosis (BV) is a common condition in women that represents an imbalance of the vaginal microflora, lactobacilli depletion, and excess growth of mainly anaerobic Gram-negative pathogens. Diagnosis is made using a series of tests or a Gram stain of a vaginal smear. Treatment with antibiotics is quite effective, but recurrences are common. A study of 55 vaginal samples from 11 postmenopausal women showed the presence of BV by the Gram stain-based Nugent scoring system, and polymerase chain reaction-denaturing gradient gel electrophoresis showed that Bacteroides or Prevotella species were the most common isolates recovered (24 of 25), with Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae also found in some samples. In one case, only Gardnerella vaginalis was found. These findings illustrate that BV remains common even among otherwise healthy women, but it is not caused solely by either Gardnerella or Mobiluncus. Use of a FemExam system (Cooper Surgical, Shelton, CT), based upon elevated pH and trimethylamine levels, to screen vaginal smears from 59 healthy women showed poor correlation with the Gram stain method. A randomized, placebo-controlled trial of these subjects showed that the lactobacilli-dominant microbiota was restored in subjects with BV but not in controls, following 2 months of daily oral intake of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14. These studies show that nucleic acid-based methods are effective at identifying bacteria responsible for BV. If such methods could be used to develop a commercially available, self-use kit, women would be much better placed to take control of their own health, for example, using medicinal food or dietary supplement products such as the clinically proven probiotic strains L. rhamnosus GR-1 and L. fermentum RC-14.