Metabolic influence of core ciliates within the rumen microbiome.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

Corrigendum: The P4 Study: Postpartum Maternal and Infant Faecal Microbiome 6 Months After Hypertensive Versus Normotensive Pregnancy.

[This corrects the article DOI: 10.3389/fcimb.2022.646165.].

Refining a Protocol for Faecal Microbiota Engraftment in Animal Models After Successful Antibiotic-Induced Gut Decontamination.

BACKGROUND: There is mounting evidence for the therapeutic use of faecal microbiota transplant (FMT) in numerous chronic inflammatory diseases. Germ free mice are not always accessible for FMT research and hence alternative approaches using antibiotic depletion prior to FMT in animal studies are often used. Hence, there is a need for standardising gut microbiota depletion and FMT methodologies in animal studies. The aim of this study was to refine gut decontamination protocols prior to FMT engraftment and determine efficiency and stability of FMT engraftment over time. METHODS: Male C57BL/6J mice received an antibiotic cocktail consisting of ampicillin, vancomycin, neomycin, and metronidazole in drinking water for 21 days ad libitum. After antibiotic treatment, animals received either FMT or saline by weekly oral gavage for 3 weeks (FMT group or Sham group, respectively), and followed up for a further 5 weeks. At multiple timepoints throughout the model, stool samples were collected and subjected to bacterial culture, qPCR of bacterial DNA, and fluorescent in-situ hybridisation (FISH) to determine bacterial presence and load. Additionally, 16S rRNA sequencing of stool was used to confirm gut decontamination and subsequent FMT engraftment. RESULTS: Antibiotic treatment for 7 days was most effective in gut decontamination, as evidenced by absence of bacteria observed in culture, and reduced bacterial concentration, as determined by FISH as well as qPCR. Continued antibiotic administration had no further efficacy on gut decontamination from days 7 to 21. Following gut decontamination, 3 weekly doses of FMT was sufficient for the successful engraftment of donor microbiota in animals. The recolonised animal gut microbiota was similar in composition to the donor sample, and significantly different from the Sham controls as assessed by 16S rRNA sequencing. Importantly, this similarity in composition to the donor sample persisted for 5 weeks following the final FMT dose. CONCLUSIONS: Our results showed that 7 days of broad-spectrum antibiotics in drinking water followed by 3 weekly doses of FMT provides a simple, reliable, and cost-effective methodology for FMT in animal research.

The P4 Study: Postpartum Maternal and Infant Faecal Microbiome 6 Months After Hypertensive Versus Normotensive Pregnancy.

OBJECTIVE/HYPOTHESIS: To explore potential differences in faecal microbiome between women, and their infants, who had normotensive pregnancies (NP) and those who had a hypertensive pregnancy (HP), either gestational hypertension (GH) or preeclampsia (PE). METHODS: This is a sub study of P4 (Postpartum Physiology, Psychology, and Paediatrics Study) and includes 18 mother-infant pairs: 10 NP and 8 HP (HP as defined by blood pressure > 140/90mmHg; of which 6 had PE, and 2 GH), six months postpartum. The participating mothers collected stool samples from themselves and their infants. 16S rRNA V3-V4 amplicons were used to study the faecal microbiome. RESULTS: The sample of women and their infants were mostly primiparous (n =16) with vaginal birth (n = 14). At the time of faecal sampling 8 women were using hormonal contraception, and one HP woman remained on an antihypertensive. All women had blood pressure < 130/80mmHg, and 10 had high BMI (> 30). All infants had started solids, 8 were exclusively breastfed, 1 exclusively formula fed and 9 both. Three infants had been exposed to a course of antibiotics. Six months postpartum, there were no significant differences in alpha or beta diversity between the gut microbiota of HP and NP women (P > 0.05). However, a statistically significant difference was detected in alpha diversity between infants following HP and NP, with lower diversity levels in HP infants (P < 0.05). It was also found that at a genus and species level, the gut microbiota of HP women was enriched with Bifidobacterium and Bifididobacterium sp. and depleted in Barnisiella and Barnesiella intestinihominis when compared to NP women (P < 0.05). Similarly, the gut microbiota of infants born from HP was enriched in Streptococcus infantis and depleted in Sutterella, Sutterella sp., Bacteroides sp. and Clostridium aldenense compared to infants born from NP (P < 0.05). DISCUSSION: While our findings are at best preliminary, due to the very small sample size, they do suggest that the presence of hypertension in pregnancy may adversely affect the maternal microbiota postpartum, and that of their infants. Further analysis of postpartum microbiome data from future studies will be important to validate these early findings and provide further evidence about the changes in the microbiota in the offspring of women following hypertensive disorders of pregnancy (HDP), including possible links to the causes of long-term cardiovascular disease, the prevalence of which is increased in women who have experienced HDP.

Gut microbiota impact on the peripheral immune response in non-alcoholic fatty liver disease related hepatocellular carcinoma.

The gut microbiota is reported to modulate the immune response in hepatocellular carcinoma (HCC). Here, we employ metagenomic and metabolomic studies to characterise gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD) related cirrhosis, with or without HCC, and evaluate its effect on the peripheral immune response in an ex vivo model. We find that dysbiosis characterises the microbiota of patients with NAFLD-cirrhosis, with compositional and functional shifts occurring with HCC development. Gene function of the microbiota in NAFLD-HCC supports short chain fatty acid production, and this is confirmed by metabolomic studies. Ex vivo studies show that bacterial extracts from the NAFLD-HCC microbiota, but not from the control groups, elicit a T cell immunosuppressive phenotype, characterised by expansion of regulatory T cells and attenuation of CD8 + T cells. Our study suggest that the gut microbiota in NAFLD-HCC is characterised by a distinctive microbiome/metabolomic profile, and can modulate the peripheral immune response.

Microbiome Understanding in Maternity Study (MUMS), an Australian prospective longitudinal cohort study of maternal and infant microbiota: study protocol.

INTRODUCTION: Pregnancy induces significant physiological and cardiometabolic changes, and is associated with alterations in the maternal microbiota. Increasing rates of prepregnancy obesity, metabolic abnormalities and reduced physical activity, all impact negatively on the microbiota causing an imbalance between the commensal microorganisms (termed dysbiosis), which may drive complications, such as gestational diabetes or hypertensive disorders. Considerable work is needed to define the inter-relationships between the microbiome, nutrition, physical activity and pregnancy outcomes. The role of the microbiota during pregnancy remains unclear. The aim of the study is to define microbiota signatures longitudinally throughout pregnancy and the first year post birth, and to identify key clinical and environmental variables that shape the female microbiota profile during and following pregnancy. METHODS AND ANALYSIS: The Microbiome Understanding in Maternity Study (MUMS) is an Australian prospective longitudinal cohort study involving 100 mother-infant pairs. Women are enrolled in their first trimester and followed longitudinally. Assessment occurs at <13+0, 20+0-24+6 and 32+0-36+6 weeks gestation, birth and 6 weeks, 6 months and 12 months postpartum. At each assessment, self-collected oral, vaginal and faecal samples are collected with an additional postpartum skin swab and breastmilk sample. Each infant will have oral, faecal and skin swab samples collected. Measurements include anthropometrics, body composition, blood pressure, serum hormonal and metabolic parameters and vaginal pH. Dietary intake, physical activity and psychological state will be assessed using validated self-report questionnaires, and pregnancy and infant outcomes recorded. Parametric and non-parametric hypothesis tests will be used to test the association between high-risk and low-risk pregnancies and their outcomes. ETHICS AND DISSEMINATION: The study received the following approval: South Eastern Sydney Local Health District Research Ethics Committee (17/293 (HREC/17/POWH/605). Results will be made available to the participants of MUMS, their families and the funding bodies; in the form of a summary document. Results for the greater maternity care community and other researchers will be disseminated through conferences, local, national and international presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: ACTRN12618000471280 (prospectively registered).

Investigation into the effect of divergent feed efficiency phenotype on the bovine rumen microbiota across diet and breed.

The relationship between rumen microbiota and host feed efficiency phenotype, for genetically divergent beef cattle breeds is unclear. This is further exacerbated when different growth stages, chemically diverse diets and production systems are considered. Residual feed intake (RFI), a measure of feed efficiency, was calculated for individually fed Charolais (CH) and Holstein-Friesian (HF) steers during each of four 70-day (excluding adaptation) successive dietary phases: namely, high-concentrate, grass silage, fresh zero-grazed grass and high-concentrate again. Rumen fluid from the ten highest- (HRFI) and ten lowest-ranking (LRFI) animals for RFI, within breed, during each dietary phase was collected using a trans-oesophageal sampler and subjected to 16S rRNA amplicon sequencing and metabolic profiling. The datasets were analysed to identify microbial and rumen fermentation markers associated with RFI status. Age, dietary phase and breed were included in the statistical model. Within breed, for each dietary phase, mid-test metabolic weight and average daily gain did not differ (P > 0.05) between HRFI and LRFI steers; however, for the initial high-concentrate, grass silage, fresh grass herbage and final high-concentrate dietary phases, HRFI HF steers consumed 19, 23, 18 and 27% more (P < 0.001) than their LRFI counterparts. Corresponding percentages for CH HRFI compared to CH LRFI steers were 18, 23, 13 and 22%. Ten OTUs were associated with RFI (q < 0.05) independent of the other factors investigated. Of these Methanomassiliicoccaceae, Mogibacteriaceae and the genus p-75-a5 of Erysipelotrichaceae and were negatively associated (q < 0.05) with RFI. The results gave evidence that microbial species could potentially be an indicator of RFI in ruminants rather than broader microbiome metrics; however, further research is required to elucidate this association.

Investigating temporal microbial dynamics in the rumen of beef calves raised on two farms during early life.

Manipulation of the rumen microorganisms during early life has emerged as a promising strategy for persistent improvement of nutrient utilisation and lowering of enteric methanogenesis. However, limited understanding of the dynamics of rumen microbial colonisation has prevented the identification of the optimum timeframe for such interventions. The present study used DNA amplicon sequencing of the 16S rRNA gene to assess bacterial and archaeal dynamics in the rumen digesta of beef calves raised on two farms from birth through to post-weaning. The colonisation patterns of both communities were influenced by age (P < 0.05) and farm of origin (P < 0.05). The bacterial community exhibited an age-wise progression during the first month of life which appeared to be partly related to diet, and settled by day 21, indicating that this may mark the boundary of a timeframe for intervention. The archaeal community appeared less sensitive to age/diet than bacteria in the first month of life but was more sensitive to farm environment. These data show that ruminal microbial composition during early life is driven by calf age, diet and local environment, and provide important fundamental information concerning the ontogeny of the rumen microbiota from birth.

16S rRNA Sequencing Reveals Relationship Between Potent Cellulolytic Genera and Feed Efficiency in the Rumen of Bulls.

The rumen microbial population dictates the host's feed degradation capacity and subsequent nutrient supply. The rising global human population and intensifying demand for animal protein is creating environmental challenges. As a consequence, there is an increasing requirement for livestock with enhanced nutrient utilization capacity in order to more efficiently convert plant material to high quality edible muscle. In the current study, residual feed intake (RFI), a widely used and a highly accepted measure of feed efficiency in cattle, was calculated for a combination of three cohorts of Simmental bulls. All animals were managed similarly from birth and offered concentrate ad libitum in addition to 3 kg of grass silage daily during the finishing period. Solid and liquid rumen digesta samples collected at slaughter and were analyzed using amplicon sequencing targeting the 16S rRNA gene utilizing the Illumina MiSeq platform. Volatile fatty acid analysis was also conducted on the liquid digesta samples. Spearman's correlation coefficient was utilized to determine the association between RFI and bacterial and archaeal taxa and inter-taxonomic relationships. The data indicate a tendency toward an increase in butyrate (P = 0.06), which corresponds with an increase in plasma ß-hydroxybutyrate concentration in low RFI (LRFI) bulls in comparison to their high RFI (HRFI) contemporaries (P < 0.05). A decrease in propionate (P < 0.05) was also recorded in the rumen of LRFI in comparison to HRFI bulls. These results indicate alternate fermentation patterns in the rumen of LRFI bulls. The data also identified that OTUs within the phyla Tenericutes, Fibrobacteres, and Cyanobacteria may potentially influence RFI phenotype. In particular, a negative association between F. succinogenes and RFI was evident. The unique cellulolytic metabolism of F. succinogenes suggests it could contribute to host efficiency by providing substrate to the host ruminant and other microbial populations (e.g., Selenomonas ruminantium, Methanobrevibacter, and Methanomassiliicoccaceae) in the rumen. This study provides evidence that bacterial OTUs within common phyla could influence ruminant feed efficiency phenotype through their role in ruminal degradation of complex plant polysaccharides or increased capability to harvest nutrients from ingested feed.

Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations.

The rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICSTM). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3-V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman's correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol.

Plane of nutrition affects the phylogenetic diversity and relative abundance of transcriptionally active methanogens in the bovine rumen.

Methane generated during enteric fermentation in ruminant livestock species is a major contributor to global anthropogenic greenhouse gas emissions. A period of moderate feed restriction followed by ad libitum access to feed is widely applied in cattle management to exploit the animal's compensatory growth potential and reduce feed costs. In the present study, we utilised microbial RNA from rumen digesta samples to assess the phylogenetic diversity of transcriptionally active methanogens from feed-restricted and non-restricted animals. To determine the contribution of different rumen methanogens to methanogenesis during dietary restriction of cattle, we conducted high-throughput mcrA cDNA amplicon sequencing on an Illumina MiSeq and analysed both the abundance and phylogenetic origin of different mcrA cDNA sequences. When compared to their unrestricted contemporaries, in feed-restricted animals, the methanogenic activity, based on mcrA transcript abundance, of Methanobrevibacter gottschalkii clade increased while the methanogenic activity of the Methanobrevibacter ruminantium clade and members of the Methanomassiliicoccaceae family decreased. This study shows that the quantity of feed consumed can evoke large effects on the composition of methanogenically active species in the rumen of cattle. These data potentially have major implications for targeted CH4 mitigation approaches such as anti-methanogen vaccines and/or tailored dietary management.

The Structural and Functional Capacity of Ruminal and Cecal Microbiota in Growing Cattle Was Unaffected by Dietary Supplementation of Linseed Oil and Nitrate.

Microorganisms in the digestive tract of ruminants differ in their functionality and ability to use feed constituents. While cecal microbiota play an important role in post-rumen fermentation of residual substrates undigested in the rumen, limited knowledge exists regarding its structure and function. In this trial we investigated the effect of dietary supplementation with linseed oil and nitrate on methane emissions and on the structure of ruminal and cecal microbiota of growing bulls. Animals were allocated to either a CTL (control) or LINNIT (CTL supplemented with 1.9% linseed and 1.0% nitrates) diet. Methane emissions were measured using the GreenFeed system. Microbial diversity was assessed using amplicon sequencing of microbial genomic DNA. Additionally, total RNA was extracted from ruminal contents and functional mcrA and mtt genes were targeted in amplicon sequencing approach to explore the diversity of functional gene expression in methanogens. LINNIT had no effect on methane yield (g/kg DMI) even though it decreased methane production by 9% (g/day; P < 0.05). Methanobrevibacter- and Methanomassiliicoccaceae-related OTUs were more abundant in cecum (72 and 24%) compared to rumen (60 and 11%) irrespective of the diet (P < 0.05). Feeding LINNIT reduced the relative abundance of Methanomassiliicoccaceae mcrA cDNA reads in the rumen. Principal component analysis revealed significant differences in taxonomic composition and abundance of bacterial communities between rumen and cecum. Treatment decreased the relative abundance of a few Ruminococcaceae genera, without affecting global bacterial community structure. Our research confirms a high level of heterogeneity in species composition of microbial consortia in the main gastrointestinal compartments where feed is fermented in ruminants. There was a parallel between the lack of effect of LINNIT on ruminal and cecal microbial community structure and functions on one side and methane emission changes on the other. These results suggest that the sequencing strategy used here to study microbial diversity and function accurately reflected the absence of effect on methane phenotypes in bulls treated with linseed plus nitrate.

Do food blogs serve as a source of nutritionally balanced recipes? An analysis of 6 popular food blogs.

OBJECTIVE: To determine whether sampled food blogs provide nutritionally balanced recipes. METHODS: Two entree recipes per season, per year (2010-2011) were selected from 6 highly ranked food blogs (n = 96). Food Processor Nutrition and Fitness software was used to analyze sodium, saturated fat, and energy content. Analysis was separated by protein type (vegetarian, poultry, red meat, and seafood). RESULTS: Recipes met energy recommendations but were excessive in saturated fat and sodium. Vegetarian and seafood recipes were significantly lower in risk nutrients compared with red meat and poultry recipes. Red meat recipes were not significantly different from poultry recipes for risk nutrients studied; poultry recipes were higher in sodium and energy compared with red meat recipes. CONCLUSIONS AND IMPLICATIONS: The public should be aware of the nutritional limitations of popular food blogs; dietitians could assist in modifying blog recipes for individuals and partner with bloggers to improve the nutritional profile of recipes.