Saccharomyces cerevisiae derived postbiotic alters gut microbiome metabolism in the human distal colon resulting in immunomodulatory potential in vitro.

The yeast-based postbiotic EpiCor is a well-studied formulation, consisting of a complex mixture of bioactive molecules. In clinical studies, EpiCor postbiotic has been shown to reduce intestinal symptoms in a constipated population and support mucosal defense in healthy subjects. Anti-inflammatory potential and butyrogenic properties have been reported in vitro, suggesting a possible link between EpiCor's gut modulatory activity and immunomodulation. The current study used a standardized in vitro gut model, the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), to obtain a deeper understanding on host-microbiome interactions and potential microbiome modulation following repeated EpiCor administration. It was observed that EpiCor induced a functional shift in carbohydrate fermentation patterns in the proximal colon environment. Epicor promoted an increased abundance of Bifidobacterium in both the proximal and distal colon, affecting overall microbial community structure. Co-occurrence network analysis at the phylum level provided additional evidence of changes in the functional properties of microbial community promoted by EpiCor, increasing positive associations between Actinobacteria with microbes belonging to the Firmicutes phylum. These results, together with a significant increase in butyrate production provide additional support of EpiCor benefits to gut health. Investigation of host-microbiome interactions confirmed the immunomodulatory potential of the applied test product. Specific microbial alterations were observed in the distal colon, with metabotyping indicating that specific metabolic pathways, such as bile acid and tryptophan metabolism, were affected following EpiCor supplementation. These results, especially considering many effects were seen distally, further strengthen the position of EpiCor as a postbiotic with health promoting functionality in the gut, which could be further assessed in vivo.

A Randomized Controlled Trial to Evaluate the Impact of a Novel Probiotic and Nutraceutical Supplement on Pruritic Dermatitis and the Gut Microbiota in Privately Owned Dogs.

Pruritic dermatitis (PD) is a common presentation of canine allergic skin diseases, with diversity in severity and treatment response due to complex etiopathogenesis. Evidence suggests the gut microbiota (GM) may contribute to the development of canine allergies. A 10-week double-blind randomized controlled trial evaluated a novel probiotic and nutraceutical blend (PNB) on clinical signs of skin allergy, health measures, and the GM of privately owned self-reported pruritic dogs. A total of 105 dogs were enrolled, with 62 included in pruritus and health analysis and 50 in microbiome analysis. The PNB supported greater improvement of owner-assessed clinical signs of PD at week 2 than the placebo (PBO). More dogs that received the PNB shifted to normal pruritus (digital PVAS10-N: <2) by week 4, compared to week 7 for the PBO. While a placebo effect was identified, clinical differences were supported by changes in the GM. The PNB enriched three probiotic bacteria and reduced abundances of species associated with negative effects. The PBO group demonstrated increased abundances of pathogenic species and reduced abundances of several beneficial species. This trial supports the potential of the PNB as a supplemental intervention in the treatment of PD; however, further investigation is warranted, with stricter diagnostic criteria, disease biomarkers and direct veterinary examination.

Effect of Saccharomyces cerevisiae Postbiotics and Essential Oil on Growth Performance and Intestinal Health of Weanling Pigs During K88 ETEC Infection.

Enterotoxigenic Escherichia coli (ETEC) is one of the major bacterial infections, causing substantial economic losses globally in the swine industry. This study aimed to investigate the impact of low Saccharomyces cerevisiae fermentation postbiotics (SCFP), high SCFP, essential oil (EO), or their combination on the growth performance and health of weanling pigs during ETEC infection. Forty-eight male weanling pigs were randomly allocated to five groups: 1) control group (CON-basal diet, n = 16); 2) low SCFP group (LSC-basal diet + 1.25 g/kg SCFP, n = 8); 3) high SCFP group (HSC-basal diet + 2 g/kg SCFP, n = 8); 4) essential oil group (EO-basal diet + 0.4 g/kg EO, n = 8); 5) the SCFP and EO combination group (SE-basal diet + 1.25 g/kg SCFP + 0.4 g/kg EO, n = 8). On day 15 of the trial, pigs in CON were divided into positive control (PC) and negative control (NC), and all pigs, except in NC, were challenged with ETEC. Under the normal condition, dietary LSC, HSC, EO, and EO all increased average daily gain (ADG) (P < 0.05), and decreased F:G ratio (P < 0.05) accompanied by decreased malondialdehyde (MDA) and increases in catalase (CAT), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) indicating enhanced anti-oxidative capacity, as well as decreased IL-2, IL-8, INF-γ, indicating mitigated systemic inflammation. During ETEC infection, all treatments alleviated ETEC-induced ADG reduction, diarrhea, damages in intestinal permeability and morphology, and down-regulation of tight junctions (Claudin1, ZO-1, and Occludin), while HSC and EO exhibited additional protections. All treatments increased CAT, T-SOD, and T-AOC, and decreased MDA in serum and jejunal mucosa at similar degrees (P < 0.05). Moreover, all treatments alleviated ETEC-induced inflammation as shown by decreased IL-6, TNF-α, INF-γ, and increased IL-4 and IL-10 in serum or jejunal mucosa (P < 0.05), and enhanced the immunity by increased serum IgG and mucosal sIgA (P < 0.05). HSC and SE further reduced mucosal INF-γ and TNF-α than LSC or EO aligning with their additional protection against diarrhea during ETEC infection. Additionally, the key gut bacteria (e.g., Terrisporobacter) related to the benefits of SCFP and EO were identified. In sum, all treatments enhanced growth performance and protected against ETEC-induced intestinal damage through the regulation of redox and immune homeostasis. HSP and SE offered extra protection during disease for their additional control of inflammation. Our study provided new insight into the use of feed additives in the context of animal health states.

Weanling pigs are vulnerable to a variety of stressors and pathogen infections. Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea and growth retardation in weanling pigs. The postbiotics, Saccharomyces cerevisiae fermentation postbiotics (SCFP), and essential oil (EO, mainly thymol, and cinnamaldehyde) were reported to exert health benefits in different sites of the intestine. However, whether SCFP and EO have dose and synergistic effects on weanling pigs, especially against ETEC infection, is incompletely understood. Our research has revealed that SCFP, EO, and their combination all enhanced the growth performance and intestinal barrier function, and reduced diarrhea of piglets, albeit to varying degrees, under both health conditions and ETEC infection. We further elucidated the disparity in the regulation of redox and immune homeostasis by SCFP, EO, and their combination contributing to their different action in distinct states. This has led to a reevaluation of the function of additives in the context of gut health and disease susceptibility.

Saccharomyces cerevisiae fermentation product improves robustness of equine gut microbiome upon stress.

Introduction: Nutritional and environmental stressors can disturb the gut microbiome of horses which may ultimately decrease their health and performance. We hypothesized that supplementation with a yeast-derived postbiotic (Saccharomyces cerevisiae fermentation product-SCFP) would benefit horses undergoing an established model of stress due to prolonged transportation. Methods: Quarter horses (n = 20) were blocked based on sex, age (22 ± 3 mo) and body weight (439 ± 3 kg) and randomized to receive either a basal diet of 60% hay and 40% concentrate (CON) or the basal diet supplemented with 21 g/d Diamond V TruEquine C (SCFP; Diamond V, Cedar Rapids, IA) for 60 days. On day 57, horses were tethered with their heads elevated 35cm above wither height for 12 h to induce mild upper respiratory tract inflammation. Fecal samples were collected at days 0, 28, and 56 before induction of stress, and at 0, 12, 24, and 72 h post-stress and subjected to DNA extraction and Nanopore shotgun metagenomics. Within sample (alpha) diversity was evaluated by fitting a linear model and between sample (beta) diversity was tested with permutational ANOVA. Results: The SCFP stabilized alpha diversity across all time points, whereas CON horses had more fluctuation (P < 0.05) at 12, 24, and 72 h post-challenge compared to d 56. A significant difference between CON and SCFP was observed at 0 and 12 h. There was no difference in beta-diversity between SCFP and CON on d 56. Discussion: Taken together, these observations led us to conclude that treatment with SCFP resulted in more robust and stable microbial profiles in horses after stress challenge.

A Grain-Based SARA Challenge Affects the Composition of Epimural and Mucosa-Associated Bacterial Communities throughout the Digestive Tract of Dairy Cows.

The effects of a subacute ruminal acidosis (SARA) challenge on the composition of epimural and mucosa-associated bacterial communities throughout the digestive tract were determined in eight non-lactating Holstein cows. Treatments included feeding a control diet containing 19.6% dry matter (DM) starch and a SARA-challenge diet containing 33.3% DM starch for two days after a 4-day grain step-up. Subsequently, epithelial samples from the rumen and mucosa samples from the duodenum, proximal, middle and distal jejunum, ileum, cecum and colon were collected. Extracted DNA from these samples were analyzed using MiSeq Illumina sequencing of the V4 region of the 16S rRNA gene. Distinct clustering patterns for each diet existed for all sites. The SARA challenge decreased microbial diversity at all sites, with the exception of the middle jejunum. The SARA challenge also affected the relative abundances of several major phyla and genera at all sites but the magnitude of these effects differed among sites. In the rumen and colon, the largest effects were an increase in the relative abundance of Firmicutes and a reduction of Bacteroidetes. In the small intestine, the largest effect was an increase in the relative abundance of Actinobacteria. The grain-based SARA challenge conducted in this study did not only affect the composition and cause dysbiosis of epimural microbiota in the rumen, it also affected the mucosa-associated microbiota in the intestines. To assess the extent of this dysbiosis, its effects on the functionality of these microbiota must be determined in future.

Repeatability and reproducibility assessment in a large-scale population-based microbiota study: case study on human milk microbiota.

BACKGROUND: Quality control including assessment of batch variabilities and confirmation of repeatability and reproducibility are integral component of high throughput omics studies including microbiome research. Batch effects can mask true biological results and/or result in irreproducible conclusions and interpretations. Low biomass samples in microbiome research are prone to reagent contamination; yet, quality control procedures for low biomass samples in large-scale microbiome studies are not well established. RESULTS: In this study, we have proposed a framework for an in-depth step-by-step approach to address this gap. The framework consists of three independent stages: (1) verification of sequencing accuracy by assessing technical repeatability and reproducibility of the results using mock communities and biological controls; (2) contaminant removal and batch variability correction by applying a two-tier strategy using statistical algorithms (e.g. decontam) followed by comparison of the data structure between batches; and (3) corroborating the repeatability and reproducibility of microbiome composition and downstream statistical analysis. Using this approach on the milk microbiota data from the CHILD Cohort generated in two batches (extracted and sequenced in 2016 and 2019), we were able to identify potential reagent contaminants that were missed with standard algorithms and substantially reduce contaminant-induced batch variability. Additionally, we confirmed the repeatability and reproducibility of our results in each batch before merging them for downstream analysis. CONCLUSION: This study provides important insight to advance quality control efforts in low biomass microbiome research. Within-study quality control that takes advantage of the data structure (i.e. differential prevalence of contaminants between batches) would enhance the overall reliability and reproducibility of research in this field. Video abstract.

Response of Microbial Community to Induced Failure of Anaerobic Digesters Through Overloading With Propionic Acid Followed by Process Recovery.

In order to effectively use microbial-based strategies to manage anaerobic digesters, it is necessary to distinguish between community shifts that are part of the natural dynamic of the system and shifts caused by environmental or operational disturbances. The objective of this research study was to evaluate the significance of changes in the microbial community of anaerobic digesters during failure in correlation to operational parameters such as an organic acid overload. Five continuously stirred 0.5 L reactors were set-up as semi-continuously-fed, mesophilic dairy manure digesters with a 30-day hydraulic retention time. After a 120-day stabilization period, two digesters were kept as controls, while the organic loading rates in the triplicate set were increased step-wise to ultimately provide a shock-load leading to failure using propionic acid spikes. Acidosis resulting in near cessation of biogas and termination of methane production occurred between 4 and 7 weeks, after which all the digesters continued to be fed only dairy manure. The shock loading of propionic acid led to an accumulation of mainly acetate and propionate, with low levels of iso-butyrate, butyrate, iso-valerate, and valerate. High-throughput Illumina sequencing of the V4 region of the bacterial and archaeal 16S rRNA gene in digester samples showed a significant change in the microbial community composition during propionic acid overload, followed by a return to the original composition with regular feedstock. Bacterial genera whose relative abundance decreased during the inhibition stage included Sedimentibacter, Syntrophomonas, TSCOR003.O20, and Marinilabiaceae, while the relative abundance of Lachnospiraceae, Ruminococcus, Mogibacteriaceae, Pyramidobacter, and Bacteroides increased. The relative abundance of dominant methanogens, Methanosarcina and Methanobacterium, although initially resistant, were decreased (from 91.71 to 12.14% and from 2.98 to 0.73%, respectively) during inhibition, while Methanobrevibacter and Methanosphaera that were prominent in the manure feedstock increased from 17.36 to 79.45% and from 0.14 to 1.12%, respectively. Shifts in bacterial and archaeal compositions, back to their pre-shock steady state after failure, highlight the digester's microbial resilience and recovery potential.

Interrelationships of Fiber-Associated Anaerobic Fungi and Bacterial Communities in the Rumen of Bloated Cattle Grazing Alfalfa.

Frothy bloat is major digestive disorder of cattle grazing alfalfa pastures. Among the many factors identified to contribute to the development of frothy bloat, the disruption of rumen microbiota appears to be of central importance. Anaerobic rumen fungi (ARF) play an important role in sequential breakdown and fermentation of plant polysaccharides and promote the physical disruption of plant cell walls. In the present study, we investigated the dynamics of ARF during the development of alfalfa-induced frothy bloat and in response to bloat preventive treatments. By sequencing the internal transcribed spacer （ITS1）region of metagenomic DNA from the solid fraction of rumen contents, we were able to identify eight distinct genera of ARF, including Neocallimastix, Caecomyces, Orpinomyces, Piromyces, Cyllamyces, Anaeromyces, Buwchfawromyces, and unclassified Neocallimastigaceae. Overall, transition of steers from a baseline hay diet to alfalfa pastures was associated with drastic changes in the composition of the fungal community, but the overall composition of ARF did not differ (p > 0.05) among bloated and non-bloated steers. A correlation network analysis of the proportion of ARF and ruminal bacterial communities identified hub fungal species that were negatively correlated with several bacterial species, suggesting the presence of inter-kingdom competition among these rumen microorganisms. Interestingly, the number of negative correlations among ARF and bacteria decreased with frothy bloat, indicating a potential disruption of normal microbial profiles within a bloated rumen ecosystem. A better understanding of fungal-bacterial interactions that differ among bloated and non-bloated rumen ecosystem could advance our understanding of the etiology of frothy bloat.

Biological observations in microbiota analysis are robust to the choice of 16S rRNA gene sequencing processing algorithm: case study on human milk microbiota.

BACKGROUND: In recent years, the microbiome field has undergone a shift from clustering-based methods of operational taxonomic unit (OTU) designation based on sequence similarity to denoising algorithms that identify exact amplicon sequence variants (ASVs), and methods to identify contaminating bacterial DNA sequences from low biomass samples have been developed. Although these methods improve accuracy when analyzing mock communities, their impact on real samples and downstream analysis of biological associations is less clear. RESULTS: Here, we re-processed our recently published milk microbiota data using Qiime1 to identify OTUs, and Qiime2 to identify ASVs, with or without contaminant removal using decontam. Qiime2 resolved the mock community more accurately, primarily because Qiime1 failed to detect Lactobacillus. Qiime2 also considerably reduced the average number of ASVs detected in human milk samples (364 ± 145 OTUs vs. 170 ± 73 ASVs, p < 0.001). Compared to the richness, the estimated diversity measures had a similar range using both methods albeit statistically different (inverse Simpson index: 14.3 ± 8.5 vs. 15.6 ± 8.7, p = 0.031) and there was strong consistency and agreement for the relative abundances of the most abundant bacterial taxa, including Staphylococcaceae and Streptococcaceae. One notable exception was Oxalobacteriaceae, which was overrepresented using Qiime1 regardless of contaminant removal. Downstream statistical analyses were not impacted by the choice of algorithm in terms of the direction, strength, and significance of associations of host factors with bacterial diversity and overall community composition. CONCLUSION: Overall, the biological observations and conclusions were robust to the choice of the sequencing processing methods and contaminant removal.

Denosumab Regulates Gut Microbiota Composition and Cytokines in Dinitrobenzene Sulfonic Acid (DNBS)-Experimental Colitis.

The pro-inflammatory mediator receptor activator of nuclear factor-kappa B ligand (RANKL) plays a significant role in the development of rheumatoid arthritis; however, its role in inflammatory bowel disease is unknown. Genome-wide association meta-analysis for Crohn's disease (CD) identified a variant near the TNFSF11 gene that encodes RANKL and CD risk allele increased expression of RANKL in specific cell lines. This study aims to elucidate if the RANKL inhibitor denosumab can reduce the severity of experimental colitis and modify the gut microbiota composition using murine dinitrobenzenesulfonic acid (DNBS)-experimental model of colitis mimicking CD. In colitic conditions, denosumab treatment significantly decreased the pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α within the colonic mucosa. Moreover, colitis was accompanied by disruption of gut microbiota, and preventative treatment with denosumab modulated this disruption. Denosumab treatment also modified the alpha- and beta diversity of colonic mucosa and fecal microbiota. These results provide a rationale for considering denosumab as a future potential therapy in CD; however, more detailed experimental and clinical studies are warranted.

Saccharomyces cerevisiae fermentation products (SCFP) stabilize the ruminal microbiota of lactating dairy cows during periods of a depressed rumen pH.

BACKGROUND: Effects of Saccharomyces cerevisiae fermentation products (SCFP) on rumen microbiota were determined in vitro and in vivo under a high and a depressed pH. The in vitro trial determined the effects of Original XPC and NutriTek (Diamond V, Cedar Rapids, IA) at doses of 1.67 and 2.33 g/L, respectively, on the abundances of rumen bacteria under a high pH (> 6.3) and a depressed pH (5.8-6.0) using quantitative PCR (qPCR). In the in vivo trial eight rumen-cannulated lactating dairy cows were used in a cross-over design. Cows were randomly assigned to SCFP treatments (Original XPC, Diamond V, Cedar Rapids, IA) or control (No SCFP) before two 5-week experimental periods. During the second period, SCFP treatments were reversed. Cows on the SCFP treatment were supplemented with 14 g/d of SCFP and 126 g/d of ground corn. Other cows received 140 g/d ground corn. During the first 4 wk. of each period, cows received a basal diet containing 153 g/kg of starch. During week 5 of both periods, the rumen pH was depressed by a SARA challenge. This included replacing 208 g/kg of the basal diet with pellets of ground wheat and barley, resulting in a diet that contained 222 g/kg DM of starch. Microbial communities in rumen liquid digesta were examined by pyrosequencing, qPCR, and shotgun metagenomics. RESULTS: During the in vitro experiment, XPC and NutriTek increased the relative abundances of Ruminococcus flavefaciens, and Fibrobacter succinogenes determined at both the high and the depressed pH, with NutriTek having the largest effect. The relative abundances of Prevotella brevis, R. flavefaciens, ciliate protozoa, and Bifidobacterium spp. were increased by XPC in vivo. Adverse impacts of the in vivo SARA challenge included reductions of the richness and diversity of the rumen microbial community, the abundances of Bacteroidetes and ciliate protozoa in the rumen as determined by pyrosequencing, and the predicted functionality of rumen microbiota as determined by shotgun metagenomics. These reductions were attenuated by XPC supplementation. CONCLUSIONS: The negative effects of grain-based SARA challenges on the composition and predicted functionality of rumen microbiota are attenuated by supplementation with SCFP.

Breastmilk Feeding Practices Are Associated with the Co-Occurrence of Bacteria in Mothers' Milk and the Infant Gut: the CHILD Cohort Study.

Gut microbiota play a critical role in infant health. It is now accepted that breastmilk contains live bacteria from endogenous and exogenous sources, but it remains unclear whether these bacteria transfer to the infant gut and whether this process is influenced by breastmilk feeding practices. Here, we show that certain bacteria, including Streptococcus spp. and Veillonella dispar, co-occur in mothers' milk and their infants' stool, and co-occurrence is reduced when infants receive pumped breastmilk. The relative abundances of commonly shared species are positively correlated between breastmilk and stool. Overall, gut microbiota composition is strongly associated with breastfeeding exclusivity and duration but not breastmilk feeding mode (nursing versus pumping). Moreover, breastmilk bacteria contributed to overall gut microbiota variation to a similar extent as other modifiers of the infant microbiome, such as birth mode. These results provide evidence that breastmilk may transfer bacteria to the infant gut and influence microbiota development.

Human milk fungi: environmental determinants and inter-kingdom associations with milk bacteria in the CHILD Cohort Study.

BACKGROUND: Fungi constitute an important yet frequently neglected component of the human microbiota with a possible role in health and disease. Fungi and bacteria colonise the infant gastrointestinal tract in parallel, yet most infant microbiome studies have ignored fungi. Milk is a source of diverse and viable bacteria, but few studies have assessed the diversity of fungi in human milk. RESULTS: Here we profiled mycobiota in milk from 271 mothers in the CHILD birth cohort and detected fungi in 58 (21.4%). Samples containing detectable fungi were dominated by Candida, Alternaria, and Rhodotorula, and had lower concentrations of two human milk oligosaccharides (disialyllacto-N-tetraose and lacto-N-hexaose). The presence of milk fungi was associated with multiple outdoor environmental features (city, population density, and season), maternal atopy, and early-life antibiotic exposure. In addition, despite a strong positive correlation between bacterial and fungal richness, there was a co-exclusion pattern between the most abundant fungus (Candida) and most of the core bacterial genera. CONCLUSION: We profiled human milk mycobiota in a well-characterised cohort of mother-infant dyads and provide evidence of possible host-environment interactions in fungal inoculation. Further research is required to establish the role of breastfeeding in delivering fungi to the developing infant, and to assess the health impact of the milk microbiota in its entirety, including both bacterial and fungal components.

Composition and co-occurrence patterns of the microbiota of different niches of the bovine mammary gland: potential associations with mastitis susceptibility, udder inflammation, and teat-end hyperkeratosis.

BACKGROUND: Within complex microbial ecosystems, microbe-microbe interrelationships play crucial roles in determining functional properties such as metabolic potential, stability and colonization resistance. In dairy cows, microbes inhabiting different ecological niches of the udder may have the potential to interact with mastitis pathogens and therefore modulate susceptibility to intramammary infection. In the present study, we investigated the co-occurrence patterns of bacterial communities within and between different niches of the bovine mammary gland (teat canal vs. milk) in order to identify key bacterial taxa and evaluate their associations with udder health parameters and mastitis susceptibility. RESULTS: Overall, teat canal microbiota was more diverse, phylogenetically less dispersed, and compositionally distinct from milk microbiota. This, coupled with identification of a large number of bacterial taxa that were exclusive to the teat canal microbiota suggested that the intramammary ecosystem, represented by the milk microbiota, acts as a selective medium that disfavors the growth of certain environmental bacterial lineages. We further observed that the diversity of milk microbiota was negatively correlated with udder inflammation. By performing correlation network analysis, we identified two groups of phylogenetically distinct hub species that were either positively (unclassified Bacteroidaceae and Phascolarctobacterium) or negatively (Sphingobacterium) correlated with biodiversity metrics of the mammary gland (MG). The latter group of bacteria also showed positive associations with the future incidence of clinical mastitis. CONCLUSIONS: Our results provide novel insights into the composition and structure of bacterial communities inhabiting different niches of the bovine MG. In particular, we identified hub species and candidate foundation taxa that were associated with the inflammatory status of the MG and/or future incidences of clinical mastitis. Further in vitro and in vivo interrogations of MG microbiota can shed light on different mechanisms by which commensal microbiota interact with mastitis pathogens and modulate udder homeostasis.

The Manitoba Personalized Lifestyle Research (TMPLR) study protocol: a multicentre bidirectional observational cohort study with administrative health record linkage investigating the interactions between lifestyle and health in Manitoba, Canada.

INTRODUCTION: Lifestyle factors, such as diet, physical activity and sleep, are associated with the development of many chronic diseases. The objective of The Manitoba Personalized Lifestyle Research study is to understand how these lifestyle factors interact with each other and with other factors, such as an individual's genetics and gut microbiome, to influence health. METHODS: An observational study of adults, with extensive phenotyping by objective health and lifestyle assessments, and retrospective assessment of early life experiences, with retrospective and prospective utilisation of secondary data from administrative health records. STUDY POPULATION: A planned non-random convenience sample of 840 Manitobans aged 30-46 recruited from the general population, stratified by sex (equal men and women), body mass index (BMI; 60% of participants with a BMI>25 kg/m2) and geography (25% from rural areas). These stratifications were selected based on Manitoba demographics. MEASUREMENTS: Lifestyle factors assessed will include dietary pattern, physical activity, cardiovascular fitness, and sleep. Factors such as medical history, socioeconomic status, alcohol and tobacco consumption, cognition, stress, anxiety, and early life experiences will also be documented. A maternal survey will be performed. Body composition and bone density will be measured by dual energy X-ray absorptiometry. Blood pressure, pulse wave velocity, and augmentation index will be measured on two consecutive days. Chronic disease risk biomarkers will be measured in blood and urine samples. DNA will be extracted for genetic analysis. A faecal sample will be collected for microbiome analysis. Participants may provide their Manitoba personal health information number to link their study data with administrative health records. ETHICS AND DISSEMINATION: Ethics approval has been obtained from the University of Manitoba Health Research Ethics Board (protocol # HS18951; 05/01/2016). Data analysis, release of results and publication of manuscripts are scheduled to start in early 2019. Additional information at www.TMPLR.ca. TRIAL REGISTRATION NUMBER: NCT03674957; Pre-results.

Integrated Analysis of Human Milk Microbiota With Oligosaccharides and Fatty Acids in the CHILD Cohort.

Background: Human milk contains many bioactive components that are typically studied in isolation, including bacteria. We performed an integrated analysis of human milk oligosaccharides and fatty acids to explore their associations with milk microbiota. Methods: We studied a sub-sample of 393 mothers in the CHILD birth cohort. Milk was collected at 3-4 months postpartum. Microbiota was analyzed by 16S rRNA gene V4 sequencing. Oligosaccharides and fatty acids were analyzed by rapid high-throughput high performance and gas liquid chromatography, respectively. Dimension reduction was performed with principal component analysis for oligosaccharides and fatty acids. Center log-ratio transformation was applied to all three components. Associations between components were assessed using Spearman rank correlation, network visualization, multivariable linear regression, redundancy analysis, and structural equation modeling. P-values were adjusted for multiple comparisons. Key covariates were considered, including fucosyltransferase-2 (FUT2) secretor status of mother and infant, method of feeding (direct breastfeeding or pumped breast milk), and maternal fish oil supplement use. Results: Overall, correlations were strongest between milk components of the same type. For example, FUT2-dependent HMOs were positively correlated with each other, and Staphylococcus was negatively correlated with other core taxa. Some associations were also observed between components of different types. Using redundancy analysis and structural equation modeling, the overall milk fatty acid profile was significantly associated with milk microbiota composition. In addition, some individual fatty acids [22:6n3 (docosahexaenoic acid), 22:5n3, 20:5n3, 17:0, 18:0] and oligosaccharides (fucosyl-lacto-N-hexaose, lacto-N-hexaose, lacto-N-fucopentaose I) were associated with microbiota α diversity, while others (C18:0, 3'-sialyllactose, disialyl-lacto-N-tetraose) were associated with overall microbiota composition. Only a few significant associations between individual HMOs and microbiota were observed; notably, among mothers using breast pumps, Bifidobacterium prevalence was associated with lower abundances of disialyl-lacto-N-hexaose. Additionally, among non-secretor mothers, Staphylococcus was positively correlated with sialylated HMOs. Conclusion: Using multiple approaches to integrate and analyse milk microbiota, oligosaccharides, and fatty acids, we observed several associations between different milk components and microbiota, some of which were modified by secretor status and/or breastfeeding practices. Additional research is needed to further validate and mechanistically characterize these associations and determine their relevance to infant gut and respiratory microbiota development and health.

Impact of Saskatoon berry powder on insulin resistance and relationship with intestinal microbiota in high fat-high sucrose diet-induced obese mice.

The present study examined the impact of Saskatoon berry powder (SBp) on insulin resistance, inflammation and intestinal microbiota in diet-induced obese mice. Male C57 BL/6 J mice were fed control diet, high fat-high sucrose (HFHS) diet or HFHS+5% SBp (HFHS+B) diet for 15 weeks. The composition of fecal bacterial community was characterized using the Illumina sequencing of V4 region of 16S rRNA gene. HFHS diet increased body weight, fasting plasma glucose, cholesterol, triglycerides, insulin, homeostatic model assessment-insulin resistance, monocyte adhesion, tumor necrosis factor-α, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, intracellular cell adhesion molecule-1, urokinase plasminogen activator and its receptor in plasma or aortae compared to the control diet. HFHS+B diet postponed the increase in body weight, suppressed HFHS diet-induced disorders in the metabolic and inflammatory variables. The ratio of Firmicutes/Bacteroidetes in the HFHS group was higher than that in the control group (P<.01), and that in the HFHS+B group was lower than that in the HFHS group (P<.05). The abundances of S24-7 family negatively correlated with body weight and tested metabolic or inflammatory variables. The results suggest that SBp attenuated HFHS diet-induced metabolic disorders and vascular inflammation in gut microbiota in mice.

Detection of fecal bacteria and antibiotic resistance genes in drinking water collected from three First Nations communities in Manitoba, Canada.

This study analyzed the microbiological quality of drinking and source water from three First Nations communities in Manitoba, Canada that vary with respect to the source, storage and distribution of drinking water. Community A relies on an aquifer and Community B on a lake as source water to their water treatment plants. Community C does not have a water treatment plant and uses well water. Quantification of free residual chlorine and fecal bacterial (E. coli and coliforms), as well as detection of antibiotic resistance genes (sul, ampC, tet(A), mecA, vanA, blaSHV, blaTEM, blaCTX-M, blaOXA-1, blaCYM-2, blaKPC, blaOXA-48, blaNDM, blaVIM, blaGES and blaIMP) was carried out. While water treatment plants were found to be working properly, as post-treatment water did not contain E. coli or coliforms, once water entered the distribution system, a decline in the chlorine concentration with a concomitant increase in bacterial counts was observed. In particular, water samples from cisterns not only contained high number of E. coli and coliforms, but were also found to contain antibiotic resistance genes. This work shows that proper maintenance of the distribution and storage systems in First Nations communities is essential in order to provide access to clean and safe drinking water.

Feeding practice influences gut microbiome composition in very low birth weight preterm infants and the association with oxidative stress: A prospective cohort study.

Knowledge about the development of the preterm infant gut microbiota is emerging and is critical to their health. Very-low-birth-weight (VLBW; birth weight, <1500 g) infants usually have special dietary needs while showing increased oxidative stress related to intensive care. This prospective cohort study assessed the effect of feeding practice on gut microbiome development and oxidative stress in preterm infants. Fecal samples were collected from each infant in the early (1-2 weeks of enteral feeding) and late (2-4 weeks of enteral feeding) feeding stages. We performed high-throughput sequencing of V3-V4 regions of the 16S rRNA gene to analyze the fecal microbiome composition of 20 VLBW preterm infants and to determine the association of gut bacterial composition with feeding practice using an oxidative stress marker (urinary F2-isoprostane). Our results showed that feeding practices in the late stage significantly influenced the gut microbiome composition and oxidative stress in preterm infants. Preterm infants fed human milk + human milk fortifier and only formula diets showed a significant increase in F2-isoprostane levels (P < 0.05) compared with those fed human milk + formula diet. The gut microbiome of the infants fed the human milk + Human milk fortifier diet showed the lower relative abundance of Veillonella (P < 0.05) compared with that of the infants fed the human milk + formula diet. The gut microbiome of the infants fed the only formula diet showed the lowest microbial diversity and the highest relative abundance of Terrisporobacter (P < 0.05) compared with the gut microbiome of the infants fed the other diets. Correlation network analysis showed that urinary F2-isoprostane level was positively correlated with Terrisporobacter and Enterobacteriaceae abundance (P < 0.05) in the preterm infants. In conclusion, these data suggest that feeding practice affects the bacterial diversity and composition in the gut microbiome and is associated with oxidative stress in VLBW preterm infants.

Composition and Variation of the Human Milk Microbiota Are Influenced by Maternal and Early-Life Factors.

Breastmilk contains a complex community of bacteria that may help seed the infant gut microbiota. The composition and determinants of milk microbiota are poorly understood. Among 393 mother-infant dyads from the CHILD cohort, we found that milk microbiota at 3-4 months postpartum was dominated by inversely correlated Proteobacteria and Firmicutes, and exhibited discrete compositional patterns. Milk microbiota composition and diversity were associated with maternal factors (BMI, parity, and mode of delivery), breastfeeding practices, and other milk components in a sex-specific manner. Causal modeling identified mode of breastfeeding as a key determinant of milk microbiota composition. Specifically, providing pumped breastmilk was consistently associated with multiple microbiota parameters including enrichment of potential pathogens and depletion of bifidobacteria. Further, these data support the retrograde inoculation hypothesis, whereby the infant oral cavity impacts the milk microbiota. Collectively, these results identify features and determinants of human milk microbiota composition, with potential implications for infant health and development.