Associations of Maternal Breastmilk microRNAs and Infant Obesity Status at 1 Year.

Infant consumption of human milk (HM) is associated with a reduced risk of overweight and obesity, but the reasons for this relationship are not completely understood. There is emerging evidence that micro RNAs (miRNAs) regulate infant development and metabolism, but the associations between HM miRNAs and infant growth remain poorly understood. We examined the relationship between HM miRNA consumption and infant obesity in 163 mother-infant dyads to determine (1) if miRNA profiles differentiate infants with obesity, and (2) if individual miRNAs accurately predicted infant obesity status at one year of age. Infant obesity was categorized as weight-for-length (WFL) Z scores or conditional weight gain (CWG) in the 95th percentile. HM miRNA profile was associated with infant age (r2 = 6.4%, p = 0.001), but not maternal obesity status (r2 = 1.5%, p = 0.87) or infant weight status (WFL Z-score) at birth (r2 = 0.6%, p = 0.4), 1 month (r2 = 0.5%, p = 0.6), or 4 months (r2 = 0.8%, p = 0.2). Nine HM miRNAs were associated with either 12-month CWG or 12-month WFL Z scores. Among these 9 miRNAs, miR-224-5p remained significant in a logistic regression model that accounted for additional demographic factors (estimate = -27.57, p = 0.004). These findings suggest involvement of HM miRNAs and particularly miR-224-5p in infant growth, warranting further investigation. To our knowledge, this is the largest study of HM miRNAs and early-life obesity and contributes to the understanding of the relationship between HM miRNAs and infant growth.

Changes in the type 2 diabetes gut mycobiome associate with metformin treatment across populations.

The human gut teems with a diverse ecosystem of microbes, yet non-bacterial portions of that community are overlooked in studies of metabolic diseases firmly linked to gut bacteria. Type 2 diabetes mellitus (T2D) is associated with compositional shifts in the gut bacterial microbiome and the mycobiome, the fungal portion of the microbiome. However, whether T2D and/or metformin treatment underpins fungal community changes is unresolved. To differentiate these effects, we curated a gut mycobiome cohort spanning 1,000 human samples across five countries and validated our findings in a murine experimental model. We use Bayesian multinomial logistic normal models to show that T2D and metformin both associate with shifts in the relative abundance of distinct gut fungi. T2D is associated with shifts in the Saccharomycetes and Sordariomycetes fungal classes, while the genera Fusarium and Tetrapisipora most consistently associate with metformin treatment. We confirmed the impact of metformin on individual gut fungi by administering metformin to healthy mice. Thus, metformin and T2D account for subtle, but significant and distinct variation in the gut mycobiome across human populations. This work highlights for the first time that metformin can confound associations of gut fungi with T2D and warrants the need to consider pharmaceutical interventions in investigations of linkages between metabolic diseases and gut microbial inhabitants. IMPORTANCE: This is the largest to-date multi-country cohort characterizing the human gut mycobiome, and the first to investigate potential perturbations in gut fungi from oral pharmaceutical treatment. We demonstrate the reproducible effects of metformin treatment on the human and murine gut mycobiome and highlight a need to consider metformin as a confounding factor in investigations between type 2 diabetes mellitus and the gut microbial ecosystem.

Investigating antibiotic free feed additives for growth promotion in poultry: effects on performance and microbiota.

The poultry industry is evolving towards antibiotic-free production to meet market demands and decelerate the increasing spread of the antimicrobial resistance. The growing need for antibiotic free products has challenged producers to decrease or completely stop using antimicrobials as feed supplements in broiler diet to improve feed efficiency, growth rate, and intestinal health. Natural feed additives (e.g., probiotics and phytobiotics) are promising alternatives to substitute antimicrobial growth promoters. The goal of our study was to characterize the effects of a Probiotic and an Essential Oils blend on broilers' performance and perform a time-series analysis to describe their excreta microbiome. A total of 320 Cobb 500 (1-day-old) chicks were raised for 21 d in 32 randomly allocated cages. Treatments consisted of 4 experimental diets: a basal diet, and a basal diet mixed with an Antibiotic (bacitracin methylene disalicylate), an essential oils blend (oregano oil, rosemary, and red pepper), or a Probiotic (Bacillus subtilis). Body weight (on 1, 10, and 21d), and feed intake (10d and 21d) were recorded and feed conversion ratio was calculated. Droppings were collected daily (1-21d) to characterize broilers' excreta microbiota by targeted sequencing of the bacterial 16S rRNA gene. The Probiotic significantly improved feed conversion ratio for starter phase 1 to 10d (P = 0.03), grower phase 10 to 21d (P = 0.05), and total period 1 to 21d (P = 0.01) compared to the Antibiotic. Feed supplements did not affect alpha diversity but did impact microbial beta diversity (P < 0.01). Age also impacted microbiome turnover as differences in alpha and beta diversity were detected. Furthermore, when compared to the basal diet, the probiotic and antibiotic significantly impacted relative abundance of Bifidobacterium (log2 fold change -1.44, P = 0.03), Intestinimonas (log2 fold change 0.560, P < 0.01) and Ligilactobacillus (log2 fold change -1.600, P < 0.01). Overall, Probiotic supplementation but not essential oils supplementation positively impacted broilers' growth performance by directly causing directional shifts in broilers' excreta microbiota structure.

Alterations of rumen and fecal microbiome in growing beef and dairy steers fed rumen-protected Capsicum oleoresin.

The microbiome has been linked to animal health and productivity, and thus, modulating animal microbiomes is becoming of increasing interest. Antimicrobial growth promoters (AGP) were once a common technology used to modulate the microbiome, but regulation and consumer pressure have decreased AGP use in food animals. One alternative to antimicrobial growth promoters are phytotherapeutics, compounds derived from plants. Capsaicin is a compound from the Capsicum genus, which includes chili peppers. Capsaicin has antimicrobial properties and could be used to manipulate the gastrointestinal microbiome of cattle. Both the rumen and fecal microbiomes are essential to cattle health and production, and modulation of either microbiome can affect both cattle health and productivity. We hypothesized that the addition of rumen-protected capsaicin to the diet of cattle would alter the composition of the fecal microbiome, but not the rumen microbiome. To determine the impact of rumen-protected capsaicin in cattle, four Holstein and four Angus steers were fed rumen-protected Capsicum oleoresin at 0 (Control), 5, 10, or 15 mg kg-1 diet dry matter. Cattle were fed in treatment groups in a 4 × 4 Latin Square design with a 21-d adaptation phase and a 7-d sample collection phase. Rumen samples were collected on day 22 at 0-, 2-, 6-, 12-, and 18-h post-feeding, and fecal swabs were collected on the last day of sample collection, day 28, within 1 h of feeding. Sequencing data of the 16s rRNA gene was analyzed using the dada2 pipeline and taxa were assigned using the SILVA database. No differences were observed in alpha diversity among fecal or rumen samples for either breed (P > 0.08) and no difference between groups was detected for either breed in rumen samples or for Angus steers in fecal samples (P > 0.42). There was a difference in beta diversity between treatments in fecal samples of Holstein steers (P < 0.01), however, a pairwise comparison of the treatment groups suggests no difference between treatments after adjusting for multiple comparisons. Therefore, we were unable to observe substantial overall variation in the rumen or fecal microbiomes of steers due to increasing concentrations of rumen-protected capsaicin. We do, however, see a trend toward increased concentrations of capsaicin influencing the fecal microbiome structure of Holstein steers despite this lack of significance.

The microbiome is the collection of microbes present in an animal's body and has been discovered to be directly connected to animal health and productivity. In production animals, such as feedlot cattle, the microbiome can be modulated by antimicrobials to promote growth, but increasing consumer pressure to reduce antimicrobial use has producers seeking alternatives. Capsaicin is a phytotherapeutic derived from chili peppers that can be used to modulate the microbiome due to its antimicrobial properties. Eight steers were fed rumen-protected Capsicum oleoresin to determine its effect on average daily gain. In addition, rumen and fecal samples were collected for microbiome testing. No differences were detected in the rumen microbiomes between cattle fed capsaicin (treatment) or those that received no capsaicin (control). While no overall effect was observed on the fecal microbiome of cattle fed different doses of capsaicin or control, we did observe changes in fecal beta diversity due to capsaicin treatment in Holstein steers fed greater doses. The fecal microbiome structure of Holsteins fed greater dosages of capsaicin differed from those fed control or low doses, as observed by the presence of two distinct clusters. This observation suggests an impact of greater doses of capsaicin treatment on microbiome structure.

Effect of Probiotic Supplementation on Intestinal Permeability in Overweight and Obesity: A Systematic Review of Randomized Controlled Trials and Animal Studies.

Overweight and obesity are associated with increased intestinal permeability, characterized by loss of gut epithelial integrity, resulting in unregulated passage of lipopolysaccharide (LPS) and other inflammatory triggers into circulation, i.e., metabolic endotoxemia. In obesity, shifts in the gut microbiome negatively impact intestinal permeability. Probiotics are an intervention that can target the gut microbiome by introducing beneficial microbial species, potentially restoring gut barrier integrity. Currently, the role of probiotic supplementation in ameliorating obesity- and overweight-associated increases in gut permeability has not been reviewed. This systematic review aimed to summarize findings from both animal and clinical studies that evaluated the effect of probiotic supplementation on obesity-induced impairment in intestinal permeability (International Prospective Register of Systematic Reviews, CRD42022363538). A literature search was conducted using PubMed (Medline), Web of Science, and CAB Direct from origin until August 2023 using keywords of intestinal permeability, overweight or obesity, and probiotic supplementation. Of 920 records, 26 eligible records were included, comprising 12 animal and 14 clinical studies. Clinical trials ranged from 3 to 26 wk and were mostly parallel-arm (n = 13) or crossover (n = 1) design. In both animal and clinical studies, plasma/serum LPS was the most common measure of intestinal permeability. Eleven of 12 animal studies reported a positive effect of probiotic supplementation in reducing intestinal permeability. However, results from clinical trials were inconsistent, with half reporting reductions in serum LPS and half reporting no differences after probiotic supplementation. Bifidobacterium, Lactobacillus, and Akkermansia emerged as the most common genera in probiotic formulations among the animal and clinical studies that yielded positive results, suggesting that specific bacteria may be more effective at reducing intestinal permeability and improving gut barrier function. However, better standardization of strain use, dosage, duration, and the delivery matrix is needed to fully understand the probiotic impact on intestinal permeability in individuals with overweight and obesity.

The Effects of Bifidobacterium Probiotic Supplementation on Blood Glucose: A Systematic Review and Meta-Analysis of Animal Models and Clinical Evidence.

Probiotic supplementation is a potential therapeutic for metabolic diseases, including obesity, metabolic syndrome (MetS), and type 2 diabetes (T2D), but most studies deliver multiple species of bacteria in addition to prebiotics or oral pharmaceuticals. This may contribute to conflicting evidence in existing meta-analyses of probiotics in these populations and warrants a systematic review of the literature to assess the contribution of a single probiotic genus to better understand the contribution of individual probiotics to modulate blood glucose. We conducted a systematic review and meta-analysis of animal studies and human randomized controlled trials (RCTs) to assess the effects of Bifidobacterium (BF) probiotic supplementation on markers of glycemia. In a meta-analysis of 6 RCTs, BF supplementation had no effect on fasting blood glucose {FBG; mean difference [MD] = -1.99 mg/dL [95% confidence interval (CI): -4.84, 0.86], P = 0.13}, and there were no subgroup differences between subjects with elevated FBG concentrations and normoglycemia. However, BF supplementation reduced FBG concentrations in a meta-analysis comprised of studies utilizing animal models of obesity, MetS, or T2D [n = 16; MD = -36.11 mg/dL (CI: -49.04, -23.18), P < 0.0001]. Translational gaps from animal to human trials include paucity of research in female animals, BF supplementation in subjects that were normoglycemic, and lack of methodologic reporting regarding probiotic viability and stability. More research is necessary to assess the effects of BF supplementation in human subjects with elevated FBG concentrations. Overall, there was consistent evidence of the efficacy of BF probiotics to reduce elevated FBG concentrations in animal models but not clinical trials, suggesting that BF alone may have minimal effects on glycemic control, may be more effective when combined with multiple probiotic species, or may be more effective in conditions of hyperglycemia rather than elevated FBG concentrations.

Changes in the Type 2 diabetes gut mycobiome associate with metformin treatment across populations.

The human gut teems with a diverse ecosystem of microbes, yet non-bacterial portions of that community are overlooked in studies of metabolic diseases firmly linked to gut bacteria. Type 2 diabetes mellitus (T2D) associates with compositional shifts in the gut bacterial microbiome and fungal mycobiome, but whether T2D and/or pharmaceutical treatments underpin the community change is unresolved. To differentiate these effects, we curated a gut mycobiome cohort to-date spanning 1,000 human samples across 5 countries and a murine experimental model. We use Bayesian multinomial logistic normal models to show that metformin and T2D both associate with shifts in the relative abundance of distinct gut fungi. T2D associates with shifts in the Saccharomycetes and Sordariomycetes fungal classes, while the genera Fusarium and Tetrapisipora most consistently associate with metformin treatment. We confirmed the impact of metformin on individual gut fungi by administering metformin to healthy mice. Thus, metformin and T2D account for subtle, but significant and distinct variation in the gut mycobiome across human populations. This work highlights for the first time that oral pharmaceuticals can confound associations of gut fungi with T2D and warrants the need to consider pharmaceutical interventions in investigations of linkages between metabolic diseases and gut microbial inhabitants.

Metformin modulates the gut microbiome in broiler breeder hens.

Broiler breeder hens, the parent stock of commercial broiler chickens, are genetically selected for rapid growth. Due to a longer production period and the focus of genetic selection on superior carcass traits in their progeny, these hens have the propensity to gain excess adipose tissue and exhibit severe ovarian dysfunction, a phenotype that is similar to human polycystic ovary syndrome (PCOS). Metformin is an antihyperglycemic drug approved for type 2 diabetes that is prescribed off-label for PCOS with benefits on metabolic and reproductive health. An additional effect of metformin treatments in humans is modulation of gut microbiome composition, hypothesized to benefit glucose sensitivity and systemic inflammation. The effects of dietary metformin supplementation in broiler breeder hens have not been investigated, thus we hypothesized that dietary metformin supplementation would alter the gut microbiome of broiler breeder hens. Broiler breeder hens were supplemented with metformin at four different levels (0, 25, 50, and 75 mg/kg body weight) from 25 to 65 weeks of age, and a subset of hens (n = 8-10 per treatment group) was randomly selected to undergo longitudinal microbiome profiling with 16S rRNA sequencing. Metformin impacted the microbial community composition in 75 mg/kg metformin compared to controls (adjusted PERMANOVA p = 0.0006) and an additional dose-dependent difference was observed between 25 mg/kg and 75 mg/kg (adjusted PERMANOVA p = 0.001) and between 50 mg/kg and 75 mg/kg (adjusted PERMANOVA p = 0.001) but not between 25 mg/kg and 50 mg/kg (adjusted PERMANOVA p = 0.863). There were few differences in the microbiome attributed to hen age, and metformin supplementation did not alter alpha diversity. Bacteria that were identified as differentially relatively abundant between 75 mg/kg metformin treatment and the control, and between metformin doses, included Ruminococcus and members of the Clostridia family that have been previously identified in human trials of PCOS. These results demonstrate that metformin impacts the microbiome of broiler breeder hens in a dose-dependent manner and several findings were consistent with PCOS in humans and with metformin treatment in type 2 diabetes. Metformin supplementation is a potentially promising option to improve gut health and reproductive efficiency in broiler breeder hens.

Quantifying the immediate response of the soil microbial community to different grazing intensities on irrigated pastures.

Grazing is known to affect soil microbial communities, nutrient cycling, and forage quantity and quality over time. However, a paucity of information exists for the immediate changes in the soil physicochemical and microbial environment in response to different grazing strategies. Soil microbes drive nutrient cycling and are involved in plant-soil-microbe relationships, making them potentially vulnerable to plant-driven changes in the soil environment caused by grazing. To test the hypothesis that variable grazing intensities modulate immediate effects on the soil microbial community, we conducted a grazing trial of three management approaches; high-intensity, short-duration grazing (HDG), low-intensity, medium-duration grazing (LDG), and no grazing (NG). Soil and vegetation samples were collected before grazing and 24 hours, 1 week, and 4 weeks after HDG grazing ended. Soil labile carbon (C) and nitrogen (N) pools, vegetation biomass, and soil microbial diversity and functional traits were determined, including extracellular enzymatic assays and high-throughput sequencing of the bacterial 16S rRNA and fungal ITS2 regions. We found that labile soil C and inorganic N increased following the LDG grazing while C-cycling extracellular enzymatic activities increased in response to HDG grazing but both total extracellular enzymatic activity profiles and soil abiotic profiles were mostly affected by temporal fluxes. The soil fungal community composition was strongly affected by the interaction of sampling time and grazing treatment, while the soil bacterial community composition was largely affected by sampling time with a lesser impact from grazing treatment. We identified several key fungal taxa that may influence immediate responses to grazing and modulate plant-soil-microbe interactions. There was strong evidence of temporal influences on soil biogeochemical variables and the soil microbiome, even within our narrow sampling scheme. Our results indicate that the soil ecosystem is dynamic and responsive to different grazing strategies within very short time scales, showing the need for further research to understand plant-soil-microbe interactions and how these feedback mechanisms can inform sustainable land management.

Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs.

As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.