Dietary Features Are Associated with Differences in the Urinary Microbiome in Clinically Healthy Adult Dogs.

Nutrition plays an important role in shaping the gut microbiome composition, although the impact of diet on the urinary microbiome (i.e., urobiome) remains unknown. The aim of this pilot study was to discover how nutritional features affect the diversity and composition of the urobiome in dogs. Dietary histories were obtained for 15 clinically healthy adult dogs, including limited nutrient (protein, fat, crude fiber), commercial diet brand, and dietary diversity profiles. The urine samples were collected via cystocentesis, followed by sequencing of the bacterial 16S rRNA gene. The data were analyzed to determine associations between major nutrients and dietary sources with the urobiome's composition. The protein, fat, and crude fiber contents had no statistically significant effect on the alpha or beta diversity. However, the beta diversity values differed (PERMANOVA; p = 0.017, R2 = 0.10) between dogs fed one commercial diet brand compared to dogs consuming any other brand. The beta diversity values also differed (p = 0.019, R2 = 0.10) between dogs consuming more diverse daily diets compared to those consuming less diverse diets (≥3 or <3 unique food sources, respectively). Overall, the results of this pilot study suggest that diet might impact the urobiome and support further exploration of the relationship between diet and the urobiome's composition in dogs.

Establishment of the uterine microbiome following artificial insemination in virgin heifers.

Introduction: The concept of a sterile uterus was challenged by recent studies that have described the microbiome of the virgin and pregnant uterus for species including humans and cattle. We designed two studies that tested whether the microbiome is introduced into the uterus when the virgin heifer is first inseminated and whether the origin of the microbiome is the vagina/cervix. Methods: The uterine microbiome was measured immediately before and after an artificial insemination (AI; Study 1; n = 7 AI and n = 6 control) and 14 d after insemination (Study 2; n = 12 AI and n = 12 control) in AI and non-AI (control) Holstein heifers. A third study (Study 3; n = 5 Holstein heifers) that included additional negative controls was subsequently conducted to support the presence of a unique microbiome within the uterus despite the low microbial biomass and regardless of insemination. Traditional bacteriological culture was performed in addition to 16S rRNA gene sequencing on the same samples to determine whether there were viable organisms in addition to those detected based on DNA sequencing (16S rRNA gene sequence). Results and discussion: Inseminating a heifer did not lead to a large change in the microbiome when assessed by traditional methods of bacterial culture or metataxonomic (16S rRNA gene) sequencing (results of Studies 1 and 2). Very few bacteria were cultured from the body or horn of the uterus regardless of whether an AI was or was not (negative control) performed. The cultured bacterial genera (e.g., Bacillus, Corynebacterium, Cutibacterium, Micrococcus, Staphylococcus, and Streptococcus) were typical of those found in the soil, environment, skin, mucous membranes, and urogenital tract of animals. Metataxonomic sequencing of 16S rRNA gene generated a large number of amplicon sequence variants (ASV), but these larger datasets that were based on DNA sequencing did not consistently demonstrate an effect of AI on the abundance of ASVs across all uterine locations compared with the external surface of the tract (e.g., perimetrium; positive control samples for environment contamination during slaughter and collection). Major genera identified by 16S rRNA gene sequencing overlapped with those identified with bacterial culture and included Cutibacterium, Staphylococcus, and Streptococcus.

The microbiome of the pregnant uterus in Holstein dairy heifers and cows assessed by bacterial culture and 16S ribosomal RNA gene sequencing.

Introduction: The possibility that there is a resident and stable commensal microbiome within the pregnant uterus has been supported and refuted by a series of recent studies. One element of most of the initial studies was that they were based primarily on 16S rRNA gene sequencing from bacteria. To account for this limitation, the current study performed both bacterial culture and 16S rRNA gene sequencing in a side-by-side manner (e.g., same tissues isolated from the same animal). Methods: The uteruses of 10 mid-pregnant (156 ± 5 d of gestation) Holstein heifers and cows were collected following slaughter. The external surface of the reproductive tract (positive control for contamination during tissue collection) as well as tissues within the pregnant uterus (placentome, inter-cotyledonary placenta, inter-caruncular endometrium, amnionic fluid, allantoic fluid, fetal abomasum content, and fetal meconium) were sampled for bacterial culture and 16S rRNA gene sequencing. Results: There were 87 unique bacterial species cultured from the external surface of the pregnant reproductive tract (contamination control) and 12 bacterial species cultured from pregnancy tissues. Six out of 10 cattle (60%) exhibited bacterial growth in at least one location within the pregnant uterus. For the metataxonomic results (16S rRNA gene sequencing), a low targeted microbial biomass was identified. Analyses of the detected amplicon sequence variants (ASV) revealed that there were: (1) genera that were prevalent on both the external surface and within the pregnant uterus; (2) genera that were prevalent on the external surface but either not detected or had very low prevalence within the pregnant uterus; and (3) genera that were either not detected or had low prevalence on the external surface but found with relatively high prevalence within the pregnant uterus. Conclusion: There are a small number of viable bacteria in the pregnant uterus. The 16S rRNA gene sequencing detected a microbial community within the pregnant uterus but with a low biomass. These results are consistent with recent studies of the pregnant bovine uterus and leave open the question of whether there is adequate microbial mass to significantly affect the biology of the normal healthy bovine pregnancy.

Metritis and the uterine disease microbiome are associated with long-term changes in the endometrium of dairy cows†.

Cows with metritis (uterine disease) during the first 1 to 2 wk postpartum have lower pregnancy rates when inseminated later postpartum (typically >10 wk). We hypothesized that metritis and the disease-associated uterine microbiome have a long-term effect on endometrial gene expression. Changes in gene expression may inform a mechanism through which disease lowers pregnancy rates. A total of 20 cows were enrolled at 1 to 2 wk postpartum to either metritis (clinical disease; n = 10) or healthy (control; n = 10) groups and randomly assigned to be slaughtered at approximately 80 d and 165 d postpartum (mid-lactation). The microbiome of the reproductive tract was sampled to confirm the presence of pathogens that are typical of metritis. In addition to the original clinical diagnosis, study cows were retrospectively assigned to uterine-disease and control groups based on the composition of their microbiome. There was no effect of early postpartum uterine disease on the uterine microbiome at mid-lactation (time of slaughter). Nonetheless, early postpartum metritis and the disease microbiome were associated with a large number of differentially-expressed genes at mid-lactation primarily in the caruncular compared with the inter-caruncular endometrium. Gene enrichment analysis identified oxidative phosphorylation as the primary pathway increased in caruncular endometrium of diseased cows whereas growth factor signaling pathways were reduced. The current study demonstrated that metritis and a uterine disease microbiome leave a sustained imprint on gene expression in the caruncular endometrium that may explain lower fertility in cows with postpartum uterine disease.

Multistrain probiotics fail to modulate the asthmatic phenotype, respiratory microbiota, and immune responses in cats.

OBJECTIVE: To determine if multistrain probiotics administered to asthmatic cats treated with anti-inflammatory glucocorticoids would attenuate the asthmatic phenotype and beneficially alter respiratory, blood, and oropharyngeal (OP) microbial communities and immune parameters versus placebo. ANIMALS: 13 client-owned asthmatic cats. METHODS: A randomized, blinded, placebo-controlled clinical trial of asthmatic cats receiving anti-inflammatory glucocorticoids with oral multistrain probiotics or placebo assessed owner-perceived improvement and airway eosinophilia at baseline and after 2 weeks of treatment. Bronchoalveolar lavage fluid (BALF), blood, OP, and rectal microbial communities were compared using 16S rRNA amplicon sequencing. Real-time PCR for transcription factors, activation markers and cytokines, and IgA ELISAs were evaluated. Statistical analyses used 2-way repeated-measures ANOVA or permutational ANOVA (significance, P < .05). RESULTS: After treatment, there were no significant differences in owner-perceived clinical signs or mean ± SEM BALF eosinophils between groups. There was a significant decrease in rectal α-diversity but not in α- or ß-diversity in BALF, blood, or OP between groups or over time. There were no significant differences in CD25, FoxP3, GATA, Helios, IL-4, IL-5, IL-10, IL-13, IL-17, IFN-γ mRNA, or serum or BALF IgA between groups or over time. CLINICAL RELEVANCE: In asthmatic cats, oral multistrain probiotics failed to improve owner-perceived signs, reduce airway eosinophilia, modify microbial community composition, or alter assessed immune responses versus placebo or over time. Longer treatment, different probiotic composition or delivery (eg, aerosolized), or larger number of cats would represent the next stages of study.

Timing of standard chow exposure determines the variability of mouse phenotypic outcomes and gut microbiota profile.

Standard chow diet contributes to reproducibility in animal model experiments since chows differ in nutrient composition, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here, we measured the impact of diet (5V5M, 5V0G, 2920X, and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE), and developmental exposure (DE)) on growth & development, metabolic health indicators, and gut bacterial microbiota profiles across genetically identical C57BL6/J mice. Diet drove differences in macro- and micronutrient intake for all exposure models. AE had no effect on measured outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight, and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. Diet-induced beta diversity profiles were sex-dependent for LE mice. Compared to AE, LE drove 9X more diet-driven differentially abundant genera, majority of which were the result of inverse effects of 2920X and 5058. Our findings demonstrate that lifetime exposure to different chow diets has the greatest impact on reproducibility of experimental measures that are common components of preclinical mouse model studies. Importantly, weaning DE mice onto a uniform diet is likely an effective way to reduce unwanted phenotypic variability among experimental models.

Systemic antibiotic treatment of cows with metritis early postpartum does not change the progression of uterine disease or the uterine microbiome at 1 month postpartum.

Background: Postpartum uterine disease (metritis) is common in dairy cows. The disease develops within 1 week after calving and is associated with microbial dysbiosis, fever, and fetid uterine discharge. Cows with metritis have a greater likelihood of developing endometritis and infertility later postpartum. Antibiotic treatment is used to relieve symptoms of metritis but the capacity of antibiotic treatment to improve fertility later postpartum is inconsistent across published studies. We hypothesized that an antibiotic has only a short-term effect on the uterine microbiome and does not change the progression of disease from metritis to endometritis. To test this hypothesis, we studied the effects of systemic antibiotic given to cows diagnosed with metritis and healthy cows early postpartum on the development of endometritis and the uterine microbiome at 1 month postpartum. Results: Cows diagnosed with metritis were compared to healthy ones in a 2 × 2 factorial design, where they were either treated with an antibiotic (ceftiofur hydrochloride) at 7 to 10 days postpartum or left untreated. Cows were slaughtered at one month postpartum and the uterus was assessed for endometritis (presence of purulent material in the uterine lumen and inflammation in the endometrium) and uterine samples were collected for bacteriology and metagenomics (16S rRNA gene sequencing). As expected, the uterine microbiome at disease diagnosis had dysbiosis of typical metritis pathogens (e.g., Fusobacterium, Bacteroides, and Porphyromonas) in diseased compared with healthy cows. At one month postpartum, there was a tendency for more endometritis in metritis cows compared with healthy but antibiotic treatment had no effect on endometritis prevalence regardless of the original disease diagnosis. Likewise, when bacteria were cultured or sequenced, there were a greater number of species (culture) or amplicon sequence variants (ASV; sequencing) in the uterine lumen of cows with metritis. However, antibiotic treatment had no effect on the prevalence of cultured species or the composition of the detected ASV. The uterine microbiome at 1 month postpartum was associated with the clinical observation of the uterus (endometritis or healthy). Conclusions: Early postpartum antibiotic treatment only provides temporary resolution of uterine dysbiosis that is not sustained long-term. Failure to resolve the dysbiosis is associated with a greater prevalence of endometritis in cows with metritis, and the occurrence of endometritis significantly impacts fertility later postpartum.

Effect size of delayed freezing, diurnal variation, and hindgut location on the mouse fecal microbiome.

Practical considerations in fecal sample collection for microbiome research include time to sample storage, time of collection, and hindgut position during terminal collections. Here, parallel experiments were performed to investigate the relative effect of these factors on microbiome composition in mice colonized with two different vendor-origin microbiomes. 16S rRNA amplicon sequencing of immediately flash-frozen feces showed no difference in alpha or beta diversity compared to samples incubated up to 9 h at room temperature. Samples collected in the morning showed greater alpha diversity compared to samples collected in the afternoon. While a significant effect of time was detected in all hindgut regions, the effect increased from cecum to distal colon. This study highlights common scenarios in microbiome research that may affect outcome measures of microbial community analysis. However, we demonstrate a relatively low effect size of these technical factors when compared to a primary experimental factor with large intergroup variability.

Temporal dynamics of the fecal microbiome in female pigs from early life through estrus, parturition, and weaning of the first litter of piglets.

BACKGROUND: Age-associated changes in the gastrointestinal microbiome of young pigs have been robustly described; however, the temporal dynamics of the fecal microbiome of the female pig from early life to first parity are not well understood. Our objective was to describe microbiome and antimicrobial resistance dynamics of the fecal microbiome of breeding sows from early life through estrus, parturition and weaning of the first litter of piglets (i.e., from 3 to 53 weeks of age). RESULTS: Our analysis revealed that fecal bacterial populations in developing gilts undergo changes consistent with major maturation milestones. As the pigs progressed towards first estrus, the fecal bacteriome shifted from Rikenellaceae RC9 gut group- and UCG-002-dominated enterotypes to Treponema- and Clostridium sensu stricto 1-dominated enterotypes. After first estrus, the fecal bacteriome stabilized, with minimal changes in enterotype transition and associated microbial diversity from estrus to parturition and subsequent weaning of first litter piglets. Unlike bacterial communities, fecal fungal communities exhibited low diversity with high inter- and intra-pig variability and an increased relative abundance of certain taxa at parturition, including Candida spp. Counts of resistant fecal bacteria also fluctuated over time, and were highest in early life and subsequently abated as the pigs progressed to adulthood. CONCLUSIONS: This study provides insights into how the fecal microbial community and antimicrobial resistance in female pigs change from three weeks of age throughout their first breeding lifetime. The fecal bacteriome enterotypes and diversity are found to be age-driven and established by the time of first estrus, with minimal changes observed during subsequent physiological stages, such as parturition and lactation, when compared to the earlier age-related shifts. The use of pigs as a model for humans is well-established, however, further studies are needed to understand how our results compare to the human microbiome dynamics. Our findings suggest that the fecal microbiome exhibited consistent changes across individual pigs and became more diverse with age, which is a beneficial characteristic for an animal model system.

Fecal dysbiosis and inflammation in intestinal-specific Cftr knockout mice on regimens preventing intestinal obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (ß diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice.NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice.

The effects of 6 wk of resistance training on the gut microbiome and cardiometabolic health in young adults with overweight and obesity.

Obesity is a known risk factor for the development of insulin resistance and other cardiometabolic disorders. Recently, the gut microbiome has been associated with obesity and subsequent health complications. Exercise has been regularly utilized as a therapeutic intervention to treat obesity and its associated comorbidities. This study examined the effects of a 6-wk resistance training exercise program (RT) on the diversity, composition, and metabolic pathways of the gut microbiome. Sedentary young adults (age 18-35 yr) with overweight and obesity (BMI 25-45 kg/m2) were recruited to participate in this randomized controlled trial. Participants were randomized to RT (n = 16), a 6-wk resistance training program (3 days/wk), or control (CT) (n = 16), a nonexercising control. Main outcomes of the study included gut microbiome measures (taxa abundances, diversity, and predicted function) and cardiometabolic outcomes [blood pressure (BP) and glucoregulation]. Increased abundances of Roseburia, a short-chain fatty acid (SCFA) producer were observed over 6 wk (W6) with RT compared with CT (group × week, P < 0.05, q < 0.25). RT also induced marginal alterations in predicted microbial metabolic and cell motility pathways compared with CT (group × week, P < 0.05, q < 0.25). However, RT did not significantly impact overall microbial diversity. Furthermore, RT resulted in higher quantitative insulin-sensitivity check index (QUICKI) and lower diastolic BP at W6 compared with CT [baseline (BL)-adjusted P < 0.05]. RT had mixed effects on the gut microbiome. Although RT increased abundances of Roseburia and induced minor changes in microbial pathways, it is important to consider these changes in the context of the overall stability observed in the microbiome composition.NEW & NOTEWORTHY Resistance training induces mixed changes in the gut microbiome, including an increase in the abundances of the Roseburia genus and minor alterations in microbial pathways. However, it is vital to interpret these changes in light of the broader context, where we observe stability in the overall microbiome composition. This stability may be attributed to the microbiome's resilience, demonstrating its capacity to withstand short-term physiological stressors.

Field study examining the mucosal microbiome in equine glandular gastric disease.

Equine glandular gastric disease (EGGD) is a common disease among athletic horses that can negatively impact health and performance. The pathophysiology of this EGGD remains poorly understood. Previous studies using controlled populations of horses identified differences in the gastric glandular mucosal microbiome associated with disease. The objective of this study was to compare the gastric microbiome in horses with EGGD and those without across multiple barns and differing management practices. We hypothesized that alterations in the microbiome of the gastric glandular mucosa are associated with EGGD. A secondary objective was to perform a risk factor analysis for EGGD using the diet and management data collected. Microbial populations of biopsies from normal pyloric mucosa of horses without EGGD (control biopsies), normal pyloric mucosa of horses with EGGD (normal biopsies) and areas of glandular mucosal disruption in horses with EGGD (lesion biopsies) were compared. Lesion biopsies had a different microbial community structure than control biopsies. Control biopsies had a higher read count for the phylum Actinomycetota compared to lesion biopsies. Control biopsies also had an enrichment of the genera Staphylococcus and Lawsonella and the species Streptococcus salivarius. Lesion biopsies had an enrichment of the genera Lactobacillus and Actinobacillus and the species Lactobacillus equigenerosi. These results demonstrate differences in the gastric glandular microbiome between sites of disrupted mucosa in horses with EGGD compared to pyloric mucosa of horses without EGGD. Risk factor analysis indicated that exercise duration per week was a risk factor for EGGD.

Effect of Sugar Beet Pulp on the Composition and Predicted Function of Equine Fecal Microbiota.

The purpose of this study is to determine the effect of the partial replacement of dietary hay with sugar beet pulp (SBP) on the composition and predicted function of the fecal microbiota of healthy adult horses. Fecal samples were collected daily for 12 days from six adult horses after removal from pasture, including a five-day acclimation period, and a seven-day period following the introduction of SBP into their diet, and compared to six untreated horses over a comparable period. Fecal DNA was subjected to 16S rRNA amplicon sequencing and a longitudinal analysis was performed comparing the composition and predicted function. While no significant treatment-associated changes in the richness, alpha diversity, or beta diversity were detected, random forest regression identified several high-importance taxonomic features associated with change over time in horses receiving SBP. A similar analysis of the predicted functional pathways identified several high-importance pathways, including those involved in the production of L-methionine and butyrate. These data suggest that feeding SBP to healthy adult horses acutely increases the relative abundance of several Gram-positive taxa, including Cellulosilyticum sp., Moryella sp., and Weissella sp., and mitigates the predicted functional changes associated with removal from pasture. Large-scale studies are needed to assess the protective effect of SBP on the incidence of the gastrointestinal conditions of horses.

The respiratory microbiota and its impact on health and disease in dogs and cats: A One Health perspective.

Healthy lungs were long thought of as sterile, with presence of bacteria identified by culture representing contamination. Recent advances in metagenomics have refuted this belief by detecting rich, diverse, and complex microbial communities in the healthy lower airways of many species, albeit at low concentrations. Although research has only begun to investigate causality and potential mechanisms, alterations in these microbial communities (known as dysbiosis) have been described in association with inflammatory, infectious, and neoplastic respiratory diseases in humans. Similar studies in dogs and cats are scarce. The microbial communities in the respiratory tract are linked to distant microbial communities such as in the gut (ie, the gut-lung axis), allowing interplay of microbes and microbial products in health and disease. This review summarizes considerations for studying local microbial communities, key features of the respiratory microbiota and its role in the gut-lung axis, current understanding of the healthy respiratory microbiota, and examples of dysbiosis in selected respiratory diseases of dogs and cats.

The contribution of maternal oral, vaginal, and gut microbiota to the developing offspring gut.

There is limited understanding of how the microbiota colonizing various maternal tissues contribute to the development of the neonatal gut microbiota (GM). To determine the contribution of various maternal microbiotic sites to the offspring microbiota in the upper and lower gastrointestinal tract (GIT) during early life, litters of mice were sacrificed at 7, 9, 10, 11, 12, 14, and 21 days of age, and fecal and ileal samples were collected. Dams were euthanized alongside their pups, and oral, vaginal, ileal, and fecal samples were collected. This was done in parallel using mice with either a low-richness or high-richness microbiota to assess the consistency of findings across multiple microbial compositions. Samples were analyzed using 16S rRNA amplicon sequencing. The compositional similarity between pup and dam samples were used to determine the contribution of each maternal source to the composition of the neonate fecal and ileal samples at each timepoint. As expected, similarity between neonate and maternal feces increased significantly over time. During earlier time-points however, the offspring fecal and ileal microbiotas were closer in composition to the maternal oral microbiota than other maternal sites. Prominent taxa contributed by the maternal oral microbiota to the neonate GM were supplier-dependent and included Lactobacillus spp., Streptococcus spp., and a member of the Pasteurellaceae family. These findings align with the microbial taxa reported in infant microbiotas, highlighting the translatability of mouse models in this regard, as well as the dynamic nature of the GM during early life.

The effect of physical and psychological stress on the oral microbiome.

Background: The oral microbiome is incredibly complex, containing a diverse complement of microbiota that has previously been categorized into 6 broad phyla. While techniques such as next-generation sequencing have contributed to a better understanding of the composition of the oral microbiome, the role it plays in human health and disease is still under investigation. Previous studies have identified that a more diverse microbiome is advantageous for health. Therefore, alterations to the physical or mental health that are of interest in this study, such as stress, are the factors that decrease microbial diversity, leading to the potential for dysbiosis and disease disposition. Intensive Surgical Skills Week (ISSW) is a hyper-realistic simulation training week for military medical students that takes place at the Strategic Operations (STOPS) facility in San Diego, CA. This training week puts students through mass causality simulations and requires them to work through distinct roles within the healthcare team, providing an almost ideal environment to assess the impact of acute stress on oral microbiome diversity. Based on the literature on stress and microbiota, we hypothesized that the high stress simulation events at ISSW will impact the composition and diversity of the oral microbiome. Methods: To investigate this hypothesis, thirty-seven (n = 37) second-or third-year medical students who are enlisted in a branch of the military and who attended ISSW in July of 2021 were included in the study. Student participants were divided into 7 teams to complete the hyper-realistic simulations (SIMs) at ISSW. A pilot of sixty-four buccal samples (n = 64) from three of the seven teams were sent for analysis at the University of Missouri Metagenomic Center. Results: We saw an overall increase in species richness at the end of ISSW when looking at all samples (n = 64). Fourteen significantly different bacteria were identified from the beginning to the end of data collection. Additionally, third year medical students appear to have a greater species richness compared to second year medical students. Further, third year medical students had a statically significant difference in their oral microbiome richness from beginning to end of data collection (p = 0.008). Conclusion: Our preliminary data indicates that physical and psychological stress can impact the composition of the oral microbiome. The analyses in this study show that using the oral microbiome as an indicator of stress is promising and may provide evidence to support stress management practices.

Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1-7).

Compromised barrier function of colon epithelium with aging is largely due to gut microbial dysbiosis. Recent studies implicate an important role for angiotensin converting enzymes, ACE and ACE2, angiotensins, and the receptors, AT1 receptor (AT1R) and Mas receptor (MasR), in the regulation of colon functions. The present study tested the hypothesis that leaky gut in aging is associated with an imbalance in ACE2/ACE and that the treatment with angiotenisn-(1-7) (Ang-(1-7)) will restore gut barrier integrity and microbiome. Studies were carried out in Young (3-4 months) and old (20-24 months) male mice. Ang-(1-7) was administered by using osmotic pumps. Outcome measures included expressions of ACE, ACE2, AT1R, and MasR, intestinal permeability by using FITC-dextran, and immunohistochemistry of claudin 1 and occludin, and intestinal stem cells (ISCs). ACE2 protein and activity were decreased in Old group while that of ACE were unchanged. Increased intestinal permeability and plasma levels of zonulin-1 in the Old group were normalized by Ang-(1-7). Epithelial disintegrity, reduced number of goblet cells and ISCs in the old group were restored by Ang-(1-7). Expression of claudin 1 and occludin in the aging colon was increased by Ang-(1-7). Infiltration of CD11b+ or F4/80+ inflammatory cells in the old colons were decreased by Ang-(1-7). Gut microbial dysbiosis in aging was evident by decreased richness and altered beta diversity that were reversed by Ang-(1-7) with increased abundance of Lactobacillus or Lachnospiraceae. The present study shows that Ang-(1-7) restores gut barrier integrity and reduces inflammation in the aging colon by restoring the layer of ISCs and by restructuring the gut microbiome.

The impact of urine collection method on canine urinary microbiota detection: a cross-sectional study.

BACKGROUND: The urinary tract harbors unique microbial communities that play important roles in urogenital health and disease. Dogs naturally suffer from several of the same urological disorders as humans (e.g., urinary tract infections, neoplasia, urolithiasis) and represent a valuable translational model for studying the role of urinary microbiota in various disease states. Urine collection technique represents a critical component of urinary microbiota research study design. However, the impact of collection method on the characterization of the canine urinary microbiota remains unknown. Therefore, the objective of this study was to determine whether urine collection technique alters the microbial populations detected in canine urine samples. Urine was collected from asymptomatic dogs by both cystocentesis and midstream voiding. Microbial DNA was isolated from each sample and submitted for amplicon sequencing of the V4 region of the bacterial 16 S rRNA gene, followed by analyses to compare microbial diversity and composition between urine collection techniques. RESULTS: Samples collected via midstream voiding exhibited significantly higher sequence read counts (P = .036) and observed richness (P = .0024) than cystocentesis urine. Bray Curtis and Unweighted UniFrac measures of beta diversity showed distinct differences in microbial composition by collection method (P = .0050, R2 = 0.06 and P = .010, R2 = 0.07, respectively). Seven taxa were identified as differentially abundant between groups. Pasteurellaceae, Haemophilus, Friedmanniella, two variants of Streptococcus, and Fusobacterium were over-represented in voided urine, while a greater abundance of Burkholderia-Caballeronia-Paraburkholderia characterized cystocentesis samples. Analyses were performed at five thresholds for minimum sequence depth and using three data normalization strategies to validate results; patterns of alpha and beta diversity remained consistent regardless of minimum read count requirements or normalization method. CONCLUSION: Microbial composition differs in canine urine samples collected via cystocentesis as compared to those collected via midstream voiding. Future researchers should select a single urine collection method based on the biological question of interest when designing canine urinary microbiota studies. Additionally, the authors suggest caution when interpreting results across studies that did not utilize identical urine collection methods.

Standardized Complex Gut Microbiomes Influence Fetal Growth, Food Intake, and Adult Body Weight in Outbred Mice.

Obesity places a tremendous burden on individual health and the healthcare system. The gut microbiome (GM) influences host metabolism and behaviors affecting body weight (BW) such as feeding. The GM of mice varies between suppliers and significantly influences BW. We sought to determine whether GM-associated differences in BW are associated with differences in intake, fecal energy loss, or fetal growth. Pair-housed mice colonized with a low or high microbial richness GM were weighed, and the total and BW-adjusted intake were measured at weaning and adulthood. Pups were weighed at birth to determine the effects of the maternal microbiome on fetal growth. Fecal samples were collected to assess the fecal energy loss and to characterize differences in the microbiome. The results showed that supplier-origin microbiomes were associated with profound differences in fetal growth and excessive BW-adjusted differences in intake during adulthood, with no detected difference in fecal energy loss. Agreement between the features of the maternal microbiome associated with increased birth weight here and in recent human studies supports the value of this model to investigate the mechanisms by which the maternal microbiome regulates offspring growth and food intake.

Ophthalmic viscoelastics commonly used in cataract surgery: A microbiota investigation.

PURPOSE: To survey commonly used, sterile ophthalmic viscoelastic materials used during routine cataract surgery for the presence of bacterial DNA and/or viable bacteria and endotoxin quantification. METHODS: Samples from three different ophthalmic viscoelastic manufacturers and three different production lots per manufacturer were collected for 16 S ribosomal ribonucleic acid (rRNA) sequencing and conventional aerobic and capnophilic bacterial culture. Other samples of viscoelastic material from the same three manufacturers were collected for endotoxin quantification using a commercially available Limulus amebocyte lysate (LAL) assay. Statistical analysis was performed using Sigma Plot 14.0, and R v4.0.2.0. Differences (p ≤ .05) between sample collection sites in total DNA concentration, microbial richness, mean intra-group distances, and endotoxin quantification alongside reagent controls were evaluated. RESULTS: Culture yielded two isolates, identified as Staphylococcus epidermidis and Bacillus megaterium. 16 S rRNA sequencing revealed no differences between brands in richness or overall composition. The most common bacterial DNA detected across all brands was Staphylococcus sp., Cutibacterium sp., Flavobacterium sp., and Lactobacillus sp. A significant difference was found between the median endotoxin concentration between Anvision and Hyvisc® viscoelastic (Anvision: 0.171 EU/mL, Hyvisc®: 0.03 EU/mL; p < .001). CONCLUSIONS: No brand-specific differences in bacterial DNA were detected in the viscoelastic materials. Staphylococcus, Cutibacterium, Flavobacterium, and Lactobacillus were the dominant contributors to the bacterial DNA detected. Although Anvision viscoelastic samples contained significantly more endotoxin than Hyvisc® viscoelastic samples, endotoxin concentrations were below the FDA limit of 0.2 EU/mL for both manufacturers. These data further the understanding of inflammatory outcomes following cataract surgery.