Fecal Microbiota Research Articles

The Effect of Drugs on the Intestinal Microbiota in Crohn's Disease.

We took advantage of a single-center cross-sectional study to investigate the effect of different drugs on intestinal microbiota and function in Crohn's disease. We studied the difference in fecal microbiota of Crohn's disease patients treated with mesalazine, azathioprine, and infliximab, as well as untreated patients, by metagenome and screened for differential microbiota. Further, we investigated functional differences in intestinal microbiota among the four groups. Through metagenomic sequencing, we found that there was no difference between the four groups in ACE and Chao1 indices, but IFX and mesalazine improved species diversity and homogeneity compared to the untreated group, as evidenced by statistically significant differences in the Shannon index, Simpson index, and pielou_evenness. In addition, beta diversity suggested a difference between groups, but the difference was not significant. Non-parametric tests revealed differences between the four groups at the phylum level, class level, and genus level. Further analysis by LEfSe analysis revealed that the level of short-chain fatty acid-producing microbiota was increased in the treated groups, while there was no difference between the treated groups when compared to each other. Finally, the KEGG database and EggNOG database revealed that there were functional differences in intestinal microbiota among the four groups, including microbial metabolism pathway, cysteine and methionine metabolism pathway, cytoskeleton, etc. Conclusions: Mesalazine, azathioprine, and infliximab can all affect the intestinal microbiota and function in patients with Crohn's disease, and the drugs may alleviate intestinal inflammation in patients with Crohn's disease by modulating the intestinal microbiota.

Fecal microbiota transplantation and gut microbiota reconstruction in patients with refractory diarrhea

Fecal microbiota transplantation and gut microbiota reconstruction in patients with refractory diarrhea

Understanding the role of the human gut microbiome in overweight and obesity.

The gut microbiome may be related to the prevalence of overweight and obesity, but high interindividual variability of the human microbiome complicates our understanding. Obesity often occurs concomitantly with micronutrient deficiencies that impair energy metabolism. Microbiota composition is affected by diet. Host-microbiota interactions are bidirectional. We propose three pathways whereby these interactions may modulate the gut microbiome and obesity: (1) ingested compounds or derivatives affecting small intestinal transit, endogenous secretions, digestion, absorption, microbiome balance, and gut barrier function directly affect host metabolism; (2) substrate availability affecting colonic microbial composition and contact with the gut barrier; and (3) microbial end products affecting host metabolism. The quantity/concentration, duration, and/or frequency (circadian rhythm) of changes in these pathways can alter the gut microbiome, disrupt the gut barrier, alter host immunity, and increase the risk of and progression to overweight and obesity. Host-specific characteristics (e.g., genetic variations) may further affect individual sensitivity and/or resilience to diet- and microbiome-associated perturbations in the colonic environment. In this narrative review, the effects of selected interventions, including fecal microbiota transplantation, dietary calorie restriction, dietary fibers and prebiotics, probiotics and synbiotics, vitamins, minerals, and fatty acids, on the gut microbiome, body weight, and/or adiposity are summarized to help identify mechanisms of action and research opportunities.

Exploring the Gut Microbiota Landscape in Cow Milk Protein Allergy: Clinical Insights and Diagnostic Implications in Pediatric Patients

Cow milk protein allergy (CMPA) is a significant health concern characterized by adverse immune reactions to cow milk proteins. Biomarkers for the accurate diagnosis and prognosis of CMPA are lacking. This study analyzed the clinical features of CMPA, and 16S RNA sequencing was used to investigate potential biomarkers through fecal microbiota profiling. Children with CMPA exhibit a range of clinical symptoms, including gastrointestinal (83% of patients), skin (53% of patients), and respiratory manifestations (26% of patients), highlighting the complexity of this condition. Laboratory analysis revealed significant differences in red cell distribution width (RDW) and inflammatory markers between the CMPA and control groups, suggesting immune activation and inflammatory responses in CMPA. Microbial diversity analysis revealed higher specific diversity indices in the CMPA group compared with those in control group, with significant differences at the genus and species levels. Bacteroides were more abundant in the CMPA group, whereas Bifidobacterium, Ruminococcus, Faecalibacterium, and Parabacteroides were less abundant. The control group exhibited a balanced microbial profile, with a predominant presence of Bifidobacterium bifidum and Akkermansia muciniphila. The significant abundance of Bifidobacterium in the control group (23.19% vs 9.89% in CMPA) was associated with improved growth metrics such as height and weight, suggesting its potential as a probiotic to prevent CMPA and enhance gut health. Correlation analysis linked specific microbial taxa such as Coprococcus and Bifidobacterium to clinical parameters such as family allergy history, weight and height, providing insights into CMPA pathogenesis. Significant differences in bacterial abundance suggested diagnostic potential, with a panel of 6 bacteria achieving high predictive accuracy (area under curve (AUC) = 0.8708). This study emphasizes the complex relationship between the gut microbiota and CMPA, offering valuable insights into disease mechanisms and diagnostic strategies.

Total alkaloids of Aconitum carmichaelii Debx alleviate cisplatin-induced acute renal injury by inhibiting inflammation and oxidative stress related to gut microbiota metabolism

BackgroundCisplatin-induced acute kidney injury (AKI) is a complex and serious clinical issue, representing a major cause of hospital-acquired AKI. Alkaloids are the main active constituents of Aconitum carmichaelii Debx, which exhibit protective effects in several kidney disease models and against other acute organ injuries. However, its activity and mechanism of action in AKI treatment remain unclear. PurposeThis study aimed to elucidate the effect of Aconitum carmichaelii Debx (ACA) in a model of cisplain-induced AKI and comprehensively investigate its underlying mechanisms. MethodsThe major alkaloids in ACA were analyzed using high-performance liquid chromatography. Blood urea nitrogen (BUN) and serum creatine levels were measured using automated biochemical instruments. 16S rRNA sequencing, short-chain fatty acid (SCFA) analysis, fecal microbiota transplantation (FMT), non-targeted metabolomics, and transcriptomics were performed to systematically identify prospective biomarkers after ACA treatment. Anti-inflammatory and anti-oxidative stress activities were monitored using ELISA and western blotting. ResultsFour main compounds (fuziline, neoline, talatisamine, and songorine) were identified in ACA. ACA significantly alleviated cisplatin-induced AKI by reducing (BUN) and serum creatine levels and improving histopathological scores. Moreover, ACA balanced cisplatin-mediated confoundments in microbial composition and function, including decreasing the levels of Escherichia-Shigella, Clostridium, and Ruminococcus, as well as increasing Ligilactobacillus, Anaerotruncus, Bacteroides and Desulfovibrio levels, accompanied by uremic toxin reduction, and augmenting serum SCFAs. The FMT experiments further confirmed that ACA exerts anti-AKI effects by affecting gut microbiota. A multi-omics study has shown that ACA regulates glutathione and tryptophan metabolism and mediates pathways that trigger inflammatory responses. Finally, ACA reduced serum levels of inflammatory factors (IL-1β, IL-6, and TNF-α), restored enzymes of the antioxidative system (SOD and CAT) and GSH values, and decreased monoester diterpene alkaloid levels in the kidney by inhibiting the expression of NF-κB pathway-related proteins and increasing Nrf2/HO-1 pathway-related protein expression. ConclusionACA protects against cisplatin-induced AKI through its anti-inflammatory and antioxidant functions, which may be associated with the restoration of gut microbiota metabolism. ACA is a potential drug for AKI and other forms of organ damage related to the disruption of the gut microbiota.

S284 Is Fecal Microbiota Transplant the Future of Small Intestinal Bacterial Overgrowth?

S284 Is Fecal Microbiota Transplant the Future of Small Intestinal Bacterial Overgrowth?

Astragalus polysaccharides alleviate DSS-induced ulcerative colitis in mice by restoring SCFA production and regulating Th17/Treg cell homeostasis in a microbiota-dependent manner

Natural polysaccharides from Astragalus membranaceus have been shown to relieve ulcerative colitis (UC). However, the mechanism and causal relationship between the gut microbiota and Astragalus polysaccharides (APS) treatment of UC are unclear. The results of the present study showed that APS ameliorated colonic injury and the disruption of the gut microbiota and restored intestinal immune homeostasis in mice with DSS-induced colitis. Meanwhile, we found that APS treatment was ineffective in antibiotic-treated colitis mice but was effective when FMT (Fecal microbiota transplantation) was performed on UC mice using APS-treated mice as donors. APS increased the proportion of relevant microbiota that produce SCFAs and both direct administration of APS and administration of APS-adjusted gut microbiota significantly promoted the production of SCFAs in colitis mice. We demonstrated that APS dually inhibited NF-κB activation via the TLR4 and HDAC3 pathways and improved the balance in Th17/Treg cells in UC mice. In conclusion, our study revealed that APS is a promising prebiotic agent for the maintenance of intestinal health and demonstrated that APS may ameliorate colitis in a gut microbiota-dependent manner.

Hesperidin and Fecal Microbiota Transplantation Modulate the Composition of the Gut Microbiota and Reduce Obesity in High Fat Diet Mice.

Obesity, which is associated with gut microbiota dysbiosis, low-grade chronic inflammation and intestinal barrier dysfunction, can cause a variety of chronic metabolic diseases. Phytochemical flavonoids have a variety of biological activities, among which there may besafe and effective anti-obesity solutions. We tested a plant-derived flavonoid hesperidin and fecal microbiota transplantation (FMT) to alleviate diet-induced obesity. High-fat diet (HFD)-fed mice were treated with hesperidin (100 and 200 mg/kg BW) and FMT. Results indicated that hesperidin had the effects of reducing obesity as indicated by reduction of body weight, fat accumulation and blood lipids, reducing inflammation as indicated by reduction of pro-inflammation factors including TNFα, IL-6, IL-1βand iNOS, and improving gut integrity as indicated by increasing colon length, reducing plasma gut permeability indicators iFABP and LBP, increased mRNA expression of mucus protein Muc2, tight junction p Claudin 2, Occludin and ZO-1 in the HFD-fed mice. The anti-obesity effects of hesperidin treatment have a dose-dependent manner. In addition, 16S rRNA-based gut microbiota analysis revealed that hesperidin selectively promoted the growth of Lactobacillus salivarius, Staphylococcus sciuri and Desulfovibrio C21_c20 while inhibiting Bifidobacterium pseudolongum, Mucispirillum schaedleri, Helicobacter ganmani and Helicobacter hepaticus in the HFD-fed mice. Horizontal feces transfer from the normal diet (ND)-fed mice to the HFD-fed mice conferred anti-obesity effects and transmitted some of the HFD-modulated microbes. We concluded that hesperidin and FMT both affect the reduction of body weight and improve HFD-related disorders in the HFD-fed mice possibly through modulating the composition of the gut microbiota.

Life on both environment in semi-aquatic frogs: Impact of aquatic microplastic (MP) from MP enrichment to growth, immune function and physiological stress

The pervasive distribution of microplastics (MPs) in aquatic ecosystems presents a significant threat to wildlife, with amphibians being particularly vulnerable due to their complex life cycles and ecological roles. This study investigates physiological and ecological impacts of aquatic MP exposure on juvenile black-spotted pond frogs (Pelophylax nigromaculatus), focusing on juvenile frog stage, history of life after metamorphosis. MP examinations in the intestine and body revealed accumulation primarily in the gastrointestinal tracts without evidence of systemic distribution. Experimental exposure to different concentrations of MPs demonstrated adverse effects on growth, physiological stress, and immune function. Notably, higher MP concentrations led to significant reductions in growth and innate immunity, indicative of compromised health. High concentrations of MPs were associated with elevated levels of corticosterone and antioxidant enzymes, indicating physiological stress. However, there was no evidence of extreme hormonal surges or imbalances in antioxidant enzyme activity, suggesting that amphibians were able to effectively cope with the levels of MPs used in the study. Changes in gastrointestinal morphology and fecal microbiota composition were observed, reflecting response of metabolic adaptation to MP exposure. At low concentrations of MPs, adaptive changes in digestive tract morphology and the maintenance of gut microbiota balance were observed, indicating that the frogs were able to manage the exposure below a certain threshold. In contrast, high concentrations of MPs had clear negative effects on amphibians, which could impact biodiversity and ecosystem stability. These findings also suggest that MPs may trigger adaptive responses at lower concentrations, while still posing significant environmental risks at higher levels.

The gut microbiotas with metabolites regulate the protective role of miR-30a-5p in myocardial infarction

IntroductionGut microbial homeostasis is closely associated with myocardial infarction (MI). However, little is known about how gut microbiota influences miRNAs-regulated MI. ObjectivesThis study aims to elucidate the connections between miR-30a-5p, MI, gut microbiota, and gut microbial metabolite-related pathways, to explore potential strategy for preventing and treating MI. MethodsWe evaluated the effects of knocking out (KO) or overexpressing (OE) miR-30a-5p on MI by assessing cardiac structure and function, myocardial enzyme levels, and apoptosis. Then, we applied 16S rDNA sequencing and metabolomics to explore how intestinal microecology and its microorganisms affect miR-30a-5p-regulated MI. ResultsThe results showed that KO exacerbated MI, whereas OE improved MI damage, compared to the wild-type (WT) mice. KO exacerbated intestinal barrier structure deterioration and further downregulated the expression of Cloudin-1, Occludin, and ZO-1 in MI mice. 16S rDNA sequencing-analyzed gut microbiome of KO and WT mice found that KO mainly reduced g_Lactobacillus. Transplanting fecal microorganisms from KO mice aggravated MI damage in WT mice. However, administering probiotics (mainly containing Lactobacillus) helped neutralize these damages. Intriguingly, fecal microbiota transplantation from OE mice reduced MI damage. Analysis of intestinal microbial metabolites in KO and WT mice found that KO may mainly affect ABC transporters. ABCC1 was identified as the target of KO-aggravated MI. Furthermore, fecal transplantation microorganisms of MI patients aggravated MI injury in mice and miR-30a-5p and ABCC1 were involved in the process. ConclusionsOur findings demonstrate that miR-30a-5p regulates MI by affecting intestinal microbiota homeostasis and targeting ABCC1. This highlights the critical importance of maintaining a healthy gut microbiota homeostasis in MI management.

Etiolated-green tea attenuates colonic barrier dysfunction and inflammation in high-fat diet-induced mice by modulating gut microbiota

Colonic barrier dysfunction and inflammation arising from dysbiosis gut microbiota (GM) are strongly associated with a high-fat diet (HFD). Yellow leaf green tea (YLGT), a novel variety of etiolated-green tea, improving the intestinal barrier and inflammation is related to the regulation of GM disorders. To explore the ameliorative mechanism of YLGT, mice were fed an HFD with or without YLGT at doses of 150, 300, and 450 mg kg−1 for 12 weeks. YLGT rectified the GM imbalance, enriched short-chain fatty acid (SCFA)-producing bacteria and gut SCFA contents, activated G protein-coupled receptors, inhibited TLR4/NF-κB signaling pathway, strengthened the tight junction, and repaired the damaged intestinal barrier. The fecal microbiota transplantation experiment further confirmed that the GM was a key element in the anti-obesity and anti-intestinal inflammation effect of YLGT. YLGT has great promise in attenuating obesity-induced intestinal dysfunction. This research provides novel insights into the new mechanism of YLGT on HFD-induced obesity.

Colonization profiles of gut microbiota in goat kids from neonatal to weaning period.

Understanding the colonization and change patterns of gut microbiota is pivotal for comprehending host health. As a newly cultured breed, the studies on the gut microbiota of Tianfu goats remain limited. This study aimed to address this gap by analyzing the microbial composition and colonization patterns of fecal samples collected from goat kids from birth to weaning. Fecal samples were collected on days 0, 7, 14, 21, 28, 35, 42, 49, 53, 55, 57, and 64, and the changes and colonization patterns of microorganisms were analyzed through high-throughput 16S rRNA sequencing. The results showed that the abundance of fecal microbiota in goat kids gradually increased over time, followed by a decrease after weaning and stabilization, with reduced individual differences. The colonization of fecal microorganisms mainly presented three different stages: days 0-14, days 21-49, and days 53-64. During the suckling period, the relative abundance of Proteobacteria (72.34%) was the highest, followed by Firmicutes (21.66%). From 21 days old, the microbiota in goat kids gradually to be diverse, with Lachnospiraceae and Ruminococcaceae being dominant. During post-weaning, Ruminococcaceae (30.98-33.34%) was becoming prominence which helpful for cellulose decomposition. LEfSe analyzed three important time points (d0 vs. d7, d7 vs. d14, d49 vs. d53, LDA score > 4 and p < 0.05), 53 microbial communities with stage differences were identified. Functional prediction using PICRUSt revealed that differential microbial communities are mainly related to carbohydrate and amino acid metabolism pathways. Overall, this study addresses the intricate relationship between ages, diets, and microbiota compositions in Tianfu goat kids, and also offering insights into microorganisms-host interactions.

Body Composition Changes Following Fecal Microbiota Transplant Therapy for Recurrent Clostridiodes Difficile Infection in a Cohort of Female Patients ≥55 Years of Age

Body Composition Changes Following Fecal Microbiota Transplant Therapy for Recurrent Clostridiodes Difficile Infection in a Cohort of Female Patients ≥55 Years of Age

S280 Safety of Fecal Microbiota Spores, Live-brpk (Formerly SER-109) in Participants With Recurrent Clostridioides difficile Infection and Comorbidities: Findings From an Integrated Analysis of Phase 3 Trials

S280 Safety of Fecal Microbiota Spores, Live-brpk (Formerly SER-109) in Participants With Recurrent Clostridioides difficile Infection and Comorbidities: Findings From an Integrated Analysis of Phase 3 Trials

Lycopene attenuates D-galactose-induced memory and behavioral deficits by mediating microbiota-SCFAs-gut-brain axis balance in female CD-1 mice

Aging impairs cognitive function, whereas nutritional intervention can delay aging and age-related diseases. Lycopene (LYC), a naturally occurring carotenoid, posses multiple health-promoting properties, including neuroprotective function. Here, the effects of LYC on memory and behavioral deficits induced by D-galactose (D-gal) treatment and the relative contribution of LYC-derived gut microbiota in these process were investigated. Results demonstrated that LYC showed effective protection on D-gal induced cognitive deficit and neuronal damage. Moreover, LYC treatment has beneficial effects on gut barrier damage, microbiota dysbiosis and levels of SCFAs in D-gal-induced subacute aging mice. Next, fecal microbiota transplantation (FMT) experiment was performed and increased SCFAs were observed in mice received stools from D-gal+LYC group when compared with D-gal-FMT group. Thus, we added SCFAs treatment served as a control group in order to evaluated whether the alterations of gut-brain axis could be attributed to LYC-reshaped gut microbiota and SCFAs. Results showed that recipient mice received SCFAs and stools from D-gal+LYC group have similar beneficial effects in improving gut and brain function, demonstrated as: improved intestinal health via elevating antioxidant enzymes contents, increasing the expressions of tight junctions proteins and protecting gut barrier, enhanced mice working memory capacity via alleviating hippocampal neurons impairment, improving synaptic function and enhancing mitochondrial function in the intestinal pseudo-aseptic mice. In conclusion, our results demonstrated that LYC-derived microbiome played a pivotal role in the regulation of cognitive functions during aging and enhanced SCFAs formation might be an important signaling molecule connecting gut microbiome and brain.

Clostridioides difficile infections: Update and therapeutic guidelines

Clostridioides difficile infection (CDI) represents a significant challenge due to its increasing incidence, severity, and treatment difficulty. Effective management requires a multifactorial approach that includes preventive strategies, prudent antibiotic use, and adapted therapeutic options. Ongoing research and innovation offer promising prospects for improving ICD management, making vigilance and informed practices essential among healthcare professionals. Two main complications of ICD are pseudomembranous colitis (PMC) and toxic megacolon. PMC involves severe colonic inflammation due to C.difficile toxins, leading to pseudomembrane formation. Diagnosis relies on clinical criteria, microbiological tests, and endoscopy. Toxic Megacolon is characterized by severe colonic dilation and systemic toxicity, requiring immediate medical intervention. ICD diagnosis combines clinical signs and microbiological tests. These tests include toxin tests, GDH antigen detection, PCR for toxin genes, and stool culture. Imaging techniques assess colonic inflammation and complications. Combined diagnostic criteria from the American Gastroenterological Association (AGA) and European guidelines emphasize integrating clinical and laboratory findings for accurate diagnosis. ICD treatment involves stopping the implicated antibiotics and starting specific antimicrobial therapy. Common treatments include mainly fidaxomicin and oral vancomycin. Fecal microbiota transplantation (TMF) is recommended for recurrent cases unresponsive to standard treatments. Bezlotoxumab, an antibody targeting C.difficile toxin B, is used to prevent recurrence in high-risk adults. ICD poses a major challenge due to its increasing incidence, severity, and difficulty in treatment. A multifactorial approach involving rigorous preventive strategies, prudent antibiotic management, and adapted therapeutic options is essential for controlling the infection. Ongoing research and innovations in treatment offer promising prospects for improving patient management. Healthcare professionals must remain vigilant and informed to ensure effective practices in combating this infection and utilizing available resources optimally.

Z-ligustilide alleviates atherosclerosis by reconstructing gut microbiota and sustaining gut barrier integrity through activation of cannabinoid receptor 2

BackgroundZ-Ligustilide (ZL) is an essential phthalide found in Ligusticum chuanxiong Hort, a commonly used traditional Chinese medicine for treating atherosclerosis (AS) clinically. ZL has been shown to be effective in treating AS. However, the underlying mechanism of ZL against AS and its potential targets remain elusive. PurposeThe purpose of this research was to assess the influence of ZL on AS and explore the role of the gut microbiome in mediating this effect. MethodsA well-established AS mouse model, apolipoprotein E deficient (ApoE-/-) mice was used to examine the effects of ZL on AS, inflammation, and the intestinal barrier. To analyze the changes in gut microbial community, we employed the 16S rRNA gene sequencing. Antibiotic cocktail and fecal microbiota transplantation (FMT) were employed to clarify the contribution of the gut microbiota to the anti-AS effects of ZL. The mechanism through which ZL provided protective effects on AS and the intestinal barrier was explored by untargeted metabolomics, as well as by validating the involvement of cannabinoid receptor 2 (CB2R) in mice and Caco-2 cells. ResultsOral administration of ZL inhibited the development of atherosclerotic lesions, improved plaque stability, inhibited the increase in serum and atherosclerotic inflammation, and improved intestinal barrier function. Fecal bacteria from ZL-treated mice induced similar beneficial effects on AS and the intestinal barrier. We used 16S RNA gene sequencing to reveal a significant increase in Rikenella abundance in both ZL-treated mice and ZL-FMT mice, which was associated with the beneficial effects of ZL. Further function prediction analysis of the gut microbiota and CB2R antagonist intervention experiment in mice and Caco-2 cells showed that the activation of CB2R resulted in the enhancement of the intestinal barrier by ZL. Furthermore, the analysis of metabolomic profiling revealed the enrichment of capsaicin upon ZL treatment, which induced the activation of CB2R in human colon epithelial cells. ConclusionOur study is the first to demonstrate that oral treatment with ZL has the potential to alleviate AS by reducing inflammation levels and enhancing the intestinal barrier function. This mechanism relies on the gut microbiota in a CB2R-dependent manner, suggesting promising strategies and ideas for managing AS. This study provides insights into a novel mechanism for treating AS with ZL.

402P Faecal microbiota transplantation: a potential novel therapeutic avenue for Duchenne muscular dystrophy

402P Faecal microbiota transplantation: a potential novel therapeutic avenue for Duchenne muscular dystrophy

Effects of a Veterinary Gastrointestinal Low-Fat Diet on Fecal Characteristics, Metabolites, and Microbiota Concentrations of Adult Dogs Treated with Metronidazole.

Antibiotics are known to cause loose stools, disrupt the fecal microbiota, and alter fecal bile acid (BA) profiles of dogs. Recovery may be aided by diet, but little research has been conducted. The objective of this study was to determine how a veterinary low-fat diet affected the fecal characteristics, metabolites, BA, and microbiota of dogs receiving antibiotics. Twenty-four healthy adult dogs [7.38 ± 1.95 yr; 7.67 ± 0.76kg body weight (BW)] were used in an 8-wk completely randomized design study. During a 2-wk baseline, all dogs were fed a leading grocery brand dry kibble diet (GBD). Over the next 2wk, dogs were fed GBD and received metronidazole orally (20mg/kg BW twice daily). At wk 4, dogs were randomly allotted to one of two treatments [GBD or Blue Buffalo Natural Veterinary Diet GI Gastrointestinal Support Low-Fat (BB)] and fed for 4wk. Fecal scores were recorded daily and fresh fecal samples were collected at wk 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter content, and metabolite and BA concentrations. Fecal microbiota populations were analyzed by 16S rRNA gene amplicon sequencing and qPCR-based dysbiosis index (DI). All data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, testing for effects of treatment, time, and treatment*time and significance set at P<0.05. Metronidazole increased (P<0.0001) fecal scores (looser stools), reduced fecal short-chain fatty acid, branched-chain fatty acid, phenol, and indole concentrations, increased primary BA concentrations, and decreased secondary BA concentrations. Metronidazole also reduced fecal bacterial alpha diversity, altered the abundance of 58 bacterial genera, and increased DI. During antibiotic recovery, change in fecal pH, dry matter percentage, and metabolite and immunoglobulin A concentrations were altered (P<0.05) by diet. Fecal BA concentrations recovered quickly for all dogs. Change in lithocholic acid was affected (P<0.0001) by diet, but other BA were not. Recovery of over 25 bacterial genera was impacted by diet (P<0.05). While many bacterial taxa returned to baseline levels after 4wk, others did not fully recover. DI and bacterial alpha diversity measures recovered quickly for all dogs, but were not impacted by diet. In conclusion, metronidazole drastically altered the fecal microbiota and metabolites of dogs. While most variables returned to baseline by wk 8, diet may be used to aid in recovery.

Multi-Omics Analysis Unravels the Impact of Stool Sample Logistics on Metabolites and Microbial Composition.

Human health and the human microbiome are inevitably intertwined, increasing their relevance in clinical research. However, the collection, transportation and storage of faecal samples may introduce bias due to methodological differences, especially since postal shipping is a common practise in large-scale clinical cohort studies. Using four different Omics layer, we determined the structural (16S rRNA sequencing, cytometric microbiota profiling) and functional integrity (SCFAs, global metabolome) of the microbiota in relation to different easy-to-handle conditions. These conditions were storage at -20 °C, -20 °C as glycerol stock, 4 °C and room temperature with and without oxygen exposure for a maximum of one week. Storage time affected the microbiota on all Omics levels. However, the magnitude was donor-dependent, highlighting the need for purpose-optimized sample collection in clinical multi-donor studies. The effects of oxygen exposure were negligible for all analyses. At ambient temperature, SCFA and compositional profiles were stable for 24 h and 48 h, respectively, while at 4 °C, SCFA profiles were maintained for 48 h. The global metabolome was highly susceptible, already changing at 24 h in non-frozen conditions. Thus, faecal microbiota was best preserved on all levels when transported as a native sample frozen within 24 h, leading to the least biased outcomes in the analysis. We conclude that the immediate freezing of native stool samples for transportation to the lab is best suited for planned multi-Omics analyses that include metabolomics to extend standard sequencing approaches.