Urinary microbiome dysbiosis is associated with an inflammatory environment and perturbed fatty acids metabolism in the pathogenesis of bladder cancer.

BACKGROUND: Bladder cancer is a common malignancy with high recurrence rate. Early diagnosis and recurrence surveillance are pivotal to patients' outcomes, which require novel minimal-invasive diagnostic tools. The urinary microbiome is associated with bladder cancer and can be used as biomarkers, but the underlying mechanism is to be fully illustrated and diagnostic performance to be improved. METHODS: A total of 23 treatment-naïve bladder cancer patients and 9 non-cancerous subjects were enrolled into the Before group and Control group. After surgery, 10 patients from the Before group were further assigned into After group. Void mid-stream urine samples were collected and sent for 16S rDNA sequencing, targeted metabolomic profiling, and flow cytometry. Next, correlations were analyzed between microbiota, metabolites, and cytokines. Finally, receiver operating characteristic (ROC) curves of the urinary biomarkers were plotted and compared. RESULTS: Comparing to the Control group, levels of IL-6 (p < 0.01), IL-8 (p < 0.05), and IL-10 (p < 0.05) were remarkably elevated in the Before group. The α diversity of urine microbiome was also significantly higher, with the feature microbiota positively correlated to the level of IL-6 (r = 0.58, p < 0.01). Significant differences in metabolic composition were also observed between the Before and Control groups, with fatty acids and fatty acylcarnitines enriched in the Before group. After tumor resection, cytokine levels and the overall microbiome structure in the After group remained similar to that of the Before group, but fatty acylcarnitines were significantly reduced (p < 0.05). Pathway enrichment analysis revealed beta-oxidation of fatty acids was significantly involved (p < 0.001). ROC curves showed that the biomarker panel of Actinomycetaceae + arachidonic acid + IL-6 had superior diagnostic performance, with sensitivity of 0.94 and specificity of 1.00. CONCLUSIONS: Microbiome dysbiosis, proinflammatory environment and altered fatty acids metabolism are involved in the pathogenesis of bladder cancer, which may throw light on novel noninvasive diagnostic tool development.

Association of microbial dynamics with urinary estrogens and estrogen metabolites in patients with endometriosis.

Endometriosis is an estrogen dependent gynecological disease associated with altered microbial phenotypes. The association among endogenous estrogen, estrogen metabolites, and microbial dynamics on disease pathogenesis has not been fully investigated. Here, we identified estrogen metabolites as well as microbial phenotypes in non-diseased patients (n = 9) and those with pathologically confirmed endometriosis (P-EOSIS, n = 20), on day of surgery (DOS) and ~1-3 weeks post-surgical intervention (PSI). Then, we examined the effects of surgical intervention with or without hormonal therapy (OCPs) on estrogen and microbial profiles of both study groups. For estrogen metabolism analysis, liquid chromatography/tandem mass spectrometry was used to quantify urinary estrogens. The microbiome data assessment was performed with Next generation sequencing to V4 region of 16S rRNA. Surgical intervention and hormonal therapy altered gastrointestinal (GI), urogenital (UG) microbiomes, urinary estrogen and estrogen metabolite levels in P-EOSIS. At DOS, 17ß-estradiol was enhanced in P-EOSIS treated with OCPs. At PSI, 16-keto-17ß-estradiol was increased in P-EOSIS not receiving OCPs while 2-hydroxyestradiol and 2-hydroxyestrone were decreased in P-EOSIS receiving OCPs. GI bacterial α-diversity was greater for controls and P-EOSIS that did not receive OCPs. P-EOSIS not utilizing OCPs exhibited a decrease in UG bacterial α-diversity and differences in dominant taxa, while P-EOSIS utilizing OCPs had an increase in UG bacterial α-diversity. P-EOSIS had a strong positive correlation between the GI/UG bacteria species and the concentrations of urinary estrogen and its metabolites. These results indicate an association between microbial dysbiosis and altered urinary estrogens in P-EOSIS, which may impact disease progression.

The Microbiome of Catheter Collected Urine in Males with Bladder Cancer According to Disease Stage.

PURPOSE: The dogma that urine is sterile has been overturned and dysbiosis of the urinary microbiome has been linked to many urological disorders. We tested the hypothesis that the urinary microbial composition may be different between men with or without bladder cancer in catheter collected urines, bladder washouts and midstream voided urines, and may be dependent on tumor staging. MATERIALS AND METHODS: Liquid samples were collected from male patients with bladder cancer, and sex and age matched nonneoplastic controls. Total DNA was extracted and processed for 16S rRNA gene sequencing. Bioinformatic analysis for microbial classification was performed to assess diversity and variations. RESULTS: The urinary microbiome associated with catheter collected urine samples of patients with bladder cancer was characterized by a significantly increased abundance of Veillonella (p=0.04) and Corynebacterium (p=0.03), and decreased Ruminococcus (p=0.03) compared to controls, with differences exacerbating with disease progression. Compared to catheterized urines, bladder cancer washouts showed the specific increase of some taxa, like Burkholderiaceae (p=0.014), whereas midstream urines were enriched in Streptococcus (p <0.0001), Enterococcus (p <0.0001), Corynebacterium (p=0.038) and Fusobacterium (p <0.0001). CONCLUSIONS: The bladder is colonized by endogenous bacteria and microbial modifications characterize the microbiome of patients with bladder cancer. Different microbial compositions can be characterized by changing sampling strategy. These results pave the way for exploring new diagnostic and therapeutic options based on the manipulation of the bacterial community.

Development and validation of GC-MS/MS method useful in diagnosing intestinal dysbiosis.

Dysbiosis is a disorder of the bacterial flora of the human digestive tract. It is usually diagnosed clinically by direct detection of an abnormal pattern of the intestinal microbiota. The intermediate diagnosis based on determining the content of microflora metabolites, considered as chemical markers of this disorder, is still rarely used. This is, among others, due to the variety of properties of compounds recognised as dysbiosis markers and as a consequence, the use of different methods for their analysis. To the best of our knowledge, there is still no analytical procedure that would allow unambiguous determination of all compounds in one procedure. In the present study, we have established a detailed method for the quantitative analysis of hydrocinnamic, citramalic, p-hydroxybenzeneacetic, tartaric, hippuric, 4-hydroxybenzoic, indoxylsulfuric, tricarballylic, 3,4-dihydroxyhydrocinnamic and benzoic acids along with DL-arabitol that employs the direct derivatization of compounds in a small volume of urine sample followed by gas chromatography - tandem mass spectrometry (GC-MS/MS). To show that the optimised method is a useful tool for chemical diagnosis of dysbiosis, it was applied for determination of the dysbiosis markers in the authentic urine samples.

Lactobacillus brevis DM9218 ameliorates fructose-induced hyperuricemia through inosine degradation and manipulation of intestinal dysbiosis.

OBJECTIVE: High fructose consumption exacerbates purine degradation and intestinal dysbiosis, which are closely related to the development of hyperuricemia. Probiotics are powerful weapons to combat metabolic disturbance and intestinal dysbiosis. Previously we isolated a Lactobacillus strain named DM9218 that could reduce the serum uric acid (UA) level by assimilating purine nucleosides. The present study aimed to evaluate the effects of DM9218 on high-fructose-induced hyperuricemia and to elucidate the underlying mechanisms. METHODS: Mice were fed a normal diet, a high-fructose diet, or high-fructose diet with DM9218. Metabolic parameters, fructose- and UA-related metabolites, and fecal microbiota were investigated. Whole-genome sequencing of strain DM9218 was also conducted. In addition, an inosine hydrolase from DM9218 was heterologously expressed in Escherichia coli, and its inosine-degrading activity was detected. RESULTS: Our results indicated that DM9218 could decrease serum UA level and hepatic xanthine oxidase activity in fructose-fed mice. It could protect against high-fructose-induced liver damage and retard UA accumulation by degrading inosine. The modulation effect of DM9218 on high-fructose-induced intestinal dysbiosis resulted in enhancement of intestinal barrier function and reduction of liver lipopolysaccharide, which was closely correlated with the down-regulation of inflammatory cytokine-stimulated xanthine oxidase expression and activity. CONCLUSIONS: Lactobacillus brevis DM9218 is a probiotic strain with the potential to ameliorate fructose-induced hyperuricemia.

Effect of a Protein Supplement on the Gut Microbiota of Endurance Athletes: A Randomized, Controlled, Double-Blind Pilot Study.

Nutritional supplements are popular among athletes to improve performance and physical recovery. Protein supplements fulfill this function by improving performance and increasing muscle mass; however, their effect on other organs or systems is less well known. Diet alterations can induce gut microbiota imbalance, with beneficial or deleterious consequences for the host. To test this, we performed a randomized pilot study in cross-country runners whose diets were complemented with a protein supplement (whey isolate and beef hydrolysate) (n = 12) or maltodextrin (control) (n = 12) for 10 weeks. Microbiota, water content, pH, ammonia, and short-chain fatty acids (SCFAs) were analyzed in fecal samples, whereas malondialdehyde levels (oxidative stress marker) were determined in plasma and urine. Fecal pH, water content, ammonia, and SCFA concentrations did not change, indicating that protein supplementation did not increase the presence of these fermentation-derived metabolites. Similarly, it had no impact on plasma or urine malondialdehyde levels; however, it increased the abundance of the Bacteroidetes phylum and decreased the presence of health-related taxa including Roseburia, Blautia, and Bifidobacterium longum. Thus, long-term protein supplementation may have a negative impact on gut microbiota. Further research is needed to establish the impact of protein supplements on gut microbiota.

Exploration of the Fecal Microbiota and Biomarker Discovery in Equine Grass Sickness.

Equine grass sickness (EGS) is a frequently fatal disease of horses, responsible for the death of 1 to 2% of the U.K. horse population annually. The etiology of this disease is currently uncharacterized, although there is evidence it is associated with Clostridium botulinum neurotoxin in the gut. Prevention is currently not possible, and ileal biopsy diagnosis is invasive. The aim of this study was to characterize the fecal microbiota and biofluid metabolic profiles of EGS horses, to further understand the mechanisms underlying this disease, and to identify metabolic biomarkers to aid in diagnosis. Urine, plasma, and feces were collected from horses with EGS, matched controls, and hospital controls. Sequencing the16S rRNA gene of the fecal bacterial population of the study horses found a severe dysbiosis in EGS horses, with an increase in Bacteroidetes and a decrease in Firmicutes bacteria. Metabolic profiling by 1H nuclear magnetic resonance spectroscopy found EGS to be associated with the lower urinary excretion of hippurate and 4-cresyl sulfate and higher excretion of O-acetyl carnitine and trimethylamine-N-oxide. The predictive ability of the complete urinary metabolic signature and using the four discriminatory urinary metabolites to classify horses by disease status was assessed using a second (test) set of horses. The urinary metabolome and a combination of the four candidate biomarkers showed promise in aiding the identification of horses with EGS. Characterization of the metabolic shifts associated with EGS offers the potential of a noninvasive test to aid premortem diagnosis.

Urinary and Fecal Metabonomics Study of the Protective Effect of Chaihu-Shu-Gan-San on Antibiotic-Induced Gut Microbiota Dysbiosis in Rats.

Accumulating evidence suggests that the gut microbiota dysbiosis and their host metabolic phenotype alteration is an important factor in human disease development. A traditional Chinese herbal formula, Chaihu-Shu-Gan-San (CSGS), has been effectively used in the treatment of various gastrointestinal (GI) disorders. The present study was carried out to investigate whether CSGS modulates the host metabolic phenotype under the condition of gut microbiota dysbiosis. The metabonomics studies of biochemical changes in urine and feces of antibiotic-induced gut microbiota dysbiosis rats after treatment with CSGS were performed using UPLC-Q-TOF/MS. Partial least squares-discriminate analysis (PLS-DA) indicated that the CSGS treatment reduced the metabolic phenotype perturbation induced by antibiotic. In addition, there was a strong correlation between gut microbiota genera and urinary and fecal metabolites. Moreover, the correlation analysis and the metabolic pathway analysis (MetPA) identified that three key metabolic pathways including glycine, serine and threonine metabolism, nicotinate and nicotinamide metabolism, and bile acid metabolism were the most relevant pathways involved in antibiotic-induced gut microbiota dysbiosis. These findings provided a comprehensive understanding of the protective effects of CSGS on the host metabolic phenotype of the gut microbiota dysbiosis rats, and further as a new source for drug leads in gut microbiota-targeted disease management.

Dysbiosis of urinary microbiota is positively correlated with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) may be associated with altered urinary microbiota in female patients. We investigated alterations of urinary microbiota in Chinese female T2DM patients, and explored the associations between urinary microbiota and a patient's fasting blood glucose (FBG), urine glucose (UGLU), age, menstrual status, and body mass index (BMI). Midstream urine was collected from 70 female T2DM patients and 70 healthy females. Microbial diversity and composition were analyzed using the Illumina MiSeq sequencing platform by targeting the hypervariable V3-V4 regions of the 16S rRNA gene. We found that bacterial diversity was decreased in T2DM patients. Increased Actinobacteria phylum was positively correlated with FBG, UGLU, and BMI; Lactobacillus abundance decreased with age and menopause; and increased Lactobacillus correlated positively with FBG and UGLU. Decreased Akkermansia muciniphila was associated with FBG and UGLU. Escherichia coli abundance did not differ between the two cohorts. Carbohydrate and amino acid metabolism was reduced in T2DM patients, which were associated with bacterial richness indices such as Chao1 and ACE. Detailed microbiota analysis of well-characterized T2DM patients and healthy controls indicate that Chinese T2DM female patients exhibit dysbiosis of urinary microbiota.

Urinary microbiome of kidney transplant patients reveals dysbiosis with potential for antibiotic resistance.

Recent studies have established that a complex community of microbes colonize the human urinary tract; however, their role in kidney transplant patients treated with prophylactic antibiotics remains poorly investigated. Our aim was to investigate the urinary microbiome of kidney transplant recipients. Urine samples from 21 patients after kidney transplantation and 8 healthy controls were collected. All patients received prophylactic treatment with the antibiotic combination trimethoprim-sulfamethoxazole. Metagenomic DNA was isolated from urine samples, sequenced using shotgun sequencing approach on Illumina HiSeq 2000 platform, and analyzed for microbial taxonomic and functional annotations. Our results demonstrate that the urine microbiome of kidney transplants was markedly different at all taxonomic levels from phyla to species, had decreased microbial diversity, and increased abundance of potentially pathogenic species compared with healthy controls. Specifically, at the phylum level, we detected a significant decrease in Actinobacteria and increase in Firmicutes due to increases in Enterococcus faecalis. In addition, there was an increase in the Proteobacteria due to increases in Escherichia coli. Analysis of predicted functions of the urinary metagenome revealed increased abundance of enzymes in the folate pathway including dihydrofolate synthase that are not inhibited by trimethoprim-sulfamethoxazole, but can augment folate metabolism. This report characterizes the urinary microbiome of kidney transplants using shotgun metagenomics approach. Our results indicate that the urinary microbiota may be modified in the context of prophylactic antibiotics, indicating that a therapeutic intervention may shift the urinary microbiota to select bacterial species with increased resistance to antibiotics. The evaluation and development of optimal prophylactic regimens that do not promote antibiotic resistance is an important future goal.

Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism.

BACKGROUND: Autism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of "control" status. METHODS: ASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2-7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway. RESULTS: Urinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B6, riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate. CONCLUSIONS: The metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

Urinary Metabolomic Approach Provides New Insights into Distinct Metabolic Profiles of Glutamine and N-Carbamylglutamate Supplementation in Rats.

Glutamine and N-carbamylglutamate can enhance growth performance and health in animals, but the underlying mechanisms are not yet elucidated. This study aimed to investigate the effect of glutamine and N-carbamylglutamate supplementation in rat metabolism. Thirty rats were fed a control, glutamine, or N-carbamylglutamate diet for four weeks. Urine samples were analyzed by nuclear magnetic resonance (NMR)-based metabolomics, specifically high-resolution ¹H NMR metabolic profiling combined with multivariate data analysis. Glutamine significantly increased the urine levels of acetamide, acetate, citrulline, creatinine, and methymalonate, and decreased the urine levels of ethanol and formate (p < 0.05). Moreover, N-carbamylglutamate significantly increased the urine levels of creatinine, ethanol, indoxyl sulfate, lactate, methymalonate, acetoacetate, m-hydroxyphenylacetate, and sarcosine, and decreased the urine levels of acetamide, acetate, citrulline, creatine, glycine, hippurate, homogentisate, N-acetylglutamate, phenylacetyglycine, acetone, and p-hydroxyphenylacetate (p < 0.05). Results suggested that glutamine and N-carbamylglutamate could modify urinary metabolome related to nitrogen metabolism and gut microbiota metabolism. Moreover, N-carbamylglutamate could alter energy and lipid metabolism. These findings indicate that different arginine precursors may lead to differences in the biofluid profile in rats.

Increased urinary indoxyl sulfate (indican): new insights into gut dysbiosis in Parkinson's disease.

INTRODUCTION: Changes in the composition of gut microflora have been associated with an increase in chronic diseases. Indican urinary concentration is one of the most common and easily assessable markers of intestinal dysbiosis. Little information is available on intestinal dysbiosis in Parkinson's disease (PD). We decided to investigate indican urinary concentrations in a cohort of PD patients. METHODS: A case-control study including PD patients (N = 68) on treatment with levodopa (PD) or on no pharmacological treatment (De Novo, DPD; N=34) and an age and gender-matched healthy control group (CTR; N=50). Main confounders, such as nutritional habits and constipation diagnosed according to Rome III criteria, were also investigated. RESULTS: Indican urinary concentrations were significantly higher in PD and DPD than in CTR (P < 0.001 and P < 0.01, respectively). In PD patients the concentrations were unrelated to the presence of constipation, whereas this symptom was associated with higher concentrations in controls (P=0.043). The frequency of dairy product consumption was also positively associated with increased concentrations (P=0.008). Predictors of indican concentrations were sought by multivariate linear regression analysis. The higher indican urinary concentrations found in both DPD (P=0.045) and PD (P=0.023) patients persisted after adjustment for age, gender, BMI, constipation and consumption of dairy products. CONCLUSIONS: Gut dysbiosis seems to be an important issue in PD, independently of the presence of constipation and starting from the early stages of the disease. The role of gut dysbiosis in the pathogenesis of PD deserves further investigation.