Dysbiosis Of Urinary Microbiome In Overactive Bladder By High Throughput Sequencing

BackgroundThe microbiota is increasingly considered an essential component of the normal physiologic state,and plays important roles in human health and disease.The female urinary tract has recently been recognized as being "non-sterile".Urinary microbiome may have an effect on lower urinary tract symptoms(LUTS).Overactive bladder(OAB)is characterized by urgency,with or without urgency incontinence,usually with frequency and nocturia,in the absence of a urinary tract infection(UTI)or other identifiable causes.Evidences suggests that OAB patients usually reported anxiety and depression.The etiology of OAB is unidentified,and OAB symptoms are rarely cured.Therefore,there is an urgent need to further investigate the pathogenesis of OAB,and identify new therapeutic targets.Our primary purpose was to characterize the female urinary microbiome associated with OAB in China and investigate relationships between the microbiome and psychological factors.We also analyzed the urinary microbiome in a paruria model produced by repeated exposure to water avoidance stress(WAS).Materials and methods1.Subject recruitment and urine collectionBetween September 2016 and March 2017,this study enrolled 30 adult OAB women(OABs)and 20 healthy female controls(HCs)at Nanfang Hospital in China.Each participant was required to finish overactive bladder symptom score(OABSS),self-rating anxiety scale(SAS)and self-rating depression scale(SDS).Urine specimens were collected by transurethral catheterization and left some for cultivation to exclude UTIs.The rest were immediately centrifuged at 16,000g for 10 minutes.The pellets were stored at-80?until further processing.2.16S rRNA gene sequencingDNA isolation was performed using a DNeasy Blood and Tissue Kit(Qiagen,Germany).PCR amplification of 16S rDNA sequences was performed using primer sets specific for V4 regions.The amplified sequences were then sequenced by Illumina MiSeq sequencer(Illumina,USA).3.Animal experimentAnimal studies were conducted using adult female Wistar rats,which were approved by the Institutional Animal Care and Use Committee of Southern Medical University.Rats were randomly assigned to stress group(exposure to water-avoidance stress for 10 days),stress-withdrawal group(exposure to the stress and then withdrawal for 2 weeks)or control group.On day 11 for stress and control groups or 2 weeks after day 11 for stress-withdrawal group,the Light-dark box transition test,lower abdominal pain threshold,micturition frequency and cystometry measurements were determined.Urine specimens were collected and processed for 16S rDNA gene sequencing.4.Statistical analysisDifferences in the baseline characteristics between patients and controls were evaluated using the Mann-Whitney U-test for continuous variables and Pearson's chi-square test for count data.Bivariate correlations were assessed using Pearson's correlation.All data were analyzed using SPSS(Version 22.0).Sequencing reads were processed using QIIME(version 1.9.0),which allowed for calculations of alpha diversity,UniFrac distances,and principal coordinate analysis(PCoA)results.To identify significantly different bacteria between groups,taxa summaries were reformatted and input into LEfSe via the Huttenhower Lab Galaxy Server.Results1.The two cohorts showed no significance difference in their demographic characteristics.The OABs had significantly higher scores on OABSS,SDS and SAS than the HCs.2.The Simpson index indicated that the diversity was also higher among the HCs.Beta diversity was calculated and a PCoA was performed to visualize the urinary microbiomes of the two cohorts The most frequently detected phylum was Firmicutes,followed by Proteobacteria.At the family level,Bifidobacteriaceae and Enterobacteriaceae were more abundant in the OABs than HCs.The LEfSe revealed that 8 taxa were over-represented in the OABs(e.g.,Proteus),and twenty-two taxa were under-represented(e.g.,reute).3.The alpha diversity indexes were all markedly lower in depression cases(P all<0.01).Higher score on SDS inversely correlated with both Shannon and Chao1 indexes.Some bacterial genera of OAB women with anxiety or depression were significantly different from those without.4.Compared to control group,the water-avoidance stress dramatically decreased in the time spent in the light area and decreased mechanical pain threshold in stress group,which were reversed in stress-withdrawal group.While there was no significant difference in the micturition frequency between the control and stress-withdrawal groups,the stress group showed significantly more micturition frequencies than control and stress-withdrawal group.The stress dramatically increased voiding contractions in both the stress and stress-withdrawal groups compared to control rats.However,the stress-withdrawal group showed less voiding contractions than the stress group.5.The bacteria diversity in stress group were dramatically higher than those of the other two groups.Almost all specimens of the control rats were dominated by Enterococcacae,whereas samples from the stress group contained more diverse bacteria.The diversity of microbiota in the stress-withdrawal group was between those of the other two groups.LEfSe identified genera differed significantly among groups:Lactococcus and Proteus were significantly increased in in the stress-withdrawal group;Halomonas,Aggregatibacter,Haemophilus,Pseudomonas and Streptococcus were significantly higher in in the stress group.Conclusion1.The aberrant urinary microbiome may serve as disease biomarkers and potential therapeutic targets of OAB.A negative correlation between depression scores and both bacterial richness and diversity in OAB patients indicated that there might be a connection between central nervous system and urinary microbiome.2.Water-avoidance stress experiments demonstrated the impact of stress on bladder function and urinary microbiome,which tended to be ameliorated after a two-week withdrawal from stress.Further research is needed to understand the connections between central nervous system,bladder and urinary microbiome.