Dysbiosis Of Urinary Microbiota In Diabetic Bladder Dysfunction By 16S RRNA Gene High Throughput Sequencing

BackgroundMicrobiota is considered to be closely related to diseases and health of human being.The traditional view of"healthy human urinary sterility"has been broken with the emergence of new technologies,and a large number of studies suggested that urinary microbiota may have an effect on lower urinary tract symptoms(LUTS).Diabetic bladder dysfunction(DBD)is one of the common lower urinary tract complications of diabetes.Due to the pathogenesis of DBD is still unclear,the overall treatment effect is not satisfactory recently.The aim of this study was to explore the differences in urine microbiota between DBD patients and normal people,to find potential bacterial markers of DBD,and to explore the relationship between changes in urine microbiota and DBD.Materials and methods1.Volunteers recruitmentFrom June 2017 to November 2017,middle-aged or older female patients diagnosed vwith type 2 diabetes in the Department of Endocrinology and Metabolism,Nanfang Hospital(DM group,32)and healthy volunteers(control group,26)were enroll in this study.2.Data collectionDemographic characteristics(age,weight,etc.),clinical characteristics(hypertension,diabetic nephropathy,etc.)and biochemical indicators(glycated hemoglobin,serum creatinine,etc.)were collected.All participants were required to complete the International Prostate Symptom Score(IPSS).3.Urine samples collection,preservation and 16S rRNA gene sequencingSamples were obtained from midstream urine(about 50ml).Some of the urine samples were left for cultivation to exclude urinary tract infections.The rest urine samples were centrifuged at 16,000g for 10 minutes.Until next processing all pellets were stored at-80℃.We used a BiOstic Bacteremia DNA Isolation(MOBIO,USA)to isolate DNA.Primer sets specific for V3-V4 regions of 16S rDNA gene were applied to PCR amplification.Illumina MiSeq sequencer(Illumina,USA)were used to sequence the amplified sequences.4.Bioinformatics analysisSequencing data was imported into QIIME and clustered into operational taxonomic units(OTUs).Based on the analysis results of OTUs,the α and β diversity comparison of urine microbiota was performed.The Linear discriminant analysis effect size(LEfSe)online tool was used to identify bacteria that were significantly different in genus levels.5.Statistical analysisMann-Whitney U test or student t test were applied for continuous variables comparison and Pearson’s chi-square test or Fisher’s exact test were used for count data analysis.Bivariate correlations were assessed using Pearson’s correlation.P<0.05 indicates that the difference was statistically significant.ResultsNo significant differences in demographic and clinical characteristics were found between the DM group and the control group except for fasting blood glucose.The total scores,storage scores and voiding scores of IPSS in the DM group were significantly higher than those in the control group(P<0.05).There were no statistical differences in the alpha diversity between the DM group and the control group,suggesting that no significant change in the diversity and abundance of urinary microbiota were presented in the DM patients.The three-dimensional graph of principal component analysis showed that the DM group and the control group were clustered into two different regions,suggesting that there was a significant difference in the structure of urnne microbiota between the two groups(β diversity).LEf’Se analysis indicated that the abundance of 10 genera such as Escherichia-Shigella and Klebsiella was elevated in the DM group.The abundance of 7 genera such as Peptoniphilus and Solitalea was elevated in the control group.Except for hemoglobin Ale(GHb),there were no significant differences in demographic and clinical characteristics between the moderate to severe LUTS group(HS group,IPSS total score of IPSS>7)and the mild LUTS group(LS group,total score of IPSS≤7).The Observed Species,Chao 1 index and ACE index in the HS group were significantly lower than those in the control group,suggesting that the abundance of urinary microbiota in HS group was decreased.The three-dimensional plots of PCoA showed statistically significant differences in urnnary microbiota beta diversity between the LS group and the HS group.The LEfSe analysis indicated that the abundances of the three genera such as Escherichia-Shigella and Canmpylobacter were increased in the HS group.However,the abundance of 16 genera such as Prevotella and Dialister were significantly higher in the LS group.Because of the significant differences in GHb were found between the LS and HS groups,we further analyzed the urinary microbiota Characteristics in DM patients with better control blood glucose(LH,GHb≤7%)and those with poor control blood glucose(HH,GHb>7).The Shannon index in HH group was significantly lower than that in LH group,and the Simpson index was significantly higher than that in LH group,suggesting that the diversity of urinary microbiota in HH group was decreased.The three-dimensional plots of PCoA showed that the differences in urinary microtiota beta diversity between the HH group and the LH group were statistically significant.LEfSe analysis suggested that the relative abundance of Escherichia-Shigella and Lactobacillus was elevated in the HH group at the genus level.The relative abundance of 10 genera such as Prevotella and Camp.ylobacter was significantly increased in the LH group.In addition,bivariate correlation analysis found that GHb was positively correlated with the total score of IPSS.ConclusionThere were significant differences in the composition of urinary microbiota between DM group and control group,HS group and LS group,as well as HH group and LH group.Changes in certain urine microbiota may be associated with the onset of DBD.The severity of LUTS might be related to the chronic state of hyperglycemia,which may contribute to the development of LUTS by affecting the urinary microbiota.