| BackgroudsSince bacterial and viral DNA sequencing technique has been applied in detecting microorganisms in urine,the concept "Bladder is Sterile" was broken down.Recently,emerging evidence demonstrated that urinary microbiota played an important role in the onset and development of some diseases.The level of urine glucose and pH value increases in patients with type 2 diabetes mellitus(T2DM),which might affects the living environment of microorganisms in the bladder and increases patient's susceptibility to urinary tract infection(UTI).Women are prone to UTI because of the special anatomical structure of urethra.Interleukin(IL)-8 has been considered a diagnostic factor of bladder inflammation.Healthy diet pattern plays an important role in improving patient's condition,which involves moderating microbiota dysbiosis and immune disorder.Due to urine composition is mainly determined by diet,it might regulate urinary microbiota and bladder inflammation.In order to prove this hypothesis,it is necessary to explore an appropriate urine specimen collection technique for urinary microbiota research.Objectives1.To explore a modified midstream urine(MMSU)collection technique for urinary microbiota research,which might prevent patients from transurethral catheterization(TUC)hurt.2.To examine alterations of urinary microbiota in female T2DM patients,and explore the associations between urinary microbiota and a patient's fasting blood glucose(FBG),urine glucose(UGLU),etc..3.To investigate the associations between the altered urinary microbiota and urinary IL-8 in female T2DM patients.4.To explore the moderating effects of diet on the relationship between urinary microbiota and IL-8 concentrations in T2DM patients.Methods1.A self-designed MMSU included:?patient was instructed disinfection procedure applied in TUC?patient disinfected the perineal area and the opening of urethral tract with iodophor sanitizer,and urinated with squatting position with continuously separating the labia majora and minora.Four sterilized centrifuge tubes labeled sequenced number 1,2,3 and 4 were used to collect urine specimen.Controlled before-and-after study was applied.Thirty women who were being delivering by cesarean-section and TUC was necessary were recruited.The first urine specimen was collected prior to the day of delivery with MMSU,and the second specimen was collected with TUC on the day of delivery.Bacterial sequencing and urine culture were performed.Urine from tube 2 and 3 was used in the study.Bacterial genomic DNA from urine specimens was isolated using magnetic beads and the urinary microbiota was assessed using Illumina MiSeq platform targeting on the 16S rRNA gene V3-V4 region.R language,STAMP and SPSS software were used to perform bioinformatic analysis and statistical analysis.The urinary microbiota in specimens collected by MMSU and TUC was compared.2.Individually matched case control study was designed.70 female T2DM patients and 70 healthy controls(HCs)were recruited.MMSU was used to collect urine specimens.The bioinfomatic and statistical analysis were similar to the second part of this study.A comprehensive assessment of bacterial diversity and community composition in T2DM patients and HCs was conducted.Influencing factors of urinary microbiota in T2DM patients such as age,FBG,UGLU,BMI and menstrual status were analysized.3.Urine specimens collected in the second part of this study were used in this part.Urinary IL-8 was determined by enzyme linked immunosorbent assay.Subsequently,the T2DM patients were separated into urine IL-8 detectable(WIL8)and undetectable(NIL8)groups,and the composition of urinary microbiota between the two groups was compared.Meanwhile,the levels of IL-8 concentrations between the "? HCs" group(those specific bacterial genera were more than or equal to the HCs)and the "<HCs"group(those specific bacterial genera were less than the HCs)was also compared.4.Chinese Food Frequency Questionnaire was used to collect patient's food intake data while home visits.Matlab software was applied to calculate nutrients intake.Variables in regression models were as follows:the relative abundance of the bacterial genus and species those were significant different between the T2DM cohort and HCs,between the WIL8 and NIL8 group,and bacterial genus those were related to IL-8 concentration in the regression model in the Part 3 were used as independent variable.IL-8 concentration was used as dependent variable,and nutrients intake was used as moderator variable.Hierarchical regression analysis was applied.Results1.No significant difference was found in the bacterial richness and diversity,and bacterial relative abundance at bacterial phylum,order and genus level between specimens collected by MMSU and TUC(p>0.05).Moreover,bacterial community at bacterial phylum,order and genus level in specimen collected by MMSU was similar to TUC.2.The main findings of urinary microbiota in female T2DM patients were as follows:?bacterial diversity and richness were decreased in T2DM patients;?bacterial phylum,genus and species of bacterial community altered significantly,including the relative abundance of Chloroflexi,Nitrospirae and Verrucomicrobia sharply decreased in T2DM patients compared to HCs(p<0.05);the relative abundance of thirty-three bacterial genera showed significant difference between the patients and HCs(p<0.05),such as Prevotella,Lactobacillus and Shuttleworthia enriched in the patients cohort,whereas Akkermansia,Faecalibacterium and Pseudomonas enriched in the HCs;the relative abundance of Akkermansia muciniphila dramatically declined in the patient cohorts;? Actinobacteria,Porphyromonas,Flavobacteriales,Desulfovibrio,Flavobacteria and Collinsella could be considered as distinguish biomarkers of T2DM patients;?Increased Actinobacteria phylum was positively correlated with FBG,UGLU and BMI;and Increased Lactobacillus and Actinobacteria were correlated positively with FBG and UGLU,while decreased Akkermansia muciniphila was associated with FBG and UGLU;?Carbohydrate and amino acid metabolism was reduced in T2DM patients,which were associated with bacterial richness indices such as Chaol and ACE.3.Of the 70 urine samples from T2DM patients,46 patients had detectable IL-8 in their urine(64.31 ± 70.43 pg/mL),while 24 patients had undetectable IL-8.Compared to the NIL8 group,11 bacterial genera increased in the WIL8 group(p<0.05),including Corynebacterium,Akkermansia,Enterococcus,etc.,whereas 10 genera,such as Faecalibacterium,Bacteroides,and Pseudomonas decreased(p<0.05).One species of Lactobacillus,Lactobacillus iners,increased obviously in the WIL8 group.The"?HCs"and'<HCs"subgroup from four bacterial genus showed significant difference in IL-8 concentration(p<0.05),such as Actinobacteria,Klebsiella,Pseudomonas and Microbacterium.In addition,18 genera contributed to the concentration of urinary IL-8 in T2DM patients.4.Fiber,vitamin(Vit)B3,vit E and water intake had moderation effects on the relationship between the relative abundance of Ruminococcus and IL-8 concentration(p<0.05).Water intake had negative moderating effect,whereas others had positive moderating effects.Cholesterol and magnesium intake had positively moderating effects on the relationship between the relative abundance of Comamonas and IL-8 concentration(p<0.05).Vit A intake had negatively moderating effect on the relationship between the relative abundance of Anaerotruncus and IL-8 concentration(p<0.05).Conclusions1.Non-contaminated and genuine midstream urine could be collected with MMSU.Therefore,it could be applied in specimen collection for urinary microbiota study and urine culture.2.T2DM female patients exhibited dysbiosis of urinary microbiota,and the alterations were related to patient's characteristics including FBG and UGLU.Moreover,the microbiota dysbiosis were correlated with metabolism disorders.Nursing interventions focusing on moderating urinary microbiota might improve patient's metabolic disorders as well.3.This study demonstrated that dysbiosis of the urinary microbiota with several key bacteria was associated with urinary IL-8 in female T2DM patients,which might be useful to explore the interactions between urinary microbiota and inflammatory responses and shed light on novel diagnosis and therapy for urinary microbiota associated with infections in T2DM patients.4.Food intake had moderation effects on the relationship between urinary microbiota and bladder inflammation.Thus,it is recommended that modulating T2DM patient's dietary pattern to increase the nutrients such as Vit A?Vit B3?Vit E and fiber intake which might improve bladder inflammation.However,water intake and food enriched with cholesterol and magnesium should be monitored to avoid food-induced bladder inflammation. |
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