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中华实验和临床感染病杂志(电子版) ›› 2023, Vol. 17 ›› Issue (03) : 164 -172. doi: 10.3877/cma.j.issn.1674-1358.2023.03.004

论著

基于16S rRNA测序分析糖尿病尿路感染者尿液菌群特征
尹九湖, 卢晓明, 孙科, 易忠权, 沈园园, 刘亚东†()   
  1. 224000　盐城市，盐城市第三人民医院泌尿外科；224000　盐城市，江苏医药职业学院临床医学院
    224000　盐城市，盐城市第三人民医院泌尿外科；224000　盐城市，南通大学第六附属医院泌尿外科
    224000　盐城市，盐城市第三人民医院内分泌科
    224000　盐城市，盐城市第三人民医院中心实验室
    224000　盐城市，盐城市第三人民医院泌尿外科
    224000　盐城市，盐城市第三人民医院泌尿外科；224000　盐城市，江苏医药职业学院临床医学院；224000　盐城市，南通大学第六附属医院泌尿外科
  • 收稿日期:2022-12-01 出版日期:2023-06-15
  • 通信作者: 刘亚东
  • 基金资助:
    江苏省卫生健康委医学科研项目(No. Z2021024); 盐城市医学科技发展计划项目(No.YK2020075); 江苏医药职业学院临床医学院科研项目(No. 20209110)

Microbiota characteristics of urine microbiota in patients with diabetic urinary tract infection based on 16S rRNA gene sequencing analysis

Jiuhu Yin, Xiaoming Lu, Ke Sun, Zhongquan Yi, Yuanyuan Shen, Yadong Liu†()   

  1. Department of Urology, The Third People’s Hospital of Yancheng, Yancheng 224000, China; Department of Central Laboratory, The Third People’s Hospital of Yancheng, Yancheng 224000, China
    Department of Urology, The Third People’s Hospital of Yancheng, Yancheng 224000, China; Yancheng Hospital, the Affiliated Hospital of Jiangsu Vocational College of Medicine;, Yancheng 224000, China
    Department of Endocrinology and Metabolic Diseases, The Third People’s Hospital of Yancheng, Yancheng 224000, China
    Department of Urology, The Third People’s Hospital of Yancheng, Yancheng 224000, China
    Department of Urology, The Third People’s Hospital of Yancheng, Yancheng 224000, China; Department of Central Laboratory, The Third People’s Hospital of Yancheng, Yancheng 224000, China; Yancheng Hospital, the Affiliated Hospital of Jiangsu Vocational College of Medicine;, Yancheng 224000, China
  • Received:2022-12-01 Published:2023-06-15
  • Corresponding author: Yadong Liu
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引用本文：

尹九湖, 卢晓明, 孙科, 易忠权, 沈园园, 刘亚东. 基于16S rRNA测序分析糖尿病尿路感染者尿液菌群特征[J/OL]. 中华实验和临床感染病杂志(电子版), 2023, 17(03): 164-172.

Jiuhu Yin, Xiaoming Lu, Ke Sun, Zhongquan Yi, Yuanyuan Shen, Yadong Liu. Microbiota characteristics of urine microbiota in patients with diabetic urinary tract infection based on 16S rRNA gene sequencing analysis[J/OL]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2023, 17(03): 164-172.

目的

探讨16S rRNA测序技术在糖尿病尿路感染者尿液菌群特征分析中的应用。

方法

收集于2021年3月至2021年9月盐城市第三人民医院泌尿外科、内分泌科门诊及住院患者的尿液样本，对糖尿病尿路感染者（DI）（12例）及非尿路感染人群（DNI）（12例）、正常人群（NOR）（9例）及单纯尿路感染者（UTI）（7例）尿液菌群16S保守区V3、V4进行测序分析。利用Alpha和Beta多样性指数分析尿液菌群的丰度和均匀度及组间差异。对不同分组进行菌群物种组成分析，利用R软件包绘制菌群间相关性热图。利用BugBase软件分析预测各组菌群表型。

结果

在Alpha多样性分析中，DI组与NOR组患者observed species指数、香农指数（shannon）、辛普森指数（simpson）以及chao1指数4个指标差异均有统计学意义（t = 2.833、P = 0.011，t = 3.619、P = 0.002，t = 2.82、P = 0.011，t = 2.69、P = 0.017）。PCoA分析4组间Beta多样性差异无统计学意义（F = 1.71、P = 0.071），DI与DNI组菌群组成差异有统计学意义（F = 2.56、P = 0.031）。在物种组成分析中，在门水平，厚壁菌门在DI组与DNI组患者尿液样本中差异有统计学意义（Z =－2.425、P = 0.014，）；在属水平，乳酸菌属（Lactobacillus）（Z =－2.175、P = 0.03）、Negativicoccus（Z =－2.685、P = 0.007）、理研菌属（Rikenella）（Z =－2.134、P = 0.033）；卟啉单胞菌属等14类菌属在DI组与NOR组差异均有统计学意义（P均< 0.05）；Vulcaniibacterium属在DI组与UTI组患者尿液样本差异有统计学意义（Z = －2.405、P = 0.019）。菌群相关性分析结果显示，DI组患者中变形菌门与放线菌门（r =－0.73、P = 0.007）、厚壁菌门（r =－0.67、P = 0.017）呈负相关。菌群表型预测结果显示，DI组与DNI组患者生物膜形成表型差异有统计学意义（Z =－2.456、P = 0.014）。

结论

DI组患者菌群丰度与均匀度较NOR组显著下降。变形菌门和放线菌门、厚壁菌门的菌群失衡，可能导致生物膜形成表型差异，进而引起糖尿病患者尿路感染易感性增加。

关键词: 糖尿病, 尿路感染, 菌群, 16S rRNA
Objective

To explore the application of 16S rRNA sequencing on analysis of the microbiota characteristics of urine flora in diabetic urinary tract infections.

Methods

The urine samples of patients with diabetic urinary tract infection (DI) (12 cases), patients with diabetic non-urinary tract infection (DNI) (12 cases), normal population (NOR) (9 cases) and patients with urinary tract infection (UTI) (7 cases) were collected from outpatients and inpatients in the Department of Urology and Endocrinology of the Third People’s Hospital of Yancheng from March 2021 to September 2021, and were sequenced and analyzed by the conserved region V3 and V4 of 16S rDNA. The indices Alpha was used to analyze the abundance and evenness of urine microbiota. The flora species composition of different groups was analyzed by beta diversity, and the heat map of correlation between the flora was drawn by R software. The phenotypes of different groups were forecasted by BugBase software.

Results

In Alpha diversity analysis, there were significant differences in observed species, shannon, simpson, chao1 between DI group and NOR group (t = 2.833, P = 0.011; t = 3.619, P = 0.002; t = 2.82, P = 0.011; t = 2.69, P = 0.017). In Beta diversity comparison, PCoA analysis showed no significant difference among the four groups (F = 1.71, P = 0.071), while patients in DI group and DNI group had significant difference in PCoA analysis (F = 2.56, P = 0.031). During the analysis of species composition, at the phylum level, Firmicutes were significantly different between DI group and DNI group (Z =-2.425, P = 0.014). At the genus level, 3 genera including Lactobacillus, Negativicoccus, Rikenella showed significant differences in the urine samples between DI group and DNI group (Z =-2.175, P = 0.03; Z =-2.685, P = 0.007; Z =-2.134, P = 0.033). Total of 14 genera including Porphyromonas were significantly different between DI group and NOR group (all P < 0.05). Vulcaniibacterium were significantly different in the urine samples of DI group and UTI group (Z =-2.405, P = 0.019). In the analysis of microbiota correlation, Proteobacteria were negatively correlated with Actinobacteria (r =-0.73, P = 0.007) and Firmicutes (r =-0.67, P = 0.017) in DI group. In the study of microbiota phenotype, biofilm formation phenotype were significantly different between DI group and DNI group (Z =-2.456, P = 0.014).

Conclusions

The abundance and evenness of microbiota in DI group were significantly lower than those in NOR group. The imbalance of Proteobacteria, Actinomycetes and Firmicutes may lead to differences in biofilm formation phenotypes, and thus increase the susceptibility to urinary tract infections of diabetic patients.

Key words: Diabetes mellitus, Urinary tract infection, Microbiota, 16S rRNA
图1 测序序列长度分布
图2 四组样本OTU分布维恩图注：每1个椭圆代表1个组。绿色代表NOR组，黄色代表DNI组，紫色代表DI组，橙色代表UTI组
表1 DI组与NOR组α多样性指标[M（P25，P75）]
α多样性指标 DI组 NOR组 Z值 P值
observed species 76（27.50，117.50） 156（111.50，295.00） －2.879 0.004
shannon 2.06（0.97，3.34） 3.75（3.19，4.93） －2.595 0.009
simpson 0.62（0.28，0.78） 0.87（0.73，0.91） －2.737 0.006
chao1 77.84（31.25，115.09） 159.88（113.43，313.38） －2.843 0.004
图3 observed species指数和香农（shannon）指数注：香农指数（shannon）稀释曲线走势趋于平坦时，说明测序数量足够，能够反应样本中绝大多数微生物群落信息。goods _coverage稀释曲线走势趋于平坦时，说明低丰度的微生物也已经被覆盖，能够反应样本中微生物群落覆盖率良好
图4 菌群分布PCoA图注：A：四组样本菌群分布，B：DI与NOR组，C：DI与DNI组，D：DI与UTI组
图5 门水平物种组成注：柱状堆叠图显示DI、DNI、NOR及UTI 4组患者尿液，在门水平丰度最高的前30门细菌。其中最具优势的5个门的菌群分别是厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）、放线菌门（Actinobacteria）、拟杆菌门（Bacteroidetes）和梭杆菌门（Bacteroidetes）
图6 属水平物种组成注：柱状堆叠图显示DI、DNI、NOR及UTI4组患者尿液，在属水平丰度最高的前30细菌。其中最具优势的5个菌属分别是加德纳菌属（Gardnerella）、乳酸菌属（Lactobacillus）、埃希菌属（Escherichia-Shigella）、葡萄球菌属（Staphylococcus）和伯克霍尔德菌属（Burkholderia）
表2 DI组与NOR组中存在差异的菌属[M（P25，P75）]
菌属 DI组（12例） NOR组（9例） Z值 P值
卟啉单胞菌属 0.00（0.00，0.02） 0.24（0.00，2.95） －2.245 0.025
动弯杆菌属 0.00（0.00，0.00） 0.01（0.00，0.58） －2.444 0.015
Ezakiella 0.00（0.00，0.01） 0.58（0.01，12.90） －2.758 0.006
Negativicoccus 0.00（0.00，0.00） 0.14（0.00，2.19） －2.494 0.013
W5053 0.00（0.00，0.00） 0.00（0.00，1.04） －2.488 0.013
丛毛单胞菌属 0.00（0.00，0.13） 0.15（0.06，0.50） －2.318 0.020
慢生根瘤菌属 0.01（0.00，0.16） 0.09（0.03，0.33） －2.160 0.031
Murdochiella 0.00（0.00，0.00） 0.00（0.00，0.48） －2.101 0.036
红蝽菌属 0.00（0.00，0.00） 0.00（0.00，0.03） －2.101 0.036
Gallicola 0.00（0.00，0.00） 0.00（0.00，0.75） －2.101 0.036
隐秘杆菌属 0.00（0.00，0.00） 0.00（0.00，0.02） －2.101 0.036
肠杆菌属 0.00（0.00，0.00） 0.00（0.00，0.02） －2.101 0.036
Tissierellia 0.00（0.00，0.00） 0.00（0.00，0.02） －2.101 0.036
黄杆菌属 0.00（0.00，0.00） 0.00（0.00，0.02） －2.489 0.013
图7 糖尿病感染组（DI）门水平相关性热图注：蓝色代表正相关，橘红色代表负相关。*：P < 0.05，**：P < 0.01，***：P < 0.001
图8 DI与DNI组生物膜表型差异
[1]
Russo T. Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an increasingly important endemic problem[J]. Microbes Infect,2003,5(5):449-456.
[2]
Foxman B. Urinary tract infection syndromes: occurrence, recurrence, bacteriology, risk factors, and disease burden[J]. Infect Dis Clin North Am,2014,28(1):1-13.
[3]
Flores-Mireles AL, Walker JN, Caparon M, et al. Urinary tract infections: epidemiology, mechanisms of infection and treatment options[J]. Nat Rev Microbiol,2015,13(5):269-284.
[4]
Wilke T, Boettger B, Berg B, et al. Epidemiology of urinary tract infections in type 2 diabetes mellitus patients: An analysis based on a large sample of 456,586 German T2DM patients[J]. J Diabetes Complicat,2015,29(8):1015-1023.
[5]
Fu AZ, Iglay K, Qiu Y, et al. Risk characterization for urinary tract infections in subjects with newly diagnosed type 2 diabetes[J]. J Diabetes Complicat,2014,28(6):805-810.
[6]
程玲玲. 糖尿病尿路感染患者尿液菌群分布及耐药性分析[J]. 临床医学,2021,41(2):52-53.
[7]
McDonald M, Kameh D, Johnson ME, et al. A head-to-head comparative phase Ⅱ study of standard urine culture and sensitivity versus DNA next-generation sequencing testing for urinary tract infections[J]. Rev Urol,2017,19(4):213-220.
[8]
Barnes HC, Wolff B, Abdul-Rahim O, et al. A randomized clinical trial of standard versus expanded cultures to diagnose urinary tract infections in women[J]. J Urol,2021,206(5):1212-1221.
[9]
Aragon IM, Herrera-Imbroda B, Queipo-Ortuno MI, et al. The urinary tract microbiome in health and disease[J]. Eur Urol Focus,2018,4(1):128-138.
[10]
Whiteside SA, Razvi H, Dave S, et al. The microbiome of the urinary tract-a role beyond infection[J]. Nat Rev Urol,2015,12(2):81-90.
[11]
Wolfe AJ, Brubaker L. "Sterile Urine" and the presence of bacteria[J]. Eur Urol,2015,68(2):173-174.
[12]
中华医学会糖尿病学分会. 中国2型糖尿病防治指南(2020年版)[J]. 国际内分泌代谢杂志,2021,37(4):311-396.
[13]
Fadrosh DW, Ma B, Gajer P, et al. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform[J]. Microbiome,2014,2(1):6.
[14]
Langille M, Zaneveld J, Caporaso J, et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences[J]. Nat Biotechnol,2013,31(9):814-821.
[15]
BrubakerL, Putonti C, Dong Q, et al. The human urobiome[J]. Mamm Genome,2021,32(4):232-238.
[16]
Jones-Freeman B, Chonwerawong M, Marcelino VR, et al. The microbiome and host mucosal interactions in urinary tract diseases[J]. Mucosal Immunol,2021,14(4):779-792.
[17]
Klein RD, Hultgren SJ. Urinary tract infections: microbial pathogenesis, host-pathogen interactions and new treatment strategies[J]. Nat Rev Microbiol,2020,18(4):211-226.
[18]
孙宇焱, 丁三珍. 女性2型糖尿病患者尿液细菌分布及其与血清IL-6的相关性[J]. 中国微生态学杂志,2021,33(10):1170-1175.
[19]
陈嘉炜, 曹瑛, 陈杨, 等. 女性Ⅱ型糖尿病患者尿液菌群特征与非感染性下尿路症状的关系[J]. 临床泌尿外科杂志,2018,33(1):16-19.
[20]
汪琳姣, 杨小华, 席巍, 等. 女性糖尿病患者泌尿系统感染的尿液菌群特征,血清CRP水平及危险因素分析[J]. 国际泌尿系统杂志,2022,42(2):287-290.
[21]
Thomas-White KJ, Kliethermes S, Rickey L, et al. Evaluation of the urinary microbiota of women with uncomplicated stress urinary incontinence[J]. Am J Obstet Gynecol,2017,216(1):55e51-55e16.
[22]
Moustafa A, Li W, Singh H, et al. Microbial metagenome of urinary tract infection[J]. Sci Rep,2018,8(1):4333.
[23]
Huang L, Li X, Zheng B, et al. Differential urinary microbiota composition between women with and without recurrent urinary tract infection[J]. Front Microbiol,2022,13:888681.
[24]
Penckofer S, Limeira R, Joyce C, et al. Characteristics of the microbiota in the urine of women with type 2 diabetes[J]. J Diabetes Complicat,2020,34(6):107561,
[25]
Liu F, Ling Z, Xiao Y, et al. Characterization of the urinary microbiota of elderly women and the effects of type 2 diabetes and urinary tract infections on the microbiota[J]. Oncotarget,2017,8(59):100678- 100690.
[26]
Nitzan O, Elias M, Chazan B, et al. Urinary tract infections in patients with type 2 diabetes mellitus: review of prevalence, diagnosis, and management[J]. Diabetes Metab Syndr Obes,2015,8:129-136.
[27]
Ghane M, Babaeekhou L, Ketabi S. Escherichia coli antibiofilm activity of kefir probiotic lactobacilli against uropathogenic (UPEC)[J]. Avicenna J Med Biotechnol,2020,12(4):221-229.
[28]
Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms[J]. Lancet,2001,358(9276):135-138.
[29]
Bjarnsholt T, Ciofu O, Molin S, al. e. Applying insights from biofilm biology to drug development--can a new approach be developed?[J]. Nat Rev Drug Discov,2013,12(10):791-808.
[30]
Eberly AR, Floyd KA, Beebout CJ, et al. Biofilm formation by uropathogenic Escherichia coli is favored under oxygen conditions that mimic the bladder environment[J]. Int J Mol Sci,2017,18(10): 2077.
[31]
Sharma D, Saharan BS. Functional characterization of biomedical potential of biosurfactant produced by Lactobacillus helveticus[J]. Biotechnol Rep (Amst),2016,11:27-35.
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