基于16S rRNA测序分析糖尿病尿路感染者尿液菌群特征

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

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。

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.