食管癌患者与健康对照者的食管菌群差异分析

Analysis of the difference in esophageal flora between patients with esophageal carcinoma and healthy controls

目的探讨食管癌患者食管微生态状况,并比较其与健康人群食管菌群的差异。方法选择2018年7月至2019年7月在十堰市太和医院就诊的82例食管癌患者和同期年龄、性别相匹配的20名健康对照者,30份食管癌病理组织根据分化程度分为低分化(8份)、中分化(9份)和高分化(13份)。采集食管癌患者和健康对照者的食管组织样本,提取样本DNA,对细菌16S rRNA V4区域行PCR扩增。运用Illumina HiSeq 4000测序平台测序,行α多样性分析和主坐标分析(PCoA),采用线性判别分析效应量(LEfSe)的线性判别分析(LDA)值筛选差异物种,ROC曲线验证随机森林模型,BugBase数据库预测食管细菌表型。采用非参数Kruskal-Wallis H检验和Wilcoxon秩和检验进行统计学分析。结果食管癌患者Chao1指数高于健康对照者[362.51(284.29,646.13)比284.83(244.31,344.74)],差异有统计学意义(Z=-2.857,P=0.004)。PCoA显示食管癌患者和健康对照者样本间距离较近,两者微生态菌群构成差异无统计学意义(P>0.05)。食管癌患者食管蓝藻菌门和疣微菌门的丰度均高于健康对照者(0.2%比0.1%、0.4%比0),而食管变形菌门、SR1菌门和TM7菌门的丰度均低于健康对照者(21.9%比34.2%、0.1%比0.2%和0.2%比0.5%),差异均有统计学意义(Q=0.090、0.077、0.010、0.026、0.001,P均<0.05)。低分化,中分化和高分化食管癌患者食管梭状芽孢杆菌纲、梭状芽孢杆菌目、巴斯德氏菌目、巴斯德氏菌科、埃肯菌属、放线杆菌属和嗜血杆菌属的相对丰度分别为28.3%、24.2%和17.0%,28.3%、24.2%和17.0%,3.2%、0.3%和5.0%,3.2%、0.3%和5.0%,0、1.5%和0.1%,0.5%、0和0.7%,1.3%、0.2%和3.9%,比较差异均有统计学意义(Q=0.579、0.557、0.390、0.711、0.768、0.768、0.768,P均<0.05)。LEfSe分析显示,食管癌患者梭杆菌目、瘤胃球菌属、气味杆菌属和S24_7科的丰度均高于健康对照者(21.5%比11.7%、0.5%比0.1%、0.1%比0、0比0),差异均有统计学意义(LDA值=2.591、2.379、2.790、2.927,P均<0.05)。ROC曲线证实随机森林模型可靠,AUC值为0.92。BugBase数据库预测显示,食管癌患者生物膜形成、致病潜力、移动元件、氧需求(厌氧菌、需氧菌、兼性菌)和氧化胁迫耐受食管细菌表型较健康对照者更丰富(P均<0.05)。结论食管癌患者的食管菌群发生改变,梭杆菌目、瘤胃球菌属、气味杆菌属和S24_7科可作为潜在的菌群标志物。

Objective To investigate the esophageal microecology in patients with esophageal carcinoma(EC),and to compare the difference in esophageal flora between patients with esophageal cancer and healthy people.Methods From July 2018 to July 2019,at Taihe Hospital,82 EC patients and 20 age-and gender-matched healthy controls during the same period were selected.The pathology of EC were divided into poorly differentiated(8 cases),moderately differentiated(9 cases)and well differentiated cancers(13 cases)according to the degree of differentiation.The esophageal tissue samples of EC patients and healthy individuals were collected.Sample DNA was extracted and the V4 region of bacterial 16S rRNA was amplified by polymerase chain reaction(PCR).Sequencing was performed by lllumina HiSeq 4000 sequencing platform.Alpha-diversity analysis and principal co-ordinates analysis(PCoA)were performed,and linear discriminant analysis(LDA)of linear discriminant analysis effect size(LEfSe)was used to screen different species.The random forest model was verified by receiver operating characteristic(ROC)curve and the esophageal bacterial phenotype was predicted by BugBase database.Non-parametric Kruskal-Wallis H test and Wilcoxon rank sum test were used for statistical analysis.Results The Chao1 index of the EC patients was higher than that of healthy controls(362.51(284.29,646.13)vs.284.83(244.31,344.74)),and the difference was statistically significant(Z=-2.857,P=0.004).The results of PCoA showed that the distance between samples of EC patients and healthy control samples was relatively close,and there was no significant difference in the composition of microecology between the two groups(P>0.05).The abundance of esophageal Cyanobacteria and Verrucomicrobia of EC patients were both higher than those of healthy controls(0.2%vs.0.1%,0.4%vs.0),while the abundances of esophageal Proteobacteria,SR1 and TM7 phylum of EC patients were lower than those of healthy controls(21.9%vs.34.2%,0.1%vs.0.2%,0.2%vs.0.5%),and the differences were statistically significant(Q=0.090,0.077,0.010,0.026 and 0.001,all P<0.05).The abundances of Clostridia,Elostridiales,Pasteurella,Pasteurellaceae,Eikenella,Actinobacillus and Haemophilus in poorly differentiated patients,moderately differentiated and higher differentiated patients were 28.3%,24.2%and 17.0%,28.3%,24.2%and 17.0%,3.2%,0.3%and 5.0%,3.2%,0.3%and 5.0%,0,1.5%and 0.1%,0.5%,0 and 0.7%,1.3%,0.2%and 3.9%,respectively,and the differences were statistically significant(Q=0.579,0.557,0.390,0.711,0.768,0.768 and 0.768,all P<0.05).LEfSe analysis showed that the abundances of Fusobacterium,Ruminococcus,Odorbacterium and S24_7 of EC patients were higher than those of healthy controls(21.5%vs.11.7%,0.5%vs.0.1%,0.1%vs.0 and 0 vs.0),and the differences were statistically significant(LDA=2.591,2.379,2.790 and 2.927,all P<0.05).The ROC curve confirmed that the random forest model was reliable and the AUC value was 0.92.BugBase database phenotypic prediction showed that the phenotype of esophageal bacteria related to biofilm formation,pathogenic potential,mobile elements,oxygen demand(aerobic,anaerobic and facultative bacteria),and oxidative stress tolerance of EC patients were more abundant than those of healthy controls(all P<0.05).Conclusions The esophageal flora of patients with esophageal cancer has changed.Fusobacterium,Ruminococcus,Odoribacterium and S24_7 may be potential biomarkers of esophageal flora.