基于16SrDNA测序技术分析重症肺炎患者肠道菌群的变化

Analysis of intestinal patients'flora changes with severe pneumonia based on 16SrDNA sequencing technology

目的用16SrDNA测序技术分析重症肺炎患者肠道菌群变化的特征.方法采用前瞻性研究方法,收集2018年6月至12月住宁夏医科大学总医院重症医学科(ICU)16例重症肺炎患者入院2 d内自然排便或灌肠所得的粪便标本,以及10例健康体检者的粪便标本,采用16SrDNA测序技术对粪便菌群进行检测并进行生物学信息分析.结果①对重症肺炎患者和健康者粪便样本进行16SrDNA测序共得到1015475条有效序列,样本序列平均有效长度为458.35 bp,总样本平均序列条数为39056.73条.②肠道菌群α多样性分析显示,与健康对照组比较,重症肺炎组患者肠道菌丰度指数Ace指数、Chao指数及肠道菌群多样性指数Shannon指数显著下降〔Ace指数:167.23(143.14,211.26)比227.71(214.53,247.05),Chao指数:152.38(138.09,182.54)比228.25(215.49,248.95),Shannon指数:2.37(1.68,2.89)比3.39(3.03,3.63),均P<0.01〕,肠道菌群多样性指数Simpson指数显著升高〔0.21(0.11,0.33)比0.07(0.06,0.12),P<0.01〕,提示重症肺炎患者肠道菌群的丰度和多样性下降.③肠道菌群β多样性分析(采用主坐标分析)显示,健康对照组菌群结构相似度高,重症肺炎组菌群结构差异较大.Adonis分析显示,重症肺炎组与健康对照组菌群群落结构存在显著差异(R2=0.061,P=0.05).④门水平差异菌分析显示,重症肺炎组厚壁菌门比例较健康对照组显著下降〔27.36(18.12,39.28)%比52.25(38.36,63.82)%,P=0.02〕,而放线菌门、互养菌门和梭杆菌门的比例均较健康对照组明显增加〔2.30(0.30,4.80)%比0.02(0.00,0.06)%,0.36(<0.01,0.57)%比<0.01(<0.01,<0.01)%,0.01(<0.01,0.08)%比<0.01(<0.01,<0.01)%,均P<0.05〕.⑤属水平差异菌分析显示,重症肺炎组有益共生菌属双歧杆菌属、瘤胃球菌属、假丁酸弧菌属、粪球菌属、毛螺菌属、普氏菌属比例均较健康对照组显著下降〔0.18(0.01,0.25)%比3.40(0.46,5.78)%,0.01(<0.01,0.29)%比2.26(0.84,4.86)%,0.01(<0.01,0.02)%比2.73(1.87,5.74)%,0.02(<0.01,0.07)%比0.80(0.50,2.32)%,<0.01(<0.01,<0.01)%比0.88(0.33,2.08)%,0.02(<0.01,0.31)%比7.74(0.07,36.27)%,均P<0.05〕;机会性致病菌埃希杆菌属及致病菌肠球菌属比例较健康对照组升高,但差异无统计学意义〔2.00(0.57,10.23)%比1.16(0.23,2.68)%,0.02(<0.01,0.42)%比<0.01(<0.01,0.04)%,均P>0.05〕;而致病菌梭杆菌属、葡萄球菌属比例较健康对照组明显升高〔0.01(<0.01,0.08)%比<0.01(<0.01,<0.01)%,0.01(<0.01,0.02)%比<0.01(<0.01,<0.01)%,均P<0.05〕.结论重症肺炎患者肠道菌群发生紊乱,表现为丰度及多样性下降,菌群群落结构发生改变,有益共生菌属减少,致病菌属增多,可能与重症肺炎疾病的发生发展有关.

Objective To investigate the characteristics of gut microbiota dysbosis in patients with severe pneumonia using 16SrDNA sequencing.Methods A prospective observational research was conducted.The stool samples retained by natural defecation or enema within 2 days after hospital were collected from 16 patients with severe pneumonia admitted to department of intensive care unit(ICU)of General Hospital of Ningxia Medical University from June to December in 2018 and 10 persons for physical exam were enrolled as the healthy control group.The 16SrDNA sequencing technology was used to detect fecal flora and analyze biological information.Results①1015475 effective sequences were obtained from the stool samples from the severe pneumonia group and the healthy control group.Using 16SrDNA method,it was found that the average effective length of the sample sequence was 458.35 bp and the average sequence number of the total samples was 39056.73.②Analysis ofαdiversity of gut microbiota showed that,compared with the healthy control group,the Ace index,Chao index and the Shannon index of gut microbiota diversity in the severe pneumonia group were significantly decreased[Ace index:167.23(143.14,211.26)vs.227.71(214.53,247.05),Chao index:152.38(138.09,182.54)vs.228.25(215.49,248.95),Shannon index:2.37(1.68,2.89)vs.3.39(3.03,3.63),all P<0.01],and the Simpson index was significantly increased[0.21(0.11,0.33)vs.0.07(0.06,0.12),P<0.01],which indicated the gut microbiota diversity of the severe pneumonia group was decreased.③Analysis ofβdiversity of gut microbiota,principal coordinate analysis(PCoA)showed that gut microbiota structural with the healthy control group was similar,while that in the severe pneumonia group was different.Adonis analysis showed that the structural of the gut microflora revealing significant differences between the severe pneumonia group and the healthy control group(R2=0.061,P=0.05).④Analysis of phylum difference gut microflora showed that,compared with the healthy control group,the proportion of Firmicutes in severe pneumonia group was decreased[27.36(18.12,39.28)%vs.52.25(38.36,63.82)%,P=0.02],the proportions of Actinobacterias,Synergistetes and Fusobacterias were increased[2.30(0.30,4.80)%vs.0.02(0.00,0.06)%,0.36(<0.01,0.57)%vs.<0.01(<0.01,<0.01)%,0.01(<0.01,0.08)%vs.<0.01(<0.01,<0.01)%,all P<0.05].⑤Analysis of genus difference gut microflora showed that,the proportions of Bifidobacterium,Ruminococcus,Pseudobutyrivibrio,Coprococcus,Lachnospira and Prevotella in the severe pneumonia group were significantly lower than those in healthy control group[0.18(0.01,0.25)%vs.3.40(0.46,5.78)%,0.01(<0.01,0.29)%vs.2.26(0.84,4.86)%,0.01(<0.01,0.02)%vs.2.73(1.87,5.74)%,0.02(<0.01,0.07)%vs.0.80(0.50,2.32)%,<0.01(<0.01,<0.01)%vs.0.88(0.33,2.08)%,0.02(<0.01,0.31)%vs.7.74(0.07,36.27)%,all P<0.05];the proportions of Escherichia and Enterococcus in the severe pneumonia group were higher than those in healthy control group,but there was no difference between the two groups[2.00(0.57,10.23)%vs.1.16(0.23,2.68)%,0.02(<0.01,0.42)%vs.<0.01(<0.01,0.04)%,both P>0.05];the proportions of Fusobacterium and Staphylococcus in severe pneumonia group were significantly higher than those in healthy control group[0.01(<0.01,0.08)%vs.<0.01(<0.01,<0.01)%,0.01(<0.01,0.02)%vs.<0.01(<0.01,<0.01)%,both P<0.05].Conclusion Gut microbiota dysbiosis in patients with severe pneumonia shows that the abundance and diversity decrease,structure of intestinal flora changes,and beneficial symbiotic bacteria decrease and pathogenic bacteria increase,which may be associated with the occurrence and development of severe pneumonia.