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[16S核糖体RNA高通量测序对重度烧伤患者早期肠道菌群变化分析及代谢功能预测的前瞻性研究]

[Prospective study on the analysis of intestinal microflora changes and prediction on metabolic function in severe burn patients at early stage by 16S ribosomal RNA high-throughput sequencing].

作者信息

Guo Z W, Song M M, Zhang J, Liu L, Yang Y X, Shao Y M, Li L B, Sun B W

机构信息

Department of Burns and Plastic Surgery, Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215002, China.

Department of Burns and Plastic Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China.

出版信息

Zhonghua Shao Shang Za Zhi. 2021 Dec 20;37(12):1122-1129. doi: 10.3760/cma.j.cn501120-20200916-00414.

Abstract

To analyze the changes of intestinal microflora and to predict the metabolic function of intestinal microflora in severe burn patients at early stage by 16S ribosomal RNA () high-throughput sequencing. In this prospective observational study, 48 patients with severe burns who met the inclusion criteria were admitted to Department of Burns and Plastic Surgery of Affiliated Hospital of Jiangsu University from January 2018 to December 2019 were included in burn group, and 40 healthy volunteers who met the inclusion criteria and underwent physical examination at the Physical Examination Center of Affiliated Hospital of Jiangsu University in the same period were included in healthy group. Fecal samples were collected from patients in burn group in about 1 week after admission and from volunteers in healthy group on the day of physical examination. The 16S rRNA V4 gene sequencing was performed in the feces of patients in burn group and volunteers in healthy group to analyze the relative abundance of various bacteria. The operational classification unit (OTU) was divided by Mothur software to analyze the dominant bacteria. The OTU number, Chao1 index, Ace index, and Shannon index of fecal microflora were analyzed by QIIME1.9.0 software. The principal component analysis for relative abundance of fecal microflora was performed by Canoco Software 5.0. The metabolic function of fecal microflora was predicted by Kyoto Encyclopedia of Genes and Genomes. Data were statistically analyzed with independent sample test, and Mann-Whitney test, and Bonferroni correction. The relative abundance of , , , , and in feces of patients in burn group was significantly higher than that of volunteers in healthy group (=-5.20, -2.37, -5.17, -4.41, -6.03, <0.05 or <0.01), and the relative abundance of , , , , and (=-8.03, -3.21, -7.63, -5.88, -8.05, -8.05, -6.77, <0.01) and other 12 species of bacteria in the feces of volunteers in healthy group was significantly higher than that of patients in burn group. The diversity of fecal microflora of volunteers in healthy group was better than that of patients in burn group, the main dominant microflora of volunteers in healthy group were , -, , - , , , , etc., and the main dominant microflora of patients in burn group were , , , and . The OTU number, Ace index, Chao1 index, and Shannon index of fecal microflora of patients in burn group were 149±47, 199±45, 190±45, 2.0±0.9, which were significantly lower than 266±57, 323±51, 318±51, 3.8±0.5 of volunteers in healthy group (=10.325, 11.972, 12.224, 11.662, <0.01). The relative abundance of fecal microflora of patients in burn group and volunteers in healthy group was clearly divided into two groups by principal component 1, and the contribution rate of principal component 1 was 32.50%, <0.01. The fecal microflora of volunteers in healthy group were more concentrated on principal component 2, the fecal microflora of patients in burn group were dispersed in principal component 2, and the contribution rate of principal component 2 was 13.44%, >0.05. The metabolic levels of alanine-aspartate-glutamate, arginine- proline, cysteine-methionine, glycine-serine-threonine, phenylalanine, tryptophan, and tyrosine in amino acid, tricarboxylic acid cycle, glucose and mannose, galactolipin, glycolysis/gluconiogenesis, starch and sucrose in carbohydrate of fecal microflora of patients in burn group were significantly lower than those of volunteers in healthy group (=-4.75, -4.54, -4.75, -4.62, -3.71, -3.28, -4.19, -3.82, -4.72, -4.35, -4.75, -4.71, <0.01). The levels of lipoic acid metabolism and coenzyme Q synthesis of fecal microflora of patients in burn group were significantly higher than those of volunteers in healthy group (=-6.07, -4.51, <0.01). The metabolic level of arachidonic acid of fecal microflora of patients in burn group was similar to that of volunteers in healthy group (>0.05). There are significant differences in intestinal microflora between severe burn patients at the early stage and healthy people, and the species and diversity of microflora are decreased, and the nutrient metabolism level is decreased in burn patients by 16S rRNA high-throughput sequencing.

摘要

通过16S核糖体RNA(rRNA)高通量测序分析重度烧伤患者早期肠道菌群的变化,并预测肠道菌群的代谢功能。在这项前瞻性观察性研究中,2018年1月至2019年12月入住江苏大学附属医院烧伤整形科、符合纳入标准的48例重度烧伤患者纳入烧伤组,同期在江苏大学附属医院体检中心进行体检、符合纳入标准的40例健康志愿者纳入健康组。烧伤组患者于入院后约1周采集粪便样本,健康组志愿者于体检当日采集粪便样本。对烧伤组患者和健康组志愿者的粪便进行16S rRNA V4基因测序,分析各种细菌的相对丰度。采用Mothur软件划分操作分类单元(OTU),分析优势菌。运用QIIME1.9.0软件分析粪便微生物群的OTU数量、Chao1指数、Ace指数和Shannon指数。使用Canoco Software 5.0软件对粪便微生物群相对丰度进行主成分分析。通过京都基因与基因组百科全书预测粪便微生物群的代谢功能。数据采用独立样本t检验、Mann-Whitney U检验及Bonferroni校正进行统计学分析。烧伤组患者粪便中双歧杆菌属、真杆菌属、乳杆菌属、肠球菌属和拟杆菌属的相对丰度显著高于健康组志愿者(t=-5.20、-2.37、-5.17、-4.41、-6.03,P<0.05或P<0.01),健康组志愿者粪便中葡萄球菌属、芽孢杆菌属、放线菌属、梭菌属、韦荣球菌属、消化链球菌属、优杆菌属等12种菌的相对丰度显著高于烧伤组患者(t=-8.03、-3.21、-7.63、-5.88、-8.05、-8.05、-6.77,P<0.01)。健康组志愿者粪便微生物群的多样性优于烧伤组患者,健康组志愿者的主要优势微生物为拟杆菌属、普氏菌属、粪杆菌属、瘤胃球菌属、双歧杆菌属、真杆菌属、乳杆菌属等,烧伤组患者的主要优势微生物为肠球菌属、葡萄球菌属、芽孢杆菌属和放线菌属。烧伤组患者粪便微生物群的OTU数量、Ace指数、Chao1指数和Shannon指数分别为149±47、199±45、190±45、2.0±0.9,显著低于健康组志愿者的266±57、323±51、318±51、3.8±0.5(F=10.325、11.972、12.224、11.662,P<0.01)。主成分1将烧伤组患者和健康组志愿者粪便微生物群的相对丰度明显分为两组,主成分1的贡献率为32.50%,P<0.01。健康组志愿者的粪便微生物群在主成分2上更为集中,烧伤组患者的粪便微生物群在主成分2上较为分散,主成分2的贡献率为13.44%,P>0.05。烧伤组患者粪便微生物群在氨基酸的丙氨酸-天冬氨酸-谷氨酸、精氨酸-脯氨酸、半胱氨酸-甲硫氨酸、甘氨酸-丝氨酸-苏氨酸、苯丙氨酸、色氨酸和酪氨酸、三羧酸循环、碳水化合物的葡萄糖和甘露糖、半乳糖脂、糖酵解/糖异生、淀粉和蔗糖等方面的代谢水平显著低于健康组志愿者(t=-4.75、-4.54、-4.75、-4.62、-3.71、-3.28、-4.19、-3.82、-4.72、-4.35、-4.75、-4.71,P<0.01)。烧伤组患者粪便微生物群的硫辛酸代谢和辅酶Q合成水平显著高于健康组志愿者(t=-6.07、-4.51,P<0.01)。烧伤组患者粪便微生物群的花生四烯酸代谢水平与健康组志愿者相似(P>0.05)。重度烧伤患者早期与健康人肠道菌群存在显著差异,菌群种类和多样性降低,通过16S rRNA高通量测序发现烧伤患者营养物质代谢水平降低。

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