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Correlation between Gut Microbiome and Disease Severity in Patients with Acute Pancreatitis: A Prospective Cross-sectional Study
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摘要:
目的 探究急性胰腺炎(acute pancreatitis, AP)患者肠道菌群变化特征及其与病情严重程度的相关性。 方法 本研究为前瞻性横断面研究。研究对象为2018年6月至2022年1月北京协和医院AP患者和健康志愿者。收集两组临床资料及粪便标本，对其肠道菌群16S rRNA进行DNA测序并进行生物信息学分析。比较两组肠道菌群差异，并采用受试者操作特征曲线分析肠道菌群与AP病情严重程度的相关性。 结果 共入选符合纳入和排除标准的AP患者60例、健康志愿者20名。AP患者中，轻症、中度重症、重症患者各20例，住院期间转入ICU 22例，未转入ICU 38例。α多样性分析显示，AP患者肠道菌群Shannon指数低于健康志愿者(P＜0.05)；β多样性分析显示，AP患者肠道菌群结构异于健康志愿者。在门、科、属、种水平的比较中，AP患者与健康志愿者肠道菌群分布均存在差异。线性判别分析结果显示，包括大肠志贺菌属、肠球菌属和肠球菌科在内的多个菌群在AP患者中呈优势分布，而布劳特氏菌属和双歧杆菌属等菌群在健康志愿者中呈优势分布。功能分析提示AP患者肠道菌群中多种氨基酸合成受阻，菌群致病性与迁移能力增强。ICU患者与非ICU患者的亚组分析亦可观察到类似变化，ICU患者肠球菌表达增多，拟杆菌表达降低。受试者操作特征曲线显示，基于表达差异菌种计算的疾病概率(probability of disease, POD)指数识别AP患者、转入ICU的AP患者的曲线下面积分别为0.996、0.743。 结论 AP患者肠道致病性菌群增多、有益菌减少。肠道菌群变化与AP病情严重程度相关，有望作为AP的新型生物标志物。 Abstract:Objective To investigate the gut microbiome composition and changes and its association with disease severity in patients with acute pancreatitis (AP). Methods This study was a prospective cross-sectional analysis. The subjects of the study were AP patients in Peking Union Medical College Hospital from June 2018 to January 2022 and healthy volunteers. The clinical data and stool samples of the two groups were collected, the 16S rRNA of the gut microbiome was DNA sequenced, and bioinformatic analysis was performed. The differences in gut microbiome between the two groups were compared, and the correlation between the intestinal flora and the severity of AP was analyzed by receiver operating characteristic(ROC) curve. Results A total of 60 AP patients and 20 healthy volunteers were enrolled. Among the AP patients, 20 were mild AP, 20 were moderately severe AP, and 20 were severe AP. During hospitalization, 22 cases were transferred to ICU, while 38 cases were not. In α diversity analysis, the Shannon index of AP patients was significant decreased compared to healthy volunteers (P < 0.05). β diversity of the two groups was significantly different. At the phylum, family, genus and species levels, there were also significant differences in the microbiome composition between the two groups. Linear discriminant analysis effect size analysis revealed that g_Escherichia-Shigella, g_Enterococcus, and f_Enterococcaceae were dominant in AP patients while g_Blautia, and g_Bifidobacterium were dominant in healthy volunteers. Function analysis found that multiple amino acid biosynthesis pathways were blocked in gut microbiome of AP patients, and potential pathogenicity and migration ability of gut microbiome increased significantly. In subgroup analysis, g_Enterococcus was increased and g_Bacteroidaceae was decreased in ICU patients compared to non-ICU patients. Based on the probability of disease index, the ROC curve showed that the area under the curve for identifying AP patients and AP patients transferred to ICU were 0.996 and 0.743. Conclusions The pathogenic bacteria increased and the beneficial bacteria decreased in the gut microbiome of AP patients. Changes in gut microbiota are related to the severity of AP disease and therefore have the potential to be used as novel biomarkers for AP. -
Key words:
- acute pancreatitis /
- gut microbiome /
- biomarker /
- functional prediction
作者贡献：韩梓莹负责数据采集与论文撰写；宋锴、范正阳、宋晓负责患者入组、数据采集；胡晓敏指导研究设计、数据分析与论文修订；吴东指导研究设计、论文修订。利益冲突：所有作者均声明不存在利益冲突 -
图 4 AP患者与健康志愿者差异菌群功能预测
A.PICRUSt2功能分析；B.BugBase表型预测
M00016：赖氨酸合成(琥珀酸-二氨基庚二酸通路)；M00026：组氨酸合成通路；M00527：赖氨酸合成(二氨基庚二酸转氨酶通路)；M00570：异亮氨酸合成通路；M00019：缬氨酸、异亮氨酸合成通路；M00855：糖原降解通路；M00845：精氨酸合成通路；M00432：亮氨酸合成通路；M00532：光呼吸作用；M00010：柠檬酸循环(2-羟戊二酸合成通路)；M00535：异亮氨酸合成通路；M00116：半醌合成通路；M00025：酪氨酸合成通路; AP: 同图 1
图 6 AP-ICU与AP-non-ICU患者肠道菌群组成及功能比较
A.属水平；B.种水平；C.PICRUSt2功能分析；AP: 同图 1；AP-ICU、AP-non-ICU：同表 1
M00165：还原磷酸戊糖循环通路(卡尔文循环)；M00017：甲硫氨酸生物合成通路：M00126：四氢叶酸生物合成通路；M00144：NADH-醌氧化还原酶通路；M00167：磷酸戊糖还原循环通路；M00854：糖原合成通路；M00345：甲醛同化(核酮糖单磷酸途径)；M00549：核苷酸糖生物合成通路；M00844：精氨酸合成通路；M00344：甲醛同化(木酮糖单磷酸通路)：M00554：核苷酸糖生物合成通路；M00168：景天酸代谢通路；M00652：万古霉素耐药相关通路
图 8 差异菌群水平变化及其与临床特征的相关性分析
A.肠球菌属在不同人群中的变化；B.耐久肠球菌在不同人群中的变化；C.差异菌群与临床特征的相关性分析结果(红色为正相关，蓝色为负相关); AP-ICU、AP-non-ICU、APACHE Ⅱ、SOFA：同表 1
表 1 住院期间AP-ICU与AP-non-ICU患者临床资料比较
指标 AP-ICU
(n=22)AP-non-ICU
(n=38)P值 男性[n(%)] 14(63.6) 17(44.7) 0.158 年龄(x±s, 岁) 45.9±14.9 46.5±15.5 0.881 BMI(x±s, kg/m2) 26.5±3.7 26.0±3.2 0.579 病因[n(%)] 0.064 胆源性 6(27.3) 20(52.9) 脂源性 15(68.2) 14(36.8) 酒精源性 1(4.5) 4(10.5) 疾病严重程度[n(%)] ＜0.001 MAP 0(0) 20(52.6) MSAP 4(18.2) 16(42.1) SAP 18(81.8) 2(5.3) SOFA评分
[M(P25, P75), 分]4.5(3.0，8.0) 0.5(0，1.0) ＜0.001 APACHE Ⅱ评分(x±s, 分) 10.0±3.7 3.3±2.5 ＜0.001 局部并发症[n(%)] 急性胰周液体积聚 22(100) 14(36.8) ＜0.001 假性囊肿 2(9.1) 4(10.5) 0.858 急性坏死物积聚 12(54.5) 2(5.26) ＜0.001 包裹性坏死 2(9.1) 0(0) 0.131 感染性坏死 7(31.8) 0(0) ＜0.001 器官衰竭[n(%)] 急性呼吸窘迫综合征 17(77.3) 5(13.2) ＜0.001 急性肾衰竭 11(50.0) 2(5.3) ＜0.001 肝脏损害 8(36.4) 3(7.9) 0.006 循环系统障碍 9(40.9) 0(0) ＜0.001 意识障碍 1(4.5) 0(0) 0.367 全身感染[n(%)] 11(50.0) 2(5.3) ＜0.001 住院时间(x±s, d) 26.2±9.7 8.8±8.5 ＜0.001 院内死亡[n(%)] 1(4.5) 0(0) 0.367 AP-ICU：转入ICU的AP患者；AP-non-ICU：未转入ICU的AP患者；BMI：体质量指数；MAP: 轻症AP；MSAP：中度重症AP；SAP：重症AP；SOFA：序贯器官衰竭评分；APACHE Ⅱ：急性生理学及慢性健康状况Ⅱ；AP: 同图 1 
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