基于培养组学的肠道菌群培养及鉴定方法研究

Culture and Identification Method of Gut Microbiota Based on Culturomics

目的探索一种适宜临床应用的肠道菌群培养及鉴定的简便、实用方法,为临床诊断肠道菌群失调、制定个性化微生态调节方案、指导抗菌药物应用提供参考.方法应用GAM培养49种厌氧菌标准菌株,应用哥伦比亚培养基培养20种需氧菌标准菌株,监测各菌种的生长趋势;通过检测粪便标本OD_(600)值,确定接种细菌的数量和体积;分别检测不同时间接种生长的粪便标本菌落数和种类,并对健康人和应用抗菌药物患者的肠道菌群进行对比分析;采用MADLI-TOF MS对生长的菌落进行鉴定.结果厌氧菌培养36 h、需氧菌培养18 h后90%以上的菌种达到生长平台期;接种5μL菌种,OD_(600)值为0.045时,厌氧培养生长的菌落数最佳;接种50μL菌种,OD_(600)值为0.050时,需氧培养生长的菌落数最佳;留样后0、1、2 h培养的菌落数和种类均多于3 h培养的菌落数和种类,差异具有统计学意义(P<0.05);对照组总菌数、厌氧菌数/需氧菌数、G^(-)菌/G^(+)菌、杆菌/球菌、益生菌占比均高于抗菌药物组,真菌数低于抗菌药物组,差异具有统计学意义(P<0.001).结论此方法可培养鉴定出人体肠道中的优势菌,获得评估肠道菌群的相关量化指标,并且成本低,操作简便,可为诊治肠道菌群失调、指导抗菌药物应用提供参考.

Objective Explore a simple and practical method suitable for gut microbiota cultivation and identification in clinical application,and provide a reference basis for clinical diagnosis of gut microbiota disorders,formulation of personalized micro-ecological adjustment programs,and guidance for the application of antibiotics.Method The GAM was used to cultivate 49 standard strains of anaerobic bacteria,and 20 standard strains of aerobic bacteria were cultivated on Columbia medium,and the growth trend of each strain was monitored;The number and volume of inoculated bacteria were determined by detecting the OD_(600) value of the stool sample;The number and types of colonies grown at different times after the stool sample are separately detected;The gut microbiota of healthy people and patients with antibiotics were compared,and MADLI-TOF MS was used to identify the growth of colonies.Results Within 36 hours culture of anaerobic bacteria and 18 hours culture of aerobic bacteria,more than 90%of the bacterial species reached the growth plateau.When inoculated with 5μL and the specimen OD_(600) value is 0.045,the number of colonies grown in anaerobic culture is the best;When inoculated with 50μL and the specimen OD_(600) value is 0.050,the number of colonies grown in aerobic culture is the best;The number of colonies and species cultured at 0 h,1 h and 2 h after sample retention were all more than those of 3 h,and the difference was statistically significant(P<0.05);Total bacteria,anaerobic bacteria/aerobic bacteria,G-bacteria/G+bacteria,bacilli/cocci,and the proportions of probiotics in the control group were all higher than those of the antibiotic group,and the number of fungi was lower than that of the antibiotic group,the difference was statistically significant(P<0.001).Conclusion This method can cultivate and identify the dominant bacteria in the human intestinal tract,and obtain relevant quantitative indicators for evaluating the gut microbiota.It has low cost and simple operation.It can provide references for the diagnosis and treatment of gut microbiota disorders and guiding the application of antibiotics.