利用培养组学技术分离培养肺部微生物群研究

Isolation and cultivation of lung microbiota with culturomics

摘要:

【目的】使用培养组学技术分离培养肺部微生物群，初步了解人体肺部可培养细菌的组成特点，建立呼吸道微生物菌库，为以后进行重点菌株的功能研究提供菌株条件。【方法】针对6个临床肺泡灌洗液样本，使用补充不同添加剂的血培养瓶对样本进行预增菌，在预增菌不同的时间点对血培养瓶内细菌进行分离培养和保藏，使用MALDI-TOF质谱和16S rRNA基因测序鉴定分离菌株。【结果】共获得101种已鉴定细菌，6株潜在新菌，2株真菌。其中细菌包括5个门、14个纲、24个目、35个科和45个属。预增菌前期的时间点分离菌种数较多，厌氧环境分离菌种多于需氧条件，在预增菌时添加羊血和瘤胃液起到良好的分离效果，本实验分离的肺部微生物群与人体其他部位（尤其是肠道）有很大的交叉。【结论】利用培养组学技术可以实现对肺部微生物群的分离培养，继续探索对肺部微生物群培养的优化方法具有现实意义。

关键词:培养组学;肺部微生物群;肺泡灌洗液;方法优化

Abstract:

[Objective] To understand the composition and characteristics of the human lung microbiota, and build a respiratory tract microbial bank which provides strains for further study, we isolated lung microbiota with culturomics.[Methods] Six clinical alveolar lavage fluid samples were collected, and blood culture bottles supplemented with different additives were used to pre-enrich the samples. The strains in the blood bottles were isolated, cultured, and preserved at different time points of the pre-enrichment. Isolated strains were identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry or 16S rRNA gene sequencing.[Results] A total of 101 known bacterial species, 6 potential new bacterial species, and 2 fungal species were obtained. The bacteria belonged to 45 genera, 35 families, 24 orders, 14 classes of 5 phyla. More isolates were obtained from the early stage of pre-enrichment, and there were more isolates in the anaerobic environment than at aerobic conditions. The additional supplement of sheep blood and rumen fluid during pre-enrichment were beneficial to strain isolation. The lung bacteria isolated in this experiment overlapped to a large extent with those from other parts (especially the intestine) of the human body.[Conclusion] Culturomics realizes the isolation of lung microbiota, and it is essential to continue to optimize the methods for isolating more bacteria from lungs.

Key words:culturomics;lung microbiota;alveolar lavage fluid;method optimization