人体肠道细菌寡营养培养组条件的优化研究

Optimization of oligotrophic culturomics for isolation of human gut microbiota

摘要:

【目的】 探讨寡营养对人体肠道细菌培养组的条件。【方法】 通过稀释富集培养基、固体平板和增菌肉汤培养基成分获得寡营养培养基。对健康人粪便样本分别用原液(0)、5、10、20、30和40倍稀释的富集培养基(添加羊血和瘤胃液的血培养瓶)连续增菌,在不同时间点(第0、3、6、9、15、27、30天)吸取增菌液,用YCFA (yeast casitone fatty acid)固体培养平板分离菌落;用YCFA增菌肉汤增菌后再次挑取单菌落,利用基质辅助激光解吸/电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF)质谱和16S rRNA基因测序鉴定菌株。通过比较上述6种寡营养条件分离肠道菌群的效果,选取富集培养基原液、稀释10倍和30倍这3种条件下分离效果较好的富集条件,与同样稀释倍数条件的固体平板和增菌肉汤分别组合成9种培养基条件,进一步优化肠道菌群的培养组条件。【结果】 在6种寡营养富集培养基中,未稀释(原液)、10倍和30倍稀释的富集培养基分离细菌的种类比其他稀释倍数多,其中10倍稀释的富集培养基分离细菌种数最多;同时除去原液,仅在寡营养条件中分离细菌为24种。在进一步优化固体培养基平板和增菌肉汤下,发现原液富集培养基-10倍稀释的固体平板和增菌肉汤培养基、10倍稀释的富集培养基-原液或10倍稀释的固体平板和增菌肉汤培养基这3个组合分离的细菌种数较多;除去原液,仅在寡营养条件中分离细菌为20种,其中10倍稀释的富集培养基-原液的固体平板和增菌肉汤培养基分离的菌种数最多。【结论】 通过稀释富集培养基、固体平板和增菌肉汤培养基成分获得的寡营养培养条件,能够分离出约40%的常规培养条件分离不到的细菌,为从人体肠道微生物群分离更多的菌种提供了有效的方法。

Abstract:

[Objective] To explore the oligotrophic culturomics for the isolation of human gut microbiota. [Methods] Oligotrophic media were obtained by diluting the enrichment medium (blood culture bottle supplemented with sheep blood and rumen fluid), yeast casitone fatty acid (YCFA) agar plate, and YCFA broth. Healthy human fecal samples were cultured with the original medium (0), 5, 10, 20, 30, and 40-fold dilutions of enrichment medium. At different time points (0, 3, 6, 9, 15, 27, and 30 d) during the enrichment, the YCFA agar plate was used to isolate colonies, and then single colonies were picked for further enrichment with YCFA broth. The strains were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing. The effects of the above six oligotrophic conditions on the isolation of gut microbiota were compared. Three enrichment media with better isolation effects, i.e., 0-, 10-, and 30-fold dilutions of the enrichment medium, were selected and combined with YCFA agar plate and YCFA broth with the same dilution factors to form nine culture conditions for the optimization of the culture conditions of gut microbiota. [Results] Among the six oligotrophic enrichment media, the 0-, 10-, and 30-fold dilutions of the enrichment medium isolated more species than the other dilutions, and the 10-fold dilution isolated the most species. Twenty-four species were only isolated by the oligotrophic media and not by the original medium. Further optimization showed that the following three combinations isolated the most bacterial species:0-fold diluted enrichment medium+10-fold diluted YCFA plate and broth, 10-fold diluted enrichment medium+0- or 10-fold diluted YCFA plate and broth. As for the three combinations, 20 species were only isolated by the oligotrophic media, and the combination of 10-fold diluted enrichment medium+0-fold diluted YCFA plate and broth isolated the largest number of species. [Conclusion] The oligotrophic culture conditions obtained by diluting the enrichment medium, YCFA agar plate, and YCFA broth can isolate about 40% of species that cannot be isolated by conventional culture conditions, which provide an effective method for isolating more species from the human gut microbiota.