The University of Technology Sydney, Australia.
St Vincent's Hospital, Sydney, Australia.
Parasitology. 2020 Jan;147(1):29-38. doi: 10.1017/S0031182019001173. Epub 2019 Sep 23.
The presence of bacterial DNA in Dientamoeba fragilis DNA extracts from culture poses a substantial challenge to sequencing the D. fragilis genome. However, elimination of bacteria from D. fragilis cultures has proven difficult in the past, presumably due to its dependence on some unknown prokaryote/s. This study explored options for removal of bacteria from D. fragilis cultures and for the generation of genome sequence data from D. fragilis. DNA was extracted from human faecal samples and xenic D. fragilis cultures. Extracts were subjected to 16S ribosomal DNA bacterial diversity profiling. Xenic D. fragilis cultures were then subject to antibiotic treatment regimens that systematically removed bacterial species depending on their membrane structure (Gram-positive or Gram-negative) and aerobic requirements. The impact of these treatments on cultures was assessed by 16S amplicon sequencing. Prior to antibiotic treatment, the cultures were dominated by Gram-negative bacteria. Addition of meropenem to cultures eliminated anaerobic Gram-negative bacteria, but it also led to protozoan death after 5 days incubation. The seeding of meropenem resistant Klebsiella pneumoniae strain KPC-2 into cultures before treatment by meropenem prevented death of D. fragilis cells beyond this 5 day period, suggesting that one or more species of Gram-negative bacteria may be an essential nutritional requirement for D. fragilis. Gram-positive cells were completely eliminated using vancomycin without affecting trophozoite growth. Finally, this study shows that genome sequencing of D. fragilis is feasible following bacterial elimination from cultures as the result of the major advances occurring in bioinformatics. We provide evidence on this fact by successfully sequencing the D. fragilis 28S large ribosomal DNA subunit gene using culture-derived DNA.
从培养物中提取的脆弱双核阿米巴原虫 DNA 中存在细菌 DNA，这对脆弱双核阿米巴原虫基因组的测序构成了重大挑战。然而，过去从脆弱双核阿米巴原虫培养物中去除细菌一直很困难，这可能是由于其对某些未知原核生物的依赖。本研究探讨了从脆弱双核阿米巴原虫培养物中去除细菌并从脆弱双核阿米巴原虫中生成基因组序列数据的方法。从人类粪便样本和异养脆弱双核阿米巴原虫培养物中提取 DNA。提取物进行 16S 核糖体 DNA 细菌多样性分析。然后，将异养脆弱双核阿米巴原虫培养物进行抗生素处理方案，根据其膜结构（革兰氏阳性或革兰氏阴性）和需氧要求有系统地去除细菌种类。通过 16S 扩增子测序评估这些处理对培养物的影响。在进行抗生素处理之前，培养物主要由革兰氏阴性菌组成。向培养物中添加美罗培南可消除厌氧革兰氏阴性菌，但在孵育 5 天后也会导致原生动物死亡。在使用美罗培南进行治疗之前，将美罗培南耐药肺炎克雷伯菌 KPC-2 菌株接种到培养物中，可防止脆弱双核阿米巴原虫细胞在此 5 天期限后死亡，这表明一种或多种革兰氏阴性菌可能是脆弱双核阿米巴原虫的必需营养物质。使用万古霉素可完全消除革兰氏阳性菌，而不会影响滋养体生长。最后，本研究表明，随着生物信息学的重大进展，从培养物中去除细菌后，脆弱双核阿米巴原虫的基因组测序是可行的。我们通过使用从培养物中获得的 DNA 成功测序脆弱双核阿米巴原虫的 28S 大亚基核糖体 DNA 基因，提供了这一事实的证据。
