Background

Many species of intestinal bacteria are present in moderate numbers in the faecal microbiota, which is dominated by obligate anaerobes. Little is known regarding the detection sensitivity of next-generation sequencing for these microbes in samples of complex microbiota.

Methods

Twenty stool samples from six healthy infants, who were followed from 1 week to 1 year of age, were previously cultured quantitatively for total population counts, as well as for counts of relevant facultative bacteria and a limited selection of obligate anaerobes that are prevalent in the neonatal microbiota. The same samples were analysed by Next-generation sequencing (NGS, pyrosequencing) of the 16S rRNA gene (V1–V3 regions; average read length, 500 nucleotides; average number of reads per sample, 30,000). We used the bacterial culture data to determine the lowest bacterial populations that could be detected by NGS. Different DNA extraction kits (QIAamp DNA Stool Mini, ZR Faecal DNA MiniPrep, and PowerSoil DNA Isolation) were compared for efficacy in extracting DNA from Gram-negative and Gram-positive Type strains.

Results

NGS yielded one read per 10⁶ CFU/g faeces of the Gram-negative commensal gut bacteria Bacteroides and Enterobacteriaceae, but only one read per 10⁸ CFU/g faeces of Gram-positive bifidobacteria. The Gram-positive facultative bacteria Enterococcus was often undetectable by DNA-based methods despite being present at >10⁶ CFU/g faeces. The DNA extraction kits tested varied considerably in their ability to extract DNA from bacterial samples, and showed considerably lower efficacies in extracting DNA from Gram-positive than from Gram-negative bacteria.

Conclusions

NGS has lower sensitivity for detecting Gram-positive bacteria than Gram-negative bacteria, due at least in part to inefficient extraction of DNA from Gram-positive bacteria. Therefore, enzymatic lysis may enhance the yield of DNA and increase the sensitivity of NGS methods for Gram-positive bacteria, and the inclusion of positive and negative controls during DNA extraction is indicated for validation purposes. The differential extraction of DNA from bacterial samples by different DNA extraction kits may limit comparability between studies on the gut microbiota. Finally, quantitative culture methods detect certain bacteria with greater sensitivity than NGS techniques, and thus culture- and DNA-based methods can be used in tandem to define the complex composition of the gut microbiota with greater accuracy.

中文翻译：

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是否所有粪便细菌的检测效率均相同？一项使用下一代测序和婴儿粪便样品定量培养的研究。

背景

肠道菌群中存在多种肠道细菌，其中以专性厌氧菌为主。对于复杂微生物群样品中这些微生物的下一代测序检测灵敏度知之甚少。

方法

从六个星期（从1周到1岁）的健康婴儿的20个粪便样本事先进行了定量培养，以检测总人口数以及相关兼性细菌数和有限选择的普遍存在的专性厌氧菌。新生儿微生物群。通过16S rRNA基因的下一代测序（NGS，焦磷酸测序）（V1-V3区域；平均读取长度，500个核苷酸；每个样品的平均读取次数，30,000）分析了相同的样品。我们使用细菌培养数据确定了NGS可以检测到的最低细菌种群。比较了不同的DNA提取试剂盒（QIAamp DNA Stool Mini，ZR粪便DNA MiniPrep和PowerSoil DNA分离）在从革兰氏阴性和革兰氏阳性菌株中提取DNA的功效。

结果

NGS产生每10一个读⁶  CFU / g的革兰氏阴性共生肠道细菌的粪便杆菌和肠杆菌，但每10只一个读⁸  CFU / g的革兰氏阳性双歧杆菌的粪便。革兰氏阳性兼性肠球菌尽管基于> 10 ⁶  CFU / g粪便存在，但通常无法通过基于DNA的方法检测到。测试的DNA提取试剂盒从细菌样品中提取DNA的能力差异很大，并且从革兰氏阳性菌中提取DNA的效率要比从革兰氏阴性菌中提取的效率低得多。

结论

NGS检测革兰氏阳性细菌的灵敏度低于革兰氏阴性细菌，这至少部分是由于从革兰氏阳性细菌中提取DNA效率低下。因此，酶促裂解可以提高DNA的产量，并提高NGS方法对革兰氏阳性细菌的敏感性，并且在DNA提取过程中包括阳性和阴性对照都可用于验证目的。通过不同的DNA提取试剂盒从细菌样品中提取DNA的差异可能会限制肠道菌群研究之间的可比性。最后，定量培养方法比NGS技术检测某些细菌的灵敏度更高，因此可以将基于培养和DNA的方法串联使用，以更准确地定义肠道菌群的复杂组成。