Ceuppens Siele, De Coninck Dieter, Bottledoorn Nadine, Van Nieuwerburgh Filip, Uyttendaele Mieke
Laboratory of Food Microbiology and Food Preservation (LFMFP), Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Laboratory of Pharmaceutical Biotechnology (LabFBT), Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
Int J Food Microbiol. 2017 Sep 18;257:148-156. doi: 10.1016/j.ijfoodmicro.2017.06.016. Epub 2017 Jun 17.
Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results.
16S rRNA（基因）扩增子测序在食品样本中的应用越来越多地用于评估微生物多样性，但它可能还有一个意想不到的优势，即无需事先定义就能同时检测出任何人类病原体。在本研究中，对16S rRNA基因的V1-V2-V3区域进行了高通量下一代测序（NGS），以鉴定新鲜罗勒叶上存在的细菌。然而，结果受到生物信息学分析流程（MEGAN、SILVAngs、QIIME和MG-RAST）变化的强烈影响，包括数据库选择（Greengenes、RDP和M5RNA）和注释算法（最佳匹配、代表性匹配和最低共同祖先）。使用默认参数的流程会导致差异。因此，使用16S rRNA（基因）扩增子测序对新鲜罗勒的微生物多样性进行估计具有指示性，但容易产生偏差。肠炎沙门氏菌的检测频率较低，在细菌序列的0.1%至0.4%之间，对应37至166条读数。然而，这一结果取决于所使用的流程：MEGAN、SILVAngs和MG-RAST检测到了沙门氏菌，但QIIME未检测到。通过实时PCR对沙门氏菌序列进行确认未成功。结果表明，从包含高变区V1-V3的16S rRNA基因的短（500bp）序列读数中获得的分类分辨率无法区分沙门氏菌与密切相关的肠杆菌物种。总之，16S扩增子测序作为食品微生物生态学研究中的标准方法，在结果分析方面需要生物信息学和微生物学方面的专业知识。它是估计细菌多样性的有力工具，但容易产生偏差。应认识到在某些细菌物种的分类分辨率方面存在的局限性或其无法检测到亚优势（致病）物种的情况，以避免对结果的过度解读。
