Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification
Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification
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DOI：
10.1016/j.scitotenv.2019.03.278
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发表时间：
2019-06-20
影响因子：
9.8
通讯作者：
Ryan, Una M.
中科院分区：
文献类型：
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作者：
Greay, Telleasha L.;Gofton, Alexander W.;Ryan, Una M.
再生废水可携带人类传染性微生物病原体，因此废水处理策略必须在再生废水用于补充饮用水和农业用水之前有效消除病原体。本研究描述了西澳大利亚四个污水处理厂（三个废物稳定池和一个使用活性污泥处理的氧化沟污水处理厂）的细菌组成。利用Illumina MiSeq平台对细菌16S rRNA （16S）基因的高变区4 （V4）进行测序。序列在USEARCH v10.0中预处理，用UNOISE3去噪为零半径分类单元（ZOTUs）。采用QIIME 2和Greengenes数据库对该菌株进行分类，并与NCBI nr/nt数据库进行交叉比对。所有污水处理厂和处理阶段（进水、中游和出水）的细菌组成以变形菌属（29.0 ~ 87.4%）为主，其中以Betaproteobacteria（9.0 ~ 53.5%）和Gamrnaproteobacteria（8.6 ~ 34.6%）居多。硝化细菌（Nitrospira spp.）仅在氧化沟污水处理厂的中间和出水中发现，反硝化和絮凝形成细菌在所有污水处理厂中都有发现，特别是在共胞菌科和红环菌科。Greengenes数据库对12种病原菌进行了分类，但与NCBI nr/nt数据库中已知含有病原菌的属和科序列的比较显示，在V4区域的数据集中仅鉴定出3种病原菌（venerupis Arcobacter, hongkong Laribacter and Neisseria canis）。重要的是，肠杆菌科的属不能被区分。在大多数情况下，Greengenes数据库划分的科级分类群与NCBI nr/nt一致，但BLAST分析发现Greengenes数据库中的分类群存在错误。本研究强调了利用NCBI nr/nt等数据库验证NGS序列分类的重要性，并建议在未来旨在鉴定肠道细菌病原体的短扩增子NGS研究中包括16S的V3区域，因为这将提高大多数（但不是全部）肠杆菌科物种的分类分辨率。(C) 2019 Elsevier B.V.版权所有
Recycled wastewater can carry human-infectious microbial pathogens and therefore wastewater treatment strategies must effectively eliminate pathogens before recycled wastewater is used to supplement drinking and agricultural water supplies. This study characterised the bacterial composition of four wastewater treatment plants (WWTPs) (three waste stabilisation ponds and one oxidation ditch WWTP using activated sludge treatment) in Western Australia. The hypervariable region 4 (V4) of the bacterial 16S rRNA (16S) gene was sequenced using next-generation sequencing (NGS) on the Illumina MiSeq platform. Sequences were pre-processed in USEARCH v10.0 and denoised into zero-radius taxonomic units (ZOTUs) with UNOISE3. Taxonomy was assigned to the ZOTUs using QIIME 2 and the Greengenes database and cross-checked with the NCBI nr/nt database. Bacterial composition of all WWTPs and treatment stages (influent, intermediate and effluent) were dominated by Proteobacteria (29.0-87.4%), particularly Betaproteobacteria (9.0-53.5%) and Gamrnaproteobacteria (8.6-34.6%). Nitrifying bacteria (Nitrospira spp.) were found only in the intermediate and effluent of the oxidation ditch WWTP, and denitrifying and floc-forming bacteria were detected in all WWTPs, particularly from the families Comamonadaceae and Rhodocyclales. Twelve pathogens were assigned taxonomy by the Greengenes database, but comparison of sequences from genera and families known to contain pathogens to the NCBI nr/nt database showed that only three pathogens (Arcobacter venerupis, Laribacter hongkongensis and Neisseria canis) could be identified in the dataset at the V4 region. Importantly, Enterobacteriaceae genera could not be differentiated. Family level taxa assigned by Greengenes database agreed with NCBI nr/nt in most cases, however, BLAST analyses revealed erroneous taxa in Greengenes database. This study highlights the importance of validating taxonomy of NGS sequences with databases such as NCBI nr/nt, and recommends including the V3 region of 16S in future short amplicon NGS studies that aim to identify bacterial enteric pathogens, as this will improve taxonomic resolution of most, but not all, Enterobacteriaceae species. (C) 2019 Elsevier B.V. All rights reserved.