Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification
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...
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| Format: | Journal Article |
| Language: | English |
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ELSEVIER
2019
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| Online Access: | http://purl.org/au-research/grants/arc/LP130100602 http://hdl.handle.net/20.500.11937/90969 |
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| author | Greay, T.L. Gofton, A.W. Zahedi, A. Paparini, A. Linge, Kathryn Joll, Cynthia Ryan, U.M. |
| author_facet | Greay, T.L. Gofton, A.W. Zahedi, A. Paparini, A. Linge, Kathryn Joll, Cynthia Ryan, U.M. |
| author_sort | Greay, T.L. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | 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 Gammaproteobacteria (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. |
| first_indexed | 2025-11-14T11:35:50Z |
| format | Journal Article |
| id | curtin-20.500.11937-90969 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T11:35:50Z |
| publishDate | 2019 |
| publisher | ELSEVIER |
| recordtype | eprints |
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| spelling | curtin-20.500.11937-909692023-05-01T08:05:05Z Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification Greay, T.L. Gofton, A.W. Zahedi, A. Paparini, A. Linge, Kathryn Joll, Cynthia Ryan, U.M. Science & Technology Life Sciences & Biomedicine Environmental Sciences Environmental Sciences & Ecology Wastewater Next-generation sequencing 16S rRNA V4 Greengenes Enterobacteriaceae RIBOSOMAL-RNA GENE SEWAGE-TREATMENT PLANTS PHYLOGENETIC ANALYSIS ACTIVATED-SLUDGE DIVERSITY DEGRADATION SALMONELLA STRAINS TOLUENE SEARCH 16S rRNA Enterobacteriaceae Greengenes Next-generation sequencing V4 Wastewater Bacteria Bacteriological Techniques Enterobacteriaceae High-Throughput Nucleotide Sequencing RNA, Bacterial RNA, Ribosomal, 16S Sequence Analysis, RNA Wastewater Western Australia Bacteria Enterobacteriaceae RNA, Bacterial RNA, Ribosomal, 16S Bacteriological Techniques Sequence Analysis, RNA Western Australia High-Throughput Nucleotide Sequencing Wastewater 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 Gammaproteobacteria (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. 2019 Journal Article http://hdl.handle.net/20.500.11937/90969 10.1016/j.scitotenv.2019.03.278 English http://purl.org/au-research/grants/arc/LP130100602 http://creativecommons.org/licenses/by-nc-nd/4.0/ ELSEVIER fulltext |
| spellingShingle | Science & Technology Life Sciences & Biomedicine Environmental Sciences Environmental Sciences & Ecology Wastewater Next-generation sequencing 16S rRNA V4 Greengenes Enterobacteriaceae RIBOSOMAL-RNA GENE SEWAGE-TREATMENT PLANTS PHYLOGENETIC ANALYSIS ACTIVATED-SLUDGE DIVERSITY DEGRADATION SALMONELLA STRAINS TOLUENE SEARCH 16S rRNA Enterobacteriaceae Greengenes Next-generation sequencing V4 Wastewater Bacteria Bacteriological Techniques Enterobacteriaceae High-Throughput Nucleotide Sequencing RNA, Bacterial RNA, Ribosomal, 16S Sequence Analysis, RNA Wastewater Western Australia Bacteria Enterobacteriaceae RNA, Bacterial RNA, Ribosomal, 16S Bacteriological Techniques Sequence Analysis, RNA Western Australia High-Throughput Nucleotide Sequencing Wastewater Greay, T.L. Gofton, A.W. Zahedi, A. Paparini, A. Linge, Kathryn Joll, Cynthia Ryan, U.M. Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification |
| title | Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification |
| title_full | Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification |
| title_fullStr | Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification |
| title_full_unstemmed | Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification |
| title_short | Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification |
| title_sort | evaluation of 16s next-generation sequencing of hypervariable region 4 in wastewater samples: an unsuitable approach for bacterial enteric pathogen identification |
| topic | Science & Technology Life Sciences & Biomedicine Environmental Sciences Environmental Sciences & Ecology Wastewater Next-generation sequencing 16S rRNA V4 Greengenes Enterobacteriaceae RIBOSOMAL-RNA GENE SEWAGE-TREATMENT PLANTS PHYLOGENETIC ANALYSIS ACTIVATED-SLUDGE DIVERSITY DEGRADATION SALMONELLA STRAINS TOLUENE SEARCH 16S rRNA Enterobacteriaceae Greengenes Next-generation sequencing V4 Wastewater Bacteria Bacteriological Techniques Enterobacteriaceae High-Throughput Nucleotide Sequencing RNA, Bacterial RNA, Ribosomal, 16S Sequence Analysis, RNA Wastewater Western Australia Bacteria Enterobacteriaceae RNA, Bacterial RNA, Ribosomal, 16S Bacteriological Techniques Sequence Analysis, RNA Western Australia High-Throughput Nucleotide Sequencing Wastewater |
| url | http://purl.org/au-research/grants/arc/LP130100602 http://hdl.handle.net/20.500.11937/90969 |