Absolute quantification of viable bacteria abundances in food by next-generation sequencing
Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbi...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-01-01
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| Series: | Current Research in Food Science |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2665927123000114 |
| _version_ | 1797798011610333184 |
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| author | Aili Kallastu Esther Malv Valter Aro Anne Meikas Mariann Vendelin Anna Kattel Ranno Nahku Jekaterina Kazantseva |
| author_facet | Aili Kallastu Esther Malv Valter Aro Anne Meikas Mariann Vendelin Anna Kattel Ranno Nahku Jekaterina Kazantseva |
| author_sort | Aili Kallastu |
| collection | DOAJ |
| description | Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota. |
| first_indexed | 2024-03-13T03:57:00Z |
| format | Article |
| id | doaj.art-77abedaa061442b89bf7118209d4262d |
| institution | Directory Open Access Journal |
| issn | 2665-9271 |
| language | English |
| last_indexed | 2024-03-13T03:57:00Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Current Research in Food Science |
| spelling | doaj.art-77abedaa061442b89bf7118209d4262d2023-06-22T05:04:54ZengElsevierCurrent Research in Food Science2665-92712023-01-016100443Absolute quantification of viable bacteria abundances in food by next-generation sequencingAili Kallastu0Esther Malv1Valter Aro2Anne Meikas3Mariann Vendelin4Anna Kattel5Ranno Nahku6Jekaterina Kazantseva7Center of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, EstoniaCenter of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, EstoniaCenter of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, Estonia; Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia tee 15, Tallinn, 12618, EstoniaCenter of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, EstoniaCenter of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, EstoniaCenter of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, Estonia; Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia tee 15, Tallinn, 12618, EstoniaCenter of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, EstoniaCenter of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, Estonia; Corresponding autho.Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota.http://www.sciencedirect.com/science/article/pii/S2665927123000114Viable bacteriaBacteria abundancesBacteria enumeration16S rRNA gene amplicon next-generation sequencingTaxonomical identification |
| spellingShingle | Aili Kallastu Esther Malv Valter Aro Anne Meikas Mariann Vendelin Anna Kattel Ranno Nahku Jekaterina Kazantseva Absolute quantification of viable bacteria abundances in food by next-generation sequencing Current Research in Food Science Viable bacteria Bacteria abundances Bacteria enumeration 16S rRNA gene amplicon next-generation sequencing Taxonomical identification |
| title | Absolute quantification of viable bacteria abundances in food by next-generation sequencing |
| title_full | Absolute quantification of viable bacteria abundances in food by next-generation sequencing |
| title_fullStr | Absolute quantification of viable bacteria abundances in food by next-generation sequencing |
| title_full_unstemmed | Absolute quantification of viable bacteria abundances in food by next-generation sequencing |
| title_short | Absolute quantification of viable bacteria abundances in food by next-generation sequencing |
| title_sort | absolute quantification of viable bacteria abundances in food by next generation sequencing |
| topic | Viable bacteria Bacteria abundances Bacteria enumeration 16S rRNA gene amplicon next-generation sequencing Taxonomical identification |
| url | http://www.sciencedirect.com/science/article/pii/S2665927123000114 |
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