Absolute quantification of viable bacteria abundances in food by next-generation sequencing: Quantitative NGS of viable microbes

Aili Kallastu; Esther Malv; Valter Aro; Anne Meikas; Mariann Vendelin; Anna Kattel; Ranno Nahku; Jekaterina Kazantseva

doi:10.1016/j.crfs.2023.100443

Absolute quantification of viable bacteria abundances in food by next-generation sequencing: Quantitative NGS of viable microbes

Curr Res Food Sci. 2023 Jan 12:6:100443. doi: 10.1016/j.crfs.2023.100443. eCollection 2023.

Authors

Aili Kallastu¹, Esther Malv¹, Valter Aro^{1

2}, Anne Meikas¹, Mariann Vendelin¹, Anna Kattel^{1

2}, Ranno Nahku¹, Jekaterina Kazantseva¹

Affiliations

¹ Center of Food and Fermentation Technologies, Mäealuse 2/4, Tallinn, 12618, Estonia.
² Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia tee 15, Tallinn, 12618, Estonia.

Abstract

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.

Keywords: 16S rRNA gene amplicon next-generation sequencing; Bacteria abundances; Bacteria enumeration; Taxonomical identification; Viable bacteria.