Influence of sampling and DNA extraction on 16S rRNA gene amplicon sequencing - Comparison of the bacterial community between two food processing plants. (April 2018)

• Record Type: Journal Article
• Authors: Witte, Anna Kristina; Leeb, Christine; Pinior, Beate; Mester, Patrick; Fister, Susanne; Bobal, Martin; Schoder, Dagmar; Rossmanith, Peter
• Is Part Of: Lebensmittel-Wissenschaft + Technologie =. Volume 90(2018)
• Page Start: 186
• Page End: 192
• Publication Date: 2018-04
• Subjects: 16S rRNA gene amplicon sequencing -- DNA extraction -- Sampling -- Dairy production -- Listeria monocytogenes; Food industry and trade -- Periodicals; Food -- Composition -- Periodicals; Microbiology -- Periodicals; Nutrition -- Periodicals; 664.005
• DOI: 10.1016/j.lwt.2017.12.027
• Languages: English
• ISSNs: 0023-6438

Abstract

Abstract: 16S rRNA amplicon sequencing is frequently used to investigate and compare microbiomes. This study focuses on the potential, applicability and limits of 16S rRNA amplicon sequencing for comparing the microbiomes of two dairy production plants, one of which experienced recurrent contamination with Listeria monocytogenes . During preceding in vitro studies, DNA extraction of Listeria monocytogenes and Salmonella enterica ser. Typhimurium with two extraction kits ( PowerSoil and NucleoSpin ) revealed differences between these exemplary bacteria and both extraction methods. When equivalent samples of the companies were processed independently with both methods, sequencing results demonstrated also bacterium specific variances. These variances do not reflect a direct correlation between Gram-positive or -negative bacteria and the performance of both kits. Despite these differences, the analysis with a non-metric multi-dimensional scaling plot showed grouping of respective samples independent of the DNA extraction method. Some operational taxonomic units (OTUs) occurred predominantly in one of the companies, but none of them generated a clear hypothesis why one of the plants had recurrent contamination with L. monocytogenes . This work provides insights into the microbiomes of the two dairy companies but advises cautions in over-interpreting sequencing results as selective DNA extraction and sampling only reflect components of prevalent bacterial communities. Highlights: Exposure of DNA extraction and sampling biases relevant for subsequent applications. Underrepresentation of Gram-positives is stronger using PowerSoil than NucleoSpin. Despite using different DNA extraction methods the basic massage is conform. Sequencing data only reflect a partial of the prevalent bacterial community. Due to various biasing factors sequencing results should be construed carefully.