AUTHOR=Azinheiro Sarah , Roumani Foteini , Costa-Ribeiro Ana , Prado Marta , Garrido-Maestu Alejandro 

TITLE=Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 13 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.931810

DOI=10.3389/fmicb.2022.931810

ISSN=1664-302X

ABSTRACT=Microbial pathogens may be present in different types of foods, thus the development of novel methods to assure consumer’s safeness is of interest. Molecular methods are known to provide sensitive, and rapid, results however, they are typically targeted approaches. In recent years, the advent of non-targeted approaches based on Next Generation Sequencing (NGS), have emerged as a rational way to proceed. This technology allows to detect several pathogens simultaneously, furthermore, with the same set of data it is possible to characterize the microorganisms in terms of serotype, virulence and/ or resistance genes, among other molecular features. In the current study a novel method for the detection of L. monocytogenes based on the “quasimetagenomics” approach was developed. Different enrichment media, and immunomagnetic separation (IMS) strategies, were compared to determine the best approach in terms of L. monocytogenes sequences generated starting from smoked salmon samples. Finally, the data generated were analyzed with a user-friendly workflow which simultaneously provided the species identification, serotype and antimicrobial resistance genes. The new method was thoroughly evaluated against a culture-based approach, using smoked salmon inoculated with L. monocytogenes as the matrix of choice. The sequencing method reached a very low limit of detection, LOD50 1.2 CFU/ 25 g, along with high diagnostic sensitivity and specificity (100 %), and a perfect correlation with the culture-based method (Cohen’s k = 1.00). Overall, the proposed method overcomes all the major limitations reported for the implementation of NGS as a routine food testing technology, and paves the way for future developments taking advantage of it.