Genomic analysis of high copy-number sequences for the targeted detection of Listeria species using a flow-through surveillance system

Beatriz Quiñones ,Veronica S De Guzman,Jaszemyn C Yambao,Bertram G Lee,David L Medin

doi:10.1007/s00203-021-02388-2

Abstract

The bacterial foodborne pathogen Listeria monocytogenes has been implicated in fresh produce outbreaks with a significant economic impact. Given that L. monocytogenes is widespread in the environment, food production facilities constantly monitor for the presence of Listeria species. To develop a surveillance platform for food processing facilities, this study conducted a comparative genomic analysis for the identification of conserved high copy sequences in the ribosomal RNA of Listeria species. Simulated folding was performed to assess RNA accessibility in the identified genomic regions targeted for detection, and the developed singleplex assay accurately detected cell amounts lower than 5 cells, while no signals were detected for non-targeted bacteria. The singleplex assay was subsequently tested with a flow-through system, consisting of a DNA aptamer-capture step, followed by sample concentration and mechanical lysis for the detection of Listeria species. Validation experiments indicated the continuous flow-through system accurately detected Listeria species at low cell concentrations.

Highlights

Foodborne pathogens are responsible for a consistent level of human illness that poses a substantial public health and economic burden, resulting in an economic burden of $15.5 billion annually (Hoffmann et al 2015)
Design strategy of the oligonucleotide-based assay for detecting Listeria species As a suitable targeted region, ribosomal RNA (rRNA) has been considered for the development of detection assays as it is found to be represented in the bacterial genome in multiple copies (Livezey et al 2013; Milner et al 2001)
Whole-genome analysis revealed that a single L. monocytogenes cell contains 6 copies of the rRNA operon (Glaser et al 2001), and expression studies estimated that approximately 600–25,000 copies of the ribosomes were detected per cell (Milner et al 2001)

Summary

Introduction

Foodborne pathogens are responsible for a consistent level of human illness that poses a substantial public health and economic burden, resulting in an economic burden of $15.5 billion annually (Hoffmann et al 2015). Listeriosis, a serious infection caused by eating food contaminated with the bacterium Listeria monocytogenes, has been recognized as an important public health problem in the United States, and the annual economic impact of listeriosis in the United States alone is estimated at over US$2.8 billion. While L. monocytogenes is considered one of the major foodborne pathogens that cause listeriosis in humans and animals (Pesavento et al 2010), other Listeria species, which include Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri, have been occasionally implicated in human clinical case reports, primarily in individuals with suppressed immune functions and/ or underlying illnesses (Korsak and Szuplewska 2016). Other Listeria species have been found in ready-to-eat, raw or unprocessed foods (Arslan and Özdemir 2020; Guerra et al 2001; Pesavento et al 2010; Soriano et al 2001; Zeinali et al 2017), as well as in food

Methods

Results

Conclusion