Detection of Listeria monocytogenes in Soft Cheese Using a Shotgun Metagenomics

Introduction:  Listeria monocytogenes is a major foodborne pathogen present in a variety of foods such as soft cheese made with pasteurized milk. The microbe causes invasive syndromes with case fatality rates as high as 30% in specific populations such as the elderly, immune-compromised populations.  Metagenomics can be applied to develop a faster and more robust method to analyze milk-based products for bacterial pathogens such as Listeria monocytogenes.

Purpose:  The objective of this study was to assess the efficacy of a metagenomics approach for strain-level detection of L. monocytogenes in soft cheese in both pre-enriched and subsequent enriched samples.

Methods: Soft cheese samples were spiked with L. monocytogenes and were processed according to the FDA BAM method and prepared for sequencing libraries.  Sequencing data was analyzed using a k-mer signature method, an in-house analytical tool, to identify indigenous microbial populations.  In addition, L. monocytogenes-specific DNA fragments were assembled and used to confirm the identity of the contaminating pathogen.

Results:  The indigenous bacteria in soft cheeses were analyzed at several time points during pre-enrichment and enrichment for both spiked and unspiked samples. Our metagenomic analysis identified L. monocytogenes specific molecular markers and enabled assembly of the L. monocytogenes genome at level as low as 1,000 CFU/g soft cheese without requiring overnight culture.

Significance: A metagenomics approach may provide a faster and more rapid method to detect and identify L. monocytogenes contamination in soft cheeses as compared to conventional culture methods, and thus, improving food safety and outbreak response efficiency.