Quasimetagenomic source tracking of Listeria monocytogenes from naturally contaminated ice cream

Abstract

Background: The more quickly bacterial pathogens responsible for foodborne illness outbreaks can be linked to a vehicle of transmission or a source, the more illnesses can be prevented. Whole genome sequencing (WGS) based approaches to source tracking have greatly increased the speed and resolution with which public health response can pinpoint the vehicle and source of outbreaks. Traditionally, WGS approaches have focused on the culture of an individual isolate before proceeding to DNA extraction and sequencing. For Listeria monocytogenes (Lm), generation of an individual isolate for sequencing typically takes about 6 days. Here we demonstrate that a hybrid, "quasimetagenomic" approach ie; direct sequencing of microbiological enrichments (first step in pathogen detection and recovery) can provide high resolution source tracking sequence data, 5 days earlier than response that focuses on culture and sequencing of an individual isolate. This expedited approach could save lives, prevent illnesses and potentially minimize unnecessary destruction of food.

Methods: Naturally contaminated ice cream (from a 2015 outbreak) was enriched to recover Listeria monocytogenes following protocols outlined in the Bacteriological Analytic Manual (BAM). DNA from enriching microbiota was extracted and sequenced at incremental time-points during the first 48 h of pre-enrichment using the Illumina MiSeq platform (2 by 250), to evaluate genomic coverage of target pathogen, Listeria monocytogenes.

Results: Quasimetagenomic sequence data acquired from hour 20 were sufficient to discern whether or not Lm strain/s were part of the ongoing outbreak or not. Genomic data from hours 24, 28, 32, 36, 40, 44, and 48 of pre-enrichments all provided identical phylogenetic source tracking utility to the WGS of individual isolates (which require an additional 5 days to culture).

Conclusions: The speed of this approach (more than twice as fast as current methods) has the potential to reduce the number of illnesses associated with any given outbreak by as many as 75% percent of total cases and potentially with continued optimization of the entire chain of response, contribute to minimized food waste.

Keywords: (qMGS); Ice cream; Listeria monocytogenes; Metagenome; Metagenomic; Microbiological enrichment; Quasimetagenome; Quasimetagenomic; Source tracking.

Fig. 1

Full (a) and abbreviated (b) phylogeny of the NCBI outbreak cluster of Listeria monocytogenes (environmental and clinical) from contaminated ice cream. a. Phylogeny of the full representative set of the NCBI outbreak associated cluster (environmental and clinical isolates associated with the 2015 listeriosis outbreak linked to ice cream). Listeria monocytogenes genomic data from pre-enrichment microbiomes from hours: 20 to 40 are shown clustering with WGS of individual isolates (blue shading) (that took five days longer to obtain). The outgroup (grey shading) is comprised of closely related genomes of Lm not associated with the outbreak. b. A phylogeny of an abbreviated set of clinical and environmental isolates associated with the 2015 listeriosis outbreak linked to ice cream. WGS genomes from the full Listeria monocytogenes enrichment protocol are shown clustering with genomic data of Listeria monocytogenes from pre-enrichment microbiomes from hours 20, 36 and 40 (blue shading). The outgroup (grey shading) is comprised of closely related genomes of Lm not associated with the outbreak. A full list of all isolates, sources and accession numbers used for the full tree and the abbreviated tree is available in Additional file 1

Fig. 2

Relative abundance of Listeria monocytogenes and other co-enriching bacterial genera throughout the 48 h of pre-enrichment for recovery of Lm from ice-cream. From h 0 to h 48, at 4 h increments, the relative abundance of Listeria monocytogenes and other co-enriching bacterial genera that grow during recovery of Listeria monocytogenes from a dairy homogenate are described (n = 4) per time-point. Taxonomy was assigned using CosmosID with their bacterial database (Rockville, MD)