Abstract
BackgroundThe 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.MethodsNaturally 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.ResultsQuasimetagenomic 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).ConclusionsThe 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.
Highlights
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
Enrichment, Deoxyribonucleic acid (DNA) extraction and sequencing Enrichment Homogenized ice cream scoop samples were added to Buffered Listeria Enrichment Broth (BLEB) according to the specifications for Listeria monocytogenes (Lm) recovery outlined in the Bacteriological Analytical Manual (BAM) [6]
Evaluating genomic coverage of Listeria monocytogenes for trace-back utility At h 20, from an average of 2.4 million sequences per replicate, an average of 1.68x coverage was obtained for Lm genome/s from ice-cream enrichments (Table 1)
Summary
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. 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. Sequence data is organized using pipelines that generate matrices of single nucleotide polymorphisms (SNPs) that can be used with a variety of algorithms to infer phylogenetic relationships This approach can differentiate highly clonal strains within serovars that differ by as few as 1 to 29 nucleotides [4]. Culture independent approaches are an alternative but still require financially and computationally impractical amounts of sequence data to achieve sufficient genomic coverage of low abundant targets that occur amidst a complex microbial ecology
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