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Whole Genome Sequencing:

Implications for the Frozen


Food Industry
Whole genome sequencing (WGS) is a high-resolution deoxyribose nucleic acid
(DNA)-based application that determines an organism’s complete DNA composition.
WGS provides large volumes of genetic data in a short amount of time and informs
genetic variations between organisms (both within and between species) with high
precision. Increasing data processing capacity and decreasing sequencing costs
make WGS a very efficient and valuable testing methodology.

WGS is the most advanced microbial testing tool available to identify pathogens
in food samples, environments, and clinical patients. The specificity of WGS-based
identification is also superior to all preceding microbial surveillance and detection
methodologies currently in use. Understandably, WGS enables an expanding list of
food safety and even food quality related applications to both industry and regulators.
Among the most exciting of WGS capabilities is identifying and defining foodborne
illnesses more rapidly and precisely, thereby limiting the potential scope and extent
of an ongoing food-related outbreak. The U.S. Centers for Disease Control and
Prevention (CDC) predicts that WGS use will result in identifying outbreaks with
greater frequency and with fewer cases per outbreak.

The capacity of WGS to define evolutionary relatedness through exact DNA


fingerprints between multiple microbial isolates is based on complex genetic data
processing and analysis. DNA sequence data from pathogens isolated from diverse
sources are chronicled into one of several WGS repositories curated in the United
States by the CDC (PulseNet), the National Center for Biotechnology Information
(NCBI) (Pathogen Detection Database-GenomeTrakr), and globally in the Genome
Microbial Identifier (GMI) program. It is well known that each of these systems
use different database mining strategies, disparate reference sequence information,
and varied sequence similarity criteria. Despite the lack of key standardization
metrics and ongoing scientific debate on best practices, the U.S. Food and Drug
Administration (FDA) and U.S. Department of Agriculture are actively transitioning
to a complete reliance on WGS for their compliance activities.

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WGS is but one among many in the repertoire of tools that food manufacturers
and regulators have at their investigative disposal, particularly in determining
whether a multiple positive Listeria monocytogenes (Lm) findings over time are
evidence of persistence of this isolate in the facility. Regulators are interested in
establishing whether positive Lm findings are “persistent” strains. As an example,
when a Lm isolate is identified in a food facility and shown to be re-isolated from the
same facility (via WGS similarity) at a different timepoint, the strain may be
considered persistent in that facility’s environment. A now well-known case of a Lm
strain found to have been embedded in a ready-to-eat (RTE) meat processing facility
in Waco, Texas, was linked to a human death, then 10 years later implicated again in
a second outbreak linked to the same facility. The two strains were shown to be
identical using WGS methods. For these reasons, regulators deem persistence of a
pathogenic isolate to imply a lack of appropriate corrective action when the
pathogen was initially found and suggest the presence of insanitary production
conditions until the period of the repeat finding.

It is now common for federal and state regulators to routinely sample and test retail
products from the marketplace, products which they consider to be of high-risk
relative to the presence of pathogens. These samples may be directly tested for the
presence of Lm using WGS methods and any positive findings may trigger a host of
investigational and compliance actions, including intensive sampling of facilities
where the product was manufactured (See What is a Swabathon). Additionally,
regulators may also take environmental and product samples during routine
inspectional facility visits, which are also tested directly for Lm using WGS methods.
Regardless of the source of a Lm isolate, regulators will be uploading whole
genome sequence data and metadata associated with each sample to their
database. Lm positive results from RTE food products are likely to result in a
Class 1 recall, if the impacted product batch is in commerce or no longer in the
manufacturer’s control.

In the unfortunate event there is WGS similarity between a clinical Lm isolate from
an individual and an isolate from an environmental sample or a food product, the FDA
has an obligation to investigate the food manufacturer and the CDC and Prevention
would initiate an epidemiological investigation. One consequence of WGS technology
and the maintenance of an ever-growing database of food-related sequences is the
possibility of retrospective attribution of Lm positive findings from foods and the
environment with previous illness cases and outbreaks.

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Standardizing WGS methodologies and incorporating principles for use of WGS
data are important prerequisites for its application as a regulatory tool. For this
reason, it is important for all frozen food manufacturers to gain an appreciation for
this technology and ask the right questions to ensure public health objectives are
met while using these methods to improve food manufacturing practices.

The following questions should be asked during an investigation to understand


how WGS analyses and sample metadata can become actionable information:
• In which locations (equipment or facility) were the Lm isolates found?
• In which zones were the Lm isolates found?
• When were the Lm isolates found? Date, time, production line, phase, etc.
• If Lm isolate is from an environmental sample, were there any food products
tested and were there any Lm positives?
• If Lm isolate is from a food product sample, was there a simultaneous or
ensuing facility sampling exercise, if so, were there any environmental Lm
positives from the facility where this food product was manufactured?
• Always request to share FDA’s analytical packet (test methods used,
chain of custody, what is the lab positive control strain?)
• How closely are the two Lm strains related (phylogenetic tree)?
° What methods were used to determine differences?
° Was a reference strain used to determine relatedness?
° What is the single nucleotide polymorphism (SNP) difference?
• What other metadata can be shared regarding the samples?
• Have any of the sequence data been associated with an ongoing human
illness case or outbreak?
° Has the sequence been associated with a previous illness or outbreak?
• What is the epidemiological information?
• How unique is the Lm isolate?
° Has it been recovered in the same or other regions?
° Has it been recovered from other environments (food or non-food)?
° Has it been recovered from other foods?
• At what point does the WGS data serve as evidence for non-compliance
or attribution?
° Discuss sporadic re-introduction of a Lm strain from raw materials, person-
nel, or other traffic

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