Food Protection Trends - July/August 2024 - 304

CASE STUDY 1
Samples may be submitted for rapid PCR testing to detect enterohemorrhagic E. coli (EHEC) or STEC. For
many rapid PCR methods, the test may use multiplexing to target DNA specific to the detection of Shiga toxin
genes (stx1
/stx2) and either intimin (eae) or hemolysis (hlyA) virulence genes characteristic of EHEC or STEC.
These primary screens may include or may be reflexed to a specific O antigen serogroup gene or a gene panel
associated with multiple serogroups of regulatory focus and most commonly associated with human foodborne
illness (O157 and non-O157 EHEC or STEC). Discrepancies may manifest, for example, when a positive EHEC
or STEC screen fails to culturally confirm an isolate or a specific serogroup, yielding a presumptively false-positive
screening result and likely yielding a costly decision to divert or destroy the product represented by the sample.
Although there can be issues with the cultural isolation method or methods, a predominant root cause
underlying problematic EHEC or STEC PCR screening is the detection of multiple targets that do not reside
within the same bacterial cell. In other words, the sample may contain various E. coli that have an stx gene and
others that harbor the eae gene or a serogroup gene, but there may be no evidence, established by cultural isolate
testing, that the sample has E. coli cells harboring all gene targets together, which would be considered the isolate
or isolates capable of causing serious foodborne illness. As an example of the natural complexity and diversity
of this issue, researchers collected 13,650 samples across 33 farms in a major leafy greens production region in
the United States. Of 1,912 samples testing positive for non-O157 STEC, 3,256 unique isolates were selected for
further characterization. Of these isolates, stx1
, stx2, and both stx1
and stx2
were detected in 63%, 74%, and 35%,
respectively, whereas the virulence genes eae and hly were observed in 25% and 79%, respectively. Only 23% of
STEC isolates belonged to the top 6 non-O157 serogroups (8).
Method developers and researchers continue to innovate new assays, targets, and workflows to minimize risk
through increased specificity.
CASE STUDY 2
While examining Listeria isolates in agricultural soil, researchers at Cornell University identified five previously
unknown and novel Listeria spp. (6). This research increases the number of known Listeria spp. that could
potentially populate a food production environment. Nonpathogenic Listeria spp. are often found coexisting in
environments that support the growth of L. monocytogenes. Food manufacturers will often analyze environmental
swabs for all Listeria spp. to verify the effectiveness of their sanitation program. If a presumptive positive result
occurs, the isolate can be speciated to determine whether pathogenic L. monocytogenes is present and corrective
actions are required.
Some unique characteristics of Listeria spp. that are closely related to L. monocytogenes were described by the
researchers. As an example, one of the novel species, Listeria immobilis, lacks motility. Motility is a common
characteristic among Listeria closely related to L. monocytogenes and is a pivotal assay in Listeria detection
methods. This lack of motility could contribute to discrepant results in analytical testing for environmental
pathogens, because traditional cultural methods used in confirmation may not propagate the newly recognized
species, despite being detected by rapid screens. In addition, because the newly identified Listeria spp. are closely
related to L. monocytogenes, existing methods of detection may misidentify them as L. monocytogenes. Some
standard identification protocols may need to be reevaluated.
304 Food Protection Trends July/August

Food Protection Trends - July/August 2024

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