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vary substantially. In dry products, for example, commercially
available lyophilized cultures may be used or wet cultures may
be dried on a carrier before inoculation of the matrix. In either
case, general criteria then include a period of equilibrium. For
dried foods, application of a dried inoculum is followed by
storage at ambient temperature for a minimum of 2 weeks. For
both raw and heat-processed foods, the inoculum is allowed to
equilibrate in the matrix for 48-72 h at 4°C or for a minimum
of 2 weeks at −20°C in the case of frozen foods (3, 43). In the
case of environmental surface matrices, AOAC International
recommends the use of microorganisms that have reached
the stationary phase (3) before inoculation of the surface and
surface drying at ambient temperature for 16-24 h before
sample collection and testing. Where feasible, the laboratory
should attempt to demonstrate the degree of stress or injury
imposed by plating the inoculum pre- and postinjury with
suitable recovery media. Another consideration is the microbial
load on the matrix. Competitive microflora may suppress
outgrowth of the target pathogen during enrichment steps. For
validation studies, nontarget microorganisms that are known
to share phenotypic characteristics with the target pathogen
are inoculated at a concentration 10 times greater than that of
the target pathogen. For validated and accredited methods, first
use in a laboratory is preceded by verification. This is generally
conducted using a small set of inoculated and uninoculated
samples and demonstrating correct performance of the method
with, usually, only a single strain of the target pathogen. First-use
verification is not within the scope of this article but does have
elements in common with matrix application studies.
In this general-interest paper, we address only methods
that have been properly validated and verified for use in
a particular laboratory. A laboratory will inevitably face
the occasional challenge of a novel matrix and the need to
determine whether available methods are fit for purpose.
This may involve additional method verification or validation
studies to ensure appropriate application of the method for
use with the new matrix (5). However, little attention to date
has addressed how to determine which strain or strains of a
target pathogen should be selected for such a study. This may
be challenging when considering the more than 2,500 serovars
for a pathogen such as Salmonella. Considerations may include
known or documented associations with the matrix of interest,
outbreaks, geographical and seasonal considerations, growth
or survival characteristics, and even access to strains for the
purpose of study.
In this paper, we discuss the relevance of these and other
considerations to the selection of strains for matrix validation
and verification studies.
WHAT AFFECTS THE STRAINS PRESENT IN A
PARTICULAR MATRIX?
Process environment
For processed food products, the facility's environment
is likely the most relevant factor for the selection of a
strain. Certain well-adapted subtypes can persist in the
processing environment and be a source of recurrent product
contamination (39). For example, Listeria monocytogenes
found in ready-to-eat (RTE) foods is often connected to
postprocess contamination from the production environment
(87). Similarly, Salmonella has been shown on equipment
within poultry houses (62), chicken processing plants
(57), and rendering plants (33), as well as to transfer from
slicing blades to tomato surfaces (88). Cross-contamination
by Salmonella within multiple production processes and
environments has been extensively reviewed by Carrasco
et al. (11) and can account for a significant fraction of the
incidence on a product, with genetically identical isolates
found on equipment at different points in the process.
Agricultural source of raw materials
Peanuts, leafy greens, and other raw agricultural
commodities may have multiple species, serotypes, and/
or subtype associations (9, 59, 75). Consideration to strain
selection could include known affinity with a particular
matrix or growing region, or it could be isolated to a
particular local farm environment. Outbreak investigations
have identified strain associations as localized, resulting
from either point sources, e.g., sewage effluent spills or
runoff from on-farm manure stockpiles, or nonpoint sources,
including wildlife and/or livestock fecal droppings (80, 81,
82). These mechanisms can introduce a particular strain to
the environment, where persistence increases the potential
for food contamination (80). However, persistence factors
are generally associated with a pathogen species rather than
a particular strain. For example, L. monocytogenes thrives
naturally in the environment and has great propensity to
contaminate raw agricultural commodities. Salmonella and
Escherichia coli O157:H7 endurance may be limited by
temperature, sunlight (UV exposure), moisture level, pH,
available nutrients, and related factors. However, if favorable
conditions exist, either pathogen, regardless of strain identity,
may persist and contaminate raw agricultural commodities
(80). As such, it is appropriate to consider inclusion of
localized and specific strains, when available, for any
commodity or matrix if the stakeholder determines that the
strain could potentially contaminate a product.
Geographical considerations
Raw agricultural commodities tend to dominate growing
regions where they are adapted to the climate. In peanuts, for
example, serotype association seems to be regional (9) (Table
1). However, the raw peanuts sampled (9) were shelled, and
the apparent presence of regional strains may be more the
result of contamination at the sheller than of the growing
region. Similarly, beef, pork, poultry, or seafood products
may be produced in particular geographical regions, each
of which may have unique prevalent serotypes or strains.
For example, Salmonella Enteritidis is most prevalent in
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