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Worlds Poultry Congress 5 - 9

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The next generation of

microbiological testing of poultry

Introduction

icrobiological testing of food products is

a common practice of food processors
to ensure compliance with food safety
criteria (Mead et al., 2010). Sampling on
its own is of limited value, but when applied regularly
at different stages of the food chain microbiology
testing can be an integral part of a quality control
program. No feasible sampling plan can assure the
absence of foodborne pathogens, but when done
on a regular basis, changes in contamination of
pathogens such as Salmonella on raw poultry can
be identified so that corrective action can be taken.

Challenges
associated
with Salmonella testing of
raw poultry
A frequently asked and difficult to answer
question is, What is the true prevalence of Salmonella
on poultry? Salmonella prevalence rates on chicken
carcasses can be increased or decreased depending
on the sampling methods used, such as changing
the portion of sample cultured after whole carcass
rinsing or changing the sample weight of neck skin
or meat sampling (Cox and Blankenship; 1975;
King et al., 2008; Simmons et al., 2003; Simmons

Whole carcass rinsing (WCR) and neck skin (NS)

maceration are the most frequently used sampling
methods to detect Salmonella from commercially
processed broilers in the U.S. and E.U., respectively.
These are nondestructive and practical methods
that result in many false-negative results. WCR
will not detect firmly-attached Salmonella and only
7.5% of the rinsate is often analyzed, whereas for
NS samples only 4% of the skin by weight of the
broiler carcass skin is frequently assayed. In 1975,
a whole carcass enrichment (WCE) method was
developed as a research tool (Cox and Blankenship;
1975). This involves incubation of the whole carcass
overnight in a preenrichment broth and this method
can recover as few as 8 inoculated Salmonella cells
per carcass 100% of the time, but is not a practical
method for routine use.
In a recent study (Cox et al., 2011b), postchill
commercial broiler carcasses were each sampled by
several methods, including neck skin, whole carcass
rinse and whole carcass enrichment. Salmonella
spp. were detected on 42.5% of the carcasses
using neck skin samples, 25% of carcasses using
whole carcass rinse samples, and on 97.5% of the
same carcasses using the whole carcass enrichment
method. Also, only one Salmonella serotype was
detected using the rinse method, whereas two
were detected using the neck skin method and 5
different serovars were recovered using the whole
carcass enrichment method. This is a major issue
with regard to attributing poultry as a vehicle of
human illness, and will be addressed in more detail
later in this section.

Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

1 - Roka Bioscience Inc., 10398 Pacific Center

Court, San Diego, CA 92121
2 - U.S. Department of Agriculture Agriculture
Research Service, Russell Research Center, 950
3 - College Station road, Athens, GA
Center for Food Safety, University of Georgia,
1109 Experiment Street, Griffin, GA 30223

et al., 2003 and Surkiewicz et al., 1969). Culturing

a smaller sample size can reduce the prevalence of
Salmonella on poultry, whereas larger-than-typical
size samples can increase the prevalence. Sampling
methods for determining Salmonella prevalence are
often based on convenience or previous experience,
and prevalence data are often presented and
compared with insufficient information regarding
testing methods.

In whole carcass rinse sampling, the rinse liquid

comes in contact with the entire surface of the
carcass if the rinse procedure is properly conducted.
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XXIV

A neck skin sample of approximately 8.3 grams is a

relatively small percentage of the total skin or surface
area of the whole carcass, so the approximate
equivalence of this sampling method observed in
several studies may imply that there are more than a
few Salmonella cells that are attached or associated
with the skin and carcass and are about as likely
to be isolated from macerating the neck skin as
from a rinse of the entire carcass. Finding a higher
Salmonella prevalence on neck skin samples than
in whole carcass rinse samples may indicate that
many of the cells are attached or associated to some
degree with the skin and are not being recovered by
rinsing the carcass. In whole carcass rinse sampling,
the first Salmonella-positive rinse is sometimes
obtained only after multiple negative rinses of the
same carcass (Izat et al., 1991 and Lillard, 1988).
Current methods were developed to obtain the
greatest number of Salmonella-positive samples
with an acceptable cost and ease of performance,
and no consideration was given to determining
the relationship of results to the risk of acquiring
human salmonellosis. Different types of samples can
contain different microbial competitors, nutrients,
and antimicrobial inhibitors, and there is a different
response regarding Salmonella growth, depending
on the degree of stress or sublethal injury that the
bacteria may have experienced; hence, sample
characteristics and history of the sample must be
considered when selecting a cultural methodology
for Salmonella detection or isolation. Even for a
relatively well-understood sample type such as a
chilled poultry carcass, there is no internationallyrecognized standard methodology for Salmonella
detection, either for sample size or microbiological
methods. Use of different cultural media is not simply
a matter of laboratory choice, with some methods
delineated by regional, national, or international
organizations.
Classical cultural techniques for Salmonella
detection generally follow a standard sequence,
including a nonselective preenrichment (which
may be used depending on whether cells are
stressed or are present in low numbers), a selective
enrichment, isolation on selective agar media,
biochemical screening and serological confirmation.
Many different media formulations have been
developed for the preenrichment, enrichment, and
selective isolation steps in the detection sequence.
A survey of diagnostic veterinary laboratories in the
United States in the mid-1990s revealed that 17
different selective enrichment media were being
used (Waltman and Mallinson, 1995). Selective
enrichment cultures were inoculated onto 14
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different plating media, with most laboratories using

two or more different types of media to increase
the likelihood or recovering Salmonella. There
was also considerable variation in the number of
colonies selected for further testing by the different
laboratories, with many selecting and identifying as
Salmonella only one colony.
Several collaborative studies have been done in
sixteen national reference laboratories in Europe
comparing the efficacy of procedures for isolating
and identifying Salmonella from feces (Voogt et
al., 2002a,b). When standardized samples were
provided to all participating labs, the results revealed
significant differences among the laboratories and
within different subsections of the study, in the ability
to detect Salmonella and identify the serotypes that
were isolated. Several modified methods were used
as the studies progressed, and new methods were
developed in the absence of an official reference
method for isolating Salmonella from fecal samples.
Discussions of Salmonella detection in control or
regulatory samples often assume accurate and
reproducible results, but the reality of results of
laboratory assays is that considerable variability can
occur (Waltman and Mallinson, 1995 and Voogt et
al., 2002a,b).
Many laboratories that routinely sample broiler
carcasses for Salmonella are primarily interested
only in determining the presence or absence of
Salmonella, so in many instances only one colonyforming unit (CFU) of presumptive Salmonella
may be selected from selective agar plates. If
that CFU is found to be Salmonella, there is no
other testing of additional colonies or serovars.
Successful Salmonella serovar isolation is a complex
multifactorial procedure (Cox et al., 2010a). Human
health and regulatory scientists have begun trying
to match-up Salmonella serotypes from human
illness with various food sources. These attribution
studies are being used to determine Salmonella
serotype significance in relation to human illness.
In most cases, Salmonella methodology procedures
focus on recovering a broad spectrum of Salmonella
serotypes and not specific serovars within a sample.
Due to the poor performance of many selective
broths and agars in recovering Salmonella from
nonclinical samples, food microbiology laboratories
usually use two or more broths and plating media to
reduce the likelihood of false-negative results (Cox
et al., 2000). Since many laboratories performing
Salmonella isolation select and identify only a single
suspect colony per sample, this complicates the use
of these data for epidemiologic and risk assessment
purposes (Gardner, 2004). The bias introduced by

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culture methods could be potentially influencing

results obtained by Salmonella surveillance systems
and hindering accurate traceback investigations
(Singer, et al., 2009).

To explore the implications of Salmonella

serotypes growing and surviving at different rates,
the binomial and multinomial probabilities of
picking serotypes present at various ratios on plates
were calculated, assuming that all picks are 100%
successful in identifying a colony of Salmonella.
It was determined that when two serotypes are
present in equal numbers, 6 colonies must be picked
to have a 95% probability of identifying all of the
serotypes present. To identify three serotypes under
the same conditions, 11 colonies must be picked.
If a serotype is outnumbered 10 to 1 by another
serotype on a plate, 32 colonies must be picked to
have a 95% probability of identifying the minority
serotype (Cason et al., 2010).
In another study involving naturally-contaminated
broiler carcasses (Cox, et al., 2011c), a total of
12 typical colonies were tested from each of 49
Salmonella-positive carcasses. The 12 colonies were
selected from two different selective plating media

Relatively small survival and growth rate

differences can produce dramatic changes in the
likelihood of selecting a colony of a particular
serotype, even when it was present in the original
sample in large numbers. Given the labor time
and expense of isolating and serotyping suspect
Salmonella colonies, better methods than those
that currently exist are needed for culturing specific
serotypes of interest.
There are also cultural methodology problems
with other types of poultry samples, and one
such example is with commercial hatchery fluff
(Cox et al., 2010b). In one study, a 300-g fluff
sample was collected and transported back to
the laboratory for analysis. Forty-one cultivation
methods (combinations that included: 2 primary
enrichment broths, 8 secondary enrichment broths,
3 second secondary enrichment broths, 3 plating
media, and 2 incubation temperatures) were
used to evaluate the Salmonella status of the fluff
sample. Five presumptive Salmonella colonies per
plate were picked and evaluated. Overall, 37 of 41
of the cultivation methods recovered Salmonella.
From the fluff samples, 455 presumptive Salmonella
isolates were evaluated. All of the Salmonella
Kentucky isolates came from one of the selective
plates (XLT4), whereas all of the S. Lille isolates
were from the other two plating media (BGS and
HE) and not XLT4. From this study, it is apparent
that cultivation methods can select for specific
Salmonella serogroups. Cultivation methods may
be enabling specific serotypes to outgrow others
which would negatively influence the probability
of recovering certain serotypes, especially when
present in lower cell numbers within the sample. This
may explain why S. Enteritidis was not obtained by
any of the 41 cultivation methods. Additional work
evaluating culture method influences on serogroup
and serotype recovery is underway, evaluating the
competition occurring among Salmonella serotypes
during cultivation.
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Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

Harvey and Price 1967, determined that

isolates of different Salmonella serotypes had
different growth characteristics even within the
same enrichment culture. In addition, they found
that the duration of incubation had a substantial
impact on S. Enteritidis recovery from enriched
samples and that 18 hours of incubation was better
than 24 hours. S. Enteritidis can be outnumbered
by other serotypes, such as S. Heidelberg (15
to 1) and S. Senftenberg (5 to 1) when mixed in
equal proportions and grown overnight (Kindu, et
al., 2004). Rostango et al., 2005 evaluated four
Salmonella culture methods and, even though the
methods were very similar, differing only in the
primary enrichment and incubation temperature, the
frequency distribution of the serotypes isolated was
different for each method. The range of agreement
between two of the methods for specific serotype
recovery ranged from 0 to 58.3%. A clear order
of dominance exists among Salmonella serotypes
within enrichment broths (Singer, et al., 2009). The
growth characteristic of S. Newport, S. Typhimurium
798, S. Enteritidis, along with S. Typhimurium 35
were compared individually and after commingling
in enrichment broth. No significant growth
difference was observed among the strains in pure
culture; however, after commingling the four strains
within the same enrichment broth, S. Newport was
determined to be the most dominant strain.

(BGSulfa and XLT4), 6 from each plate. From the 49

positive carcasses, 7 of these samples had only one
serotype, 17 had two, 18 had three, 6 four, and
one had five different serotypes. Therefore, over
85% of the Salmonella-positive broiler carcasses
had multiple serotypes and more than likely many
other serotypes went undetected. On 11 of the 49
samples, the two plating media yielded the same
serotypes, whereas for the other 38 positive samples
there were different serotypes isolated from the two
different plating media. This limited study reveals
the variability of detecting individual serotypes even
when only two plating media are used.

Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

XXIV

Swedish scientists have indicated that the

elimination of Salmonella from poultry feed is
imperative since this is considered by them to be one
of the most critical sources of contamination. Hence,
it is important to better understand the influence of
the degree of injury that occurs in the presence of
various environmental and processing stresses on
the subsequent detection of Salmonella, including
the stress occurring in preenrichment recovery
media. In a recent study by Cox et al., 2011a, an
assortment of finished poultry feeds (broiler, broiler
breeder, layer, turkey) and feed ingredients (corn,
soybean, wheat, etc.) were incubated for 24 and
48 hours in four different preenrichment broths
[lactose (North, 1961), buffered peptone, M-9
(Gomez, 1973) and Universal Preenrichment (Bailey
and Cox, 1992)]. After incubation, the pH of the
LB and BP enrichment cultures was determined to
be as low as 3.9. M-9 and Universal Preenrichment
media provided the greatest buffering capacity.
With these two preenrichment media for finished
feeds and ingredients, the pH was rarely below 5.0.
In food and feed processing, foodborne
pathogens such as Salmonella are likely to be
exposed to multiple stresses either sequentially or
concurrently. For example, with commercial animal
(poultry) feeds there is initially a heating process
or processes (conditioning and possibly pelleting)
followed by a dry stress, since the water activity
of feeds and feed ingredients must be kept low to
prevent mold growth. Then, when these products
are placed in a liquid preenrichment medium such
as LB and BPW, severe acid injury and a significant
amount of cell death can occur. Salmonella present
in these types of samples will probably be in low
numbers then after being subjected to one or more
of these stresses will more than likely go undetected.
Although injury of Salmonella induced by heat,
irradiation or freeze drying has been extensively
studied (Mackey, 2000), the mechanisms of acidinduced injury of bacteria are poorly understood
(Liao et al., 2003). Blankenship (Blankenship, 1981)
studied acid injury and recovery of S. Bareilly in acid
conditions at as low as pH 4.0. At the lowest pH,
he observed 14% death and 98% of the survivors
were injured. The injured cells lost their ability
to produce many of the expected biochemical
reactions such as H2S production and lysine
decarboxylase. Expression of these traits is necessary
to recognize and identify Salmonella on plating
media for identifying and selecting presumptive
isolates. Therefore, even though Salmonellae are
not killed, the extent of the acid injury could result
in Salmonellae being undetected on the media
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routinely used for their isolation. Considering the

various fermentable substrates that are in feed and
feed ingredients coupled with the large number of
bacteria, particularly Enterobacteriaceae, present in
commercial feed (Cox et al., 1983), an assortment of
acids is produced in a relatively short period of time
in a preenrichment medium with very little buffering
capacity, such as Lactose Broth (LB) and Buffered
Peptone Water (BPW). Of relevance, the U.S. Food
and Drug Administration, who is responsible for
regulating animal feeds and feedstuffs in the USA
recommends through its Bacteriological Analytical
Manual the use of LB for preenrichment (Food and
Drug Administration, 2011). In the E.U., the ISO
recommends the use of Buffered Peptone Water for
preenrichment for the detection of Salmonella in
animal feed (ISO, 2002). Of the feed samples tested
in this study, preenrichment cultures with these two
media (LB and BPW) resulted in the lowest pH values
of the preenrichment media evaluated due to a lack
of buffering capacity.
Not only the media used but also the presence of
fermentable substrates in the sample being tested
can influence the resulting pH of preenrichment
cultures and thus the recovery of Salmonella. In a
recent study in Spain (Torres, et al., 2001), from
a total of 3844 samples from 523 different feed
mills, Salmonella was isolated from approximately
3% of the feed samples and from 12.5% of the
dust samples in the feed mill. The question remains,
were there really more positive dust samples or did
the low pH of the preenrichment cultures of the
feed samples or perhaps sequential stresses on the
Salmonella during processing the feed produce falsenegative results from the feed and dust samples?
An expert consensus on the most appropriate
sampling sites and methods for determining the
Salmonella status of poultry flocks and poultry
meat has never been reached, because comparing
results between different methods is not a simple
matter (Mead, 2010). The lack of uniformity in
pre- and post-harvest sampling methods can lead
to misinterpretation of results when comparing
Salmonella prevalence across different studies. In
addition, there are inherent weaknesses present
in many frequently used methods, such as those
discussed in this article. More research is needed to
better understand the weaknesses that are present
in our laboratory methods for isolating Salmonella,
particularly from poultry and poultry-related
samples.

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The next generation of

testing foods for microbial
pathogens

To exploit the high copy number of rRNA in a

diagnostic assay one needs to amplify a highly
structured target. Although reverse transcriptase
polymerase chain reaction (RT-PCR) can efficiently
amplify rRNA, genomic DNA must be efficiently
eliminated by DNAse I treatment followed by
inactivation of the DNAse 1 before the RT-PCR
reaction occurs. A highly sensitive method for
amplifying rRNA without DNAse I treatment is the
use of Transcription-Mediated Amplification (TMA),
an isothermal amplification method (Hill, 2001).
There are two basic versions of TMA, i.e., a
two-primer and a single primer reaction (Becker,
et al., 2008). The mechanism of single-primer TMA
(RTMA) is shown in Figure 1.
In the first step of RTMA, a primer (primary
oligonucleotide) and a 2-0-methyl blocker

Detection
of
Salmonella enterica
by a ribosomal RNAbased
molecular
assay
Although the ribosome is a highly
conserved structure which is essential
for protein synthesis in all cells, the
rRNA within it contains stretches of

Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

In addition to the limitations described above,

traditional cultural methods for isolating foodborne
bacterial pathogens from foods have many other
weaknesses, including an extended time (several
days) to complete, the need for considerable
personnel time, and are subject to technician error
and variability. Molecular methods for pathogen
detection in foods can have several advantages
over cultural techniques, including reduced time
to completion, reduced labor needs, improved
precision of assay results, and less likelihood of
operator variability and potential error. In addition,
molecular methods are conducive to automation,
and several pathogen assays may be performed on
the same platform. Many molecular assays used
today are based on detection of DNA which has
limitations including low copy number within a cell
and its inability to serve as an indicator of a viable cell
owing to its persistence in dead cells. As
an alternative to DNA-based detection
technology, methods to detect rRNA
have been developed. rRNA-based
pathogen detection methods have
many advantages over DNA-based or
protein-based assays. These include:
(1) bacterial cells contain large
amounts of rRNA (100-1000 copies/
cell) vs. typically one copy of DNA per
cell, (2) rRNA is found in all bacterial
cells at all stages of development, and
(3) hierarchical levels of detection are
possible allowing the discrimination of
bacteria at the kingdom, genus and
species levels (Hogan, 2004).

gene sequences that vary considerably between

different organisms. This unique property of rRNA
makes it attractive for use as a target in diagnostic
assays for the detection of different microorganisms
(Kohne, 1998).

Figure 1 - Mechanism of RTMA, a single primer version of TMA.

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XXIV
oligonucleotide are bound to the RNA target.
Reverse transcriptase extends the primer until it
encounters the blocking oligonucleotide at which
point it dissociates (steps 2-4). During extension the
underlying RNA is removed by the RNase H activity
of the reverse transcriptase. In step 5, the resultant
cDNA copy of rRNA binds an oligonucelotide
containing a single-stranded T7 promoter at its 5
end and a blocking group at its 3 end. The 3 end
of the cDNA copy of rRNA then acts as a primer
to yield a cDNA copy of rRNA containing a doublestrandedT7 promoter (step 6). In step 7, a second
enzyme, T7 RNA polymerase, initiates transcription
from the double-stranded promoter yielding many
RNA copies of the cDNA with the same sequence
as the original rRNA target molecule (step 8). These
molecules (amplicons) then reenter the amplification
cycle (step 9) producing more cDNA copies of rRNA
containing a double-stranded T7 promoter at one
end (step 12). For each cycle (steps 6-12), 100 to
1000- fold amplification occurs in 30 to 60 minutes.
After 3 to 4 cycles, all primer are consumed resulting
in a 109-1012-fold amplification. TMA is very efficient
in amplifying RNA because its amplicons are RNA. As
a result of these high amplification efficiencies TMA
amplification sensitivities with rRNA vary between
10 to 100 copies per reaction, thereby allowing the
detection of single bacterial cells.
Amplification methods are typically inhibited by a
number of substances, including enrichment broths
as well as components of many environmental or
food samples. A common method to minimize

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inhibitors is to use only a very small amount of sample

in the amplification reaction. Unfortunately, by using
a small sample size, sensitivity is adversely affected.
Alternatively, target nucleic acid can be captured onto
beads and the inhibitory substances subsequently
washed away. Commonly used methods either bind
nucleic acids to beads by electrostatic forces or absorb
them onto the bead. A drawback of these methods
is their lack of specificity since other molecules also
bind to the bead, thereby necessitating the elution
of the nucleic acid from the bead before its addition
to an amplification reaction. An alternative method is
specific target capture (Weisburg et al., 2000) which
is delineated in Figure 2.
Superparamagnetic beads, 1 micron in diameter,
are coated with oligo dT14 tails. A sample suspected
of containing a desired nucleic acid is incubated at
60 C in the presence of the beads. The target in
single-stranded form binds to a chimeric capture
probe in solution containing a specific capture
sequence at its 5 end and an oligo A30 tail at its
3 end. For RNA, the specific capture sequence is
comprised of 2-0-methyl nucleotides, which have a
high affinity and specificity for RNA (Majlessi et al.,
1998). At this concentration step of the chimericcapture probe (5 picomoles is used), hybridization
of the target to the capture probe is quite rapid,
occurring within minutes. Typically, the reaction
time allowed for capture of the target is 10 to 30
minutes, at which point the temperature is reduced
to room temperature allowing the chimeric- capture
probe/target complex to be captured onto the bead
by the formation of AT base pairs between the dT14
tails on the bead and the dA30 tail on the capture
probe. The beads are then pulled to the side of the
tube and all of the liquid is removed. One or more
washing steps, consisting of the addition of wash
solution, bead release, vortexing and recapture
of the beads to the side of the tube, ensues.
Amplification reagents are then added into the tube
and amplification occurs without elution of the
target from the bead. Target bound to the beads
amplifies by steps 1 to 3 on the bead, and then cDNA
is released into solution where further amplification
takes place (steps 4-12). The resultant amplicon can
be detected in real-time using molecular beacons
(Piatek, et al., 1998) or torches (Becker et al., 2002),
or at the end of amplification using acridinium ester
(AE)-labeled probes (Nelson et al., 1996).

The application of AE-labeled probes to detect

TMA amplification reactions is represented in Figure
3. A single-stranded probe, 12 to 25 bases in length,
Figure 2 - Capture of a target sequence by the specific is covalently labeled with AE using a non-nucleotide
linker. Addition of basic hydrogen peroxide triggers
target capture system.

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Figure 4 - Steps for processing by the Roka RNA detection assay, an

environmental or food sample. A sample is enriched in media and
then transferred into a sample transport tube where it is lysed and the
released nucleic acid is stabilized for up to 5 days at room temperature.
When placed onto the Atlas instrument, the transfer tube is subjected
to target capture, amplification and detection by AE-labeled probes in
a fully automated protocol.

The basic steps in which this RNAbased detection technology is used

by Roka Bioscience to detect bacteria
in environmental or food samples
are shown in Figure 4. Samples are
first enriched in a medium to enable
growth of target bacteria. A sample
of the enrichment culture is then
placed into a sample transport tube.
The tube contains a solution that lysis
the bacteria thereby releasing nucleic
acids. This solution immediately
inactivates nucleases which stabilizes
the nucleic acids for up to 5 days at
room temperature. Once the sample
is placed into the transport tube,
it can be put onto an instrument
(Atlas) that fully automates specific
target capture, TMA amplification,
and chemiluminescent detection. This
instrument provides results for the
first sample after 3.5 hours, and the
results for each subsequent sample is
obtained every minute thereafter.
Area: Food Safety August 07

Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

The ability of a target to

protect AE from hydrolysis
forms the basis of a simple
homogenous assay to detect
nucleic acid targets termed
the hybridization protection
assay (Arnold et al., 1989). In
this protocol, a large amount
of AE-labeled probe is added
to a solution containing target
to rapidly form a double helix
between the probe and target.
The resultant solution is then
heated to 60 C to hydrolyze AE
and the subsequent injection of
basic hydrogen peroxide to the
solution results in the emission
of light only from AE-labeled
Figure 3 - Mechanism of the hybridization protection assay.
probes bound to the target.
Remarkably, when bound to a
a rapid chemiluminescent emission (2 seconds) of
target containing as little as one mismatch, the AE of
light from AE. In contrast, if the AE-labeled probe
an AE-labeled probe is hydrolyzed by a weakly alkaline
is first exposed to a weakly alkaline solution, the AE
solution when the mismatch is close to the AE moiety
moiety is hydrolyzed and fails to emit light when
(Nelson et al., 1996). Hence, no light is emitted from
exposed to basic hydrogen peroxide. Although AE
a mismatched target even under conditions in which
attached to a single-stranded probe is hydrolyzed,
the AE-labeled probe is bound to the mismatched
when the probe hybridizes to a target there is a lack
target. Because mismatches induce hydration of the
of water within the grooves of the resultant double
double helix, they can induce hydrolysis of the AE
helix thereby preventing hydrolysis of AE (Becker et
moiety of an AE-labeled probe, thereby endowing
al., 1999).
AE-labeled probes with exceptional specificity
(Becker et al., 1999).

Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

XXIV

A major goal of food testing is to reduce the

time of enrichment cultures. Enrichment times
for cultures applied to a molecular assay can be
reduced by using as much of the enrichment culture
in the assay as possible. Capture techniques that
circulate enrichment culture over beads coated
with antibodies specific for a target bacterium can
concentrate as much as 50 ml of enrichment culture
(Warren et al., 2007). However, these methods are
low throughput and expensive and as such are best
used for pooling samples. Methods that capture
nucleic acids from enrichment cultures typically
use only 1 ml or less of enrichment culture and are
therefore required to grow bacteria as rapidly as
possible in order to have sufficient target available
for detection. A major challenge is to reduce the lag
phase times during enrichment in order to achieve
exponential growth as rapidly as possible. Little or
no growth during the lag phase is typical of injured
cells in food and environmental samples, and the
lag phase can be hours long depending on the
condition and number of target bacteria, type of
stressful condition that injured the bacteria, and the
microenvironment of a food sample.
To decrease enrichment times of injured
Salmonella, many commercial media were screened
for their ability to reduce the lag phase and grow
injured Salmonella rapidly. Nine different media
were examined: buffered peptone water (BPW),
universal pre-enrichment broth (UPB), lactose
broth (LB), tryptic soy broth (TSB), tryptic soy broth
supplemented with yeast extract (TSBE), brain heart
fusion (BH1), RapidChek SELECT Salmonella Media
System (RAPID), nutrient broth (NB) and nutrient

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broth 2 (NB2).Twenty-five-gram samples of fresh

ground beef (27% fat) or lettuce were inoculated
with 1.2 or 1.6 CFUs of Salmonella Heidelberg,
respectively, and held at 4 C for 2 days. Growth
of Salmonella at 35 C was monitored at 8, 12
and 24 hours using a TMA-based Salmonella assay.
This assay targets 23S rRNA and combines specific
target capture, TMA amplification, and AE-labeled
probes to detect Salmonella. The assay has a limit of
detection of 250 CFU of Salmonella/ml, which is 40
times more sensitive than assays that target genomic
DNA and also has 100% sensitivity and 100%
specificity against 75 inclusive and 30 exclusive
bacteria as defined by the Association of Official
Analytical Chemists (AOAC) (results not shown). The
results of the best-performing media for Salmonella
enrichment culture were UPB, BPW, TSB and LB
(Tables 1 and 2). Of these, UPB, BPW and TSB
provided the most rapid growth of Salmonella in
both lettuce and ground beef samples. Of all of the
media examined, NB and NB2 performed the worse.
Although Salmonella grows more rapidly in UPB, the
TMA results of 12-hour enrichment cultures in UPB
included some false-negative findings. The failure
to detect Salmonella under these conditions could
be due to the growth of background bacteria that
suppress Salmonella growth or that suppresses TMA
amplification by competing for Salmonella primers.
Higher incubation temperatures in UPB were
evaluated and Salmonella was detected in 12 hours
in all of the inoculated lettuce samples incubated in
UPB at 42 C (Table 3). Similar results were observed
for ground beef (results not shown). Acceptable
results were also obtained for a variety of foods that
were incubated at 42 C in BPW (results not shown).

Table 1 - Comparison of different enrichment media using ground beef inoculated with 1.2 CFU of S.
Heidelberg/25 g that had been stored at 4 C for 2 days before inoculation. Inoculated samples are denoted
as S1, S2, etc., whereas uninoculated control samples are denoted as NC1 or NC2. TMA results in colored
boxes were positive for amplification.

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Table 2 - Comparison of different media using lettuce inoculated with 1.6 CFU/25 g of S. Heidelberg that
had been stored at 4 C for 2 days before inoculation to injure cells. Inoculated samples are denoted as S1,
S2, etc., whereas noninoculated control samples are denoted as NC 1 or NC2. TMA results in colored boxes
were positive for amplification

35 oC

Percent of samples positive

120

39 oC

42 oC

44 oC

100
100

100

100

100

100

91

80

90
70

60
56

40
20

55
27

11
0
8 hr 12 hr 24 hr 8 hr

12 hr 24 hr 8 hr 12 hr 24 hr 8 hr

12 hr 24 hr

Time
Table 3 - Comparison of different enrichment temperatures in UPB. Lettuce was inoculated with 0.7 CFU of
S. Newport/25g that had been stored at 4 oC for 2 days before inoculation.
Ten different foods, 2 nonperishable and 8
perishable, including fresh raw chicken and deli
cooked chicken, were inoculated with injured
Salmonella according to standard AOAC Research
Institute Performance Tested Methods and incubated
in UPB at 42 C for 12 hours (perishables) or 24
hours (nonperishables) to determine sensitivity and
specificity of the Salmonella assay. Salmonella was
detected in all foods, with a sensitivity and specificity
of 100% (Table 4). To address the performance of
the assay on environmental samples, 415 sponges

Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

Percent positive per temperature and time

from the surfaces of 10 different food production

environments were enriched in UPB at 42 C for 24
h. The assay detected Salmonella in these naturallycontaminated environmental samples obtained with
sponges, with a sensitivity and specificity of 98%
and 99%, respectively (Table 5). Hence, targeting
the rRNA enables this assay to detect Salmonella in
food and environmental samples after as little as 12
hours (perishables) or 24 hours (nonperishables and
environmental samples) after enrichment culture.

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Table 4 - Results of different food types inoculated with S. Typhimurium. Each food was inoculated with a
level of Salmonella that resulted in fractional positives, then 25 or 375 g (composite) of each food type was
stored at 4 C for 48 to 72 hours (perishables) or at room temperature for 2 weeks (nonperishables). Each
sample was enriched in UPB broth at 42 C for 12 hours (perishables) or 24 hours (nonperishables).
Composites
Food
category

Enrichment
Time

POS
Culture

Neg
Culture

POS
Assay

Neg
Assay

Sensitivity

Specificity

Fresh raw
ground beef

Perishable

12 hours

16

13

13

100

100

Fresh Lettuce

Perishable

12 hours

12

100

100

Cocoa
powder

Nonperishable

24 hours

10

100

100

Food
category

Enrichment
Time

POS
Culture

Neg
Culture

POS
Assay

Neg
Assay

Sensitivity

Specificity

Fresh raw
ground beef

Perishable

12 hours

14

100

100

Frozen raw
ground beef

Perishable

12 hours

16

13

13

100

100

Fresh Lettuce

Perishable

12 hours

64

39

25

39

25

100

100

Fresh raw
chicken*

Perishable

12 hours

16

13

13

100

100

Deli cooked
chicken

Perishable

12 hours

16

100

100

Cookie dough

Perishable

12 hours

14

10

10

100

100

Whole dried
egg

Nonperishable

24 hours

19

16

16

100

100

Food

Michael M. Becker, Nelson Cox, Kristin Livezey, Michele Wisniewski and Michael P. Doyle

Non-composites
Food

*natural contamination

Table 5 - Summary of results of environmental samples

tested for Salmonella. A total of 415 samples were collected
from 10 different regions by four independent collaborators.
The 10 types of food production environments that were
sampled included dry food mixture, grain snack, peanuts,
dairy, flour, snacks, spices, food-grade oil, frozen foods, and
flakes. All environmental samples were incubated at 42 C
in UPB broth for 24 hours. FNRate = false-negative results;
FPRate = false-positive results.
Culture
Positive

Negative

Positive

56

Negative

354

Total

Sensitivity (%)

Specificity (%)

415

98

99

FN Rate (%)

FP Rate (%)

SEN
Assay

Concluding comments
The dilemma of using new molecular
pathogen detection procedures for foods
is that although they are more rapid, easy
to perform, can be highly automated, and
be more cost effective than traditional
cultural methods, not having an isolate for
subtyping could adversely affect foodborne
disease surveillance and food attribution
systems for the near term. However, for
routine foodborne bacterial analyses, this
new generation of molecular microbiological
testing should greatly simplify protocols,
reduce labor costs, reduce the likelihood of
laboratory contamination and error, and
enhance the precision of results, of todays
food microbiology laboratory.

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