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June 12, 2008
Salmonellosis Outbreak from Certain Tomatoes
Information for State Regulatory Agencies
Questions & Answers
Tomato Testing Guidance for Salmonella Soak Method for Salmonella Analysis in Tomatoes
Questions & Answers
- How can a state regulatory agency provide information to be considered for
placement on the FDA list of areas not associated with the Salmonella Saintpaul outbreak?
Contact either or
- What is involved in the process FDA has employed to
determine whether tomatoes grown or harvested in a particular state, territory
or country is listed as not associated with the outbreak?
Because each area is unique, the process actually
involves a combination of one or more of the following criteria:
- date of initial commercial harvest of tomatoes in the area;
- whether tomatoes were shipped intrastate;
- types of tomatoes produced;
- distribution patterns of tomatoes produced; (whether tomatoes
were shipped to states with
confirmed illnesses);
- preliminary data from FDA tracebacks;
- information from the USDA Agricultural Marketing Service; and
- other relevant information provided by the states.
- What period of time has CDC identified as the onset
of this outbreak?
Mid April to May 27.
- Why is it taking FDA so long to determine the source
of this outbreak of Salmonella Saintpaul?
One of our many challenges involving a product like
tomatoes is that there is no product code, no "sell by" date, no markings on
the tomato in most cases. Another is that this product must be traced from the
producer, to the distributor, out to suppliers (which could be multiple), and
epidemiological evidence collected and analyzed, all of which takes time.
In addition, records need to be obtained at each step of the
process to document the source traceback. The traceback can be further
complicated by a lack of records or incomplete records, or in some cases, huge
volumes of records that need to be reviewed for key information. All of this
information then needs to be analyzed at each step of the traceback to
determine the next step (back) in the trace. Some tracebacks have as many as 3
to 8 steps that must be documented and analyzed.
- What
scientific analysis is done on the tomatoes to determine whether they are
implicated in the current outbreak?
The method being used is a modified version of FDA's
Bacteriological Analytical Manual (BAM) method for detection of Salmonella.
In short, the method includes a soaking step in the analysis; the tomatoes are
submerged in a growth medium that allows for the Salmonella that might
be present on the surface or in the fissures of the fruit to multiply. Once
the sample has been incubated in the growth medium, a sample of the growth
medium is taken, and then enriched to further grow the Salmonella. Then,
a rapid assay is used to detect any presence of Salmonella. Once a
colony is isolated from a rapid assay positive sample, that colony is tested to
determine its serotype and its Pulse Field Gel Electrophoresis pattern; the
PFGE pattern is also referred to as a DNA fingerprint. These tests allow
epidemiologists to determine if the isolated Salmonella is the same type
as implicated in the current outbreak (in this case, Saintpaul). The last two
tests are critical for gathering information necessary to determine the extent
of the outbreak.
Tomato Testing Guidance for Salmonella Soak Method for Salmonella Analysis in Tomatoes
References
Sample Collection
For
tomatoes, a sample will consist of ten (10) sub-samples; each sub-sample will
consist of approximately 454 grams (1 pound).
Analysis of Whole Tomatoes
- The method that
will be used for the Tomato outbreak is derived from the BAM and the
Import Produce Assignment and utilizes a soak method.
- This analysis is for whole, intact
tomatoes. Care should be taken to keep the tomatoes intact. Cut or sliced
tomatoes would be analyzed by the BAM method for comminuted or cut fruit
(chapter five, section C - 23).
- This procedure involves wet compositing
of incubated sample pre-enrichments as opposed to dry compositing as described
in the BAM.
Controls
Please note: Serology for Salmonella
Saintpaul is Salmonella Group B. Use a different serology group for the
lab positive Salmonella control.
-
Pre-sample preparation: Do not rinse the tomatoes, even if
there is visible dirt. Examine the tomatoes "as is".
- Sub-sample soak preparation: For each individual
sub-sample (e.g., approximately 454 g tomatoes), place contents into a sterile
plastic Bag, approximately 12 x 18 inches. Add enough Universal Preenrichment
Broth (UPB) to allow the tomatoes to float (in the absence of UPB,
lactose broth can be substituted). This volume of
UPB should be 1.5 times the weight of the tomatoes. For instance, tomatoes
weighing 454 g will need a volume of approximately 675 ml UPB to float. Add
more UPB if necessary. Place the plastic bag, with tomatoes and UPB broth,
into an appropriate container, for support during incubation. Allow the
open-end flap of the plastic bag to "fold over" so as to form a secure, but not
air-tight, closure during incubation.
- Incubate sub-samples at 35°C for 24 ± 2 h.
- After pre-enrichment, tomato sub-samples are to
be selectively enriched as described below. The selective enrichment strategy
is dependent on whether the BAM culture method or a rapid method is to be used.
- For the Salmonella culture (method 2000.06), and
rapid VIDAS screen method (AOAC 2004.03) use Rappaport-Vassiliadis (RV) medium
and tetrathionate (TET) broth.
- After incubation, manually mix the contents of
the bags containing the tomatoes and the pre-enrichments. The sub-sample
pre-enrichments are then to be "wet composited". From each of five incubated
sub-samples, remove 1.0 ml UPB pre-enrichment and combine into a tube or
flask containing 50 ml TET broth.
For the
other five incubated sub-samples, repeat the above compositing.
- Incubate the two TET broth composites at 35°C for 24 ± 2 h.
- In addition to the above compositing, these
sub-sample pre-enrichments are to be sub-cultured into RV medium. From each of
five incubated sub-samples, remove 0.1 ml UPB pre-enrichment and place
into a tube containing 50 ml RV medium.
For the other five incubated sub-samples, repeat the above compositing.
- Incubate the two RV medium
composites at 42 ± 0.2°C in a circulating,
thermostatically-controlled water bath for 24 ± 2 h.
- After incubation of the RV and TET composites,
continue as directed in the BAM Online.
- If a sample is a "can't rule out" based on the
rapid assay, then perform confirmation analysis as described in the BAM Online.
Serotyping
For Salmonella
serotyping, all bacterial cultures should be prepared and submitted according
to the directions specified in the Bacteriological Analytical Manual (BAM).
PFGE
This strain of Salmonella has proven to be problematic in
the past for PFGE. Some steps to prevent problems:
-
Freshly made thiourea should be added to the running buffer.
The thiourea protocol has been posted in the FDA PulseNet e-room in a folder
named Thiourea (All PFGE analysts should have access to that e-room). The
thiourea protocol is also available from the CDC.
-
If band smearing still occurs at the normal concentration of thiourea,
an increase in the concentration may be required. Some labs have reported
using four times the normal concentration.
-
If the PFGE lab has an isolate that required thiourea from past PFGE
analyses, a plug from that isolate could be run as a "thiourea control".
Sample Collection/Aseptic Sampling
- For tomatoes, a sample will
consist of ten (10) sub-samples; each sub-sample will consist of 454
grams (1 pound).
- Collect all
samples ASEPTICALLY; see IOM, Chapter 4, Section 4.3.6.
NOTE: It is important that each sub-sample be collected into a
separate bag, and controls (i.e., open and unopened collection bags, and
unopened sterile disposable gloves) be submitted with the sample. All controls
must be identified and placed in the container with the sample.
Salmonellosis Outbreak from Certain Tomatoes -
Questions & Answers for Consumers and Industry June 9, 2008