Salmonellosis Outbreak from Certain Tomatoes
Information for State Regulatory Agencies

References

• Bacteriological Analysis Method Salmonella method, Section C - 23 Preparation of foods for isolation of Salmonella  - Tomatoes
• Import Produce Assignment (DFPG #08-09)  Attachment D: Soak Method for Salmonella Analysis in Tomatoes

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.

1. Pre-sample preparation:  Do not rinse the tomatoes, even if there is visible dirt.  Examine the tomatoes "as is".
2. 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.
3. Incubate sub-samples at 35°C for 24 ± 2 h. 
4. 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.
5. 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.
6. 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. 
7. After incubation of the RV and TET composites, continue as directed in the BAM Online.
8. 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:

1. 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.
2. 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.
3. 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.