IAFP 2009 Program Book and Abstract Book - AB32

Significance: This is the first study proving the incidence of melamine in milk powder and infant formula sold in East Africa. This circumstance is so much the worse, taking into consideration that milk powder is used as food aid for millions of African people, especially malnourished infants and young children or people living with HIV or AIDS. T3-01 Comparison of Different Pre-enrichment Strategies for the Recovery of Salmonella from Internally Contaminated Red Round Tomatoes MINDI D. RUSSELL, Andrew P Jacobson and Thomas S. Hammack . FDA/CFSAN, College Park, MD, USA Introduction: In the past decade, there have been 13 tomato-related Salmonella outbreaks in the U.S. that resulted in thousands of foodborne illnesses and multiple deaths. Internalization of foodborne pathogens in fresh produce is of particular concern to the food safety community. Purpose: Comparison of two alternative pre-enrichment procedures (quartered and stomached) against the Bacteriological Analytical Manual (BAM) Salmonella culture method (whole soak) for the detection and isolation of Salmonella from internally contaminated red round tomatoes. Methods: Red round tomatoes were purchased from a local grocery store. Tomatoes were inoculated by injecting 50 µL of one of two Salmonella serovars (e.g. S. Saintpaul and S. Weltevreden) in the stem scar or side perpendicular to the stem scar. All tomatoes were disinfected twice with ethanol, allowed to air dry overnight, and held for 4 days at 4–6°C following inoculation. Twenty tomatoes each were quartered, stomached, and soaked (total of 120 analyzed). Quartered and stomached tomatoes were pre-enriched in Universal Preenrichment at a 1:1 sample-to-broth ratio. Soaked (whole) tomatoes were pre-enriched at a 1:1.5 sample-to-broth ratio. Samples were incubated for 24 h at 35°C. The BAM Salmonella culture method was followed thereafter (tomatoes were treated as a low microbial load food). Results: Findings from tomatoes injected in the stem and side indicate that the alternative methods of quartering (79 positive) and stomaching (80 positive) were more effective than the BAM soak method (59 positive) for the recovery of Salmonella from internally contaminated tomatoes. Significance: This study found that the alternative pre-enrichment methods of quartering and stomaching were equivalent to one another and superior to the current BAM Salmonella culture method. These results address the need to develop improved methodology for the detection of internalized Salmonella in tomatoes. T3-02 Rapid Detection of Salmonella Typhimurium from Spiked Lettuce and Tomatoes Using Real-time Reverse DSC Transcriptase-polymerase Chain Reaction NATHAN MILLER, P Michael Davidson and Doris H. D’Souza . University of Tennessee, Food Science & Technology, Knoxville, TN, USA Introduction: Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid and sensitive assays to help control outbreaks. Reverse-transcriptase PCR (RT-PCR) detects the presence of mRNA (shorter half-life than DNA), with greater potential of detecting viable cells. Rapid real-time methods using fluorescent dyes and probes simultaneously detect and confirm the presence of target nucleic acid, eliminating the need for gel electrophoresis. Purpose: The objective of this research was to rapidly detect Salmonella Typhimurium from spiked lettuce and tomatoes using real-time RT-PCR. Methods: Washed and ultraviolet light treated lettuce (~25 g) and tomato (~100 g) samples were inoculated with high (108 to 106 CFU) and low (103 to 101 CFU) Salmonella Typhimurium overnight cultures. Samples were then rinsed or hand massaged with 225 ml 0.05 M glycine-saline buffer containing 0.05% Tween and 3% beef extract. Un-inoculated washed produce and sterile buffer were used as negative controls; with S. Typhimurium as a positive control. RNA was extracted from each sample, using the Qiagen RNeasy Mini Kit. RT-PCR was carried out using a SYBR Green I RT-PCR kit with previously described Salmonella-specific invA gene primers and an internal amplification control (IAC) to eliminate false negatives. Reaction conditions were RT at 50°C/30 min; PCR at 95°C/45 s, 58°C/45 s, 72°C/45 s for 40 cycles followed by melt temperature™ analysis in a BioRad iCycler to determine specific invA product (Tm = 87.5°C) and IAC (Tm=82°C). To improve detection sensitivity of low inocula, spiked lettuce and tomatoes were pre-enriched in buffered peptone water for 8 h at 37°C, followed by RNA extraction and RT-PCR detection. Each experiment was repeated twice. Results: Real-time RT-PCR after 8-h pre-enrichment, Qiagen RNA extraction and the SYBR Green I kit gave Salmonella detection up to 103 CFU from lettuce and 104 CFU from tomatoes. Without enrichment, detection limits were 106 CFU for lettuce and 107 CFU for tomatoes. Sensitive and rapid detection of Salmonella from spiked lettuce and tomatoes could still be obtained within one day (~ 2 working shifts). Significance: This assay shows tremendous potential for the sensitive and rapid detection of Salmonella from lettuce and tomatoes within 24 h for routine testing and real-world scenarios. T3-03 Development of Quantitative Real-time PCR Method for the Detection and Characterization of Toxigenic Clostridium difficile DEENA BERMUDEZ and Siddhartha Thakur NCSU, Population Health and Pathobiology, Raleigh, NC, USA Introduction: The increased prevalence of Clostridium difficile associated diarrhea (CDAD) in humans and the recent detection of the pathogen in retail meat products has necessitated the development of rapid detection methods. The pathogenicity locus (PaLoc) codes two toxins, enterotoxin A (tcdA) and cytotoxin B (tcdB), which are responsible for the majority of CDAD cases. It is important to develop a method that will target these toxins genes for detecting toxigenic strains of C. difficile. Technical 32 — Abstract Book

IAFP 2009 Program Book and Abstract Book

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