Andrew P. Jacobson

Detection of live Salmonella sp. cells in produce by a TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA.

ABSTRACT Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 103 CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 105 and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDAs Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/∼ebam/bam-5.html , 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers.

Microbiological Quality of Bagged Cut Spinach and Lettuce Mixes

ABSTRACT Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log10 CFU/g and a broad range of <4 to 8.3 log10 CFU/g. Most probable numbers (MPN) of ≥11,000 /g coliforms were found in 55 samples, and generic Escherichia coli bacteria were detected in 16 samples, but no E. coli count exceeded 10 MPN/g.

Evaluation and comparison of rapid methods for the detection of Salmonella in naturally contaminated pine nuts using different pre enrichment media.

Foodborne outbreaks, involving pine nuts and peanut butter, illustrate the need to rapidly detect Salmonella in low moisture foods. However, the current Bacteriological Analytical Manual (BAM) culture method for Salmonella, using lactose broth (LB) as a pre enrichment medium, has not reliably supported real-time quantitative PCR (qPCR) assays for certain foods. We evaluated two qPCR assays in LB and four other pre enrichment media: buffered peptone water (BPW), modified BPW (mBPW), Universal Pre enrichment broth (UPB), and BAX(®) MP media to detect Salmonella in naturally-contaminated pine nuts (2011 outbreak). A four-way comparison among culture method, Pathatrix(®) Auto, VIDAS(®) Easy SLM, and qPCR was conducted. Automated DNA extraction techniques were compared with manual extraction methods (boiling or InstaGene™). There were no significant differences (P > 0.05) among the five pre enrichment media for pine nuts using the culture method. While both qPCR assays produced significantly (P ≤ 0.05) higher false negatives in 24 h pre enriched LB than in the other four media, they were as sensitive as the culture method in BPW, mBPW, UPB, and BAX media. The VIDAS Easy and qPCR were equivalent; Pathatrix was the least effective method. The Automatic PrepSEQ™ DNA extraction, using 1000 μL of pre enrichment, was as effective as manual extraction methods.

Relative Effectiveness of the Bacteriological Analytical Manual Method for the Recovery of Salmonella from Whole Cantaloupes and Cantaloupe Rinses with Selected Preenrichment Media and Rapid Methods

Soak and rinse methods were compared for the recovery of Salmonella from whole cantaloupes. Cantaloupes were surface inoculated with Salmonella cell suspensions and stored for 4 days at 2 to 6 degrees C. Cantaloupes were placed in sterile plastic bags with a nonselective preenrichment broth at a 1:1.5 cantaloupe weight-to-broth volume ratio. The cantaloupe broths were shaken for 5 min at 100 rpm after which 25-ml aliquots (rinse) were removed from the bags. The 25-ml rinses were preenriched in 225-ml portions of the same uninoculated broth type at 35 degrees C for 24 h (rinse method). The remaining cantaloupe broths were incubated at 35 degrees C for 24 h (soak method). The preenrichment broths used were buffered peptone water (BPW), modified BPW, lactose (LAC) broth, and Universal Preenrichment (UP) broth. The Bacteriological Analytical Manual Salmonella culture method was compared with the following rapid methods: the TECRA Unique Salmonella method, the VIDAS ICS/SLM method, and the VIDAS SLM method. The soak method detected significantly more Salmonella-positive cantaloupes (P < 0.05) than did the rinse method: 367 Salmonella-positive cantaloupes of 540 test cantaloupes by the soak method and 24 Salmonella-positive cantaloupes of 540 test cantaloupes by the rinse method. Overall, BPW, LAC, and UP broths were equivalent for the recovery of Salmonella from cantaloupes. Both the VIDAS ICS/SLM and TECRA Unique Salmonella methods detected significantly fewer Salmonella-positive cantaloupes than did the culture method: the VIDAS ICS/SLM method detected 23 of 50 Salmonella-positive cantaloupes (60 tested) and the TECRA Unique Salmonella method detected 16 of 29 Salmonella-positive cantaloupes (60 tested). The VIDAS SLM and culture methods were equivalent: both methods detected 37 of 37 Salmonella-positive cantaloupes (60 tested).

Evaluation of methods to prepare samples of leafy green vegetables for preenrichment with the Bacteriological Analytical Manual Salmonella culture method.

Three sample preparation procedures, soak, stomach, and blend, were evaluated using the Bacteriological Analytical Manual Salmonella culture method with eight types of leafy green produce. In the soak method, test portions were added to lactose broth without homogenization; in the stomach method, test portions were stomached with lactose broth; and in the blend method, test portions were blended with lactose broth. Twenty artificially contaminated test portions were analyzed with each procedure in individual experimental trials. The number of test portions identified as positive were compared among the procedures. Statistically significant differences were identified with Fishers exact two-tailed F test (P < 0.05). Where differences did occur (P < 0.05), the soak procedure was the most effective or was at least as effective as homogenization by either blending or stomaching. Statistically significant differences most frequently occurred with romaine lettuce and cabbage; for these items, blending was significantly less effective than the soak procedure. Overall, for all of the produce types examined, results showed that the soak procedure was more effective than either of the homogenization procedures in recovering Salmonella from leafy green produce. Of the 540 test portions examined by each sample preparation method, 344 were positive for the presence of Salmonella by soaking, 293 by stomaching, and 232 by blending. We recommend that the soak procedure replace homogenization for the analysis of leafy green produce because the soak procedure is more productive than homogenization by either blending or stomaching of the leafy green produce types as reported herein.