Evaluation of Culture and Pcr Methods for Detecting Salmonella in Artificially Inoculated Alfalfa Seeds

Evaluation of Culture and Pcr Methods for Detecting Salmonella in Artificially Inoculated Alfalfa Seeds

Authors;  Liao, Ching-Hsing; Shollenberger, Lisa

Submitted to: Journal Of Food Protection
Publication Acceptance Date: April 12, 2002
Publication Date: N/A
Publisher's URL: LETTERS IN APPLIED MICROBIOLOGY 2003, 36, 152-156

Interpretive Summary: An increasing number of outbreaks of human illness have been attributed to the consumption of raw sprouts over the past decade. Previous research indicated that the pathogens (disease-causing bacteria such as Salmonella) associated with the outbreaks most likely came from the seeds used for sprouting. Monitoring the pathogens in seeds thus becomes an important part of the safety assurance programs in sprout production. In this study, we evaluated the efficacy of cultural and molecular (PCR) methods for detection of Salmonella in alfalfa seeds. Three factors known to affect the sensitivity of detection method, including culture media, native bacteria, and seed constituents, were investigated. We found that five agar media commonly used for detection of Salmonella exhibited different degrees of sensitivity. One of them, named SRV, was 100-fold more sensitive than the others for detecting the pathogen in samples containing large numbers of native bacteria. We also found that presence of seed extracts in assay mixtures reduced the sensitivity of PCR method for detection of Salmonella by almost 1,000-fold. These results provide useful information for laboratories developing new and better products for detection of human pathogens on seeds, sprouts, and fresh produce.

Technical Abstract: Native microflora from five different alfalfa seed lots were examined and total aerobic plate count of each seed lot was found to range 3 to 5 log CFU per gram of seed. Growth of indigenous bacteria in selective enrichment media including Rappaport-Vassiliadis (RV), selenite cystine (SC), or tetrathionate (TT) broth was partially inhibited. However, growth of Salmonella Mbandaka strain S14 in pre-enrichment (BPW) and selective enrichment broth (RV or SC) was not significantly affected by the presence of alfalfa seeds and native bacteria associated with the seeds. The sensitivity of detecting Salmonella on five indicator agar media containing large numbers of native competing bacteria was evaluated. The semi-solid RV medium (SRV) was about 100- to 1,000-fold more sensitive than Xylose- Lysine-Tergitol 4 (XLT4), Hektoen Enteric Agar, Brilliant Green Agar, and Bismuth Sulfite Agar. The detection limit as measured by the ratio between native bacteria and strain S14 was 106 to 1 on SRV and 103 to 1 on other agar media. However, to detect fewer than 10 Salmonella cells on XLT4, native bacteria in the sample had to be reduced to 103 CFU or lower. The sensitivity of detecting Salmonella by PCR was greatly reduced by the presence of alfalfa seed homogenates. The minimal number of Salmonella detectable by PCR was less than 10 CFU in the absence of seed homogenates and 1,000 CFU in the presence of seed homogenates.