Isolation of Salmonella From Alfalfa Seed and Demonstration of Impaired Growth of Heat Injured Cells in Seed Homogenates

Isolation of Salmonella from Alfalfa Seed and Demonstration of Impaired Growth of Heat-Injured Cells in Seed Homogenates

Ching-Hsing Liao, William F. Fett

Journal Of Food Protection
Publication Request Approval Date: December 5, 2001
Publication Date: N/A
Publisher’s URL: JFP 64: 1110-1115 (2001)/INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 82 (2003) 245-253

Interpretive Summary: Outbreaks of human illness associated with the consumption of raw sprouts have become more frequent over the past decade. Public health investigators determined that the pathogens (disease-causing bacteria such as Salmonella) responsible for the outbreaks most likely originated from the seeds used for sprouting. However, attempts by investigators to isolate the pathogen from the implicated seeds in the outbreaks were not always successful. In this report, an improved method is described for isolating Salmonella from alfalfa seeds associated with three major sprout-related outbreaks in 1998 and 1999. Despite the fact that the seeds had been stored in the laboratory for at least 2-3 years, we were able to isolate Salmonella from all six seed lots examined in this study. The pathogen was recovered at significantly higher frequencies by using the improved method compared to the conventional method. We also found that cells of Salmonella on contaminated seeds were most likely injured and required a longer time to resuscitate in culture broth containing seed constituents. The new technique and knowledge generated from this study is useful for public health investigators and food microbiologists monitoring the microbial safety of seeds, sprouts, and fresh produce.

Technical Abstract: Three major foodborne outbreaks of salmonellosis in 1998 and 1999 were linked to the consumption of raw alfalfa sprouts. In this report, an improved method is described for isolating Salmonella from alfalfa seed lots, which had been implicated in these outbreaks. From each seed lot, eight samples each containing 25 g of seed were tested for the presence of Salmonella by the standard BAM (Bacteriological Analytical Manual) procedure and by a modified method applying two successive pre-enrichment steps. Depending on the seed lot, 1-4 out of 8 samples tested positive for Salmonella by the standard procedure and 2-7 out of 8 samples tested positive by the modified method. Thus, the use of two consecutive pre- enrichments led to a higher detection rate than a single pre-enrichment step. The results suggested that Salmonella on contaminated seeds were injured and failed to fully resuscitate in pre-enrichment broth containing seed homogenates. Responses of heat-injured Salmonella cells grown in three alfalfa seed homogenates and in buffered peptone water (BPW) were investigated. While untreated cells appeared to grow at about the same rate in BPW and in seed homogenates, heat-injured cells required approximately 0.5 to 4.0 h longer to resuscitate in seed homogenates than in BPW. Together, these results reveal that alfalfa seed homogenate present in pre- enrichment broth hinders the repair and growth of heat-injured cells. This study also shows that an additional pre-enrichment step increases the frequency of isolation of Salmonella from naturally contaminated seeds, possibly by alleviating the toxic effect of seed homogenates on repair or growth of injured cells.