Isolation of Salmonella from alfalfa seed and demonstration of impaired growth of heat-injured cells in seed homogenates

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 isolation of 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 US FDA Bacteriological Analytical Manual (BAM) procedure and by a modified method applying two successive pre-enrichment steps. Depending on the seed lot, one to four out of eight samples tested positive for Salmonella by the standard procedure and two to seven out of eight samples tested positive by the modified method. Thus, the use of two consecutive pre-enrichment steps led to a higher detection rate than a single pre-enrichment step. This result indirectly suggested that Salmonella cells on contaminated seeds might be injured and failed to fully resuscitate in pre-enrichment broth containing seed components during the first 24 h of incubation. Responses of heat-injured Salmonella cells grown in buffered peptone water (BPW) and in three alfalfa seed homogenates were investigated. For preparation of seed homogenates, 25 g of seeds were homogenized in 200 ml of BPW using a laboratory Stomacher and subsequently held at 37 °C for 24 h prior to centrifugation and filtration. While untreated cells grew at about the same rate in BPW and in seed homogenates, heat-injured cells (52 °C, 10 min) required approximately 0.5 to 4.0 h longer to resuscitate in seed homogenates than in BPW. This result suggests that the alfalfa seed components or fermented metabolites from native bacteria hinder 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.