foodborne illnesses [27]. The first recognized sprout-related outbreak due to E. coli O157:H7 occurred in Japan in 1996 and was associated with contaminated Daikon radish sprouts. To date this is the largest recorded foodborne outbreak due to contaminated sprouts worldwide with well over 7000 confirmed cases [28,29]. The first recorded sprout-related outbreak of foodborne illness in the U.S. due to contamination with E. coli O157:H7 was in 1997 [14]. Contaminated sprout seed is thought to be the primary source of the pathogens responsible for most sprout-related outbreaks of foodborne illness [4,5]. This conclusion is based on direct isolation of pathogens from seed of implicated lots and/or epidemiological evidence.

Several studies have indicated that salmonella and E. coli O157:H7 present initially on artificially as well as naturally contaminated seed have the potential to increase up to 10,000-fold on sprouts propagated at 20 to 30°C. The majority of growth of salmonella and E. coli O157:H7 on sprouting seed occurs during the first 48 hours. For sprouts grown from artificially inoculated seed, maximum populations of salmonella and E. coli O157:H7 ranging from 5 to 8 log10 colony-forming units (CFU)/g have been reported [30-39]. The maximum pathogen population obtained was not dependent on the initial inoculum level present on the seed [36]. For comparison, populations of total aerobes reported for sprouts typically range from 7 to 9 log10 CFU/g [30,40-42]. For salmonella on alfalfa, the doubling time was estimated at 47 minutes during the initial rapid growth phase and growth was not dependent on pathogen serovar, isolation source, or virulence [33]. Populations of salmonella and E. coli O157:H7 were stable from 48 hours to harvest at 3 to 5 days and then declined only slightly during subsequent storage of contaminated alfalfa sprouts at 5 to 9°C for 6 to 10 days [33,34,37]. Populations of B. cereus on sprouts grown from naturally contaminated alfalfa and mung bean seed reached approximately 4 log10 CFU/g [43]. The maximum pathogen populations attained during germination and growth of naturally contaminated seed under commercial practice may be several log10 units less than that for artificially inoculated seed [44]. Maximum populations of salmonella attained on alfalfa sprouts grown from two different lots of naturally contaminated seed were only 2 to 4 log10 MPN/g for salmonella. The reduced growth may be due to several factors. The first is the much lower overall contamination levels on naturally contaminated seed when compared to even the lowest initial pathogen populations utilized for laboratory studies. Second, pathogen populations on naturally contaminated seed may contain a higher percentage of injured cells. Third, differing methods of irrigation and increased irrigation frequency employed in commercial operations may affect the final pathogen populations attained. Interestingly, salmonella serovars attach more tightly to surfaces of alfalfa sprouts than do strains of E. coli O157:H7 and the difference in strength of attachment was proposed to explain, at least in part, the greater number of outbreaks of foodborne illness associated with contaminated sprouts due to salmonella [39].

Studies in several independent laboratories have indicated that bacterial human pathogens can be internalized in sprouts. By use of immunofluorescence and scanning immunoelectron microscopy, E. coli O157:H7 was located in stomata and the vascular system of radish sprouts grown from inoculated seed [45]. Bioluminescent Salmonella Montevideo and various salmonella serovars expressing the autofluorescent green-fluorescent protein were also located in the internal tissues of mung bean and alfalfa sprouts, respectively, after inoculation of seed or roots [38,45,46]. The mode of entry of bacterial human pathogens into plants remains unknown, but it is likely due to passive uptake at the site of injury where lateral roots emerge [46,48], as salmonella and E. coli O157:H7 have not been reported to excrete cell-wall-degrading enzymes (e.g., pectinases or cellulases) that might facilitate active entry. Pathogens may form biofilms on sprout surfaces and/or become part of biofilms produced by native microorganisms [49,50] (Figure 8.1).

FIGURE 8.1 Biofilm consisting of native bacteria on the surface of an alfalfa sprout hypocotyl.

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