Numerous field studies have revealed that irrigation water can contaminate the surface of plants; however, the occurrence of pathogen internalization is unclear. This study was conducted to determine the sites of Escherichia coli O157:H7 contamination and its survival when the bacteria were applied through spray irrigation water to either field-grown spinach or lettuce. To differentiate internalized and surface populations, leaves were treated with a surface disinfectant wash before the tissue was ground for analysis of E. coli O157:H7 by direct plate count or enrichment culture. Irrigation water containing E. coli O157:H7 at 10(2), 10(4), or 10(6) CFU/ml was applied to spinach 48 and 69 days after transplantation of seedlings into fields. E. coli O157:H7 was initially detected after application on the surface of plants dosed at 10(4) CFU/ml (4 of 20 samples) and both on the surface (17 of 20 samples) and internally (5 of 20 samples) of plants dosed at 10(6) CFU/ml. Seven days postspraying, all spinach leaves tested negative for surface or internal contamination. In a subsequent study, irrigation water containing E. coli O157:H7 at 10(8) CFU/ml was sprayed onto either the abaxial (lower) or adaxial (upper) side of leaves of field-grown lettuce under sunny or shaded conditions. E. coli O157:H7 was detectable on the leaf surface 27 days postspraying, but survival was higher on leaves sprayed on the abaxial side than on leaves sprayed on the adaxial side. Internalization of E. coli O157:H7 into lettuce leaves also occurred with greater persistence in leaves sprayed on the abaxial side (up to 14 days) than in leaves sprayed on the adaxial side (2 days).

Several sources of contamination of fresh produce by Escherichia coli O157:H7 (O157) have been identified and include contaminated irrigation water and improperly composted animal waste; however, field studies evaluating the potential for internalization of O157 into leafy greens from these sources have not been conducted. Irrigation water inoculated with green fluorescent plasmid-labeled Shiga toxin-negative strains (50 ml of 10(2), 10(4), or 10(6) CFU of O157 per ml) was applied to soil at the base of spinach plants of different maturities in one field trial. In a second trial, contaminated compost (1.8 kg of 10(3) or 10(5) CFU of O157 per g) was applied to field plots (0.25 by 3.0 m) prior to transplantation of spinach, lettuce, or parsley plants. E. coli O157:H7 persisted in the soil up to harvest (day 76 posttransplantation) following application of contaminated irrigation water; however, internalized O157 was not detected in any spinach leaves or in roots exposed to O157 during the early or late growing season. Internalized O157 was detected in root samples collected 7 days after plants were contaminated in mid-season, with 5 of 30 samples testing positive for O157 by enrichment; however, O157 was not detected by enrichment in surface-disinfected roots on days 14 or 22. Roots and leaves from transplanted spinach, lettuce, and parsley did not internalize O157 for up to 50 days in the second trial. These results indicate that internalization of O157 via plant roots in the field is rare and when it does occur, O157 does not persist 7 days later.

Environmental pests may serve as reservoirs and vectors of zoonotic pathogens to leafy greens; however, it is unknown whether insect pests feeding on plant tissues could redistribute these pathogens present on the surface of leaves to internal sites. This study sought to differentiate the degree of tissue internalization of Escherichia coli O157:H7 when applied at different populations on the surface of lettuce and spinach leaves, and to ascertain whether lettuce-infesting insects or physical injury could influence the fate of either surface or internalized populations of this enteric pathogen. No internalization of E. coli O157:H7 occurred when lettuce leaves were inoculated with 4.4 log CFU per leaf, but it did occur when inoculated with 6.4 log CFU per leaf. Internalization was statistically greater when spinach leaves were inoculated on the abaxial (underside) than when inoculated on the adaxial (topside) side, and when the enteric pathogen was spread after surface inoculation. Brief exposure (∼18 h) of lettuce leaves to insects (5 cabbage loopers, 10 thrips, or 10 aphids) prior to inoculation with E. coli O157:H7 resulted in significantly reduced internalized populations of the pathogen within these leaves after approximately 2 weeks, as compared with leaves not exposed to insects. Surface-contaminated leaves physically injured through file abrasions also had significantly reduced populations of both total and internalized E. coli O157:H7 as compared with nonabraded leaves 2 weeks after pathogen exposure.

Aims: Three soils that varied in their physicochemical characteristics and microbial diversity were inoculated with Escherichia coli O157:H7 and Salmonella to determine the relative impact of abiotic and biotic factors on the pathogens' survival when the soil was held at 25°C. Methods and Results: Three soils that were classified as having low, medium and high microbial diversity were divided into two batches for adjustment to 20% of water-holding capacity and to 40% of water-holding capacity. Soils were inoculated with both green fluorescent-labelled E. coli O157:H7 and red fluorescent-labelled Salmonella (5 log CFU g À1 dry weight) and held at 25°C.Pathogens inoculated into an acidic soil died off within 9 weeks, whereas they were still detected in the other two soils by enrichment culture after 18 weeks. Moisture did not affect inactivation of E. coli O157:H7, but did affect Salmonella inactivation in soil having the greatest organic load and microbial diversity. Using multiple linear regression analysis, 98Á7% of the variability in the inactivation rate for E. coli O157:H7 was explained by a model that included the variables of initial pH and electrical conductivity. Salmonella's inactivation rate was predicted by a model that included pH and initial cell numbers of copiotrophic and oligotrophic bacteria. Conclusion: This study provided evidence of specific properties that impact inactivation of E. coli O157:H7 and Salmonella in soils at 25°C. Significance and Impact of the Study: Identification of factors influential in the die-off of enteric pathogens will assist in developing guidelines for safe intervals between field contamination events and planting or harvesting of fresh-cut produce crops.

Freshly harvested Georgia-grown cantaloupes (Cucumis melo L. var. reticulatus cv. Athena and Atlantis) were spot inoculated with 100 μl of a five-strain mixture of Salmonella enterica serovar Poona (9 log CFU/ml) at the stem scar and on the netted rind and then subjected to no treatment (control) or a 6-min treatment (tank only) in water, 120 ppm of chlorine (pH 7.0), 1% levulinic acid plus 0.1% sodium dodecyl sulfate (SDS; pH 3.0), or 2% levulinic acid plus 0.2% SDS (pH 3.0). The log reduction for the tank-only treatments was 0.31, 0.59, 1.32, and 1.37 log CFU/g at the stem scar and 0.97, 1.59, 2.06. and 3.37 log CFU/g on the netted rind for water, chlorine, 1% levulinic acid plus 0.1% SDS, and 2% levulinic acid plus 0.2% SDS, respectively. A greater log reduction was observed for the cantaloupe surface tissue with the water, chlorine, and 2% levulinic acid plus 0.2% SDS treatments when additional sanitizer (2 ml) and brushing (to simulate cantaloupes tumbling over brushes on the processing line) were added to the dump tank treatment. The stem scar tissue reductions were 0.90, 1.69, and 1.53 log CFU/g, whereas the netted rind reductions were 1.56, 2.50, and 4.47 log CFU/g after treatment with water, chlorine, and 2% levulinic acid plus 0.2% SDS, respectively. These data suggest that 2% levulinic acid plus 0.2% SDS is effective for reducing Salmonella on the netted rind surface of cantaloupes. However, neither 2% levulinic acid plus 0.2% SDS nor 120 ppm of chlorine substantially reduced Salmonella on stem scar tissue.