Absence of internalization of Escherichia coli O157:H7 into germinating tissue of field-grown leafy greens.

Both growth chamber and field studies were conducted to investigate the potential for Escherichia coli O157:H7 to be internalized into leafy green tissue when seeds were germinated in contaminated soil. Internalized E. coli O157:H7 was detected by enrichment in both spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.) seedlings when seeds were germinated within the growth chamber in autoclaved and nonautoclaved soil, respectively, contaminated with E. coli O157:H7 at 2.0 and 3.8 log CFU/g, respectively. Internalized E. coli O157:H7 populations could be detected by enumeration within leafy green tissues either by increasing the pathogen levels in the soil or by autoclaving the soil. Attempts to maximize the exposure of seed to E. coli O157:H7 by increasing the mobility of the microbe either through soil with a higher moisture content or through directly soaking the seeds in an E. coli O157:H7 inoculum did not increase the degree of internalization. Based on responses obtained in growth chamber studies, internalization of E. coli O157:H7 surrogates (natural isolates of Shiga toxin-negative E. coli O157:H7 or recombinant [stx- and eae-negative] outbreak strains of E. coli O157:H7) occurred to a slightly lesser degree than did internalization of the virulent outbreak strains of E. coli O157:H7. The apparent lack of internalized E. coli O157:H7 when spinach and lettuce were germinated from seed in contaminated soil (ca. 3 to 5 log CFU/g) in the field and the limited occurrence of surface contamination on the seedlings suggest that competition from indigenous soil bacteria and environmental stresses were greater in the field than in the growth chamber. On the rare occasion that soil contamination with E. coli O157:H7 exceeded 5 log CFU/g in a commercial field, this pathogen probably would not be internalized into germinating leafy greens and/or would not still be present at the time of harvest.

Internalization of Escherichia coli O157:H7 following spraying of cut shoots when leafy greens are regrown for a second crop.

Both spinach and lettuce were grown to harvest, cut, and then regrown after spraying the cut shoots with irrigation water contaminated with Escherichia coli O157:H7. Plant tissue was collected on the day of spraying and again 2 and 14 days later for analysis of total and internalized E. coli O157:H7 populations. Internalization of E. coli O157:H7 occurred on the day of spraying, and larger populations were internalized as the level in the spray increased. Tissue repair was slow and insufficient to prevent infiltration of E. coli O157:H7; internalized E. coli O157:H7 in shoots cut 5 days prior to exposure to E. coli O157:H7-contaminated water were not significantly different from levels in shoots cut on the same day of spraying with contaminated water (P > 0.05). Two days after spraying plants with a high level of E. coli O157:H7 (7.3 log CFU/ml), levels of internalized E. coli O157:H7 decreased by ca. 2.6 and 1.3 log CFU/g in Tyee and Bordeaux spinach, respectively, whereas populations of internalized E. coli O157:H7 decreased very little (ca. 0.4 log CFU/g) in lettuce plants that had been sprayed either on the same day as cutting or 1 day after cutting. When cut plants were sprayed with irrigation water at a lower contamination level (4.5 log CFU/ml), internalized E. coli O157:H7 was not detected in either spinach or lettuce plants 2 days later and therefore would not likely be of concern when the crop was harvested.

Surface and internalized Escherichia coli O157:H7 on field-grown spinach and lettuce treated with spray-contaminated irrigation water.

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).

Infrequent internalization of Escherichia coli O157:H7 into field-grown leafy greens.

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.

Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water.

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 infections associated with lettuce and other leaf crops have occurred with increasing frequency in recent years. Contaminated manure and polluted irrigation water are probable vehicles for the pathogen in many outbreaks. In this study, the occurrence and persistence of E. coli O157:H7 in soil fertilized with contaminated poultry or bovine manure composts or treated with contaminated irrigation water and on lettuce and parsley grown on these soils under natural environmental conditions was determined. Twenty-five plots, each 1.8 by 4.6 m, were used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but treated with contaminated water) and five replication plots for each treatment. Three different types of compost, PM-5 (poultry manure compost), 338 (dairy manure compost), and NVIRO-4 (alkaline-stabilized dairy manure compost), and irrigation water were inoculated with an avirulent strain of E. coli O157:H7. Pathogen concentrations were 10(7) CFU/g of compost and 10(5) CFU/ml of water. Contaminated compost was applied to soil in the field as a strip at 4.5 metric tons per hectare on the day before lettuce and parsley seedlings were transplanted in late October 2002. Contaminated irrigation water was applied only once on the plants as a treatment in five plots for each crop at the rate of 2 liters per plot 3 weeks after the seedlings were transplanted. E. coli O157:H7 persisted for 154 to 217 days in soils amended with contaminated composts and was detected on lettuce and parsley for up to 77 and 177 days, respectively, after seedlings were planted. Very little difference was observed in E. coli O157:H7 persistence based on compost type alone. E. coli O157:H7 persisted longer (by > 60 days) in soil covered with parsley plants than in soil from lettuce plots, which were bare after lettuce was harvested. In all cases, E. coli O157:H7 in soil, regardless of source or crop type, persisted for > 5 months after application of contaminated compost or irrigation water.

Fate of Salmonella enterica serovar Typhimurium on carrots and radishes grown in fields treated with contaminated manure composts or irrigation water.

Three different types of compost, PM-5 (poultry manure compost), 338 (dairy cattle manure compost), and NVIRO-4 (alkaline-pH-stabilized dairy cattle manure compost), and irrigation water were inoculated with an avirulent strain of Salmonella enterica serovar Typhimurium at 10(7) CFU g(-1) and 10(5) CFU ml(-1), respectively, to determine the persistence of salmonellae in soils containing these composts, in irrigation water, and also on carrots and radishes grown in these contaminated soils. A split-plot block design plan was used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but with contaminated water applied) and five replicates for a total of 25 plots for each crop, with each plot measuring 1.8 x 4.6 m. Salmonellae persisted for an extended period of time, with the bacteria surviving in soil samples for 203 to 231 days, and were detected after seeds were sown for 84 and 203 days on radishes and carrots, respectively. Salmonella survival was greatest in soil amended with poultry compost and least in soil containing alkaline-pH-stabilized dairy cattle manure compost. Survival profiles of Salmonella on vegetables and soil samples contaminated by irrigation water were similar to those observed when contamination occurred through compost. Hence, both contaminated manure compost and irrigation water can play an important role in contaminating soil and root vegetables with salmonellae for several months.

Persistence of Salmonella enterica serovar typhimurium on lettuce and parsley and in soils on which they were grown in fields treated with contaminated manure composts or irrigation water.

There are many sources of pathogen contamination of vegetable crops in the field that include manure used as fertilizer and irrigation water. An avirulent strain of Salmonella enterica serovar Typhimurium was added to three different types of composts-PM-5 (poultry manure compost), 338 (dairy manure compost), and NVIRO-4 (alkaline stabilized dairy manure compost)-and irrigation water at 10(7) colony forming units (cfu)/g and 10(5) cfu/mL, respectively, to determine under field conditions the persistence of salmonellae in soils treated with these composts or irrigation water, and also on leaf lettuce and parsley grown on such treated soil. Contaminated compost was applied to soil in the field as a strip at a rate of 4.5 metric tons/hectare on the day before lettuce and parsley seedlings were transplanted. Contaminated irrigation water was applied only once on the plants at the rate of 2 liters per plot on the same day after the seedlings were transplanted. Twenty-five plots, each measuring 1.8 x 4.6 meters, were used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but applied with contaminated water) and five replication plots for each treatment. Salmonella persisted for 161 and up to 231 days in soils amended with contaminated composts on which lettuce and parsley, respectively, were grown, and was detected for up to 63 days and 231 days on lettuce and parsley, respectively. The type of contaminated compost had minimal effect on the persistence of S. Typhimurium in soil. Occurrence of Salmonella on vegetables and survival in soil on which these vegetables were grown, irrespective of source of contamination through irrigation water or compost, were similar, suggesting both contaminated manure compost and irrigation water can play important roles in contaminating soil and vegetables with Salmonella for an extended period of time.