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During the early phases of aerobic composting of animal manures, pathogens are inactivated primarily from the accumulation of heat produced by indigenous microbial activity. When compost materials are not exposed to these lethal temperatures, the required holding time needed to obtain a pathogen-free product that may be applied to fields is unknown. Consequently, a series of studies examined whether the carbon amendment (wheat straw, peanut hulls, rice hulls, and pine needles) added to animal manures affected survival of either Salmonellaor E. coliO157:H7 during storage of compost mixtures at sublethal temperatures (20 to 40°C). Pathogens consistently survived for longer periods of time in compost mixtures prepared with pine needles than compost mixtures prepared with either of the other three carbon amendments. Pathogen inactivation in wheat straw- or peanut hull-amended compost mixtures was dependent on the target pathogen, moisture level, and storage temperature. Moisture levels in wheat straw-amended compost mixtures stored at 40°C had no effect on inactivation of E. coliO157:H7. In contrast, wheat straw-amended mixtures stored at 30 to 35°C and equilibrated to suboptimal moisture contents (30 to 40%) were less effective for inactivating pathogens compared with drier (25% moisture) or moister (60% moisture) mixtures. In peanut hull-amended compost mixtures, inactivation of E. coliO157:H7 was affected minimally by moisture levels, whereas Salmonellasurvival increased as the moisture level was decreased. The different inactivation responses of Salmonellaand E. coliO157:H7 in compost mixtures prepared with wheat straw or peanut hulls and equilibrated to different moisture levels suggest that there are different mechanisms for inactivation. Hence, developing reliable guidelines relying on time-temperature for holding of compost mixtures at sublethal temperatures will be challenging and, perhaps, not possible.

Preharvest internalization of Escherichia coliO157:H7 into the roots of leafy greens is a food safety risk because the pathogen may be systemically transported to edible portions of the plant. In this study, both abiotic (degree of soil moisture) and biotic (E. coliO157:H7 exposure, presence of Shiga toxin genes, and type of leafy green) factors were examined to determine their potential effects on pathogen internalization into roots of leafy greens. Using field soil that should have an active indigenous microbial community, internalized populations in lettuce roots were 0.8 to 1.6 log CFU/g after exposure to soil containing E. coliO157:H7 at 5.6 to 6.1 log CFU/g. Internalization of E. coliO157:H7 into leafy green plant roots was higher when E. coliO157:H7 populations in soil were increased to 7 or 8 log CFU/g or when the soil was saturated with water. No differences were noted in the extent to which internalization of E. coliO157:H7 occurred in spinach, lettuce, or parsley roots; however, in saturated soil, maximum levels in parsley occurred later than did those in spinach or lettuce. Translocation of E. coliO157:H7 from roots to leaves was rare; therefore, decreases observed in root populations over time were likely the result of inactivation within the plant tissue. Shiga toxin–negative (nontoxigenic) E. coliO157:H7 isolates were more stable than were virulent isolates in soil, but the degree of internalization of E. coliO157:H7 into roots did not differ between isolate type. Therefore, these nontoxigenic isolates could be used as surrogates for virulent isolates in field trials involving internalization.

In the past decade, leafy greens have been implicated in several outbreaks of foodborne illness, and research has focused on contamination during preharvest operations. Concerns have been raised that internalization of pathogens into the edible tissue occurs where postharvest chemical interventions would be ineffective. This study was initiated to measure the degree and fate of Escherichia coliO157:H7 internalized in the phyllosphere tissue of leafy greens when spray conditions, inoculum level, and type of leafy green were varied. Two spraying treatments were applied: (i) spraying individual spinach or lettuce leaves on plants once with a high dose (7 to 8 log CFU/ml) of E. coliO157:H7 and (ii) spraying spinach, lettuce, or parsley plants repeatedly (once per minute) with a low dose (2.7 to 4.2 log CFU/ml) of E. coliO157:H7 over a 10- to 20-min period. With the high-dose spray protocol, no significant differences in the prevalence of internalization occurred between Shiga toxin–negative E. coliO157:H7 isolates and virulent isolates (P >0.05), implying that the Shiga toxin virulence factors did not influence internalization or the subsequent fate of those populations under these test conditions. Significantly greater internalization of E. coliO157:H7 occurred in spinach leaves compared with lettuce leaves when leaves were sprayed once with the high-dose inoculum (P >0.05), whereas internalization was not observed in lettuce leaves but continued to be observed in spinach and parsley leaves following repeated spraying of the low-dose inoculum. Based on these results, it is surmised that a moisture film was generated when spraying was repeated and this film assisted in the mobilization of pathogen cells to plant apertures, such as stomata. E. coliO157:H7 cells that were internalized into spinach tissue using a low-dose repeat-spray protocol were temporary residents because they were not detected 2 days later, suggesting that plant-microbe interactions may be responsible.

Both spinach and lettuce were grown to harvest, cut, and then regrown after spraying the cut shoots with irrigation water contaminated with Escherichia coliO157:H7. Plant tissue was collected on the day of spraying and again 2 and 14 days later for analysis of total and internalized E. coliO157:H7 populations. Internalization of E. coliO157: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. coliO157:H7; internalized E. coliO157:H7 in shoots cut 5 days prior to exposure to E. coliO157: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. coliO157:H7 (7.3 log CFU/ml), levels of internalized E. coliO157:H7 decreased by ca. 2.6 and 1.3 log CFU/g in Tyee and Bordeaux spinach, respectively, whereas populations of internalized E. coliO157: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. coliO157: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.