Ian D. Flitcroft

Preharvest internalization of Escherichia coli O157:H7 into lettuce leaves, as affected by insect and physical damage.

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.

Precision turfgrass management: challenges and field applications for mapping turfgrass soil and stress

Spatial and temporal variation of soil, climate, plants and irrigation requirements are challenges for modern agriculture and complex turfgrass sites. Precision agriculture (PA) evolved to improve site-specific management based on obtaining site-specific information. The focus of this concept paper is on the emerging area of precision turfgrass management (PTM) with attention given to: (a) comparing the concepts of PTM and PA in terms of driving forces and challenges that must be addressed for PTM to progress in science and practice and (b) discussion of specific field mapping applications (purposes) for different turfgrass situations such as golf courses, sod production fields and sports fields. The field applications relate to site-specific management of irrigation, salinity, fertilizer application and cultivation. To illustrate the potential for PTM, different approaches that may be necessary for PTM compared to PA are discussed. The initial factor that hindered the adoption of PTM has been the lack of mobile sensor platforms that can determine both key soil and plant properties for turfgrass situations. This paper concentrates on PTM field applications that involve mapping of both soil and plant attributes, in contrast to only optical sensing mapping.

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.

Fate of Escherichia coli O157:H7 and Salmonella in soil and lettuce roots as affected by potential home gardening practices

BACKGROUND The survival and distribution of enteric pathogens in soil and lettuce systems were investigated in response to several practices (soil amendment supplementation and reduced watering) that could be applied by home gardeners. RESULTS Leaf lettuce was grown in manure compost/top soil (0:5, 1:5 or 2:5 w/w) mixtures. Escherichia coli O157:H7 or Salmonella was applied at a low or high dose (10(3) or 10(6) colony-forming units (CFU) mL(-1) ) to the soil of seedlings and mid-age plants. Supplementation of top soil with compost did not affect pathogen survival in the soil or on root surfaces, suggesting that nutrients were not a limiting factor. Salmonella populations on root surfaces were 0.7-0.8 log CFU g(-1) lower for mid-age plants compared with seedlings. E. coli O157:H7 populations on root surfaces were 0.8 log CFU g(-1) lower for mid-age plants receiving 40 mL of water compared with plants receiving 75 mL of water on alternate days. Preharvest internalization of E. coli O157:H7 and Salmonella into lettuce roots was not observed at any time. CONCLUSION Based on the environmental conditions and high pathogen populations in soil used in this study, internalization of Salmonella or E. coli O157:H7 into lettuce roots did not occur under practices that could be encountered by inexperienced home gardeners.

Internalization and fate of Escherichia coli O157:H7 in leafy green phyllosphere tissue using various spray conditions.

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 coli O157: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. coli O157: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. coli O157: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. coli O157: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. coli O157: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. coli O157: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.

Biotic and Abiotic Variables Affecting Internalization and Fate of Escherichia coli O157:H7 Isolates in Leafy Green Roots

Preharvest internalization of Escherichia coli O157: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. coli O157: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. coli O157:H7 at 5.6 to 6.1 log CFU/g. Internalization of E. coli O157:H7 into leafy green plant roots was higher when E. coli O157: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. coli O157: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. coli O157: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. coli O157:H7 isolates were more stable than were virulent isolates in soil, but the degree of internalization of E. coli O157: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.

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.

Survival of Salmonella or Escherichia coli O157:H7 during holding of manure-based compost mixtures at sublethal temperatures as influenced by the carbon amendment.

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 Salmonella or E. coli O157: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. coli O157: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. coli O157:H7 was affected minimally by moisture levels, whereas Salmonella survival increased as the moisture level was decreased. The different inactivation responses of Salmonella and E. coli O157: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.

Manure Source and Age Affect Survival of Zoonotic Pathogens during Aerobic Composting at Sublethal Temperatures

Heat is the primary mechanism by which aerobic composting inactivates zoonotic bacterial pathogens residing within animal manures, but at sublethal temperatures, the time necessary to hold the compost materials to ensure pathogen inactivation is uncertain. To determine the influence of the type of nitrogen amendment on inactivation of Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7 in compost mixtures stored at sublethal temperatures, specific variables investigated in these studies included the animal source of the manure, the initial carbon/nitrogen (C:N) ratio of the compost mixture, and the age of the manure. Salmonella and L. monocytogenes were both inactivated more rapidly in chicken and swine compost mixtures stored at 20°C when formulated to an initial C:N ratio of 20:1 compared with 40:1, whereas a C:N ratio did not have an effect on inactivation of these pathogens in cow compost mixtures. Pathogen inactivation was related to the elevated pH of the samples that likely arises from ammonia produced by the indigenous microflora in the compost mixtures. Indigenous microbial activity was reduced when compost mixtures were stored at 30°C and drier conditions (<10% moisture level) were prevalent. Furthermore, under these drier conditions, Salmonella persisted to a greater extent than L. monocytogenes, and the desiccation resistance of Salmonella appeared to convey cross-protection to ammonia. Salmonella persisted longer in compost mixtures prepared with aged chicken litter compared with fresh chicken litter, whereas E. coli O157:H7 survived to similar extents in compost mixtures prepared with either fresh or aged cow manure. The different responses observed when different sources of manure were used in compost mixtures reveal that guidelines with times required for pathogen inactivation in compost mixtures stored at sublethal temperatures should be dependent on the source of nitrogen, i.e., type of animal manure, present.