2009 Annual Report
1a.Objectives (from AD-416)
1. Determine the prevalence, diversity, and quantity of bacteria associated with organic as compared to conventional fresh produce in Maryland and Virginia.
2. Determine colonization and survival rates of bacterial pathogens associated with selected organic and conventional fresh produce.
3. Investigate the mechanism(s) of introduction and transference of E. coli O157:H7 to lettuce and leafy greens during growing, harvest, postharvest handlings and processing operations.
4. Determine the persistence and survival of Escherichia coli O157:H7 on fresh and fresh-cut leafy green produce.
5. Compare the extent of enteric pathogen transmission by a chewing insect, Colorado Potato Beetle, in organically- and conventionally-grown produce.
1b.Approach (from AD-416)
A farm-based investigation will be conducted to assess the prevalence, diversity, and quantity of epi- and endophytic saprophytes and gastroenteric bacteria associated with organic as compared to conventional fresh produce. Bacterial analyses will include use of microbial cultural and molecular methods to characterize the enteric and saprophytic microflora on fresh market produce types most commonly reported in foodborne illness outbreaks: tomatoes, salad greens: lettuce, arugula, mesculin, spinach, beets; herbs: basil, cilantro, parsley; strawberries; cucurbits: cucumbers and melons. In addition, practices at each farm will be identified and characterized to associate analytical results with microbial quality of farm inputs and operations. Bacterial analysis will include total aerobic heterotrophic bacteria, enteric bacteria (coliforms), Aeromonas spp., Bacillus cereus, C. perfringens, E. coli, Enterococcus spp., L. monocytogenes, and Salmonella. Microbial community analysis of epi- and endophytic bacteria by DGGE will be used for tomato fruit and salad greens from organic and conventional farms. Growth chamber and field plot experiments will be conducted to establish the pathogen concentrations required to establish populations that survive on different plants (e.g., tomatoes, greena, basil, scallions, carrots, and strawberries). Survival of pathogens (Aeromonas, EHEC, L. monocytogenes, and Salmonella) on/in plants grown in organic and conventional soils with composted or aged manure (spike with pathogens) and irrigated with contaminated river water or sprayed with compost tea will be determined by enrichment and enumeration of rhizosphere, cortical root tissue, and/or on the edible portion of plants. Organic practices that can prevent pathogen growth in compost tea and internal plant tissues will be evaluated in growth chamber and field studies with tomato. Finally, the potential for a chewing insect, Colorado Potato Beetle, to transfer bacterial pathogens to the surface and interior tissue of tomato plants and initiate systemic colonization will be investigated for organic and conventional production methods.
Studies were conducted to determine the antimicrobial properties of essential oils in controlling Salmonella in organic soil. Vinegar and eugenol significantly reduced Salmonella populations, although the effectiveness varied with concentration and serovar. Root uptake and internalization of E. coli O157:H7 were studied in spinach grown in soil and in hydroponic media. E. coli O157:H7 was internalized in plants grown hydroponically in the presence of initial high populations. However, no internalized E. coli O157:H7 were recovered from spinach plants grown in soil, although individual cells were observed microscopically in root tissue. Studies were conducted to evaluate the efficacy of the California Leafy Green Marketing Agreement (LGMA). Spinach plants were spray irrigated (in a growth chamber) with water contaminated with EHEC (enterohemorrhagic E. coli) or APEC (avian pathogenic E. coli) at levels recommended by the LGMA. No E. coli were recovered on days 1 or 2 after three separate irrigation events, suggesting that LGMA standards for irrigation water are adequate to prevent introduction of pathogenic E. coli under the conditions evaluated. Studies were conducted to assess attachment of Salmonella and E. coli O157:H7 to intact and damaged produce surfaces. E. coli O157:H7 and Salmonella attached preferentially to damaged leafy greens over intact leaves. Most pathogen attachment occurred within the first hour of exposure, suggesting the need for an immediate intervention to prevent contamination. Salmonella attachment to green cabbage surfaces was significantly lower than attachment to romaine or iceberg lettuce. Studies were conducted to assess bacteriophage treatment as an intervention to kill E. coli O157:H7 attached to spinach harvester blades. Bacteriophages reduced E. coli O157:H7 on harvester blades within 10 min of exposure. The expression of food-borne E. coli O157:H7 virulence genes was investigated. Expression of virulence genes was similar for cells grown in ground beef or on lettuce. Refrigerated or abusive storage of lettuce did not influence expression of virulence genes. The effects of leaf age, ultraviolet exposure and inoculation position were examined for their effect on E. coli O157:H7 survival on spinach. Neither the age of the spinach leaf or ultraviolet exposure affected the persistence of E. coli O157:H7 over 28 days of incubations. A non-pathogenic E. coli O157:H12, which contains no virulence factors, was investigated as a surrogate for E. coli O157:H7 for use in field trials. The surrogate strain showed similar growth and survival characteristics on lettuce. The effect of biofumigation on reduction of E. coli and salmonellae in lettuce and spinach production soils was tested. Results show significant promise for elimination of pathogens from fecal contamination in soils. The growth and survival of E. coli and salmonellae in hornworm larvae and Colorado potato beetle larvae were evaluated on tomato leaves. Results indicate that within 24 hr of an initial low dose (less than 1000 cells) insect frass contains more than 10 million cells of E. coli or salmonellae per gram.
Uptake and internalization of E. coli O157:H7 in spinach cultivated in soil and hydroponic media. This was the first internalization study to use chromosomally transformed fluorescent E. coli cells to provide for the more sensitive detection of bacterial cells in spinach tissues. The chromosomal integration of the green fluorescent protein (gfp) gene was successfully accomplished into four nalidixic acid resistant E. coli strains: two O157:H7 strains from produce outbreaks, 4407 and 5279, one O157:H7 strain from a beef-associated outbreak, 86-24h11, and a non-pathogenic commensal isolate (HS). No E. coli populations were detected by spiral plating from homogenized internal tissues of baby spinach plants grown in pasteurized soils grown over 28 days, but individual cells were microscopically observed in the root tissue. Cells of E. coli O157:H7 86-24h11 applied to hydroponic medium at a high population were recovered by spiral plating from the shoot tissues of spinach plants in hydroponic media after 14 and 21 days. No differences in internalization ability of E. coli O157:H7 strains from produce or ground beef outbreaks were observed. It appears unlikely that root uptake is a significant route of contamination of leafy greens.
Assessment of recommended irrigation water standards for leafy greens. The California Leafy Green Marketing Agreement (LGMA) recommends that water containing less than 235 MPN (most probable number) E. col/100 ml be used in irrigating leafy greens in the field. However, no evaluation of these standards has been undertaken. EHEC (enterohemorrhagic E. coli) or APEC (avian pathogenic E. coli) was sprayed on spinach plants at low levels (85 and 189 CFU/100ml, respectively) in water every seven days over the course of 14 days. Recovery of E. coli populations on day 0 ranged from 0.3–3.6 MPN EHEC/spinach plant, and 1.28-2.19 MPN APEC/spinach plant. No E. coli were recovered on days 1 or 2 after irrigation. E. coli were only detected immediately after irrigation. Repeated irrigation of spinach plants with water complying with these standards did not increase the persistence of E. coli on foliar surfaces. These data indicate that these irrigation water standards are sufficiently stringent to limit the introduction and persistence of pathogenic E. coli on the surface of spinach plants cultivated in growth chambers.
Biofilm formation and bacteriophage inactivation of Escherichia coli O157:H7 on spinach harvester blades. Contaminated harvesting equipment can be a source of contamination for produce. No studies have examined the ability of E. coli O157:H7 to form biofilms on harvester blades, or the effect of bacteriophages to kill them. A cocktail of five nalidixic acid-resistant E. coli O157:H7 isolates was used in spinach extract containing a piece of spinach harvester blade (2 x 1") to study biofilm formation. E. coli O157:H7 adherence to blades incubated at dynamic temperature (simulated field settings) was significantly higher than the populations of E. coli O157:H7 recovered at room temperature. Prolonged incubation of 48 h at dynamic or ambient temperature did not affect E. coli O157:H7 adherence to spinach harvester blade. Spraying of spinach harvester blade with a cocktail of bacteriophages reduced E. coli O157:H7 by 2.5 log CFU within 10 min of application. Further exposure (2 h) of bacteriophage reduced E. coli O157:H7 by 4 log CFU on blades. This study will help farmers in controlling cross-contamination of spinach with potential pathogens during spinach harvesting operations.
Virulence expression of E. coli O157:H7 on lettuce and in ground beef. Outbreaks of infections associated with leafy greens in 2006 saw more patients hospitalized and develop symptoms of hemolytic uremic syndrome than in previous outbreaks associated with other commodities. Research was conducted to determine if E. coli O157:H7 cells were 'primed' to cause illness by growing O157-cells in ground beef or on cut iceberg lettuce, extracting bacterial RNA, and performing a reverse transcriptase real time PCR method to measure the levels of virulence factors. Expression of E. coli O157:H7 virulence genes were similar when grown in ground beef or on lettuce. E. coli O157:H7 cells were not more 'primed' to cause illness on refrigerated lettuce stored under aerobic conditions for 7 days at 4 deg C or 2 days at 15 deg C. These results indicated that survival/growth of E. coli O157:H7 on lettuce does not provide it with additional virulence properties that may cause more human illness when compared to ground beef.
Attachment of foodborne pathogens to intact and damaged produce surfaces. Bacterial attachment is the first step for pathogens in their survival and persistence on new surfaces. Attachment of Salmonella and E. coli O157:H7 on intact and damaged produce (green cabbage, iceberg lettuce, romaine lettuce) surfaces was studied. Most pathogen attachment occurred during the first hour of exposure to produce surface. Attachment to produce surfaces among E. coli O157:H7 and Salmonella spp. was variable. Populations of strongly attached E. coli O157:H7 on intact and damaged surfaces were significantly higher than loosely attached E. coli O157:H7. Salmonella attachment to cabbage was significantly lower than the attachment to romaine or iceberg lettuce. Most E. coli O157:H7 and Salmonella were attached to damaged surface at levels 0.4-1 log CFU above populations on intact surfaces. To develop effective strategies to minimize the risk of foodborne disease caused by this organism, it is essential to examine initial stages of bacterial attachment to various plant tissues.
Survival of E. coli O157:H7 on spinach plant grown in high tunnel. High tunnel cultivation is a low-cost season extending strategy for leafy greens and other vegetables. Various factors affecting the survival of bacterial pathogens could be different for leafy greens grown in high tunnels from open field farms. We inoculated spinach grown in high tunnels with a non-pathogenic E. coli O157:H7 strain and the survival of the inoculated cells was monitored for 4 weeks. E. coli O157:H7 population dropped precipitously within 24 hrs of inoculation, followed by steady decline during the course of the experiment to undetectable or barely detectable 28 days after inoculation. Leaf age, inoculation position and reduced ultraviolet light exposure (using different tunnel cover film plastic materials) did not significantly affect the survival of E. coli O157:H7 on the plants. This research indicates that spinach grown in high tunnels does not support the survival of E. coli O157:H7 even though UV exposure is reduced in high tunnels.
Evaluation of non-pathogenic E. coli O157:H12 as surrogate for pathogenic E. coli. An environmental isolate of E. coli O157:H12 was obtained from a stream in Baltimore County, Maryland, and characterized for its suitability as a non-pathogenic surrogate for E. coli O157:H7 in field experiments with leafy greens. E. coli O157:H12 showed similar growth rates to E. coli O157:H7 in lettuce extracts. Molecular analysis by real time PCR indicates that the O157:H12 strain did not have any virulence factors that E. coli O157:H7 has, but does retain the O157 antigen (rfb) gene. Standard recovery methods for O157:H7 E. coli (mEHEC broth and MacConkey agar) were also able to recover E. coli O157:H12. These tests provide initial indications that E. coli O157:H12 can be used as a non-pathogenic surrogate in field trials for E. coli O157:H7 strain and can be identified using standard methods.
Soil biofumigation. The effect of biofumigation on reduction of E. coli and salmonellae in lettuce and spinach production soils was tested in a field-replicated trial for the first time in late summer 2008. Soil cassettes were artificially contaminated with nonpathogenic strains and buried at two depths prior to solarization treatments. Several commercial products and one crop residue treatment were included in the biofumigation plots. Results with single rate applications show significant promise for elimination of E. coli O157:H7 and salmonellae from fecal contamination in soil using field biofumigation. The experiment will be repeated in 2009 season to obtain a second year of data.
Insect transmission of pathogens. The growth and survival of E. coli and salmonellae in two major crop pests, hornworm larvae (Manduca sexta) and Colorado potato beetle (Leptinotarsa decemlineata) larvae, were evaluated in replicated laboratory studies on tomato leaves. Insects were reared in the laboratory and 3rd instar stages were selected for assays. Results indicate that within 24 hr of an initial low dose (less than 1000 cells) insect frass contains more than 10 million cells of E. coli or salmonellae per gram. Additional tests with several different stages of low and high dosages are underway along with studies to determine if these organisms survive the insect molting process and their fate if the insect dies. Amplification and spread of bacteria in frass on tomato foliage initially contaminated by various sources (water and wildlife feces) are being tested.
Antimicrobial properties of essential oils against Salmonella in organic soil. Essential oils have been studied to control pathogens on produce surfaces. This is the first study on role of oils to control Salmonella in soil, one of the important sources of preharvest contamination of produce with pathogens. The efficacy of essential oils (ecotrol, eugenol, cinnamanaldehyde) and vinegar in killing Salmonella spp. in organic soil was studied. Vinegar and eugenol at 0.5 percent significantly reduced Salmonella in soil. Salmonella populations in soil were reduced by up to 5 log CFU/g after 28 days of incubation using vinegar or eugenol. The bactericidal effect of cinnamanaldehyde was not evident at the 0.5 or 2 percent level. Overall, S. Negev was the most sensitive of Salmonella serovar evaluated in the study. This study shows the potential use of oils in organic produce environments to effectively reduce Salmonella populations in soil.
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Sharma, M., Ingram, D.T., Patel, J.R., Millner, P.D., Hull, A., Donnenberg, M. 2009. A novel approach to investigate the uptake and internalization of Escherichia coli O157:H7 in spinach cultivated in soil and hydroponic media. Journal of Food Protection. 72(7):1513-1520.
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