Progress 03/01/10 to 03/01/14
Outputs
Target Audience: The scientific community Changes/Problems: The experiments were limited to two fresh produce (lettuce and spinach). The orignal intention was to target more produce but determination was made that it would be better to focus resource and time on these two produce to be more effective. What opportunities for training and professional development has the project provided? Five undergraduate students were trained and worked in this project under the supervision of a laboratory technician (Vijayalakshmi Mantripragada). How have the results been disseminated to communities of interest? Study findings were disseminated as publications and were also presented in poster and oral presentations in the Soil Science Society of America annual meetings and at the Georgia Organics Conference during the duration of the study. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objectives 1: Experiments were conducted to test the general hypothesis that the plant rhizosphere support E. coli growth and survival, but presence of E. coli in the rhizosphere is not linked to a predictable occurrence in the external phyllosphere population. A recombinant light emitting strain was used to visualize occurrence in radish and lettuce. Cherry Belly Radish and Romaine lettuce seeds were grown in test tubes containing agar with Hoagland’s nutrient solution. Prior to use in the agar systems, the seeds were inoculated with E. coli O157:H7 lux by soaking in 7 log CFU/mL buffered cell suspension. Bacteria inoculated on the seeds were enumerated by vortexing the contaminated seeds in buffer followed by serial dilution and plating onto LB media with the appropriate antibiotic supplement. Uninoculated seeds (control) were treated same way but soaked in sterile buffer. The distribution and metabolic activity of E. coli cells were monitored with a photon counting camera. Images of the treatment and control plants were obtained in a light tight chamber at 3, 5 and 14 days post seeding. E. coli cells were enumerated from whole plants by homogenization in a stomacher bag followed by serial dilution and spread plating. Sequential imaging of seedlings grown from the contaminated seeds showed bioluminescence from their surfaces up to 14 days post seeding. This indicates the presence of metabolically active E. coli in the plant systems. Light was first observed from the roots of the radish seedlings three days post seeding. The light intensity increased between d 3 and d 5. This is indicative of increased E. coli activity with time on the plant surface. The recovery profile for E. coli removed from radish and lettuce indicated that E. coli increased in number during germination. There was 1.62 and 0.82 logs increase of E. coli population after 7 days post seeding for radish and lettuce, respectively. The rhizosphere luminescence from E. coli confirms the bacteria are metabolizing carbon, and that E. coli is capable of using plant derived carbon and reproducing in the rhizosphere. These findings also show that the bacteria will grow from very low levels of contamination as the carbon materials released at leaf and radical emergence will support metabolism. Overall, we find that E. coli is mobile in the plant system and that the bacteria are responding to the plant rhizosphere like other soil bacteria. Objective 2: Experiments were designed to study factors that might explain the persistence of E. coli and Salmonella in soils and water. Experiments were also designed to evaluate the transferability of these pathogens to lettuce when they were introduced to soil via water or manure. Soils were collected from farms under contrasting farming practices in GA, TX and CA. The soils were initially profiled for differences in microbial diversity and function. The soils were adjusted to two moisture levels (20 and 40% of water holding capacity, WHC) and inoculated with the two pathogens at a concentration of about 5 log CFU per g of soil. The presence of the pathogens in the soils was tested by enrichment after their concentration fell below the detection limit of spread-plating. On weeks zero, three and six, the soils were additionally tested for changes in microbial diversity and function. Studies were also carried out to investigate the role of metal pollutants on the interaction between E. coli and protozoa in water. This study tested the hypothesis that pollutants are adversely affecting protozoans more than E. coli, leading to decreased grazing of E. coli by protozoans. Bioluminescent E. coli O157:H7 and Tetrahymena pyriformis (T. pyriformis) were used as model organisms. The toxicity of four metals (Zn, Cu, Ni and Cr) towards E. coli, T. pyriformis and the interaction between the two were assayed. To evaluate the transferability of E. coli to lettuce under different scenarios, cells were combined with manure and added directly to soil, or cells were combined with manure and irrigation water and then added to soil. In all cases, cells were added to achieve 7 log CFU/g soil. Lettuce was grown in a 12 cm diameter pot containing the treated soil. The control soil did not initially receive any E. coli. The bulk soil, rhizosphere and phyllosphere were sampled at predetermined intervals to represent the early (d 15), middle (d 27, d 32) and late (d 41, d 50) growth stages of the plants. The pathogens showed different survival times in the three soil types at the two moisture levels. E. coli cells were detected in TX and CA soils until week 9 without enrichment. In GA soil, they cannot be detected without enrichment after the first week of incubation. Salmonella were detected in TX and CA soils until week 6 without enrichment but in GA soil they were not detected without enrichment after the first week. High soil moisture (40% WHC) was detrimental to the persistence of the pathogens. The pathogens were detected with enrichment until week 18 but mainly in the soils at 20% WHC. There were significant and negative correlations between E. coli persistence and the abundance of oligotrophic soil bacteria (PCC - Pearson Correlation Coefficient = -0.491), biomass (PCC = -0.503), urease activity (PCC = -0.463), electrical conductivity (PCC = -0.489). There were significant and negative correlations between Salmonella persistence and the concentration of oligotrophic soil bacteria (PCC = -0.564), biomass (PCC = -0.471), urease activity (PCC = -0.453), electrical conductivity (PCC = -0.442). The study suggests that soils with diverse and active microbial community would be more antagonistic towards externally introduced human pathogens. For the E. coli-T. pyriformis interaction study, it was shown that the toxicity effect was more sever on T. pyriformis than E. coli. EC50 values of Zn for E. coli and T. pyriformis were 39.9 and 37.4 ppm, respectively. The results from the interaction studies indicated that the ingestion of E. coli by T. pyriformis was largely limited due to the toxicity of the metals. Overall, our results suggest that contaminants in the environment might decrease the predation stress on E. coli, which might prove to be beneficial for a long term survival of E. coli in the environment. Introducing E. coli to soil via manure or a combination of manure and irrigation water resulted in high numbers of E. coli in the bulk soil and rhizosphere of lettuce where they persisted for several weeks. Post-harvest, the numbers of E. coli in the rhizosphere in soil receiving the bacteria via manure or via manure and irrigation water combined were 5.20 log and 5.08 log CFU/g, respectively while the numbers were 5.52 and 5.74 log for the bulk soil samples. As compared to the initial inoculum size (7.22 logs), the decrease in E. coli number in the soil on d 41 was less than 2 logs for both treatments. The method of application of the bacteria to soil did not significantly affect (p = 0.709) the bacterial number in either location (p = 0.154). E. coli was also detected on the phyllosphere of lettuce following its introduction to soil via either manure or manure and irrigation water combined. The average bacterial number on the lettuce phyllosphere samples for both modes of introduction was 2.5 log CFU/g on d 15 and were not statistically different. While E. coli persisted in the bulk soil and rhizosphere throughout the study, they were not detected on phyllosphere samples on or after day 27. Overall, these data suggest that E. coli can persist in the rhizosphere and that introduction level will alter the ultimate rate of decline.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Erickson, M.C., Habteselassie, M.Y., Liao, J., Mantri, V., Webb, C.C., Davey, L. and Doyle, M. 2014. Identification of factors for use as predictors of human enteric pathogen survival in soil. Journal of Applied Microbiology 116:335-349.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Cannon, J.L., Erickson, M.C. and Habteselassie, MY. 2012. The likelihood of cross?contamination of head lettuce by E. coli O157:H7, Salmonella and norovirus during hand harvest and recommendations for glove sanitizing and use. Western Food Safety Summit, Hartnell College, Salinas, CA. May, 2012.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Smith, C. Ma, C., Roache, K., Mantri, V., Habteselassie, M., Gaskin, J., Harrison, J. and Cannon, J. 2012. Are organisms that can cause foodborne illnesses suppressed in organically managed soils. In Georgia Organics Conference. Feb. 24-25, 2012, Columbus, GA. 2.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2010
Citation:
Habteselassie, M. and Turco, R. 2010. Influence of inorganic contaminants on E. coli and its predator: Implications to the long-term survival of E. coli in water. In Annual Meetings Abstracts. ASA, CSSA, and SSSA, Madison, WI.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Habteselassie, M.Y., Bischoff, M., Applegate, B., Reuhs, B. and Turco, R. 2010. Understanding the role of agricultural practices in potential colonization and contamination by E. coli in rhizospheres of fresh produce. Journal of Food Protection 73:2001-2009.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: In 2012, further experiments were done to evaluate the persistence of E. coli and Salmonella spp in different soil types. Soils of varying properties were collected from California (CA), Texas (TX) and Georgia (GA) for the study. The soils were initially profiled for differences in microbial diversity and function by using the ribosomal intergenic spacer analysis (RISA) and enzyme assays, respectively. The enzyme assays were for urease, phosphatase and glucosidase. The soil microbial biomass and the abundance of the copiotrophic and oligotrophic bacterial communities were also determined, in addition to some basic soil properties (e.g., pH, electrical conductivity, texture, organic matter). The soils were adjusted to two moisture levels (20 and 40% of water holding capacity, WHC) and inoculated with the two pathogens at a concentration of about 5 log CFU per g of soil. The soils were incubated for up to 18 weeks and sampled regularly to test for the inoculated pathogens. The presence of the pathogens in the soils was tested by enrichment after their concentration fell below the detection limit of spread-plating. On weeks zero, three and six, the soils were additionally tested for changes in microbial diversity and function, and the correlation of these soil properties with the persistence of the pathogens was evaluated. Statistical analyses were done to determine the significance of the differences in pathogen survival among the soils. Two undergraduate students were trained and worked in this project under the supervision of a laboratory technician (Vijayalakshmi Mantripragada). PARTICIPANTS: Dr. Marilyn Erickson, UGA's Center for Food Safety, UGA Griffin Campus; Vijayalakshmi Mantripragada, Crop and Soil Sciences, UGA Griffin Campus; Hao Zhang, UGA Griffin Campus; Jenny Dang, UGA Griffin Campus TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The pathogens showed different survival times in the three soil types at the two moisture levels. E. coli cells were detected in TX and CA soils until week 9 without enrichment. In GA soil, however, they cannot be detected without enrichment after the first week of incubation. Salmonella were detected in TX and CA soils until week 6 without enrichment but in GA soil they were not detected without enrichment after the first week. The data suggest that high soil moisture (40% WHC) was detrimental to the persistence of the pathogens, considering same soil type. The pathogens were detected with enrichment until week 18 but mainly in the soils at 20% WHC. There were significant and negative correlations between E. coli persistence and the abundance of oligotrophic soil bacteria (PCC - Pearson Correlation Coefficient = -0.491, P < 0.0001), biomass (PCC = -0.503; P = 0.0075), urease activity (PCC = -0.463; P = 0.013), electrical conductivity (PCC = -0.489; P = 0.0082). There were significant and negative correlations between Salmonella persistence and the concentration of oligotrophic soil bacteria (PCC = -0.564, P < 0.0001), biomass (PCC = -0.471; P = 0.013), urease activity (PCC = -0.453; P = 0.015), electrical conductivity (PCC = -0.442; P = 0.018). The study suggests that soils with diverse and active microbial community would be more antagonistic towards externally introduced human pathogens. The data is being further analyzed for publications and dissemination.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: More experiments were done in the laboratory to evaluate the survival of bacterial pathogens (Salmonella spp) and norovirus in soils that were managed under contrasting farming practices. Previous studies mainly focused on the study of E. coli survival and transmission in agar system that is convenient for tracking studies without the need for destructive sampling. For this round of studies we used soils that were managed organically and conventional for more than four years. These soils were inoculated with several strains of tagged Salmonella spp. and norovirus and incubated for several weeks while their survival was monitored several time periods. A number of soil parameters were concurrently measured to see if the functional and genetic diversity of the soil microbial community is related to the survival of the introduced Salmonella spp. and norovirus strains. Studies were also carried out to investigate the role of metal pollutants on the interaction between E. coli and protozoa. This study tested the hypothesis that pollutants are adversely affecting protozoans more than E. coli, leading to decreased grazing of E. coli by protozoans. This decrease in protozoan grazing of E. coli might explain why we are seeing E. coli strains survive in the environment for an extended period of time even though the assumption has been that they die off quickly once they are introduced into the environment. Escherichia coli (E. coli) O157:H7, which is marked with the lux gene cassette and Tetrahymena pyriformis (T. pyriformis) were used as model organisms. The toxicity of four metals (Zinc, Zn; Copper, Cu; Nickel, Ni and Chromium, Cr) towards E. coli, T. pyriformis and the interaction between the two were assayed in a 96 well microtiter plates. Three undergraduate students were initially trained and worked in these projects under the supervision of a laboratory technician (Vijayalakshmi Mantripragada). Some of the study findings were also presented in poster and oral presentations in the Soil Science Society of American annual meetings and at the Georgia Organics Conference in 2010 and 2011. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The survival study of Salmonella and norovirus revealed that both of these microorganisms decline rapidly in organic and conventional soils. Use of synthetic fertilizer resulted in the largest decline. The better survival of the Salmonella and norovirus strains in a sterilized soils indicated that the indigenous microbial community had an adverse effect on the introduced microorganisms. For the E. coli-T. pyriformis interaction study, it was shown that the toxicity effect was more sever on T. pyriformis than E. coli. EC50 values of Zn for E. coli and T. pyriformis were, for example, 39.9 and 37.4 ppm, respectively. This indicates that one needs a smaller doze of Zn for T. pyriformis than E. coli to see a similar level of toxicity. The results from the interaction studies based on bioluminescence signal from the E. coli strain and use of two differentially lysing enzymes, indicated that the ingestion of E. coli by T. pyriformis was largely limited due to the toxicity of the metals. Overall, our results suggest that contaminants in the environment might decrease the predation stress on E. coli, which might prove to be beneficial for a long term survival of E. coli in the environment.
Publications
- 1. C. Smith, C. Ma, K. Roache, V. Mantri, M. Habteselassie, J. Gaskin, J. Harrison, and J. Cannon. Are organisms that can cause foodborne illnesses suppressed in organically managed soils Georgia Organics Conference. Feb. 24-25, 2012, Columbus, GA. 2. Habteselassie, M. and Turco, R. Influence of inorganic contaminants on E. coli and its predator: Implications to the long-term survival of E. coli in water. In Annual Meetings Abstracts. ASA, CSSA, and SSSA, Madison, WI. 3. Habteselassie, M.Y., Bischoff, M., Applegate, B., Reuhs, B. and Turco, R. 2010. Understanding the role of agricultural practices in potential colonization and contamination by E. coli in rhizospheres of fresh produce. Journal of Food Protection 73:2001-2009.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Experiments were conducted to test the general hypothesis that the plant rhizosphere support E. coli growth and survival, but presence of E. coli in the rhizosphere is not linked to a predictable occurrence in the external phyllosphere population. A recombinant light emitting strain (E. coli O157:H7 lux) was used to visualize occurrence in radish and lettuce. Cherry Belly Radish and Romaine lettuce seeds were grown in test tubes containing agar (20 mL) with 1X Hoagland's nutrient solution. Seeds were incubated in the dark until germination and followed by incubation in a growth chamber with a 16/8 hrs photoperiod at a temperature of 23.5oC. Relative humidity was maintained between 41 to 50%. Prior to use in the agar systems the seeds were inoculated with E. coli O157:H7 lux by soaking approximately 3 to 5 g of seeds in 7 log CFU/mL 20 mL 50 mM K-phosphate buffered cell suspension for 20 minutes. They were air dried for 2 hours on sterile filter paper under room temperature. Bacteria inoculated on the seeds were enumerated by vortexing the contaminated seeds in 10 mL K-phosphate buffer for 1 minute followed by serial dilution and plating onto LB media with the appropriate antibiotic supplement. Uninoculated seeds (control) were soaked in sterile 50 mM K-phosphate buffer for 20 minutes, air dried as described and enumerated as above. The distribution and metabolic activity of E. coli O157:H7 lux cells were monitored with a photon counting camera. Images of the treatment and control plants were obtained in a light tight chamber at 3, 5 and 14 days post seeding. E. coli O157:H7 lux were enumerated from whole plants by homogenization in a stomacher bag with a stomacher for 2 minutes at 260 rpm followed by serial dilution and spread plating as previously described. Luminescence of the colonies was used as confirmation of the presence of the inoculated E. coli O157:H7 lux cells. Enumeration after homogenization with stomacher represents mainly the surface bound cells. To test the stability of the plasmid based lux maker, an isolated colony of E. coli O157:H7 lux was inoculated into 50 mL LB broth with kanamycin (100 ug/mL) and incubated at 37oC. After 24 hrs of incubation, 1 mL of the culture was inoculated into two 250 mL flasks containing 50 mL LB liquid media with or without kanamycin and incubated at 37oC for 24 hrs. One mL aliquots were sequentially transferred from each flask to fresh LB media every day for 10 successive days and incubated at 37oC for 24 hrs. To determine the rate of segregative loss of pFSP102 in the absence of selective pressure, 1 mL samples were serial diluted and plated on LB agar with kanamycin at the time of transfer. PARTICIPANTS: This project has significantly benefited from the collaboration with two researchers at Purdue University, West Lafayette, IN. These collaborators are Dr. Ron F. Turco (Laboratory for Soil Microbiology, College of Agriculture Lilly Hall of Life Sciences, 915 W. State Street, Purdue University, West Lafayette, IN. 47907) and Dr. Bruce Applegate (Department of Food Science, 754 Agriculture Mall Dr., West Lafayette, IN 47907). TARGET AUDIENCES: Information from this project will be made available to producers and state and federal regulatory agents as well as other interested individuals. Results will be presented at professional meetings (e.g., American Society of Microbiology) and published in peer reviewed journals. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The main focus of this study was to look at the role of rhizosphere in the early colonization and establishment of E. coli on the plant system. The lux-based system was used for in situ tracking of the distribution and activity of bacteria on rhizosphere and its transfer to the phyllosphere during the early stage of growth. Sequential imaging of both radish and lettuce seedlings grown from seeds inoculated with E. coli O157:H7 lux showed bioluminescence from their surfaces up to 14 days post seeding. This indicates the presence of metabolically active E. coli O157:H7 lux in the plant systems. Light was first observed from the roots of the radish seedlings three days post seeding. The light intensity increased between d 3 and d 5. This is indicative of increased E. coli O157:H7 lux activity with time on the plant surface. Plants were not imaged after day 14 because their growth was restricted due to the size of the test tubes in which they were grown. The recovery profile for E. coli O157:H7 lux removed from radish and lettuce indicated that E. coli O157:H7 lux increased in number during germination. The numbers of E. coli O157:H7 lux introduced on the seeds before germination was 6.56 and 6.49 log CFU/g for radish and lettuce, respectively. The numbers were 8.18 and 7.31 log CFU/g 7 days after seeding for radish and lettuce, respectively. This is 1.62 and .082 logs increase of E. coli O157:H7 population after 7 days post seeding for radish and lettuce, respectively. The agar growth system is a direct means to track the early stage growth and distribution of E. coli O157:H7 lux on plants such as radish and lettuce. Moreover, the agar growth system is similar to commercial sprouting systems and the findings from this study show that bacterial growth, as indicated via light detection and enumeration, in these nutrient rich rhizosphere environments is possible. The E. coli O157:H7 lux response clearly demonstrates not simply survival but metabolic activity in the plant rhizosphere. This approach is useful for in situ tracking of distribution and activity of bacteria such as E. coli on the plant roots, avoids the need for destructive sampling and allows us to visualize the active population and where they are located. The rhizosphere luminescence from the E. coli confirms the bacteria are metabolizing carbon and this confirms the E. coli is capable of using plant derived carbon and more importantly, reproducing in the plant rhizosphere. High levels of bacterial luminescence associated with the root demonstrate the potential of the rhizosphere to support metabolism of E. coli O157:H7 during seed germination and early stage plant growth and raises questions about the contribution of the rhizosphere to long-term stability of E. coil in soil. These findings also show that the bacteria will grow from very low levels of contamination as the carbon materials released at leaf and radical emergence will support metabolism. Overall, we find that E. coli is mobile in the plant system and that the bacterium is responding to the plant rhizosphere like other soil bacteria.
Publications
- No publications reported this period
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