Source: UNIVERSITY OF NEBRASKA submitted to NRP
ACCUMULATION OF ANTIBIOTIC RESISTANT SALMONELLA IN LETTUCE AFTER IRRIGATION USING RECYCLED WATER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0223716
Grant No.
2011-67019-20052
Cumulative Award Amt.
$500,000.00
Proposal No.
2010-03583
Multistate No.
(N/A)
Project Start Date
Jan 1, 2011
Project End Date
Dec 31, 2015
Grant Year
2011
Program Code
[A1411]- Foundational Program: Agricultural Water Science
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
(N/A)
Non Technical Summary
Water scarcity occurs to many parts of the US and the rest of the world. As a sector, agriculture consumes ~40% of the total water supplies. Given the increasing scarce water sources, alternative water sources are needed for agriculture irrigation. One of such sources is treated municipal and agriculture wastewaters, as they are abundant and inexpensive. Over the past few decades, successful examples in the US and other countries demonstrate that using recycled water for irrigation is a win-win situation. The use of recycled water saves fresh water that can be used for other purposes. In the meantime, using recycled water for irrigation reduces discharge of wastewater effluents into receiving waters. However, the use of recycled water for irrigation also raises food safety concerns. Even after proper treatment, recycled waters still contain trace level chemical and microbial contaminants. These contaminants could enter the cropland through irrigation using recycled water, and subsequently enter food vegetables. Among the contaminants are pathogens. Once entering vegetables, pathogens are hard to remove, as common washing steps will be ineffective to them. If vegetables contaminated with internalized pathogens, particularly antibiotic resistant (ABR) pathogens, are consumed by human beings, medical attention will be needed. So far, little is known about the internal accumulation of ABR pathogens in vegetables irrigated using recycled water. Therefore, the purposes of this study are to quantify the internal accumulation of ABR pathogens in vegetables irrigated using recycled water, investigate the mechanisms controlling the internalization process, and find solutions to minimize the accumulation.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1110210116030%
7121430116070%
Knowledge Area
111 - Conservation and Efficient Use of Water; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables; 0210 - Water resources;

Field Of Science
1160 - Pathology;
Goals / Objectives
Recycled water from municipal and agricultural sources is often used as an alternative to supplement increasingly scarce fresh water sources for irrigation of food crops. Due to limitations in treatment methods, recycled water often contains trace level contaminants, such as antibiotics, metals, and pathogens. During irrigation these chemical and microbial constituents are introduced to cropland, where they can either be degraded, or persist and accumulate in soil. Among the persistent constituents are antibiotic resistant pathogens (ABR pathogens) and antibiotics. Pathogens can acquire antibiotic resistance genes (ABR genes) both in recycled water prior to irrigation and in soil after exposure to antibiotics introduced to soil during irrigation. ABR pathogens can gain rapid and widespread entry to food crops, colonize internally, and become endophytes. Endophytic pathogens, such as ABR Salmonella, cannot be eliminated during washing and pose a food safety concern of consuming vegetables that are eaten raw. Currently, little is known about the fate of ABR Salmonella in soil or their accumulation in fresh vegetables. This knowledge is critical in predicting the public health impacts of fresh vegetables irrigated using recycled water. The overall objective of the project is to understand and minimize the accumulation of ABR pathogens in vegetables irrigated using recycled water. The central hypothesis is that accumulation of ABR pathogens occurs in vegetables that are irrigated with recycled water, and the accumulation is positively correlated with the abundance of ABR pathogens in the rhizosphere. Three supporting Research Objectives have been established: 1) determine the fate of ABR Salmonella in soil after irrigation using recycled water, 2) quantify the accumulation of ABR Salmonella in lettuce, and 3) develop best management practices to minimize ABR Salmonella accumulation in lettuce. Completion of Research Objective #1 will reveal the mechanism governing the proliferation of ABR Salmonella in soil after irrigation using recycled water. Given that the majority of the endophytic bacteria originate from the rhizosphere, understanding the fate of the ABR Salmonella in soil is critical in predicting the abundance of the pathogen in vegetables. Research Objective #2 will generate quantitative information on the accumulation of ABR Salmonella in lettuce with respect to abundance as well as resistance level. The quantitative information is critical to evaluate the health risk of consuming fresh vegetables irrigated using recycled water. Research Objective #3 will yield practical solutions that can minimize the accumulation of ABR Salmonella in vegetables. Development of these solutions is critical in promoting the use of recycled water for irrigation, because addressing food safety concerns over the accumulation of pathogens is critical in gaining public acceptance of vegetables irrigated using recycled water.
Project Methods
To achieve Research Objective #1, we establish the following working hypothesis. Irrigation with recycled water introduces both Salmonella and human and/or veterinary antibiotics into soil. In the soil environment, a subpopulation of Salmonella acquires ABR genes due to the exposure to antibiotics that are biologically active and becomes antibiotic resistant. Later, the ABR Salmonella subpopulation accumulates in the rhizosphere around vegetable roots. To test the working hypothesis, we will first quantify ABR Salmonella and antibiotics in recycled water from typical municipal and agricultural sources. Surface water from the Platte River and ground water from the Ogallala aquifer will also be included in the analysis as controls. Antibiotics will be measured using LC/MS/MS, while Salmonella will be quantified using both culture-based and culture-independent methods. Then, recycled water will be applied to cropland using drip irrigation at typical irrigation rates. Subsequently, we will monitor the levels of ABR Salmonella and antibiotics in soil after repeated irrigation. For Research Objective #2, we establish the following working hypothesis. ABR Salmonella can gain entry to vegetable plants through roots, colonize the plants internally, and become endophytes. During endophytic growth, the abundance and resistance level of ABR Salmonella will increase. In the meantime, the growth of vegetables will be negatively affected by these endophytic pathogens. To test the working hypothesis, we will first monitor the abundance of Salmonella and ABR Salmonella in lettuce using a culture-based method designed for endophyte studies. Then, we will take a closer look at the resistance level of the endophytic ABR Salmonella in lettuce. Isolates grown on antibiotic-amended agar plates will be subject to MIC tests. Finally, we will correlate the abundance of endophytic Salmonella with lettuce growth and yield. To achieve Research Objective #3, the following working hypothesis is established. The accumulation of ABR Salmonella in lettuce will be positively correlated with the abundance of ABR Salmonella in the rhizosphere. Therefore, best management practices designed to minimize the abundance of the pathogen in soil can minimize the accumulation of the pathogen in vegetables. To test the working hypothesis, we will irrigate lettuce using waters with various blending ratios of recycled water to clean water. Efforts will be made to correlate the blending ratios with quantitative information on endophytic Salmonella and lettuce growth/yield. In addition, we will test irrigation schedules involving recycled water and clean water alternately. In this experiment, recycled water will be avoided at certain lettuce growth stages that are more susceptible to pathogen internalization than the others.

Progress 01/01/11 to 12/31/15

Outputs
Target Audience:The target audiences of this project include food safety professionals, agricultural and environmental engineers, as well as food crop producers. We made presentations on our findings at professional conferences, and published papers on peer reviewed journals. Research findings were also taught as examples in classroom to graduate and undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students have graduated with their Ph.D. degree based on their work on this project. Dr. J. Brett Sallach is currently a postdoctoral associate at Michigan State University, and Yuping Zhang is currently a postdoctoral associate at the Environmental Protection Agency. Both students have presented at regional and national conferences, and published papers as first author on peer reviewed journals. One undergraduate student, Christian Jewett, joined Dr. Li's laboratory as a UNL UCARE (Undergraduate Creative Activities and Research Experiences) student and worked with Yuping Zhang on the project. He has been admitted to graduate school to continue his academic career. Jason Thomas is another undergraduate student that worked in Dr. Li's laboraty as a UCARE student and as a McNaire scholar. He is currently a Ph.D. student in Plant Science at the University of Minnesota. How have the results been disseminated to communities of interest?Research findings were primarily disseminated to the research communities through papers on peer reviewed journals and presentation on scientific conferences. The journal publications and conference presentations are reported in the Product section of this final report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Three research objectives were established in the original proposal are: 1) determine the fate of ABR Salmonella in soil after irrigation using recycled water, 2) quantify the accumulation of ABR Salmonella in lettuce, and 3) develop best management practices to minimize ABR Salmonella accumulation in lettuce. To achieve Objective #1 we have conducted several experiments to assess the levels of antimicrobials and Salmonella in soil after irrigation using recycled water. All of the experiments were conducted in a greenhouse to simulate field conditions. Different irrigation scenarios were included: one time irrigation with recycled water vs. repeated irrigation with recycled water throughout the lettuce growth. In addition, soil samples from different depths were collected to assess the effects of irrigation method on the distribution of Salmonella in soil. Furthermore, the levels of antimicrobials and Salmonella in soil were quantified in various soil types (clay, sandy clay, and sandy clay loam) and under various soil moisture levels (control, mild drought, and severe drought). Finally, the impacts of enteric pathogens in irrigation water on the microbial community in the rhizosphere soil was evaluated. Under Objective #2, several experiments have been completed to assess the potential of internalization of antimicrobials and Salmonella in lettuce. The internalization experiment was first conducted under optimal growth condition and then repeated under drought condition to investigate how this environmental stressor may affect the internalization. Similar to objective #1, the level of antimicrobials and Salmonella internalized in lettuce was also tested in three soil types and under three soil moisture levels. In addition to what was proposed in the original proposal, additional experiments were conducted to assess the potential accumulation of antimicrobials in food crops other than lettuce, e.g., tomato, onion, etc. Also, the global modulation of bacteria and plant protein expression after Salmonella internalized lettuce was investigated. Finally, we characterized the microbial communities in the root of two lettuce cultivars grown in different soils and harvested at different stages after repeated irrigation using recycled wastewater. Under Objective #3, we tested whether the internalization frequency and the internalization concentration of antimicrobials and Salmonella were affected by the age of the lettuce plants. In other words, is lettuce at certain age more susceptible to the internalization of these contaminants than lettuce at other ages? Additionally, we tested whether irrigation with clean water following irrigation with recyclec wastewater would reduce the antibiotic and pathogen concentrations in plants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, Y., R. Nandakumar, S. Bartelt-Hunt, D. Snow, L. Hodges, and X. Li. 2014. Quantitative proteomic analysis of the Salmonella-lettuce interaction. Microbial Biotechnology, 7(6): 630-637.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sallach, J.B.#, Zhang, Y.#, D. Snow, L. Hodges, X. Li, and S. Bartelt-Hunt. 2014. Concomitant uptake of antimicrobials and Salmonella in soil and into lettuce following wastewater irrigation. Environmental Pollution, 197: 269-277. (# equal contribution)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, Y.#, Sallach, J.B.#, L. Hodges, D.D. Snow, S.L. Bartelt-Hunt, K.M. Eskridge, and X. Li. 2016. Effects of Soil texture and drought stress on antibiotic and salmonella uptake by lettuce following wastewater irrigation. Environmental Pollution, 208: 523-531. (# equal contribution)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sallach, J.B., D.D. Snow, L. Hodges, X. Li, and S.L. Bartelt-Hunt. 2015. Development and comparison of four methods for the extraction of antibiotics from a vegetative matrix. Environmental Toxicology and Chemistry, 35: 889-897.
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Zhang, Y., C. Jewett, J. Gilley, S. Bartelt-Hunt, D. Snow., L. Hodges, and X. Li. Microbial community in the rhizosphere and the root of lettuce as affected by Salmonella-contaminated irrigation water.
  • Type: Journal Articles Status: Other Year Published: 2016 Citation: Sallach, J.B., D.D. Snow, L. Hodges, X. Li, and S.L. Bartelt-Hunt. Modeling uptake of antibiotics into root, shoot, and fruit compartments of edible vegetables. In preparation.
  • Type: Journal Articles Status: Other Year Published: 2016 Citation: Sallach, J.B., D.D. Snow, L. Hodges, X. Li, and S.L. Bartelt-Hunt. The influence of soil texture on the uptake of antibiotics by lettuce from irrigation water. In preparation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sallach, J.B., D.D. Snow, L. Hodges, X. Li, and S.L. Bartelt-Hunt. 2015. Influence of soil texture on uptake of antibiotics in wastewater irrigated lettuce. 250th ACS National Meeting, Boston, MA, August 16-20.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sallach, J.B., S.L. Bartelt-Hunt, D.D. Snow, X. Li, and L. Hodges. 2015. The influence of soil texture on the uptake of antibiotics by lettuce from irrigation water. AEESP Conference, New Haven, CT, June 13-16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zhang, Y., X. Li, S.L. Bartelt-Hunt, D.D. Snow, and L. Hodges. 2015. Effects of soil texture on the fate and transport of Salmonella in the soil-plant ecosystem following wastewater irrigation. AEESP Conference, New Haven, CT, Jun 13-16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sallach, J.B., S.L. Bartelt-Hunt, D.D. Snow, X. Li, and L. Hodges. 2014. Accumulation of Antimicrobials in Soil and Subsequent Uptake into Lettuce Following Wastewater Irrigation. Antibiotics in Agriculture: State of the Science, Oracle, AZ, August 5-8.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zhang, Y., R. Nandakumar, S.L. Bartelt-Hunt, D.D. Snow, L. Hodges, and X. Li. 2014. Quantitative proteomic analysis of the Salmonella-lettuce interaction. 114th American Society for Microbiology General Meeting, Boston, MA, May 17-20.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhang, Y., J.B. Sallach, X. Li, S.L. Bartelt-Hunt, D.D. Snow, and L. Hodges. 2013. Internalization of chemical and microbial contaminants in lettuce through wastewater irrigation. 68th Soil and Water Conservation Society Annual Conference, Reno, NV, July 21-24.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sallach, J.B. S.L. Bartelt-Hunt, D.D. Snow, X. Li, L. Hodges, and Y. Zhang. 2013. Potential for antibiotic uptake by lettuce from irrigation with recycled water. AEESP 50th Anniversary Conference, Golden, CO, Jul 14-16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhang, Y., S.L. Bartelt-Hunt, D.D. Snow, L. Hodges, and X. Li. 2013. Internalization of Salmonella into lettuce leaves through irrigation water. AEESP 50th Anniversary Conference, Golden, CO, July 14-16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhang, Y., S.L. Bartelt-Hunt, D.D. Snow, L. Hodges, and X. Li. 2013. Internalization of Salmonella into lettuce leaves through irrigation water. 5th Quadrennial IWA Specialty Conference on Microbial Ecology and Water Engineering, Ann Arbor, MI, July 7-10.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Sallach, J.B., S.L. Bartelt-Hunt, D.D. Snow, X. Li, and L. Hodges. 2012. Evaluation of four extraction methods on characterization of antibiotic uptake by lettuce. 33rd Annual SETAC North American Meeting, Long Beach, CA, November 11-15.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhang, Y. and X. Li. 2013. Internalization of Salmonella into lettuce following irrigation using recycled wastewater. Nebraska Water Environment Association Fall Conference, Kearney, NE, November 6-8.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Zhang, Y, J.B. Sallach, X. Li, D.D. Snow, L. Hodges, and S.L Bartelt-Hunt. 2012. Accumulation of Salmonella enterica serovar infantis in soil and lettuce after irrigation using treated wastewater. Land Grant and Sea Grant National Water Conference, Portland, OR, May 20-24.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Zhang, Y, J.B. Sallach, X. Li, D.D. Snow, L. Hodges, and S.L. Bartelt-Hunt. 2012. Accumulation of Salmonella enterica serovar infantis in soil and lettuce after irrigation using treated wastewater. 112th American Society for Microbiology General Meeting, San Francisco, CA, June 16-19.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Zhang, Y., J.B. Sallach, L. Hodges, D.D. Snow, S.L. Bartelt-Hunt, and X. Li. 2011. Accumulation of antibiotic resistant Salmonella in lettuce after irrigation using recycled water. 3rd Water for Food Conference, Lincoln, NE, May 1-4.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Sallach, J.B., Y. Zhang, L. Hodges, D.D. Snow, X. Li, and S.L. Bartelt-Hunt. 2011. Potential antibiotic uptake by soil and crops from irrigation with recycled water. 3rd Water for Food Conference, Lincoln, NE, May 1-4.
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: J. Brett Sallach. Fate and transport of antibiotics in the agroecosystem: Uptake of antibiotics by plants. December 2015, Department of Civil Engineering, University of Nebraska-Lincoln.
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Yuping Zhang. Internalizatin of Salmonella in lettuce leaves after irrigation using recycled wastewater. August 2015, Department of Civil Engineering, University of Nebraska-Lincoln.


Progress 01/01/14 to 12/31/14

Outputs
Target Audience: The target audiences of this project include food safety professionals, agricultural and environmental engineers, as well as food crop producers. We made presentations on our findings at professional conferences, and published papers on peer reviewed journals. Research findings were also taught as examples in classroom to graduate and undergraduate students. Changes/Problems: The project has been approved for a 1-year no cost extension. The new end time will be December 31st 2015. What opportunities for training and professional development has the project provided? All PDs (Drs. Li, Bartelt-Hunt, Snow, and Hodges) meet bi-weekly with the two Ph.D. students, Ms. Yuping Zhang and Mr. J. Brett Sallach. Dr. Xu Li and Yuping Zhang meet every week to discuss research progress on the microbiological side of the project. Drs. Shannon Bartelt-Hunt and Daniel Snow meet with J. Brett Sallach weekly to discuss the research progress on the chemical side of the project. Both Zhang and Sallach have defended their Ph.D. proposals and are on track to graduate in summer 2015. Both Ph.D. students have made oral and poster presentations at national conferences. A greenhouse technician Molly Phemister has been working on the project since Summer 2013. In 2014, one undergraduate student, Christian Jewett, worked in Dr. Xu Li's laboratory as a UNL UCARE (Undergraduate Creative Activities and Research Experiences) student. How have the results been disseminated to communities of interest? Research findings were primarily disseminated to the research communities through papers on peer reviewed journals and presentation on scientific conferences. The journal publications and conference presentations are reported in the Product section of this progress report. What do you plan to do during the next reporting period to accomplish the goals? The project team plans to finish analyzing all of the data collected so far and prepare manuscripts to report the data. Additionally, a carefully designed greenhouse experiment will be conducted to collect various data to parameterize the uptake of antibiotics in plants, the ultimate goal being the development of a model for prediction of antibiotic uptake. A lecture module is being developed to incorporate knowledge obtained as part of this project for a civil engineering graduate course.

Impacts
What was accomplished under these goals? With increasingly scarce fresh water sources, recycled wastewater becomes an important source for agricultural irrigation. In this project, we assessed the potential health risks of using recycled wastewater in irrigating food crops by measuring the accumulation of chemical and microbial contaminants in soil and crop. The results from the project will help develop best management practices to minimize such health risks. Three research objectives were established in the original proposal are: 1) determine the fate of ABR Salmonella in soil after irrigation using recycled water, 2) quantify the accumulation of ABR Salmonella in lettuce, and 3) develop best management practices to minimize ABR Salmonella accumulation in lettuce. To achieve Objective #1 we have conducted several experiments to assess the levels of antimicrobials and Salmonella in soil after irrigation using recycled water. All of the experiments were conducted in a greenhouse to simulate field conditions. Different irrigation scenarios were included: one time irrigation with recycled water vs. repeated irrigation with recycled water throughout the lettuce growth. In addition, soil samples from different depths were collected to assess the effects of irrigation method on the distribution of Salmonella in soil. Finally, the levels of antimicrobials and Salmonella in soil were quantified in various soil types (clay, sandy clay, and sandy clay loam) and under various soil moisture levels (control, mild drought, and severe drought). Under Objective #2, several experiments have been completed to assess the potential of internalization of antimicrobials and Salmonella in lettuce. The internalization experiment was first conducted under optimal growth condition and then repeated under drought condition to investigate how this environmental stressor may affect the internalization. Similar to objective #1, the level of antimicrobials and Salmonella internalized in lettuce was also tested in three soil types and under three soil moisture levels. In addition to what was proposed in the original proposal, additional experiments were conducted to assess the potential accumulation of antimicrobials in food crops other than lettuce, e.g., tomato, onion, etc. Also, the global modulation of bacteria and plant protein expression after Salmonella internalized lettuce was investigated. Finally, we characterized the microbial communities in the rhizosphere soil and in the root of two lettuce cultivars grown in different soils and harvested at different stages. Under Objective #3, we tested whether the internalization frequency and the internalization concentration of antimicrobials and Salmonella were affected by the age of the lettuce plants. In other words, is lettuce at certain age more susceptible to the internalization of these contaminants than lettuce at other ages? Additionally, we tested whether irrigation with clean water following antibiotic exposure and subsequent uptake would reduce internalized concentrations.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, Y., R. Nandakumar, S. Bartelt-Hunt, D. Snow, L. Hodges, and X. Li. 2014. Quantitative proteomic analysis of the Salmonella-lettuce interaction. Microbial Biotechnology, 7(6): 630-637.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sallach, J.B.#, Zhang, Y.#, D. Snow, L. Hodges, X. Li, and S. Bartelt-Hunt. 2014. Concomitant uptake of antimicrobials and Salmonella in soil and into lettuce following wastewater irrigation. Environmental Pollution, 197: 269-277. (# equal contribution)
  • Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Sallach, J., D. Snow, L. Hodges, X. Li, and S. Bartelt-Hunt. Development and comparison of four methods for the extraction of antibiotics from vegetative plant matrices.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Sallach, J.B. et al. Influence of soil type on the transport and uptake of antibiotics by lettuce in a soil production system. In preparation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sallach, J.B., Bartelt-Hunt, S.L., Snow, D.D., Li, X., Hodges, L. (2014). Accumulation of Antimicrobials in Soil and Subsequent Uptake into Lettuce Following Wastewater Irrigation. Antibiotics in Agriculture: State of the Science, Oracle, AZ, August 5-8, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zhang, Y., R. Nandakumar, S. Bartelt-Hunt, D. Snow, L. Hodges, and X. Li. 2014. Quantitative proteomic analysis of the Salmonella-lettuce interaction. 114th American Society for Microbiology General Meeting, Boston, MA, May 17-20.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Zhang, Y., J.B. Sallach, D. Snow, L. Hodges, S. Bartelt-Hunt, and X. Li. Effects of soil type and soil moisture content on antibiotics and Salmonella uptake by lettuce following wastewater irrigation. In preparation.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Zhang, Y. et al. Soil type determines the structure of the microbial community in soil and in lettuce root following wastewater irrigation. In preparation.


Progress 01/01/13 to 12/31/13

Outputs
Target Audience: The target audiences of this project include researchers that study food safety and producers that grow food crops. We have made presentations on our findings so far at professional conferences, such as the International Water Association Specialty Conference on Microbial Ecology and Water Engineering, Association of Environmental Engineering and Science Professors Annual Conference, Soil and Water Conservation Society Annual Conference, and Nebraska Water Environment Association Annual Conference. We will continue publishing our findings on scientific journals so that they become available to broader audiences in the scientific community. Changes/Problems: The project was initially scheduled to start in January 2011 and end in December 2014. Due to the need to renovate the greenhouse at UNL to meet the Biosafety Level 2 (BSL-2) containment requirements for human pathogen work, all the greenhouse experiments were not started until May 2012. Although we conducted preliminary experiments in the laboratory while waiting for the greenhouse renovation, this inevitably delayed our progress to complete all the research tasks before the initial deadline. Therefore, the project team would like to ask for a 1-year no-cost extension for the project from 1/1/2015 to 12/31/2015. Thank you for your consideration. What opportunities for training and professional development has the project provided? All PDs (Drs. Li, Bartelt-Hunt, Snow, and Hodges) meet bi-weekly with the two Ph.D. students, Ms. Yuping Zhang and Mr. J. Brett Sallach. Dr. Xu Li and Yuping Zhang meet every week to discuss research progress on the microbiological side of the project. Drs. Shannon Bartelt-Hunt and Daniel Snow meet with J. Brett Sallach weekly to discuss the research progress on the chemical side of the project. A new greenhouse technician Molly Phemister was hired to work on the project starting Summer 2013. Two undergraduate students, Jason Thomas and Ziming Feng, worked in Dr. Xu Li’s laboratory. Both undergraduate researchers are financially supported by the UNL UCARE (Undergraduate Creative Activities and Research Experiences) program. Jason Thomas is also a McNair Scholar. The UNL McNair Scholars program recruits 10-15 students campus-wide every year, and aims to increase numbers of underrepresented students in doctoral programs. One undergraduate student Crystal Ratliff worked on the project in Summer and Fall 2013. Ms. Crystal Ratliff was supported by the UNL REU program in summer 2013 and by the project in fall 2013. Mr. J. Brett Sallach had his Ph.D. dissertation proposal defense in summer 2013. His Ph.D. committee include all project PDs and Dr. Yusong Li of the UNL Department of Civil Engineering. Ms. Yuping Zhang plans to have her Ph.D. dissertation proposal defense in early Spring 2014. Both graduate students have made oral and poster presentations at national conferences. How have the results been disseminated to communities of interest? Research findings were primarily disseminated to the research communities through papers on peer reviewed journals and presentation on scientific conferences. The journal publications and conference presentations are reported in the Product section of this progress report. What do you plan to do during the next reporting period to accomplish the goals? Complex microbial communities may exist on plant surface and inside plants. The presence of plant pathogens could potentially affect the internalization and survival of human pathogens. Several studies show that the microflora on/in postharvest vegetables has antagonistic effects on human pathogens. However, little is known about how plant-associated microflora affects human pathogens on crop surface at the field production stage. Hence, we plan to investigate how plant-associated microflora affects the internalization of Salmonella into lettuce. As a non-host environment for Salmonella, plants expose the internalized human pathogens to a wide range of stresses. Salmonella cells that survived the plant environment could potentially develop resistance to antimicrobials due to the “cross-resistance” phenomenon. By cross-resistance, exposure to one environmental stress results in resistance to another environmental stress. Antimicrobial is a relevant environmental stress, because during irrigation antimicrobial residues in recycled wastewater are introduced to soil and subsequently accumulate in plants. Hence, we plan to characterize the antimicrobial resistance level of internalized Salmonella. In 2014, we anticipate completing the experiments to evaluate the uptake of antimicrobials as a function of soil type and vegetable type. We will use a mathematical model to describe antimicrobial uptake into various varieties of produce, and we plan to use this model to make predictions about antimicrobial uptake under a variety of conditions. We also plan to evaluate whether sulfamethazole degrades within the plant after uptake.

Impacts
What was accomplished under these goals? Three research objectives were established in the original proposal are: 1) determine the fate of ABR Salmonella in soil after irrigation using recycled water, 2) quantify the accumulation of ABR Salmonella in lettuce, and 3) develop best management practices to minimize ABR Salmonella accumulation in lettuce. To achieve Objective #1 we have conducted several experiments to assess the levels of antimicrobials and Salmonella in soil after irrigation using recycled water. All of the experiments were conducted in a greenhouse to simulate field conditions. Different irrigation scenarios were included: one time irrigation with recycled water vs. repeated irrigation with recycled water throughout the lettuce growth. In addition, soil samples from different depths were collected to assess the effects of irrigation method on the distribution of Salmonella in soil. Finally, the levels of antimicrobials and Salmonella in three soil types (clay, sandy clay, sandy clay loam) were measured after a one time irrigation with recycled water. Under Objective #2, several experiments have been completed to assess the potential of internalization of antimicrobials and Salmonella in lettuce. The internalization experiment was first conducted under optimal growth condition and then repeated under drought condition to investigate how this environmental stressor may affect the internalization. Similar to objective #1, the level of antimicrobials and Salmonella internalized in lettuce was also tested in three soil types (clay, sandy clay, and sandy clay loam). In addition to what was proposed in the original proposal, additional experiments were conducted to assess the potential accumulation of antimicrobials in food crops other than lettuce, e.g., tomato, onion, etc. Also, the global modulation of bacteria and plant protein expression after Salmonella internalized lettuce was investigated. Finally, we studied whether internalized antimicrobials may affect the antimicrobial resistance level of internalized Salmonella. Under Objective #3, we tested whether the internalization frequency and the internalization concentration of antimicrobials and Salmonella were affected by the age of the lettuce plants. In other words, is lettuce at certain age more susceptible to the internalization of these contaminants than lettuce at other ages?

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Zhang, Y., R. Nandakumar, S. Bartelt-Hunt, D. Snow, L. Hodges, and X. Li. 2014. Quantitative proteome analysis of the Salmonella-lettuce interaction. Microbial Biotechnology, in press.
  • Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Zhang, Y., L. Hodges, D. D. Snow, S. L. Bartelt-Hunt, K. M. Eskridge, and X. Li. Internalization of Salmonella into drought-stressed lettuce. In revision.
  • Type: Journal Articles Status: Other Year Published: 2014 Citation: J. B. Sallach, Zhang, Y., D. Snow, L. Hodges, X. Li, and S. Bartelt-Hunt. Bioaccumulation of Salmonella enterica serovar Infantis in soil and lettuce irrigated using contaminated water. In preparation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhang, Y., J.B. Sallach, X. Li, S. Bartelt-Hunt, D. Snow, and L. Hodges. 2013. Internalization of chemical and microbial contaminants in lettuce through wastewater irrigation. 68th Soil and Water Conservation Society Annual Conference, Reno, NV, July 21-24.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sallach, J.B., S. Bartelt-Hunt, D. Snow, X. Li, L. Hodges, and Y. Zhang. 2013. Potential for antibiotic uptake by lettuce from irrigation with recycled water. AEESP 50th Anniversary Conference, Golden, CO, July 14-16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhang, Y., S. Bartelt-Hunt, D. Snow, L. Hodges, and X. Li. 2013. Internalization of Salmonella into lettuce leaves through irrigation water. AEESP 50th Anniversary Conference, Golden, CO, July 14-16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhang, Y., S. Bartelt-Hunt, D. Snow, L. Hodges, and X. Li. 2013. Internalization of Salmonella into lettuce leaves through irrigation water. 5th Quadrennial IWA Specialty Conference on Microbial Ecology and Water Engineering, Ann Arbor, MI, July 7-10.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Protocol development A comparative study was performed to determine the extraction efficiency of four methods to extract antimicrobials from a lettuce matrix: freeze and thaw, maceration, ultrasonication, and microwave assisted solvent extraction. Lettuce samples spiked with known quantities of four antimicrobials (i.e., oxytetracycline, sulfamethoxazole, lincomycin, and ciprofloxacin) were used in this study. Based on initial results on detection limits and analyte recovery, ultrasonication is the preferred method yielding consistent recovery and low detection limits. We are actively working to improve the method. A protocol for proteomic analyses was tested in collaboration with the UNL Proteomics and Metabolomics Core Facilities. The purpose of developing the proteomic protocol was to elucidate the molecular mechanisms Salmonella enterica serovar Infantis (S. infantis) uses to adjust to the change of environmental conditions after the cells move from soil into lettuce. One major challenge in developing the protocol was to enrich bacterial proteins in total protein extracts, which contain both bacterial protein and plant protein. After the homogenization of lettuce leaves containing endophytic S. infantis, total protein extracts were dominated by plant protein. To adequately investigate the bacterial proteome using 2D-LC-MS/MS, we have established two strategies: (1) separate bacteria cells from plant tissues prior to protein extraction; and (2) using alternative experimental procedure to increase endophytic S. infantis concentration in lettuce leaves. We have tested the first strategy and will test the second strategy this semester. Practical knowledge for policy and decision-makers One experiment was conducted to determine the rate of internalization of S. infantis into lettuce. Field conditions were simulated in this experiment: lettuce grown in soil was repeatedly irrigated with water containing S. infantis in a greenhouse. Results showed that the percentage of lettuce infected with endophytic Salmonella was low (5%) despite repeated irrigation with water containing 1E5 or 1E7 CFU/mL Salmonella. A second experiment was conducted to determine the effect of drought on the internalization efficiency. After a one-time irrigation with water containing 1E8 CFU/mL Salmonella, 56% of the lettuce plants that had just experienced drought stress and 42% of the unstressed lettuce plants were detected positive, and the endophytic Salmonella concentrations were up to 6.1E3 and 1.1E3 CFU/g fresh weight for the two groups, respectively. Detailed knowledge obtained from these two experiments are reported in the OUTCOMES/IMPACTS section below. Facility The renovation project on a new greenhouse space to meet BSL-2 containment requirements for human pathogens was completed in May 2012. Since then, all lettuce experiments have been conducted in the new greenhouse. This is one the few facilities in the nation that is suitable for BSL-2 human pathogen work. PARTICIPANTS: The PDs on the project include Drs. Xu Li, Shannon Bartelt-Hunt, Daniel Snow, and Laurie Hodges. All PDs have met bi-weekly with the two Ph.D. students, Ms. Yuping Zhang and Mr. J. Brett Sallach. Dr. Xu Li and the Ph.D. student Yuping Zhang meet every week to discuss research progress on the microbiological side of the project. Drs. Shannon Bartelt-Hunt and Daniel Snow meet with the Ph.D. student Mr. J. Brett Sallach weekly to discuss the research progress on the chemical side of the project. Greenhouse technician Brent Schmoker left the position in August 2012, and a new greenhouse technician George Teichmeier was hired to work on the project starting November 2012. Two undergraduate students, Jason Thomas and Ziming Feng, have been working in Dr. Xu Lis laboratory. Both undergraduate researchers are financially supported by the UNL UCARE (Undergraduate Creative Activities and Research Experiences) program. Jason Thomas is also a McNair Scholar. The UNL McNair Scholars program recruits 10-15 students campus-wide every year, and aims to increase numbers of underrepresented students in doctoral programs. Dissertation committees were formed for the two Ph.D. students. Ms. Yuping Zhangs committee includes Drs. Xu Li and Shannon Bartelt-Hunt from the UNL Department of Civil Engineering, Dr. Daniel Snow from the School of Natural Resources, and Dr. Jim Alfano from the Department of Plant Pathology. Mr. J. Brett Sallachs committee include Drs. Shannon Bartelt-Hunt, Xu Li, and Yusong Li from the UNL Department of Civil Engineering, Dr. Laurie Hodges from the Department of Agronomy and Horticulture, and Dr. Daniel Snow. Both students are scheduled to take their Ph.D. comprehensive exam this year (i.e., proposal defense). In addition, both graduate students have made oral and poster presentations at national conferences. TARGET AUDIENCES: The target audiences of this project include researchers that study food safety and producers that grow food crops. We have made presentations on our findings so far at professional conferences. We will publish our findings on scientific journals so that they become available to broader audiences in the scientific community and in the profession. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Knowledge Instrument detection limits (IDLs) determine the sensitivity of the instrument and are specific towards each compound analyzed. We have established IDLs for the four antimicrobials of interest. Ideally, IDL for these chemicals should be in the range of 100-250 ρg which we have achieved for all compounds evaluated. Method Detection Limits (MDLs) are used to determine the sensitivity of the method. This is accomplished by spiking clean matrix (lettuce) with a known amount of analyte. Ten replicates are processed for each method. Four unique extraction methods are studied to determine extraction potential in the vegetative matrix. Data from the MDL study is used to determine two important characteristics of the method: the actual method detection limit and percent recoveries. A Salmonella enterica serovar Infantis strain was used in this study. Seeds of lettuce cultivar Green Star and Green Salad Bowl were germinated and grown in potting soil. Synthetic wastewaters containing low and high (1E5 vs. 1E7 CFU/mL) levels of Salmonella were used to sub-irrigate lettuce from Week 2 till harvest. Results show that six lettuce plants, 5 from the lettuce cultivar Green Salad Bowl and 1 from Green Star, out of 120 plants (5%) were detected positive for endophytic Salmonella. When irrigated with 1E5 CFU/mL, the Salmonella in bottom soil was ~1E5 CFU/g soil wet weight (ww) from all three harvests. In comparison, the Salmonella in top soil were less than 1E4 CFU/g soil ww, especially towards later harvests. In a drought experiment, 30 out of 72 non-stressed plants (42%) and 40 out of 72 drought-stressed plants (56%) were detected positive for endophytic Salmonella. Salmonella concentration based on fresh weight (CFU/g fw) between the two groups was significantly different. There was no significant different in soil Salmonella concentration between the treatment and control group (p=0.9021), suggesting concentration of Salmonella in soil did not contribute to differential internalization between the two groups. Actions Our experiments with S. infantis could potentially lead to changes in the practice of growing lettuce in the field. One of our findings is that drought could lead to higher internalization rate of S. infantis in lettuce. In an earlier experiment, we found that lettuce plants grown without fertilizer had a higher internalization rate than those grown with fertilizer. Taken together, our results suggest that lettuce plants grown under sub-optimal conditions seem more vulnerable than those grown under optimal conditions. As a result, it is recommended to maintain optimal watering and fertilizing schedules for lettuce in the field, as health plants tend to better defend the invasion of human pathogens than their unhealthy counterparts. Conditions When optimal watering and fertilizing schedules are maintained in the field, contamination of human pathogens through root uptake will be kept at a low level. As a result, the food contamination of endophytic human pathogens will be minimized. Producers and food processing professionals can focus their efforts on minimizing surface contamination of food crops by epiphytic human pathogens.

Publications

  • Zhang, Y., X. Li, S.L. Bartelt-Hunt, D.D. Snow, and L. Hodges. 2013. Accumulation of Salmonella enterica serovar Infantis in soil and lettuce sub-irrigated using contaminated water. In preparation.
  • Zhang, Y., X. Li, S.L. Bartelt-Hunt, D.D. Snow, and L. Hodges. 2013. Drought increases the rate of accumulation of Salmonella enterica serovar Infantis in lettuce. In preparation.
  • Sallach, J.B., S.L. Bartelt-Hunt, D.D. Snow, D. Cassada, X. Li and L. Hodges. 2013. Comparison of extraction methods for antimicrobials in plant tissue. In preparation.
  • Sallach, J.B., S.L. Bartelt-Hunt, D.D. Snow, X. Li, and L. Hodges. 2012. Evaluation of four extraction methods on characterization of antibiotic uptake by lettuce. 33rd Annual SETAC North American Meeting, Long Beach, CA, November 11-15.
  • Zhang, Y, J.B. Sallach, X. Li, D.D. Snow, Laurie Hodges, and S. Bartelt-Hunt. 2012. Accumulation of Salmonella enterica serovar infantis in soil and lettuce after irrigation using treated wastewater. 112th American Society for Microbiology General Meeting, San Francisco, CA, June 16-19.
  • Zhang, Y, J.B. Sallach, X. Li, D.D. Snow, Laurie Hodges, and S. Bartelt-Hunt. 2012. Accumulation of Salmonella enterica serovar infantis in soil and lettuce after irrigation using treated wastewater. Land Grant and Sea Grant National Water Conference, Portland, OR, May 20-24.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Activities (Team building) Two Ph.D. students were recruited to work on the project. Ms. Zhang has been working with Dr. Li on the microbial aspects of the project. Mr. Sallach has been working with Drs. Bartelt-Hunt and Snow on the chemical aspects of the project. A technician was hired to work with Dr. Hodges on developing and implementing procedures to grow lettuce in greenhouse. Activities (Protocol development) Four antibiotics were selected for the project: oxytetracycline, lincomycin, sulfamethoxazole, and ciprofloxacin. A multi-drug resistant Salmonella isolate was transferred from Dr. Lisa Durso's laboratory at the USDA Agroecosystem Management Research Unit located to Dr. Xu Li's environmental engineering laboratory. The isolates were subsequently serotyped and characterized. We developed analytical methods for quantification of antibiotics and antibiotic resistant Salmonella (ABR Salmonella) from soil and plant materials. To quantify antibiotics, the following parameters have been evaluated: collection methods, extraction methods, pre-treatment, and LC/MS methods. To quantify ABR Salmonella, the following parameters have been studied: selective medium, extraction methods, extraction solution, and recovery rate. In addition, an irrigation procedure was developed and optimized. Activities (Preliminary experiments) Beginning in August 2011, an initial experiment was performed to evaluate our abilities to quantify antibiotics and ABR Salmonella in soil and plant tissues. From this preliminary trial, plants were grown in order to determine the amount of material expected at each harvest (weeks 4, 6, and 10). A control group of plants was watered with synthetic wastewater only, and a treatment group was watered with synthetic wastewater containing antibiotics and ABR Salmonella. In October 2011, we initiated an experiment that included five lettuce cultivars: Arugula (A), Green Salad Bowl (B), Red Deer Tongue (D), Royal Oak Leaf (R), and Green Star (S). The objective of this experiment was to determine (1) how the concentration of Salmonella in irrigation water affects the growth of various lettuce cultivars; and (2) how the concentration of Salmonella in irrigation water affects the accumulation of Salmonella in soil and in various lettuce cultivars. In December 2011, we initiated an experiment to compare methods to extract antibiotics from plant materials. Our current extraction method relies on the freezing and thawing of plant material to lyse cell walls. In this experiment we will further test the following five methods: freeze/thaw; microwave assisted solvent extraction, sonication, freeze drying, and maceration. Products (Facility preparation) The project team has been working with the university higher administrative (Dr. David Jackson, Interim Dean and Director of the Institute of Agriculture and Natural Resources (IANR) at UNL) to extensively renovate an existing greenhouse and convert it to laboratory space suitable for human pathogen work. The project team met with the UNL facility management team and a contractor to design the new greenhouse space. The renovation project is expected to complete in March 2012. PARTICIPANTS: Individuals The senior personnel on this project include Xu Li, Daniel Snow, Laurie Hodges, and Shannon Bartelt-Hunt. Xu Li (PD) coordinated the efforts of the key personnel on the project. He organized bi-weekly project meetings and initiated frequent e-mails and face-to-face interactions among project personnel. He has also supervised a Ph.D. student, Yuping Zhang, to work on the microbial aspects of the project. Daniel Snow (co-PD) and Shannon Bartelt-Hunt (co-PD) supervised a Ph.D. student, J. Brett Sallach, to work on the chemical aspects of the project. Laurie Hodges (co-PD) supervised a professional technician, Brent Schmoker, to develop and implement procedures for growing lettuces in greenhouse. Yuping Zhang worked with Xu Li on optimizing methods to quantify antibiotic resistant Salmonella in soil and plant tissues. She tested various experimental parameters that may affect the recovery rates of Salmonella from the two environmental matrices. She also investigated how the concentration of Salmonella in irrigation water may affect the growth of five lettuce cultivars and affect the internal accumulation of Salmonella in lettuces at various growth stages. J. Brett Sallach worked with Daniel Snow and Shannon Bartelt-Hunt on developing and optimizing the extraction and quantification methods for four antibiotics in soil and plant samples. He was also involved in developing the irrigation protocol and developing randomized designs for growing lettuces in greenhouse. Brent Schmoker has been responsible of growing and watering lettuce in the greenhouse. Partner Organization The project team has been working closely with Dr. Lisa Durso of the USDA Agroecosystem Management Research Unit located on UNL campus. The mutli-drug resistant Salmonella strain was originally isolated in Dr. Durso's laboratory and then transferred to Xu Li's environmental engineering laboratory. Through Dr. Durso, the project team got in touch with a microbiological laboratory at the USDA Meat Animal Research Center (MARC) at Clay Center, NE, where the Salmonella isolate were serotyped. Collaborators and Contracts The project team has been working closely with Dr. David Jackson, Interim Dean and Director of the Institute of Agriculture and Natural Resources (IANR) at UNL, to extensively renovate an existing greenhouse and convert it to laboratory space suitable for human pathogen work. The team met with the UNL facility management team and an outside contractor to design the new greenhouse space. During this process, a UNL IBC special committee was formed to oversee the design and make sure it compliant with all biosafety requirements. Training or Professional Development Two graduate students and one professional technician have been trained by this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in Knowledge The goal of the preliminary experiment initiated in August 2011 was to develop and optimize the chemical extraction and detection protocols. In this experiment, a control group of lettuce plants were irrigated with synthetic wastewater and a treatment group of lettuce plants were irrigated with synthetic wastewater containing the four antibiotics. Plants were grown in the greenhouse and harvested at 4 and 6 weeks. From the results, three of the four analytes, oxytetracycline, sulfamethoxazole and ciprofloxacin, were detected in extracts from plant tissues. Lincomycin was the one antibiotic that was not detected in any of extracts at either the four or six week harvest period. The results of the preliminary investigation motivated the next phase of this study currently in progress, which was to optimize the methods for extraction of antibiotics from plant material. Five lettuce cultivars, Arugula (A), Green Salad Bowl (B), Red Deer Tongue (D), Royal Oak Leaf (R), and Green Star (S), were grown in an experiment started in October 2011. Each lettuce cultivar included a control group and a treatment group. On Days 29 and 39, half of the pots from the five treatment groups were irrigated using synthetic wastewater containing antibiotics as well as 105 CFU/mL (low level) ABR Salmonella, while the other half of the pots from the treatment groups were irrigated using synthetic wastewater containing antibiotic and 107 CFU/mL (high level) ABR Salmonella. The "control", "low", and "high" pots were all watered three times before harvesting. High concentration of ABR Salmonella in irrigation water affected lettuce growth. Results showed that the effects of the Salmonella concentration in irrigation water have different impacts on different lettuce cultivars. At the early growth stage of lettuce (Day 29 to Day 39), the concentration of Salmonella in irrigation water did not appear to have a significant impact on the growth of lettuce. The only exception was the cultivar R. The R plants in the pots that received irrigation wastewaters containing a high level of Salmonella died before the end of the three irrigation events. At the later growth stage of lettuce (Day 39 to Day 47), cultivars A, B and R were not affected by the Salmonella concentration in irrigation water. In contrast, cultivar D that was irrigated with synthetic wastewaters containing Salmonella grew more plant biomass than the control group. Young lettuces were more prone to internally accumulating ABR Salmonella than lettuces that are more mature. The lettuces harvested on Day 39 and Day 47 had both been watered three times before harvesting. No endophytic ABR Salmonella were detected in the lettuce harvested on Day 47. For the lettuce samples harvested on Day 39, endophytic ABR Salmonella were detected in three cultivars irrigated with high concentration of Salmonella (i.e., 107 CFU/mL). Cultivars B, D, and S appeared to contain 1.62E+04, 2.72E+05, and 3.70E+03 CFU per gram of fresh plant weight, respectively. No endophytic ABR Salmonella were detected in cultivar A.

Publications

  • No publications reported this period