Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
NIFA-BARD COLLABORATIVE: MECHANISMS OF SALMONELLA ADAPTATION TO THE LETTUCE PHYLLOSPHERE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1012174
Grant No.
2017-67017-26180
Project No.
CA-D-PLS-2378-OG
Proposal No.
2016-10371
Multistate No.
(N/A)
Program Code
A1331
Project Start Date
Feb 15, 2017
Project End Date
Feb 14, 2022
Grant Year
2017
Project Director
Melotto, M.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences
Non Technical Summary
Healthy eating of fresh fruits and vegetables is part of an integrated strategy to decrease the risk of serious diseases. Fresh produce consumption has been increasing worldwide in the last few decades and concomitantly, an expansion of the fresh market over recent years has resulted in a wide variety of fruit and fresh produce being available throughout the year. At the same time, the number of foodborne outbreaks related to consumption of fresh or minimally processed produce has been increasing. More than 9 million foodborne illnesses in the United States are estimated to be caused by major pathogens each year. A recent analysis of illnesses in 4,589 outbreaks that occurred between 1998 and 2008 revealed that approximately 51% (4.9 million illnesses) were attributed to plant commodities, whereas ~42% (4.0 million illnesses) were attributed to land animal commodities. Among the 17 commodities analyzed, leafy vegetables were associated with the majority of illnesses (2.2 million; 22%). In particular, enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica are the most common causal agents of foodborne illness associated with the consumption of fresh leafy vegetables. In the US, non-typhoidal Salmonella is the leading cause of hospitalizations and death from foodborne illnesses. Human pathogens can be introduced into the primary food production chain by different sources, such as low-quality irrigation water, use of contaminated organic fertilizers, or close proximity to livestock operations. Fresh produce can become contaminated during production, at the processing/packing stage, and/or during preparation. Pre-harvest contamination may become a post-harvest disaster resulting in the introduction of pathogens into the processing plant, establishment of biofilms on food-contact surfaces, and subsequent cross-contamination of produce lots to be distributed at national or international scales, leading to multi-national outbreaks. Thus, Good Agricultural Practice, Good Handling Practices, Good Manufacturing Practice, and Hazard Analysis of Critical Control Points remain the cornerstone of food safety management along the production chain (from farm to fork) as part of "multiple hurdle" approaches to limit produce contamination. Unlike foods of animal origin, fresh or ready-to-eat (RTE) produce, such as leafy vegetables, cannot undergo thermal processes to inactivate human pathogens. Decontamination treatments rely mainly on the use of wash water and disinfectant treatments, usually hypochlorite. Although suspensions of foodborne pathogens, such as E. coli and S. enterica are highly susceptible to commercial disinfectants at the recommended doses and exposure times, disinfectants fail to completely eradicate pathogens from fresh produce. Consequently, the lack of an efficient kill-step is a risk factor and one of the great challenges facing the fresh produce industry. There are several plausible explanations for tolerance of plant-associated bacteria to sanitation procedures, including (a) formation of biofilms on the plant surface, (b) residing on surface-protected niches and wounds, and (c) endophytic localization.It has become evident that post-harvest sanitation is not sufficient for the complete elimination of foodborne pathogens from produce. Thus, prevention of pre-harvest contamination on the farm becomes one of the most important steps in reducing human health risk and improving food safety.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011430104040%
7121430110040%
7124010104020%
Goals / Objectives
The goal of this study is to evaluate the risk associated with colonization of the plant with Salmonella and to provide the scientific basis required to reduce plant's colonization by this pathogen through characterization of the molecular and physiological mechanisms that enable Salmonella to colonize vegetable crops. The specific objectives of the project are to: 1) Examine colonization efficiency of lettuce by outbreak-associated Salmonella serovars (OASS) 2) Evaluate the natural variations in colonization of various lettuce cultivars by Salmonella.3) Elucidate the mechanism(s) of Salmonella persistence inside the leaf.
Project Methods
Methods in objective 1: we will determine the ability of Salmonella strains to adhere to and penetrate lettuce leaf tissue, as well as to survive in the leaf intercellular space. Methods in objective 2: we will evaluate two main traits relevant to produce safety, namely incidence of internalization and endophytic persistence of Salmonella strains in various lettuce cultivars. We have chosen to test these two traits because: a) measuring incidence of internalization also takes into account the combined ability of the bacteria to attach and penetrate the leaf tissue, and b) epiphytic bacteria might be more susceptible to disinfection procedures.Methods in objective 3: we will identify and characterize Salmonella mutant strains with differential ability to persist in lettuce leaves. All mutant strains with altered endophytic persistence will be tested for their ability to induce callose deposits in the leaves as well as their growth in apoplast wash fluid.

Progress 02/15/20 to 02/14/21

Outputs
Target Audience:Many events were postponed or moved to a virtual platform this year. We have delivered a few webinar presentations in scientific meetings such as the International Association for Food Protection (IAFP) Annual Meeting and the Symposium titled "Improving safety and quality of fresh produce" at the American Chemical Society National Meeting & Exposition. Changes/Problems:The COVID-19 pandemic has significantly impacted our ability to complete this project in a timely manner. The no-cost extension until 2022 should allow us to complete the project as planned. What opportunities for training and professional development has the project provided?The PIs continue to provide training opportunities for undergraduate and graduate students, Jr. specialists, and post-doctoral fellows through this project. During this period, our training efforts were focused on written communication of research and data analyses. How have the results been disseminated to communities of interest?Results of this project have been presented to professionals in the food safety industry, the student populations, and scientific communities in the form of virtual seminars. Peer-reviewed articles in open access journals have been published. Please see products above. What do you plan to do during the next reporting period to accomplish the goals?There are no changes to the approved non-cost extension of the project.

Impacts
What was accomplished under these goals? IMPACT: despite the implementation of extensive control measures to produce and deliver safe read-to-eat leafy greens, serious disease outbreaks associated with consumption of contaminated leaves continue to occur. Prevention of pre-harvest contamination on the farm has become one of the most important steps in reducing human health risk and improving food safety. During this reporting period, we have made significant progress towards addressing each objective as follows: The objectives of this project are to (1) Examine colonization efficiency of lettuce by outbreak-associated Salmonella serovars (OASS); (2) Evaluate the natural variations in colonization of various lettuce cultivars by Salmonella; and (3) Elucidate the mechanism(s) of Salmonella persistence inside the leaf. Objectives 2 and 3 are completed and several peer-reviewed articles have been already published or are under review for publication as previously reported. However, in March 2020 our labs were closed by university mandate and compliance with public safety authorities due to COVID-19. During this reporting period, most of the work has been done remotely and our facilities are operating at minimum capacity. This unforeseen event significantly affected our ability to complete the experiments under Objective 1. Specifically, the competition assays that rely on sequencing and extensive bioinformatics analyses could not be completed. Nevertheless, over 300 leaf samples were subjected to inoculation, recovery, and growth of bacteria. PCR to generate representation of over 200 different barcodes that we had integrated into over 10 different bacterial strains was performed. We are in the process of interpreting this dataset and troubleshooting the experiments. Thus far, the barriers to interpretation remain the high variability in the estimated bacterial titers in the leaves, and the complexity when a single mixture of barcoded strains is introduced into multiple types of leaves (i.e., different lettuce cultivars). Another activity planned for 2020 was a visit of Dr. Shlomo Sela to USA, so that our team could finalize the writing of project reports and manuscripts for publication. This meeting would solidify our collaborative efforts. Due to travel restrictions, this visit had to be postponed. At this time, a no-cost extension was approved until February 2022 to allow us complete Objective 1. If travel restrictions are lifted during this NCE, Dr. Sela will travel to California.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Li Y, Salazar JK, He Y, Desai P, Porwollik S, Chu W, Paola PS, Tortorello ML, Juarez O, Feng H, McClelland M, Zhang W. 2020. Mechanisms of Salmonella Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts. Front Microbiol. 11:582202.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jayeola V, McClelland M, Porwollik S, Chu W, Farber J, Kathariou S. 2020 Identification of Novel Genes. 2020. Mediating Survival of Salmonella on Low-Moisture Foods via Transposon Sequencing Analysis. Front Microbiol 11:726.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Nguyen BD, Cuenca V M, Hartl J, G�l E, Bauer R, Meile S, R�thi J, Margot C, Heeb L, Besser F, Escriva PP, Fetz C, Furter M, Laganenka L, Keller P, Fuchs L, Christen M, Porwollik S, McClelland M, Vorholt JA, Sauer U, Sunagawa S, Christen B, Hardt WD. 2020. Import of Aspartate and Malate by DcuABC Drives H2/Fumarate Respiration to Promote Initial Salmonella Gut-Lumen Colonization in Mice. Cell Host & Microbe 27:922-936.e6.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chakraborty S, Liu L, Fitzsimmons L, Porwollik S, Kim JS, Desai P, McClelland M, Vazquez-Torres A. 2020. Glycolytic reprograming in Salmonella counters NOX2-mediated dissipation of ?pH. Nature Commun 2020 11:1783.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fitzsimmons LF, Liu L, Kant S, Kim JS, Till JK, Jones-Carson J, Porwollik S, McClelland M, Vazquez-Torres A. 2020. SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome. mBio 11:e03397-19.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Oblessuc PR, Melotto M. 2020. A simple assay to assess Salmonella enterica persistence in lettuce leaves after low inoculation dose. Front Microbiol 11:1516.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Melotto M, Brandl MT, Jacob C, Jay-Russell M, Micallef SA, Warburton M, Van Deynze A. 2020. Breeding crops for enhanced food safety. Front Plant Sci 11:428.


Progress 02/15/19 to 02/14/20

Outputs
Target Audience:We have delivered several presentations to a diverse audience and in several formats. All of these presentations were by invitation, indicating the broad interest in our project. 1) Participation in the One Health Initiative: - PI Melotto continues to deliver seminars at the NAU-UC Davis Education Conference on One Health for Food Safety and Security. This is an annual conference held at UC Davis or Nanjing Agricultural University, China to educate undergraduate and graduate students on One Health. They represent the colleges of Resources and Environmental Sciences, Horticulture, Plant Protection, Veterinary Medicine, Life Science, Agro-grassland Sciences, Food Science and Technology, and Animal Science and Technology (~100 attendees). 2) Formal Classroom Instruction: - PI Melotto developed a course on Microbiology and Safety of Fresh Produce aimed at discussing and understanding what it takes to deliver safe-to-eat food. The course has been offered to undergraduate and graduate students once a year. Students come from a diverse background and ethnical groups. (~45 students during this report period) - co-PI McClelland directed a Microbiology course that included epidemiology of infection from food products. One lecture had an emphasis on Salmonella. The course will be offered each Spring Quarter. About 1/3 are URM or first in their family to go to college (~15 PhD students) - co-PI Sela instructed a course for MSc and PhD students on biofilms in the food industry. One lecture discussed Salmonella on fresh produce. 3) Presentations to the Fresh Produce Industry: - co-PI Sela has participated in an EIT-Food partners meeting and discuss the topic of fresh produce contamination with several international food industry representatives. 4) Presentations to the Scientific Community: In 2019, the PIs delivered the research presentations to scientists, professors, and students at the following conferences: - One Health for Food Systems Conference: Integrating Veterinary, Food, Animal and Agricultural Sciences. Sponsored by Nanjing Agricultural University and Hosted by WIFSS, University of California, Davis. - 4th International Workshop on Interactions between Crop Plants and Human Pathogens. European Cooperation in Science & Technology (COST Action). Vienna, Austria. PI Melotto was the Keynote Speaker - Webinar to the USDA Plant Breeding for Food Safety Meeting. Washington, D.C. - Plant Health 2019: Annual Meeting of the American Phytopathological Society. Cleveland, Ohio, USA - NIFA Workshop: Breeding Crops for Enhanced Food Safety. UC Davis, CA. USA. 5) Hosting Workshops: - Satellite Meeting titled "The Interaction between Plants and Human Pathogens" during the Biannual Meeting of the International Society for Molecular Plant-Microbe Interactions (IS-MPMI) Glasgow, Scotland. Co-organized by Maeli Melotto and Dr. Nicola Holden - NIFA Workshop, "Breeding for Enhanced Crop Safety", University of California, Davis. Co-organizers: Maeli Melotto, Michele Jay-Russell, and Allen Van Deynze. - A COST supported symposium on: Understanding the Relationship between Human Pathogens and Plant's Production Systems is co-organized in Israel by co-PI Sela together with S. Yaron during March, 24-25, 2020 Changes/Problems:In the original Objective 2, we have proposed to use lettuce cultivars grown in the US and Israel. However, the major Israeli seed company, which sells most of the cultivars, did not response to our request to obtain seeds of various lettuce cultivar, apparently because it did not want to be associated with any study related to lettuce and Salmonella. Consequently, we revised the objective, as described above. What opportunities for training and professional development has the project provided?The PIs continue to provide training opportunities for undergraduate and graduate students, Jr. specialists, and post-doctoral fellows through this project. The multi-disciplinary nature of this research, including crop biology, microbiology, biotechnology, microscopy, as well as the engagement in scientific writing for publications and oral communication in conferences are crucial for the professional development of these young scientists. How have the results been disseminated to communities of interest?Results of this project has been presented to professionals in the food safety industry, the student populations, and scientific communities in the form of public seminars, Q&A sessions, and panel discussions. Multiple peer-reviewed articles in open access journals have been published. Please see products above. What do you plan to do during the next reporting period to accomplish the goals?There are no changes to the approved non-cost extension of the project.

Impacts
What was accomplished under these goals? IMPACT: despite the implementation of extensive control measures to produce and deliver safe read-to-eat leafy greens, serious disease outbreaks associated with consumption of contaminated leaves continue to occur. Prevention of pre-harvest contamination on the farm has become one of the most important steps in reducing human health risk and improving food safety. During this reporting period, we have made significant progress towards addressing each objective as follows: Objective 1: We have not been able to complete Objective 1 due to difficulties with the bacterial competition assays in vivo. Both the PI and co-PI's labs are working together to streamline the protocol to recover enough bacterial DNA from infected plant tissue over the course of infection. The main difficulty has been to optimize the bacterial inoculation procedure that closely mimics natural leaf tissue contamination. As such, we are still testing several inoculation doses with mixed inocula to promote competition among different strains of Salmonella. This was the basis for the request for a non-cost extension of the project. Objective 2: Evaluate the natural variations in colonization of various lettuce cultivars by Salmonella. This objective is completed and the main findings are reported in the newly published article by Jacob and Melotto (2020). We were able to use lettuce cultivars from the US to run the experiments. However, we could not use lettuce cultivar from Israel because the major Israeli seed company did not respond to our request for seeds. Consequently, we revised the objective and studied another important topic related to Salmonella colonization, i.e. the factors that affect Salmonella internalization in lettuce. In this case, we used commercial lettuce for the experiments. We found that Salmonella grown in Luria Bertani broth devoid of NaCl (LBNS), or in diluted LB (0.5×LB) internalized lettuce leaf better (62 ± 5% and 59 ± 7%, respectively) compared to bacteria grown in LB (15 ± 7%). Growth under non-aerated conditions also enhanced Salmonella internalization compared to growth under aerated conditions. Growth temperature of 25 and 37°C did not affect STm internalization, however, growth at 42°C, significantly augmented leaf internalization. These findings highlights the complexity of bacterial internalization process and may provide partial explanation for the variable, sometimes-contrasting results reported in the literature regarding stomatal internalization by Salmonella and E. coli. These findings are described by Kroupitski et al. (2019). Jacob C, Melotto M. 2020. Human pathogen colonization of lettuce dependent upon plant genotype and defense response activation. Front Plant Sci 10:1769. Kroupitski Y, Gollop R, Belausov E, Pinto R and Sela (Saldinger) S. 2019. Salmonella enterica growth conditions influence lettuce leaf internalization. Front. Microbiol. 10:639. Objective 3: Elucidate the mechanism(s) of Salmonella persistence inside the leaf. We investigated the involvement of five universal-stress proteins (USPs) genes in Salmonella leaf internalization following growth in LBNS medium. USPs are part of superfamily of conserved genes, which are induced by environmental stressors and are considered to be part of the bacterial stress response. Knockout mutations in ydaA, yecG, ybdQ, and uspAB, but not in ynaF, significantly reduced Salmonella internalization compared to the wild-type strain, without affecting bacterial attachment or motility. Transduction of the mutations back to the parent strain confirmed the linkage between the mutations and the internalization phenotype. These findings support a specific role of these universal-stress genes in lettuce leaf internalization. Most studies on the endophytic colonization of foodborne pathogens in plants utilize the surface disinfection technique to distinguish between surface attached and internalized bacteria. During this period, we have also compared endophytic colonization of Salmonella using both surface inactivation and confocal microscopy in leaves of lettuce, tomato, and Arabidopsis. We found that the level of endophytic colonization depends on the type of disinfectants used and the exposure time. In some cases, discrepancies were observed between the two analytical approaches. These findings calls for standardization of the methods used to assess Salmonella (and perhaps other foodborne pathogens) internalization in order to obtain rational conclusions in studies examining endophytic colonization. A manuscript to report these findings is in preparation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Gupta SK, Sharma P, McMillan EA, Jackson CR, Hiott LM, Woodley T, Humayoun SB, Barrett JB, Frye JG, McClelland M. 2019. Genomic comparison of diverse Salmonella serovars isolated from swine. PLoS One. 14(11):e0224518. McMillan EA, Gupta SK, Williams LE, Jov� T, Hiott LM, Woodley TA, Barrett JB, Jackson CR, Wasilenko JL, Simmons M, Tillman GE, McClelland M, Frye JG. 2019. Antimicrobial Resistance Genes, Cassettes, and Plasmids Present in Salmonella enterica Associated With United States Food Animals. Front Microbiol. 10:832. Khilyas IV, Tursunov KA, Shirshikova TV, Kamaletdinova LK, Matrosova LE, Desai PT, McClelland M, Bogomolnaya LM. 2019. Genome Sequence of Pigmented Siderophore-Producing Strain Serratia marcescens SM6. Microbiol Resour Announc. 8(18). pii: e00247-19. Subramaniam S, M�ller VS, Hering NA, Mollenkopf H, Becker D, Heroven AK, Dersch P, Pohlmann A, Tedin K, Porwollik S, McClelland M, Meyer TF, Hunke S. 2019. Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium. PloS One. e0211584. PMID: 30716090 Kroupitski Y, Gollop R, Belausov E, Pinto R and Sela (Saldinger) S. 2019. Salmonella enterica growth conditions influence lettuce leaf internalization. Front. Microbiol. 10:639. Reinink P, Buter J, Mishra VK, Ishikawa E, Cheng T-Y, Willemsen PT, Porwollik S, Brennan PJ, Heinz E, Dougan G, van Els CA, Cerundolo V, Napolitani G, Yamasaki S, Minnaard AJ, McClelland M, Moody B, Van Rhijn I. 2019. Discovery of immunogenic trehalose phospholipids in Salmonella species reveals functional convergence with mycobacteria. J. Experimental Medicine 216:757-771. Oblessuc PR, Bisneta MV, Melotto M. 2019. Common and unique Arabidopsis proteins involved in stomatal susceptibility to Salmonella enterica and Pseudomonas syringae. FEMS Microbiol Letters 366:fnz197. Roy D, Melotto M. 2019. Stomatal response and human pathogen persistence in leafy greens under preharvest and postharvest environmental conditions. Postharvest Biol. Tech. 148:76-82.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Montano J, Rossidivito G, Torreano J, Porwollik S, Sela S, McClelland M, Melotto M. 2020. Salmonella enterica serovar Typhimurium 14028s genomic regions required for colonization of lettuce leaves. Front Microbiol 11:6. doi:10.3389/fmicb.2020.00006 Oblessuc PR, Matiolli CC, Melotto M. 2020. Novel molecular components involved in callose-mediated defense against human pathogens. BMC Plant Biology 20:16. doi:10.1186/s12870-019-2232-x Jacob C, Melotto M. 2020. Human pathogen colonization of lettuce dependent upon plant genotype and defense response activation. Front Plant Sci 10:1769. doi: 10.3389/fpls.2019.01769
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Oblessuc PR, Matiolli CC, Melotto M. 2019. Plant and human bacterial pathogens trigger similar SA-mediated defense in Arabidopsis. In: Book of Abstracts, XVIII International Congress on Molecular Plant-Microbe Interactions, Glasgow, Scotland. Jacob C, Melotto M. 2019. Differential plant defense responses of lettuce genotypes against human pathogenic bacteria. In: Book of Abstracts, XVIII International Congress on Molecular Plant-Microbe Interactions, Glasgow, Scotland. Van Deynze A, Melotto M, Jay-Russell M. 2019. NIFA: Breeding for food safety. Annual Meeting of the National Association of Plant Breeders. August 25-29, Pine Mountain, GA, USA.


Progress 02/15/18 to 02/14/19

Outputs
Target Audience:We have delivered several presentations to a diverse audience and in several formats. All of these presentations were by invitation, indicating the broad interest in our project. 1) Participation in the One Heath Initiative: - PI Melotto was a panelist at the "One Health Youth Conference" hosted by the Western Institute for Food Safety and Security (WIFFS) at UC Davis on January 24, 2019. The panel was formed by four scientists representing the various aspects of the One Health Concept (i.e., plant health, soil health, animal and human health, environmental health). The audience comprised of undergraduate students from the US and China (~60 attendees). - PI Melotto delivered a seminar at the "One Health for Food Safety and Security" Conference at UC Davis on January 29, 2019. This is an annual conference held at UC Davis or Nanjing Agricultural University, China to educate undergraduate and graduate students on One Health. (~100 attendees). 2) Formal Classroom Instruction: - PI Melotto developed a course on Microbiology and Safety of Fresh Produce aimed at discussing and understanding what it takes to deliver safe-to-eat food. The course has been offered to undergraduate and graduate students in the 2019 winter quarter. Students come from a diverse background and ethnical groups. (~40 students) - co-PI McClelland directed a Microbiology course that included epidemiology of infection from food products. One lecture had an emphasis on Salmonella. The course will be offered each Spring Quarter. About 1/3 are URM or first in their family to go to college (~15 PhD students) - co-PI Sela instructed a course for MSc and PhD students on biofilms in the food industry. One lecture discussed Salmonella on fresh produce. 3) Fresh Produce Industry: - PI Melotto provided research updates during the statewide UC-ANR Vegetable Crops Program Meeting held at UC Davis on November 26-27, 2018. Farmer advisors, extension specialists, and professionals in the state of California attended the meeting. (~60 attendees) 4) Scientific Community: - In 2018, PI Melotto delivered four research presentations to scientists, professors, students, and the Pioneer R&D department highlighting the results of this project. (~50 attendees and live streaming to remote audience). - co-PI Sela has delivered four research presentations to professors and industry representatives visiting ARO on his research on Salmonella-plant interactions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At UC Davis, three undergraduate students, three PhD students, and one postdoctoral fellow are being trained in this project. At ARO, one MSc student and one postdoctoral fellow are being trained in this project. How have the results been disseminated to communities of interest?Results of this project has been presented to professionals in the food safety industry, the student populations, and scientific communities in the form of public seminars, Q&A sessions, and panel discussions. Two manuscripts have been published or accepted for publication in peer reviewed journals. Two other manuscripts are currently being reviewed. What do you plan to do during the next reporting period to accomplish the goals?There are no changes to the approved project initiation document.

Impacts
What was accomplished under these goals? IMPACT: despite the implementation of extensive control measures to produce and deliver safe read-to-eat leafy greens, serious disease outbreaks associated with consumption of contaminated leaves continue to occur. Prevention of pre-harvest contamination on the farm has become one of the most important steps in reducing human health risk and improving food safety. During this reporting period, we have made significant progress towards addressing each objective as follows: Objective 1: Our current collection of OASS has 58 isolates. PI McClelland's group generated barcoded clones of 29 isolates, including 8 or more barcoded clones for 20 isolates. Additionally, barcoded clones from 8 strains isolated from cattle were created, that will act as non-plant isolate controls. PI Melotto's group inoculated lettuce cultivar Salinas with 18 isolates to identify bacterium population growth patterns in planta. So far, we have found that 1, 7, and 10 isolates have a positive, negative, or neutral growth, respectively, indicating that some Salmonella serovars may pose a greater risk to contaminating lettuce leaves than other. In the coming year, we will perform competition assays to dissect the bacterium population dynamics in leaves (i.e. relative replication rate) and determine the relative fitness of each strain in the phyllosphere. Objective 2: We have completed the analyses of 11 commercial cultivars of lettuce for their response to the S. enterica serovar Typhimurium 14028s. Plants were grown under controlled environmental conditions and tested at two developmental stages and growth conditions; 3 week-old plants grown in peat moss pellets and 4-week old plants grown in pots. Leaf surface characteristics and bacterial attachment were evaluated using the second fully expanded leaf of younger plants. Bacterial persistence in the apoplast was assessed in plants of both developmental stages using two methods of inoculation: syringe-infiltration and dipping inoculation. Steps for leaf colonization (i.e., attachment, internalization, and persistence) of by both bacteria varied significantly among soil-grown lettuce genotypes. Different kinetics of bacterial survival in the intercellular space suggests that lettuce genotypes exhibit pre-existing and/or activated responses associated with variation in their apoplastic environment. These results were presented at the Plant Biology Annual Meeting hosted by the American Society of Plant Biologists in Montreal, Canada (Jacob and Melotto, 2018). The establishment of robust and relevant phenotypic differences using robust protocols is key for further studies aimed at elucidating the underlying molecular processes causing the phenotypic variation. We have selected two lettuce genotypes that showed the most resistance and the most susceptibility to STm 14028s to elucidate the mechanism(s) underlying these processes in the next reporting period. Objective 3: Leaf internalization is one of the first steps for a pathogen to colonize the apoplast. co-PI Sela's group determined that sub-optimal bacterium growth conditions in fact increases STm internalization into lettuce leaves. For instance, enhanced bacterial internalization through stomata was observed when growing STm in 0.5x LB, LB-without salt, or LB at 25oC and LB at 42oC as compared to growing the bacterium at its optimum temperature of 37oC. Furthermore, we have identified seven multi gene deletion (MGD) mutants of STm 14028s that show increased population titers inside lettuce leaves and one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. Interestingly, a mutant that lacks the SPI-1 and some adjacent genes did not have impaired endophytic behaviors when compared to the wild type bacterium. Additionally, we observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce leaf callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not the Mut3, was significantly impaired in the leaf apoplast wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. A manuscript reporting these results is in the second round of revision by the journal Applied and Environmental Microbiology. Montano J, Rossidivito G, Torreano J, Porwollik S, Sela S, McClelland M, Melotto M. 2019. Salmonella enterica serovar Typhimurium 14028s genomic regions required for colonization of lettuce leaves. Appl Environm Microbiol (second round of revision).

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jacob C, Melotto M. 2018. Lettuce genotypic variation affects the interaction between the plant and the human pathogens Salmonella enterica Typhimurium 14028s and Escherichia coli O157:H7. Plant Biology Annual Meeting, American Society of Plant Biologists. July 14-18, 2018, Montreal, Canada
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Oblessuc PR, Roy D, Bisneta MV, Matiolli CC, Melotto M. 2018. Genetic basis for Arabidopsis guard cell responses to human and plant pathogens. Pioneer Plant Science Symposia Series, April 16. University of California, Davis, CA. USA.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Subramaniam S, M�ller VS, Hering NA, Mollenkopf H, Becker D, Heroven AK, Dersch P, Pohlmann A, Tedin K, Porwollik S, McClelland M, Meyer TF, Hunke S. 2019. Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium. PloS One. e0211584. PMID: 30716090
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Reinink P, Buter J, Mishra VK, Ishikawa E, Cheng T-Y, Willemsen PT, Porwollik S, Brennan PJ, Heinz E, Dougan G, van Els CA, Cerundolo V, Napolitani G, Yamasaki S, Minnaard AJ, McClelland M, Moody B, Van Rhijn I. 2019. Discovery of immunogenic trehalose phospholipids in Salmonella species reveals functional convergence with mycobacteria J. Experimental Medicine (in press).


Progress 02/15/17 to 02/14/18

Outputs
Target Audience: Nothing Reported Changes/Problems:Although this project was scheduled to start in February 2017, funds were allocated to the PI in August 2017. Funds for the Israeli PI, Dr. Shlomo Sela, were released in November 2017. We were able to recruit personnel to start the project after funding began. We anticipate remaining on schedule to the end of the project, that is three years after the budget release. What opportunities for training and professional development has the project provided?Two PhD students at UC Davis and one MSc student at The Volcani Center are being trained in this project. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?There are no changes to the approved project initiation document.

Impacts
What was accomplished under these goals? IMPACT: Healthy eating of fresh fruits and vegetables is part of an integrated strategy to decrease the risk of serious diseases. Fresh produce consumption has been increasing worldwide in the last few decades and concomitantly, an expansion of the fresh market over recent years has resulted in a wide variety of fruit and fresh produce being available throughout the year. At the same time, the number of foodborne outbreaks related to consumption of fresh or minimally processed produce has been increasing. Unlike foods of animal origin, fresh or ready-to-eat (RTE) produce, such as leafy vegetables, cannot undergo thermal processes to inactivate human pathogens. Consequently, the lack of an efficient kill-step is a risk factor and one of the great challenges facing the fresh produce industry. Prevention of pre-harvest contamination on the farm has become one of the most important steps in reducing human health risk and improving food safety. In this first five months of this project reported here (please see Changes/problem section), PD Melotto secured a faculty fellowship from the Jewish National Fund to visit the Israeli PD, Dr. Sela, and discuss the project logistic and the exchange of research material between our groups. We have obtained university's authorization to work with outbreak-associated strains of pathogenic Salmonella (objective 1) and obtained a large collection of lettuce genotypes to start objective 2. We have recruited and trained new personnel to work in a BSL2 environment at both UC Davis and The Volcani Center. Progress towards achieving each objective is described below. Objective 1: we have initiated experiments to test the ability of various OASS to survive on leaves of the lettuce cultivar Salinas. Analysis are underway to compare and contrast the population dynamic within leaves among the serovars. Objective 2: lettuce genotypes have a remarkable phenotypic plasticity in response to growing conditions. During this period, we have been able to establish the optimum conditions to allow the cultivation of the different lettuce genotypes, including some wild relatives. Additionally, we have adjusted the bacterial inoculation protocol that works for all lettuce genotypes. Objective 3: we have not started this objective yet.

Publications