MICROBIAL INTERACTIONS IN HOST-ASSOCIATED COMMUNITIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007300
• Project No.: FLA-SWS-005474
• Project Start Date: Aug 24, 2015
• Project End Date: Dec 31, 2018

Project Director
Meyer, JU, .

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Soil and Water Sciences

Non Technical Summary
Over the last decade, scientists have come to realize that interactions between higher organisms and their microbial associates are central to the health of plants and animals. Most of these discoveries were made in animal models of human diseases. Significantly less is known about the role of microbes in health and stability of aquatic ecosystems and in ensuring microbiological safety of foods. Therefore, with this project, we will focus on the characterization of food safety outcomes in Salmonella-tomato interactions; the roles of novel types of signal interactions in Medicago-Rhizobium symbiosis; and promoting health of coral-associated microbial communities. In Florida, addressing these three questions is important as they represent pressing needs of the state, in which marine ecosystems are critical for tourism and fisheries; vegetable production has been affected by outbreas of foodborne illness and pasture-based beef production requires wise investment of resources into forages. We anticipate that upon completion of this work, safer crop production practices will be identified, opportunities to improve health of marine ecosystems will be defined and the ability of forage legumes (such as alfalfa and medics) to form more efficient symbioses will be described. In process, at least two cohorts of post-doctoral scientists will be better prepared for independt careers, graduate and undergraduate students will gain valuable research experience.

Animal Health Component

0%

Research Effort Categories

Basic

70%

Applied

25%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	0199	1040	60%
201	0199	1040	20%
112	0330	1070	20%

Knowledge Area
112 - Watershed Protection and Management; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0199 - Soil and land, general; 0330 - Wetland and riparian systems;

Field Of Science
1040 - Molecular biology; 1070 - Ecology;

Keywords

salmonella-tomato interactions

legume-rhizobium symbiosis

coral microbiology

Goals / Objectives
Host-associated microbial communities play central roles in health and stability of agricultural and natural ecosystems. This project focuses on three models: (1) Salmonella-tomato interactions; (2) Medicago-Rhizobium symbiosis; and (3) coral-associated microbial communities. These three system are directly relevant to the state's agricultural production and natural ecosystems. Further characterization of interactions in these three models will also offer an opportunity to define evolutionarily conserved mechanisms by which diverse host-associated microbial communities are structured and maintained.All three of the models on which my research focuses have important impacts on the economy of Florida. The number of produce-associated outbreaks of salmonellosis is on the rise and is comparable to the outbreaks associated with the consumption of meats and poultry. Fourteen major foodborne pathogens cause damage to the US economy at a rate of $14.1 billion per year. Conservatively, produce-related outbreaks account for 18.1 and 16.6 % of all E. coli O157:H7 and Salmonella outbreaks respectively. The outbreaks of foodborne illness linked to the consumption of fruits and vegetables led to the ranking of produce as the 4th riskiest food, estimated to cause over 1 million illnesses annually. Based on USDA ERS data, tomato is the most consumed fresh vegetable: in 2009, Americans purchased 18.7 lbs of fresh tomatoes per capita. Florida ranks first (or second, depending on the metric) nationally in the acreage, production, and value of fresh market tomato. In the 2010-2011 growing season, 30,700 acres were under cultivation for the fresh tomato maret. Fresh market tomatoes comprise about 40% of Florida's fresh market vegetable cash receipts. About 33,000 workers are directly involved in tomato production and harvest each year. The issue of food safety represent one of the greatest threats to sustainability and profitability of Florida tomato industry. Based on USDA ERS data, following the June-August 2008 outbreak of salmonellosis caused by S. enterica sv. St. Paul (which was initially wrongly blamed on tomatoes from Florida) the price of tomatoes at the point of first sale dropped from 56.8 cents/lb in June to 25.6 cents/lb in August. This nearly obliterated Florida tomato industry. Promoting safety of produce from Florida will help avoid future attribution errors and is seen as one of the major components of increasing competitiveness of Florida tomatoes on international markets. The goal of this project is to contribute to the development of the comprehensive approach to improving microbiological safety of tomatoes through cultivar selection, minor changes in the production practices combined with education.Based on USDA ERS data, Medicago spp are important pasture legumes in temperate and arid climates. M. sativa (alalfa) alone accounts for 60% of all hay produced in the U.S., and is the fourth most important crop in the United States both in terms of acreage and production value. M. truncatula (barrel medic) and Melilotus spp (sweet clover) are gaining recognition as promising annual pasture legumes, especially in arid regions, or as a winter cover crop/green mulch in warmer climates or as emergency annual hay crops. The symbiosis with Sinorhizobium contributes direct, timely and self-regulating amounts of nitrogen needed for growth of Medicago spp in the field. Better understanding of the steps required to establish, fine-tune and maintain the symbiosis should contribute to the development of superior inoculum strains or to management practices that optimize symbiotic benefits.In Florida, coral reefs contribute at least $2.9 billion to local economies annually, and provide employment for approximately 39,000 people. Florida reefs are a major tourist attraction: visitors spend $460-1,087/person/trip. Florida coral reefs provide habitat for over 4,000 fish species and support commercial and recreational fishing industries which generate ~ $60 million. The capitalized reef user value in southeast Florida is $8.5 billion.OBJECTIVESThe goal of this project is to define mechanisms of interactions within host-associated microbial communities, focusing specifically on the interactions of co-evolved commensals/symbionts and opportunistic pathogens. Even though interest in microbiome research has exploded recently, little is still known about the mechanisms by which host-associated microbiota contribute to the health of any host.Objective 1. The overall goal of this objective is to promote safety of vegetables by assessing how intrinsic crop properties and agronomic practices affect persistence of human pathogens in the production environment and also affect vulnerability of crops to accidental contamination post-harvest. A better understanding of these parameters and their interactions will promote microbiological safety of produce.Objective 2. We hypothesize that bacterial small regulatory RNA and post-transcriptional changes in gene expression mediated by the RNA-binding protein Hfq play central roles in maintaining the productive symbiotic relationship. Therefore, the goal of this objective is to characterize contribution of Hfq to the symbiotic regulation of rhizobial small regulatory RNA genes. Levels of srRNA gene expression within nodules will be characterized by qRT-PCR and expression patterns determined with reporter fusions. In vitro experiments will focus on comparing stability of complexes formed by Hfq with target bacterial mRNA and bacterial srRNA, and will determine the kinetics of each molecule's binding. Eventually, we plan to characterize the entire Symbiotic Hfq Regulon by co-immunoprecipitation of the non-coding and mRNA bound to Hfq within bacteroids.Objective 3. A recognition of the unique roles of the eukaryotes' microbial associates in evolution, development and responses to stressors helped define the term "meta-organism" or a "holobiont" as a complex multi-organismal symbiosis. Long-lived colonial invertebrates, such as corals, which lack adaptive immunity are thought to rely extensively on their associated microbiota for nutrient acquisition and defense, and the composition of coral-associated microbial communities correlates with the health status of the holobiont. We hypothesize that the co-evolved "primary" commensals of corals provide functions needed for the fitness of the entire holobiont and its responses to environmental stressors. We further hypothesize that, under some conditions, commensals either escape regulation by the host and themselves contribute to the appearance of the disease signs or are outcompeted by opportunists which invade the holobiont. This hypothesis is based on the observations that shifts in the composition of the coral-associated microbiome correlate with environmental stress and disease and that members of the coral microbiome produce substances that inhibit virulence behaviors in pathogens (e.g., surface colonization, attachment, enzymes involved in host mucus degradation). Therefore, the third objective of this proposal is to define the effect of environmental drivers on the physiology and biochemistry of the holobiont and to evaluate resilience of the host when functionality of the associated microbiome is affected by environmental stressors (e.g. increased temperature).

Project Methods
Even though the three models are quite distinct, it will be possible to accomplish the objectives using the same techniques. Global changes in the transcriptome profiles and enzymatic activities in the native microbiota will be defined by RNA-seq and functional assays to delineate the outcomes of the signal exchange in host-microbe/microbe interactions during the invasion of the host microbiota by opportunistic pathogens. Shifts in the composition of microbiota and changes in the abundance of functions in the metagenomes will be established with Next-Gen sequencing. Transcriptional and translational reporters in the wild type and mutant backgrounds will be constructed and their regulation will be tested as appropriate. These experiments will be carried out at the University of Florida Genetics Institute. Samples will also be collected in the Florida Keys Marine Sanctuary and at the Carribow Cay Smithsonian Marine Station.

Progress 08/24/15 to 12/31/18

Outputs
Target Audience:?During the reporting period, our research and outreach efforts reached a diverse audience of scientists, the general public, and industry. The PD presented a research seminarat the Center for Produce Safety Research Symposium (Charlotte, NC). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Julie Meyer, June 2018. Center for Produce Safety Research Symposium, Oral presentation: "Comparative genomics analysis and physiological assessment of the avirulent Salmonella surrogate relevant to food safety." (Charlotte, NC). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Project Director, Dr. Maxim Teplitski is no longer with the University of Florida and is not able to complete this project. We are closing his Hatch project.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Gao, M., Benge, A., Mesa, J., Javier, R., Liu, FX. (2018). Use of RNA immunoprecipitation method for determining Sinorhizobium meliloti RNA-Hfq protein associations in vivo. Biological Procedures Online 2018 20:8
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Teplitski M and de Moraes MH. 2018. Of mice and men... and tomatoes: comparative genomic analysis of Salmonella interactions with its diverse hosts. Trends in Microbiology 26:748-754.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: de Moraes MH, Becerra-Soto E, Gonz�les IS, Desai P, Chu W, Porwollik S, McClelland M, Teplitski M. 2018. Genome-wide comparative functional analyses reveal adaptations of Salmonella sv. Newport to a plant colonization lifestyle. Frontiers in Microbiology 9:877

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:During the reporting period, our research and outreach efforts reached a diverse audience of scientists, the general public, and industry. The PD presented research seminars at the Center for Produce Safety Research Symposium (Boulder, CO) and at the Protect Our Reefs Foundation Board Meeting (Key West, FL). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Julie Meyer, April 2017. Protect Our Reefs Foundation Meeting, oral presentation: "Microbe-microbe interactions in the establishment of Black Band Disease." (Key West, FL). Julie Meyer, June 2017. Center for Produce Safety Research Symposium, Oral presentation: "Comparative genomics analysis and physiological assessment of the avirulent Salmonella surrogate relevant to food safety." (Denver, CO). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under Objective 1, we completed a pangenomic analysis of more than 2,000 serovars of Salmonella and identified the novel gene papA (for Plant Associated Protein A) that promotes growth of Salmonella in tomatoes. Under Objective 3, we completed a comparative metagenomics study of the destructive polymicrobial disease, Black Band Disease, from Caribbean corals.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Meyer, JL, VJ Paul, L Raymundo, M Teplitski. 2017. Comparative metagenomics of the polymicrobial Black Band Disease of corals. Frontiers in Microbiology 8:618.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: de Moraes MH, Desai P, Porwollik S, Canals R, Perez DR, Chu W, McClelland M, Teplitski M. 2017. Salmonella persistence in tomatoes requires a distinct set of metabolic functions identified by transposon insertion sequencing. Applied and Environmental Microbiology 83:e03028-16.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: de Moraes MH, Becerra-Soto E, Gonz�les IS, Desai P, Chu W, Porwollik S, McClelland M, Teplitski M. 2018. Genome-wide comparative functional analyses reveal adaptations of Salmonella sv. Newport to a plant colonization lifestyle. Frontiers in Microbiology.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Teplitski M and de Moraes MH. 2018. Of mice and men... and tomatoes: comparative genomic analysis of Salmonella interactions with its diverse hosts. Trends in Microbiology.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:During the reporting period, our research and outreach efforts reached a diverse audience of scientists (domestically and internationally), general public and the industry. The PD presented research seminars at USDA-ARS Western Regional Research Center (Albany, CA), UC Davis Post-harvest Physiology Group (Davis, CA), 2016 Florida Association for Food Protection Annual Education Conference (Crystal River, FL), Protect Our Reefs Foundation Board Meeting (Key West, FL), Xavier University (New Orleans, LA), Colorado State University (Fort Collins, CO), Centro de Investigaciones Biotecnológicas del Ecuador, Escuela Superior Politecnica del Litoral (Guayaquil, Ecuador), Universidad de Cuenca (Cuenca, Ecuador). Web-based outreach materials (in English and Spanish) have been developed. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A post-doctoral scientist (Dr. Julie Meyer) and two graduate students were directly involved in this project, and five undergraduate students contributed to the project as volunteers. As a part of this umbrella project, the PD developed a mentoring program, "Tropical Connections" through which 9 female post-doctoral scientists were coached through preparation of a portfolio for application for a faculty position, and 8 more female post-docs are enrolled in the Tropical Connections program this year. As an indirect benefit of the Tropical Connections program, we were able to develop a series of professional development workshops that were attended by ~40 graduate students, post-docs and early career scientists in UF, University of Hawarii, Escuela Politecnica del Litoral (Ecuador) and University of Cuenca (Ecuador). Contributions of students involved in this project were recognized with the following awards: 2016 A. George receives USDA NIFA Graduate Student Fellowship 2016 M.H. de Moraes recieves Doris Lowe and Earl and Verna Lowe Scholarship 2016 R. Blaustein receives Doris Lowe and Earl and Verna Lowe Scholarship 2016 A. George receives Doris Lowe and Earl and Verna Lowe Scholarship 2016 M.H. de Moraes receives Dennis Carpenter Memorial Fellowship from AGRI Council 2015 M.H. de Moraes receives William C. and Bertha M. Cornett Fellowship 2015 M.H. de Moraes receives Sam Polston Scholarship Award for Outstanding Performance in Graduate Studies 2015 R. Blaustein earns 2nd place for a poster presentation at ASM (SE Branch) meeting How have the results been disseminated to communities of interest?The PD conducted the following seminars/workshops: Teplitski, M. (Seminar) Of mice and men... and tomatoes: comparative and functional genomics of Salmonella's alternate lifestyles. USDA-ARS Western Regional Research Center, May 6, 2016. Albany, CA Teplitski, M. (Presentation) An avirulent Salmonella surrogate strain: construction, validation and potential uses. 2016 Florida Association for Food Protection Annual Education Conference. May 11-13, 2016. Crystal River, FL Teplitski, M. (Presentation) Microbiome transitions leading to the black band disease in corals. Protect Our Reefs Foundation Board Meeting, April 28, 2016. Key West, FL Teplitski, M. (Presentation) Future of biology education: flipped, blended or shaken up? Biology Department, Xavier University, May 3, 2016. New Orleans, LA. Teplitski, M. (Presentation) Sustainable future: staying relevant through the 21st century. Horticulture and Landscape Architecture Department, Colorado State University, April 26, 2016. Fort Collins, CO Teplitski, M. (Seminar) Food security and food safety challenges in the 21st century. Centro de Investigaciones Biotecnológicas del Ecuador, Escuela Superior Politecnica del Litoral, June 27-28, 2016, Guayaquil, Ecuador Teplitski, M. (Workshop) Research program management and grant writing workshop. Escuela Superior Politecnica del Litoral, June 27-28, 2016, Guayaquil, Ecuador Teplitski, M. (Workshop) Research program management and grant writing workshop. Universidad de Cuenca, June 23-24, 2016, Cuenca, Ecuador Teplitski, M. (Workshop) Food Safety Workshop: survey, detection and control of food- and water-borne pathogens. Universidad de Cuenca, June 20-22, 2016, Cuenca, Ecuador Teplitski, M., Paredes, C., Sosa, D. (Round-table discussion) Careers of women in sciences: challenges and professional development opportunities. ESPOL, June 15, 2016, Guayaquil, Ecuador. Teplitski, M. (Workshop) Food- and waterborne pathogens: survey and detection. ESPOL, June 8, 2016, Guayaquil, Ecuador. Teplitski, M. (Workshop) Indicator organisms. ESPOL, June 9, 2016, Guayaquil, Ecuador. Teplitski, M. (Seminar) Of mice and men... and tomatoes: comparative and functional genomics of Salmonella's alternate lifestyles. UC Davis Post-harvest Physiology Group. May 5, 2016. Davis, CA. Teplitski, M. (Seminar) Of mice and men.... and tomatoes: functional genomics of Salmonella's alternate lifestyles. Co-sponsored by the departments of Molecular Genetics and Plant Pathology, the Ohio State University. April 18, 2016. Columbus, OH Teplitski, M. (Seminar) Dysbioses: microbial perspective on coral disease. October 1, 2015. Smithsonian Marine Station, Ft. Pierce, FL. Teplitski, M. (Training session) Food safety considerations on small farms and urban gardens. September 21, 2015, Gainesville, FL Teplitski, M. (Seminar) Of Mice and men... and tomatoes: unveiling hidden lives of human pathogens. Milken Institute School of Public Health, George Washington University. March 23, 2015. Washington, DC Teplitski, M. (Seminar) Salmonella interactions with crop plants: mechanisms and practical consequences. Food Science and Human Nutrition Department, University of Florida. Feb 10 2015. Gainesville, FL The following extension publications were released: Marvasi, M., Teplitski, M., and G.J. Hochmuth. 2015. Contribución de las prácticas de producción de cultivos y las condiciones climáticas a la seguridad microbiológica de los tomates y pimientos. SL417/SS630 https://edis.ifas.ufl.edu/ss630 Marvasi, M., Teplitski, M., and G.J. Hochmuth. 2015. Impacto de las variedades de tomate y su estado de madurez en la susceptibilidad a Salmonella. SL416/SS629 https://edis.ifas.ufl.edu/ss629 What do you plan to do during the next reporting period to accomplish the goals?Our research group will continue to characterize interactions within host-associated microbial communities, signal and nutrient exchange within microbial communities and the food safety consequences of these interactions.

Impacts
What was accomplished under these goals? Under Objective 1, we have carried out a comprehensive assessment of the genes that model human pathogens (Salmonella sv. Typhimurium and Salmonella sv Newport) use to colonize tomatoes and tomatoes that are impacted by a post-harvest spoilage organism (such as soft rot pathogen Erwinia carotovora). In parallel with these concerted research efforts, we also conducted a series of outreach activities, including seminars, workshops and on-line videos to promote both the better understanding of mechanisms underlying the ability of human pathogens to persist on plants and the consumer/industry acceptance of the pre- and post-harvest production practices that promote produce safety. Under Objective 2, we characterized interactions of a major rhizobial regulatory protein, Hfq, with symbiotically-relevant mRNA and sRNA. We determined, for the first time, that Hfq associates with the major regulator of nitrogen fixation and thus contribute to the efficiency of nitrogen fixation. On-going work focuses on understanding the contribution of the plant signals to this regulation. Under Objective 3, we carried out a comprehensive assessment of the coral microbiomes that are impacted by a devastating polymicrobial disease called "black band disease". We also carried out chemical ecology analysis and identified novel chemical signals that contribute to the structuring of the black band disease community. We documented how members of the native coral microbiota respond to these chemicals produced within the black band consortium

Publications

• Type: Book Chapters Status: Published Year Published: 2016 Citation: Teplitski, M., Krediet, C.J., Meyer, J.L, Ritchie, K.B. 2016. Microbial interactions on coral surfaces and within the coral holobiont, in The Cnidaria, past, present and future. The world of Medusa and her sisters. Eds. Goffredo, S. and Dubinsky, Z. Springer-Verlaag
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Meyer, J.L., Rodgers, J.M., Dillard, B.A., Paul, V.J., Teplitski, M. 2016. Epimicrobiota associated with the decay and recovery of Orbicella corals exhibiting dark spot syndrome. Front Microbiol 7:893
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Meyer, J.L., Gunasekera, S.P., Scott, R.M., Paul,V.J., Teplitski, M. 2016. Microbiome shifts and the inhibition of quorum sensing by Black Band Disease cyanobacteria. ISME J 10(5):1204-16
• Type: Journal Articles Status: Published Year Published: 2016 Citation: de Moraes M.H., Chapin, T.K., Ginn, A., Wright, A.C., Parker, K., Hoffman, C., Pascual D.W., Danyluk, M.D., Teplitski, M. 2016. Development of an avirulent Salmonella surrogate for modeling pathogen behavior in pre- and post-harvest environments. Appl Env Microbiol 82(14):4100-11
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Meador, D.P., P.R. Fisher, C.L. Guy, P.F. Harmon, N.A. Peres, Max Teplitski. 2016. Using a dehydrated agar to estimate microbial water quality for horticulture irrigation. J Enviro Qual: 45(4):1445-51
• Type: Journal Articles Status: Published Year Published: 2015 Citation: George, A.E., Salas Gonzalez, I., Lorca, G.L., Teplitski, M. 2015. Contribution of the Salmonella enterica KdgR regulon to persistence of the pathogen in vegetable soft rots. Appl Env Microbiol: 82: 1353-1360
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Marvasi M, de Moraes MH, Salas-Gonzalez I, Porwollik S, Farias M, McClelland M, Teplitski M. 2016. Involvement of the Rcs regulon in the persistence of Salmonella Typhimurium in tomatoes. Environ Microbiol Rep. 2016 Aug 25 10.1111/1758-2229.12457.

Progress 08/24/15 to 09/30/15

Outputs
Target Audience:This project broadly focuses on the role of host-associated microbial communities in public and ecosystem health. We are focusing on three model systems, including persistence of human pathogens in the environment and their interactions with edible plants, polymicrobial diseases of corals and nitrogen-fixing legume-rhizobium symbiosis. Therefore, the audience for this project is also quite diverse and includes other scientists, industry stakeholders, ecosystem managers, general publich and students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported professional development of 3 post-doctoral scientists, three graduate and 12 undergraduate students. It also led to a multi-state collaborative project Tropical Connections: fostering careers of women scientists in agricultural sustainability and water resource management in the tropics (2015-2017), funded by Elsevier foundation. Tropical Connections aims to foster careers of 20 female post-doctoral scientists. The first cohor of 10 female post-docs is about to graduate, and two of the participants have already secured external funding to further their research and professional goals How have the results been disseminated to communities of interest?The PI presented discoveries made by the group members at 101st Southeastern Branch Meeting of ASM, Kennesaw GA. November 13-15, 2015; Smithsonian Marine Station, Ft. Pierce, FL. October 2, 2015; Milken Institute School of Public Health, George Washington University; March 23, 2015. Washington, DCSoutheastern Microbiology Summit - Joint Meeting of Southeastern and Florida Branches of ASM, Ponte Vedra, Fl, September 5-7, 2014; Florida Association for Food Protection Annual Educational Conference, Clearwater Beach, FL, May 28-30, 2014; Joint (8-University) seminar, UNAM, Cuernavaca, Mexico. Nov 18, 2014. We also developed an excercise which could be a component of an undegraduate microbiology classroom. It is a three-week laboratory activity that can complement any microbiology teaching laboratory to expand students' knowledge of the ecology of human enteric pathogens outside of their animal hosts. Throughout this exercise, students learn that edible plant parts could be a natural habitat for enteric bacteria such as non-typhoidal strains of Salmonella enterica. Upon completion of the laboratory experience, students are able to: 1) Develop testable hypotheses addressing the ability of a human pathogen, Salmonella enterica, to colonize and proliferate in vegetables; 2) Determine that different vegetables support the growth of Salmonella to different extents; 3) Conduct statistical analysis and identify any significant differences. A detailed description of the activity was described in Marvasi, M., Chouhudry, M., Teplitski, M. 2015. Laboratory activity that focuses on the proliferation of Salmonella in vegetables. J Microbiol Biol Edu 16(2):230-6 What do you plan to do during the next reporting period to accomplish the goals?We are planning to further characterize the role of host-associated microbial communities in public and ecosystem health. We also have collaborations in place with the scientists at the Smithsonian Marine station and Carribean Coral Reef Ecosystem Program, NOAA and Southeastern Marine Sanctuaries. We conduct regular meetings with the stakeholders to fine-tune research directions as well as research questions

Impacts
What was accomplished under these goals? Objective 1. We have characterized impacts of crop production practices on microbiological safety of tomatoes and peppers. We have also carried out analysis of the genetic mechanisms that allow a model human pathogen, such as Salmonella, to colonize oysters. Salmonella has been isolated from 7.4 to 8.6% of raw oysters in the United States. This represents a significant risk for food-borne illness. Nevertheless, little is known about the factors that influence colonization of oysters (or other shellfish) by Salmonella. We screened an in vivo promoter probe library inside live oysters and identified 19 unique regions as differentially regulated during colonization. Perhaps not surprisingly, known Salmonella virulence genes were identified in this screen. We also tested the hypothesis that Salmonella genes involved in cell-to-cell signaling would be required for the colonization of oyster and their associated microbial communities. Even though in vitro Salmonella perceived signals from oyster bacteria, this ability did not contribute to its fitness within oysters. Objective 2. We are currently working on defining the role of small RNA in the legume-rhizobium symbiosis. Key mutants and reporters have been constructed and assays are currently in progress. Objective 3. Our studies of the coral black band disease (BBD), supported the conclusion that it is a polymicrobial disease consortium dominated by the filamentous cyanobacterium Roseofilum reptotaenium. Roseofilum displaces members of the epibiotic microbiome. We carried out characterization of the 16S ribosomal genes both normal surface microbiomes and BBD consortia on Caribbean corals. We found that the microbiomes of healthy corals were dominated by Gammaproteobacteria, in particular Halomonas spp., and were remarkably stable across distance, depths and time. In contrast, the microbial community structure in BBD consortia was more variable and more diverse. Nevertheless, deep sequencing revealed that members of the disease consortium were present in every sampled surface microbiome of boulder corals (Montastraea, Orbicella) and brain (Pseudodiploria) corals, regardless of the health status. Within the BBD consortium, we identified a cyanobacterial secondary metabolite (lyngbic acid). Lyngbic acid strongly inhibited quorum sensing (QS) in the Vibrio harveyi QS reporters. The effects of this naturally occurring QS inhibitor on bacterial regulatory networks potentially contribute to the structuring of the interactions within BBD consortia. These results were reported by Meyer et al., 2014. We have also carried out genome sequencing of Halomonas meridiana, a dominant member of the coral commensal microbiota. The genome of strain R1t3 reflects its ability to utilize a wide range of carbon sources, including gene homologues for six different glycoside hydrolases as well as other genes with potential functions in coral mucus colonization.

Publications

• Type: Book Chapters Status: Awaiting Publication Year Published: 2015 Citation: 1. Teplitski, M., Krediet, C.J., Meyer, J.L, Ritchie, K.B. 2015. Microbial interactions on coral surfaces and within the coral holobiont, in The Cnidaria, past, present and future. The world of Medusa and her sisters. Eds. Goffredo, S. and Dubinsky, Z. Springer-Verlaag (in press)
• Type: Book Chapters Status: Accepted Year Published: 2015 Citation: 2. Marvasi, M., OConnor, G.A. & Teplitski M. 2015. Use of tetracyclines and ?-lactams in agriculture: Fate in the environment and occurrence of antibiotic-resistance determinants in Gupta, V.K., Zeilinger-Migsich, S., Ferreira Filho, E.X., Duran, C. & Purchase, D. (eds) Microbial Applications  Recent Advancements and Future Developments. Berlin: De Gruyter.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Cox, C.E., Wright, A.C., McClelland M., Teplitski, M. 2015. Influence of Salmonella enterica sv. Typhimurium ssrB on colonization of Eastern oyster, Crassostrea virginica, revealed in a promoter-probe screen. Appl Env Microbiol pii: AEM.02870-15.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Meyer, J.L., Gunasekera, S.P., Scott, R.M., Paul,V.J., Teplitski, M. 2015. Microbiome shifts and the inhibition of quorum sensing by Black Band Disease cyanobacteria. ISME J doi: 10.1038/ismej.2015.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Meyer, J.L, Dillard, B.A., Rodgers, J.M., Ritchie, K.B., Paul, V.J., Teplitski, M. 2015. Draft genome sequence of Halomonas meridian R1t3 isolated from the surface microbiome of the Caribbean Elkhorn coral Acropora palmata. Stand Genom Sci 10:75
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Marvasi, M., Chouhudry, M., Teplitski, M. 2015. Laboratory activity that focuses on the proliferation of Salmonella in vegetables. J Microbiol Biol Edu 16(2):230-6