Source: OHIO STATE UNIVERSITY submitted to
FOODBORNE MICROBES IN FOOD SAFETY, QUALITY AND PUBLIC HEALTH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0189860
Grant No.
(N/A)
Project No.
OHO00208
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 1, 2007
Project End Date
Aug 31, 2013
Grant Year
(N/A)
Project Director
Wang, HU.
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Food Science & Technology
Non Technical Summary
The rapid emergence of antibiotic resistant pathogens threats public health and causes significant financial loss to the society. The food chain serves as the most important avenue introducing microorganisms including antibiotic resistant bacteria to human through daily food intake. The purpose of the proposed study is to investigate both the total microbial flora and antibiotic resistant microbial population in foods and the molecular mechanisms involved in horizontal gene transmission and microbial ecosystem development. Results from the study are essential for the development of counter strategies to combat this major challenge in food safety and public health.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5034010107050%
5034010110050%
Knowledge Area
503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1070 - Ecology; 1100 - Bacteriology;
Goals / Objectives
Food microbiology has historically focused on the study of pathogens or toxin producers causing acute (and sometimes chronic) diseases, spoilage agents affecting food quality, and starter cultures essential for food fermentation or used as probiotics with potential health benefit(s) to the host. Hence, reducing or eliminating pathogenic and spoilage microbes through the farm-to-fork food safety continuum, and improving the performance of starter cultures and probiotics for product quality, specific health benefit(s) and industrial financial assurance, have been the main themes for the food microbiology research for years. However, it has become increasingly apparent that these organisms account for only a small proportion of the microflora associated with foods; diverse microbial species within the environment, foods, and the host interact with one another in ways that are only beginning to be elucidated. For instance, on a daily basis, humans can easily consume 10*10 CFU microorganisms, with up to 10*6 CFU harboring some sort of antibiotic resistance. The vast majority of these organisms are commensal rather than pathogenic. Some of the fermentation starter cultures and even probiotic strains can become carriers of the resistance genes. These organisms have ample opportunity to shape the host ecosystem by interacting with the existing host microflora through various forms of signal exchange, serving as donors for horizontal gene transfer, or becoming part of the host ecosystem, and their impact on the general health of the public and the importance in disease progression are yet to be revealed. Our objective for this proposed 6-year study is to continue investigating the microbial ecology associated with the food chain and to reveal the importance of microbial progression to food safety and quality, as well as the contribution of commensal organisms to ecosystem development, disease progression, and resistance to antimicrobial agents.
Project Methods
We will development a rapid, high throughput detection platform to identify and quantify microorganisms in complex ecosystems such as samples collected from dairy production and processing environments and to investigate the roles of horizontal gene transfer-susceptible foodborne commensal bacteria in transmitting antibiotic resistance genes from the environment to the host. We will continue investigating molecular mechanisms involved in biofilm formation and ecosystem development and the microbial interactions with food matrices, microbes and hosts within such ecosystems involving the fermentation starter culture lactic acid bacteria, the spoilage agent Pseudomonas spp., and the pathogen Listeria monocytogenes. Results from this proposed study will likely generate novel understandings regarding the roles of foodborne microbes on food safety and public health and lead to changes in food industry practices.

Progress 09/01/07 to 08/31/13

Outputs
Target Audience: Scientific community, govenmentagencies, industry and general public including consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project genreated multiple training opportunities for graduate and undergraduate students, post-doctoral fellows and visiting scientists. How have the results been disseminated to communities of interest? By perr-reviewed publication, presentations at local, national and interantional conferences, by presentations as invited speakers at various institutions, reports and scientiifc input submitted to industry, government agencies, and press-releases to both the professional community and the gerenal public. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Following identification ofthe food chain as a major avenue disseminating antibiotic resistance (AR) to human and animal hosts due to commensal bacteria, Wang and collaborators worked with US dairy industry (informed starter culture companies, sponsored project by Dairy Managemen Inc,and in collaboration with US Swiss Cheese Consortium for plant investigation) for AR risk factors in dairy fermentation and made recommendation to the industry(to DMI). By 2010, theabundance of AR gene pool in most retail cheese productshad been significantly reduced (2 logs or more).The Wang lab further successfully identified oral antibiotic administration as a critical risk factor for antibiotic resistance (AR) development and amplification in human and animal gut, and the resistant bacteria and AR gene-rich feces further impacts the AR ecology in the envirionment and therefore the global ecosystem. Wang lab further investigated AR ecology in aquaculture products and environment, and zoo animals. The findings provided solid scientific foundation todevelop targetetd mitigationstrategies to combat antibiotic AR in the global ecosystem across the food chain, human, animals and the environment, and with major impact on publc health, food safety, human medicine and food animal production. Wang lab also developed microbial detection, identification and tracking platforms for microbial causing agents, which enabled the team toeffectively address food safety, quality and public health challengesacross the food chain (fromfarm to fork) and in hosts (human and animals). Theresearch findings havehave social (human beings and animals worldwide) and economic (multibillions) impacts. Wang also led international efforts provided scientific input for government regulatory agencies,impacted funding directionsofUSDA-ARS (5-year strategic plan) and NIFA (food safety RFA). Both Wang and Leelabs have produced a long list of publications, which contributed to scientific understanding in related fields, and disseminated the kowledge to stakeholders.

Publications


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

    Outputs
    OUTPUTS: Dr. Wang conducted research to fill in critical knowledge gaps for antibiotic resistance (AR) mitigation, including AR development in 1) humans, 2) lab animals, 3) pet and zoo animals, 4) cheese fermentation, 5) aquaculture production, 6) food animals, and 7) mechanisms in AR persistence; 8) received additional funding from Pew Foundation and Ohio Soybean Board; 9) organized and delivered food safety symposium in China; 10) presented food safety seminar at multiple Chinese institutes; 11) published a paper on AR in aquaculture products from China; 12) trained two scientists from China, 4 Ph.D. 1 M.S. and 1 undergraduate students; 13) delivered multiple talks at local, regional and international events; 14) built strategic collaboration with China Scholarship Council for workforce training; 15) two students selected by ASM as Young Investor at ASM general meeting; 16) one student won ASM travel award; 17) one student chosen to give oral presentation at IFT general meeting outstanding student research session; 18) published a paper on rapid detection of Listeria, with two more to be submitted; 19) submitted a paper on microbial biofilm, currently in revision; 20). served as co-chair and chair for OARDC seeds grant program; 21) served as Counselor, Food Microbiology Division, American Society for Microbiology. 1) Dr. Lee established a research area of environmental aspect of food safety, especially understanding the linkage of water, climate and fresh produce safety, and has active research projects funded by Food Innovation Center, OARDC, OWDA and EPA etc; Conducted research about 2) impact of climate change on Salmonella and hepatitis A virus internalization in fresh produce; 3) influence of phytopathogen infection (lettuce mosaic virus) on pathogen internalization (Salmonella and HAV); 4) ozonation as an environmental friendly treatment of fresh produce for improving wastewater quality; 5) pathogen internalization in bean sprout when the water is contaminated; 6) UV treatment of internalized Salmonella in fresh produce; 7) microbial source tracking of animal and human fecal contamination in fresh produce; 8) the impact of land use and climate change on microbial transmission in agricultural water; published papers on 9)impact of extreme weather on Salmonella internalization was published; 10) bacterial profiles of fish between storage conditions was published; 12) rapid detection of spore contamination in food packages and food powers using tiered approach of ATP bioluminescence and real-time PCR was published ; 13) ozonation as a clean technology for fresh produce industry and environment was published in Journal of Applied Microbiology; 14) host-specific microbial source tracking for rapid identification of fecal contamination in fresh produce was published; 15) localized Salmonella internalization in green onion was accepted for publication; 16) UV inactivation of internalized Salmonella in fresh produce was accepted for publication; 17) Two food safety-related presentation were made at the IFT meeting and IAFP meeting. PARTICIPANTS: Wang HH is the PI and Lee Jiyoung is co-PI of the project. TARGET AUDIENCES: Scientific community, industry, government agenicies, and internaitonal organizations, general public. PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    Research findings from Dr. Wang's lab changed the scientific understanding of AR development, dissemination and persistence, and triggered the strategic changes in scientific investigation, mitigation strategies and industry approaches. Her research directly contributed to AR mitigation in fermented dairy products (cheese and yogurt), a multi-billion-dollar industry worldwide. Data from her current projects on AR development in humans and animals are anticipated to have major impact on medical treatment for infectious diseases, affecting multi million people, multibillions of food animals and multi-billion-dollar healthcare costs. Her efforts also impacted government policies. The finding on proper detection of viable microbial cells is important for the food products.Furthermore, revealing the RNA stability issue will impact proper data interpretation on many RNA-based studies. The work on Listeria monocytogenes biofilms advanced scientific understanding of the molecular mechanism, and practical application of biofilm removal. Her activities also helped training workforce, advancing the "One University" mission and contributed to improving food safety in the global food chain. Dr. Lee has established an emerging area of food safety that is linked to climate change, especially extreme weather events, and water contamination, which is anticipated to draw a lot of attention in food safety and public health areas since more intensified extreme weather events become frequent due to increased global warming. She studies the sources of microbial contamination, their transport and fate mechanisms, and ecological link to environmental determinants using molecular tools, microbial community analysis and statistical approach. Dr. Lee worked on 'Impact of climate change on Salmonella hepatitis A virus internalization in fresh produce'(PI: Lee, co-Investigators: J. Li, M. Kleinhenz, D. Gardner, funded by Food Innovation Center). The group found that Salmonella were more internalized via root uptake when the plants (lettuce and green onion) underwent extreme weather event stresses and infected by phytopathogen. Some of the result was presented at IFT meeting (2012) and a paper was published in Food Research International. Three more papers were published or under revision. Dr. Lee also conducted research about 'environmental friendly' treatment of pathogen reduction, such as ozone and UV for fresh produce. For example,'Ozonation as a Clean Technology for Environment and Fresh Produce Industry: Disinfection Efficiency and Wastewater Quality'(PI: J Lee, co-PI: A. Yousef and Z. Bohrerova, funded by Ohio Water Development Authority) was completed. Her group found that ozonation of fresh produce is more beneficial in disinfecting spores and the wastewater quality was improved by using ozone when compared with chlorine. The result was published in Journal of Applied Microbiology. Dr. Lee and her research team (P. Grewal and Z. Bohrerova) started a new project (funded by Food Innovation Center) studying about window-based hydroponic food production systems for improving local food security and promoting health in urban environments.

    Publications

    • 8.J. Lee, S. Agidi, C. Lee, and J. Marion. Arcobacter in Lake Erie beaches: an emerging gastrointestinal illness agent and its contamination sources. International Water Association Water Congress, Busan, Korea, September 2012.
    • 9.C. Ge, C. Lee and J. Lee. A link between climate change and food safety: the impact of extreme weather events on pathogen internalization in green onion via contaminated irrigation. International Water Association Water Congress, Busan, Korea, September 2012.
    • 10.J. Lee. Who polluted the water Multiple genetic markers and pyrosequencing approaches for understanding health risks in US beaches. Jiangsu Institue of Parasitic Diseases, Wuxi, China, Aug 2012.
    • 11.C. Rea and J. Lee. Linkages between avian-associated pathogens and human health in wetland and recreational water environments of Lake Erie. [Abstract]. EcoHealth 2012, Kunming, China, October 2012.
    • 12.J. Lee, S. Agidi, C. Lee, and J. Marion. An emerging gastrointestinal pathogen associated with human-originated contamination sources in Lake Erie beach waters. [Abstract]. 4th International EcoSummit, Columbus, September 2012.
    • 13.C. Ge, J. Lee. Use of UV-C light and Chemical Sanitizers to Inactivate Internalized Salmonella Typhimurium in Iceberg Lettuce. [Abstract]. International Association for Food Protection Annual Meeting. Providence, Rhode Island, July 2012.
    • 14.J. Lee and C.S. Lee. Cyanobacteria and Legionella in Freshwater Beaches : a Hidden Public Health Risk under Current Beach Monitoring Systems. [Abstract]. American Society for Microbiology 112th General Meeting, San Francisco, June 2012.
    • 15.C. Lee, J. Marion and J. Lee. The potential association between toxin-producing Cayanobacteria and fecal contamination s in Lake Erie beaches beach waters. [Abstract]. American Society for Microbiology 112th General Meeting, San Francisco, June 2012.
    • 16.C.S. Lee and J. Lee. Application of Microbial Source Tracking for Rapid Detection of Animal- and Human-Specific Fecal Contamination in Fresh Produce. [Abstract]. American Society for Microbiology 112th General Meeting, San Francisco, June 2012.
    • 17.B. Layton, Y. Cao, E. Ballest, A. Farnleitner, M. Gourmelon, J. Lee, W. Meijer, R. Noble, L. Peed, G. Reischer, L. Van De Werfhorst, A. Boehm, P. Holden, J. Jay, O. Shanks, J. Griffith. Performance of human fecal-associated PCR-based methods in the California Source Identification Protocol Project. [Abstract]. American Society for Microbiology 112th General Meeting, San Francisco, June 2012.
    • 18.C. Ge, Z. Bohrerova, J. Lee. Inactivation of Internalized Salmonella Typhimurium in fresh produce with combined application of UVC and chemical sanitizers. [Abstract]. Institute of Food Technologists Annual Meeting, Las Vegas, June 2012
    • 19.S.H. Park, V.M. Balasubramaniamab, S. K. Sastry, J. Lee. Inactivation of Bacillus amyloliquefaciens and Bacillus stearothermophilus Spores using Combined Pressure, Electric field, and Heat Treatment. [Abstract]. Institute of Food Technologists Annual Meeting, Las Vegas, June 2012.
    • 20.J. Lee. Microbial Population Structure in Freshwater Beaches Revealed by Pyrosequencing and Its Public Health Implications. [Abstract]. International Meeting of the Microbiological Society of Korea, Seoul, Korea, May 2012.
    • 21.J. Lee. Who Polluted the Water The Impact of Beach Water Contamination on Human Health, 'Area of Excellence in Urban Water Infrastructure, Policy, and Infectious Diseases' University of Illinois-Chicago, March 2012.
    • 22.J. Lee. The Link between Climate Change and Water & Food Safety: Question#1, Do Extreme Weather Events Affect Fresh Produce Safety Division of Environmental Health Sciences, Ohio State University, Columbus, Ohio, Feb 2012.
    • 23.J. Lee. Impact of Contaminated Stormwater on Inland Beaches: A Public Health Perspective. Presented at Ohio Water Quality and Wastewater Conference, Columbus, Ohio, Feb 2012.
    • 24.J. Lee. Rapid Determination of On-site Wastewater Treatment Efficiency with Human-specific IMS/ATP and qPCR. Ohio Water Quality and Wastewater Conference, Columbus, Ohio, Feb 2012.
    • 25.J. Lee. Does Extreme Weather Events Affect Fresh Produce Safety Ohio Water Quality and Wastewater Conference, Columbus, Ohio, Feb 2012.
    • 1.Xiao L, Zhang L, Wang HH. 2012. Critical issues in detecting viable Listeria monocytogenes cells by real-time reverse transcriptase PCR. J Food Prot. 75(3):512-517.
    • 7.Huang Y, Zhang L, Ye L, Wang WF, Tiu L, Wang HH. 2012. Antibiotic Resistance in Aquaculture Products from Domestic Production. IFT annual meeting oral presentation (session on outstanding student research).
    • 8.Wang HH. 2012. Enhancing Food Safety and Public Health through Global Collaboration. Shanghai Academy of Agriculture Sciences. 2/29/2012.
    • 9.Wang HH. 2012.Strategic Collaboration in Food Safety Research, Education and Outreach. Fudan University. 11/6/2012.
    • 10.Wang HH. 2012. Strategic Collaboration in Food Safety Research, Education and Outreach. Shanghai Ocean University. 11/6/2012.
    • 11.Wang HH. 2012 Strategic Collaboration in Food Safety Research, Education and Outreach. China International Food Safety and Quality Conference + Expo 2012. 11/8/2012.
    • 12.Wang HH. 2012. Organism and Society: Both Sides of the Microbial World. Shanghai Academy of Agricultural Sciences. 1/21/2013.
    • 13.Wang HH. 2012. Combating the Transmission of Antibiotic Resistance through the Global Food Chain. USDA-ISE PI meeting. 5/16/2012.
    • 14.Wang HH. 2012. From food to health: the global ecological approach. Sina-ASM annual meeting. 6/16/2012.
    • 2.Ye L, Zhang L, Li X, Shi L, Huang Y, Wang HH. 2012. Antibiotic resistant bacteria associated with retail aquaculture products from Southern China. J Food Prot. (In print).
    • 3.Wang WF, Kawatra K, Buckley TJ, Wang HH. 2012. Houseflies as Potential Vectors for Antibiotic Resistant Bacteria. ASM General Meeting Oral Presentation (Young Investigator, by Wang WF).
    • 4.Zhang L, Li Y, Li X, Lavin S, Wang HH. 2012. Antibiotic Resistance in Non-Food Animals: Impact of Animal Hosts on the Resistance Ecology. ASM General Meeting Oral Presentation (Young Investigator, by Zhang L).
    • 5.Ye L, Zhang L, Li XH, Shi L, Wang HH. 2012. Prevalence of antibiotic resistance in aquaculture products: assessments of samples from Southern China. ASM General Meeting poster presentation.
    • 6.Shao Y, Wang HH. 2012. Prevalence and Diversity of Antibiotic Resistant Bacteria in Soil Samples in and around a Poultry Farm. ASM General Meeting poster presentation.
    • 15.Wang HH. 2012. Antibiotic Resistance and Targeted Mitigation-What's our chance To Ohio Congressional Fellows. 8/10/2012.
    • 16.Wang HH. 2012. Essentials to Effectively Deliver the Global Food Safety Impact:Input from a Principal Investigator. USDA-National Institute of Food and Agriculture. 10/25/2012.
    • 17.Wang HH. 2012. Minimizing Antibiotic Resistance Transmission through the Food Chain. University of Michigan School of Public Health. 11/16/2012.
    • 18.Wang HH. 2012. From food to health: the global ecological approach. Dept Food Sci & Tech Seminar. 5/3/2012.
    • 19.Wang HH. 2012. The power of cutting-edge research, strategic education and communication. PHPID annual meeting. 4/27/2012.
    • 1.C. Ge, Z. Bohrerova, J. Lee. 2012. Inactivation of internalized Salmonella Typhimurium in lettuce and green onion using combination of ultraviolet C irradiation and chemical disinfectants. Journal of Applied Microbiology (in press)
    • 2.C. Ge, C. Lee, J. Lee. 2012. Localization of Viable Salmonella Typhimurium Internalized through the Surface of Green Onion during Pre-harvest. Journal of Food Protection (in press)
    • 3.M.Y. Cheun, S. Liang, J. Lee*. 2012. Toxin-producing Cyanobacteria in Freshwater: A Review of their Problems, Impact on Drinking Water Safety, and Efforts for Protecting Public Health. Journal of Microbiology (in press)
    • 4.S. Agidi, S. Vedachalam, K. Mancl, J. Lee. 2012. Rapid determination of fecal bacteria in an onsite wastewater reuse system with human-specific IMS/ATP and qPCR. Journal of Environmental Management (in press)
    • 5.C.S. Lee, J. Lee. 2012. Application of host-specific source tracking tools for rapid identification of fecal contamination in fresh produce by human and livestock. DOI 10.1002/jsfa.5855
    • 6.C. Lee, S.Agidi, J. Marion, J. Lee. 2012. Arcobacter in Lake Erie beach waters: an emerging gastrointestinal pathogen linked with human-associated fecal contamination. Applied and Environmental Microbiology. 778:16:5511-5519.
    • 7.J. Marion, J. Lee, J. Wilkins, S. Lemeshow, E. C. Lee, Waletzko, T. Buckley. 2012. In Vivo Phycocyanin Flourometry as a Rapid Screening Tool for Predicting Elevated Microcystin Concentrations at Inland Beaches. Environmental Science & Technology. 46:8:4523-4531.


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

    Outputs
    OUTPUTS: Dr. Wang conducted research to fill in critical knowledge gaps for antibiotic resistance (AR) mitigation, including AR development in 1) humans, 2) lab animals, 3) pet and zoo animals, 4) cheese fermentation, 5) aquaculture production, and 6) mechanisms in AR persistence,7) targeted mitigation in dairy fermentation, funded by USDA-ISE, DMI, and OARDC. Received funding from Ohio Soybean Board to examine AR in domestic aquaculture production, and the impact of aquaculture practice on food safety.Led several initiatives for strategic changes on AR mitigation and policies including 8)synthesized and submitted expert input on NARMS 5-year strategic plan; 9) organized and delivered the symposium on the impact of globalization on US food safety at ASM General Meeting;10)organized an AR special issue for Appl Envrion Microbiol,for a comprehensive picture on AR and mitigation; 11) published the Commentary paper in AEM on the direction to combat AR. 12) worked with campus leadership to inform legislators, for targeted investment to facilitate AR mitigation in medicine and the food chain;13)delivered many invited talks at local, regional and international events; 14) 4 Ph.D. students, 2 post-doctoral associates, 1 undergraduate student received training on the subject. 15) Continued studies on rapid detection of microbes, including better detection of viable cells n food. 16) Revealed a molecular attribute in Listeria Monocytogenes biofilm formation. Two more Ph.D. students received training in these areas. 17) Helped the OSU China Gateway food safety effortby establishing a joint graduate training program between China (funded by Chinese Overseas'scholarship program) and U.S., and a joint food safety center between OSU and Fudan Univ. 18) Advised one exchange Ph.D. student and one post-doctoral visiting scientist from China. 19) 4 Ph.D. dissertation, 8 peer-reviewed papers and book chapters (including one in press, 2 accepted) were published. One patent was awarded, one provisional patent was filed. Dr. Lee established a research area of environmental aspect of food safety, in understanding the linkage of water, climate and fresh produce safety, and has active research projects funded by CAPPS, Food Innovation Center, OARDC, and OWDA; She examiend 20)the impact of climate change on Salmonella and hepatitis A virus internalization in fresh produce; 21)the influence of phytopathogen infection on pathogen internalization; 22)ozonation as an environmental friendly treatment of fresh produce for improving wastewater quality; 23)pathogen internalization in bean sprout when the water is contaminated; 6)developed a high-throughput detection tool for bacterial spores in food package and food powder; 24)microbial source tracking of animal and human fecal contamination in fresh produce and its health impact; 25)disinfection of pathogens in fresh produce using UV; 26)the impact of land use and climate change on microbial contamination in agricultural water. She published 3 papers and 3 presentations, and one more paper and one more conference presentation were submitted. Provided training opportunities for 6 students and postdoctoral fellows. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Scientists, government agencies, industry and the general public PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

    Impacts
    Research findings from Dr. Wang's lab changed the scientific understanding of antibiotic resistance development, dissemination and persistance, and triggered the strategic changes in investigation and mitigation direction and approaches. Her research directly contributed to AR mitigation in fermented dairy products (cheese and yogurt), a multi-billion-dollar industry worldwide. Data from her current projects on AR development in humans and animals are anticiapted to have major impact on medical treatment for infectious diseases, affecting multi million people and multi-billion-dollar healthcare cost every year. Her effort on revealing a comprehensice scientific picture of AR and facilitating the development and implementaion of innovative, targeted mitigation has led to strategic changes in U.S. governemnt (USDA-ARS 5-year strategic plan), international organization (WHO/FAO risk assessment documentation), and industry practice (dairy, starter culture, probiotics). The finding on proper detection of viable microbial cells is very important for improving the safety and quality of food products.Furthermore, revealing the RNA stability issue will impact proper data interpretation on many RNA-based studies. The work on Listeria monocytogenes biofilms advanced scientific understanding of the molecular mechanism, and practical applicaion of biofilm removal. Her activities also helped training workforce, advancing the "One University" mission and contributed to improving food safety in the global food chain. Dr. Lee has established an emerging area of food safety that is linked to climate change, especially extreme weather events, and water contamination, which is anticipated to draw a lot of attention in food safety and public health areas since more intensified extreme weather events become frequent due to increased global warming. She studies the sources of microbial contamination, their transport and fate mechanisms, and ecological link to environmental determinants using molecular tools, microbial community analysis and statistical approach. Dr. Lee worked on Impact of climate change on Salmonella hepatitis A virus internalization in fresh produce. The group found that Salmonella were more internalized via root uptake when the plants (lettuce and green onion) underwent extreme weather event stresses and infected by phytopathogen. Some of the result was presented at IFT meeting (July 2011) and a paper was published in Food Research International. Another result will be presented at International Water Association Conference in 2012. Dr. Lee also conducted research about "environmental friendly" treatment of pathogen reduction, such as ozone and UV for fresh produce. For example, "Ozonation as a Clean Technology for Environment and Fresh Produce Industry: Disinfection Efficiency and Wastewater Quality" has been studied. Her group found that ozonation of fresh produce is more beneficial in disinfecting spores and the wastewater quality was improved by using ozone when compared with chlorine. The result was presented at National Environmental Health Association Annual meeting in June 2011 and a manuscript was submitted in October 2011.

    Publications

    • 4. Zhang L, Kinkelarr D, Huang Y, Li Y. Li XJ, Wang HH.. 2011. Antibiotic resistance: are we born with it Appl. Environ. Microbiol. 77: 7134-7141.
    • 5. Wang HH, Schaffner DW. 2011. Antibiotic resistance and mitigation: how much do we know and where should we go from here Appl. Environ. Microbiol. 77:7053-7059.
    • 6. Xiao L, Zhang L, Wang HH. 2011. Critical issues in detecting viable Listeria monocytogenes cells by real-time RT-PCR. J. Food Prot.(In press).
    • 7. Wang HH, McEntire JC, Zhang L, Li XJ, Doyle M. 2011. Antimicrobial resistance in the food chain. OIE Scientific and Technical Review, Vol. 31 (1). (Accepted).
    • Patents 8. Wang HH, Luo H. 2011.Patent awarded 10/18/2011. patent #8038990. Compositions and methods for the prevention and removal of biofilms on inert and biological surfaces.
    • 9. Wang HH. 2011. Antibiotic and antimicrobial compositions for Targeted and Personalized Treatment. Tech ID # 2012-078.
    • Conference abstracts 10.Zhang L, Kinkelaar. D, Huang Y, Wang HH. 2011. Acquired antibiotic resistance: drug, food Exposure or are we born with it ASM general meeting. Program book p.94, session 31, #346.
    • 11.Wang HH. 2011. The global impact on seafood safety. ASM general meeting symposium speaker. Program book p.58. Session 168.
    • 12.Xiao L, Wang HH. 2011. Selective detection of viable spoilage Pseudomonas spp. using Propidium Monoazide-coupled Taqman real-time PCR. IAFP annual meeting. (Graduate student research competition finalist).
    • Presentations 13.Wang HH. Wang HH. 2011. Enhancing International Collaboration on Food Safety. Jiang Nan University College of Food Science, Wuxi, China. 09/21/2011
    • 14.Wang HH. 2011. Food Safety: From Problem Detection to Mitigation. Nan Jing Agriculture University. Nan Jing, China. 09/22/2011
    • 15.Wang HH. 2011. Food Safety: From Problem Detection to Mitigation. Shanghai Society for Microbiology, Division of Food Microbiology. 09/26/2011.
    • 16.Wang HH. 2011. From hazard detection to formulating health: cultivating the next generation of life engineer. Nestle R&D Center, Marysville, OH. 2/11/2011.
    • 17.Wang HH. 2011. From hazard detection to effective mitigation. OVIFT annual meeting, 3/10/2011.
    • 18.Wang HH. 2011. Impact of globalization of seafood safety. ASM general meeting symposium invited speaker. 5/23/2011.
    • 19.Wang HH. 2011. From hazard detection to formulating health: cultivating the next generation of life engineer. Nestle R&D Center, Marysville, OH. 2/11/2011.
    • 20.Wang HH. 2011. From hazard detection to effective mitigation. OVIFT annual meeting, 3/10/2011.
    • 21.Wang HH. 2011. Impact of globalization of seafood safety. ASM general meeting symposium invited speaker. 5/23/2011.
    • Art work 22. Wang HH, Zhao A. 2011. (Concept creator, image designer)."Antibiotic Resistance: Can We Solve the Puzzle"Magazine cover, Cover page, Applied and Environmental Microbiology Special Issue on Antibiotic Resistance. Vol 77 issue 20.
    • Lee Peer-reviewed papers 1.J. Rosenblum, C. Ge, Z. Bohrerova, A. Yousef, and J. Lee. 2011 Ozonation as a Clean Technology for Fresh Produce Industry and Environment: Sanitizer Efficiency and Wastewater Quality. (under review) (submitted in October, 2011)
    • 2.W. Ratphitagsanti, E. Park, C.S. Lee, R-Y. A. Wu, J. Lee. 2012. High-throughput detection of spore contamination in food packages and food powers using tiered approach of ATP bioluminescence and real-time PCR. LWT-Food Science & Technology 46:1:341-348. DOI: 10.1016/j.lwt.2011.09.003
    • 3.C. Ge, C.S. Lee, Z. Yu and J. Lee. 2012. Comparison of Bacterial Profiles of Fish between Storage Conditions at Retails Using DGGE and Banding Pattern Analysis: Consumer's Perspective. Food and Nutrition Sciences (in press)
    • 4.C. Ge, C. Lee, and J. Lee. 2011. The Impact of Extreme Weather Events on Salmonella Internalization in Lettuce and Green Onion. Food Research International (in press) doi: 10.1016/j.foodres.2011.06.054
    • Abstract and Conference Presentations 5.C. Ge and J. Lee. 2011. Salmonella Internalization Affected by Water Stress. . Institute of Food Technologists Annual Meeting. New Orleans, July, 2011.
    • 6.W. Ratphitagsanti and J. Lee. 2011. Rapid assessment of disinfection efficiency of ozone, chlorine dioxide and chlorine using Bacillus spores as a protozoa surrogate. National Environmental Health Association Meeting, Columbus, Ohio, June 2011.
    • 7.C. Ge, J. Rosenblum, Z. Bohrerova, A. Yousef and J. Lee. 2011. Ozonation for environmental friendly treatment of fresh produce and improved wastewater quality, National Environmental Health Association Meeting, Columbus, Ohio, June 2011.
    • Wang HH: Papers, books and chapters: 1. Zhang L, McEntire JC, Newsome RL, Wang HH. 2011. Antimicrobial resistance. In Food Microbiology: Fundamentals and Frontiers, Fourth Edition (Doyle MP, Buchanan RL, eds). ASM Press. (Accepted).
    • 2. Li XH, Li YL, Alvarez V, Harper WJ, Wang HH. 2011. Antibiotic resistance mitigation in dairy fermentation. Appl. Environ. Microbiol. 77:7171-7175.
    • 3. Li XH, Alvarez V, Harper WJ, Wang HH. 2011. Identification of a persistent, TA-independent tetracycline resistance-encoding plasmid from a dairy Entercococcus faecium isolate. Appl. Environ. Microbiol. 77:7096-7103.


    Progress 01/01/10 to 12/31/10

    Outputs
    OUTPUTS: Wang HH: 1. Served as Program Organization Committee Member, symposium organizer and speaker at 2010 China International Food Safety & Quality Conference + Expo (CIFSQ); 2. Provided expert opinion in country brief meeting in China for FDA, CDC , USDA, CSPI senior food safety officers; 3.Served as USDA-ISE and ARS panel member, provided expert input for USDA's international science education and ARS food safety molecular biology programs; 4. As Chair of the Food Microbiology Division, ASM, provided leadership to the society; 5.Organized the International Consortium on Antibiotic Resistance Mitigation and Food Safety (ICARM) with 15 units from 9 states in the US and 6 units from China for coordinated efforts among government, academic and industry to control antibiotic resistance in the global food chain. Submitted a $2 million consortium proposal to USDA-NIFSI program. 6. As OARDC Competitive Grant panel member, provided expert input for funding decisions; 7.As 2010 USDA-MOA scholar exchange program food safety team leader, organized the team visit to multiple academic, government and industrial units in China, presented food safety symposia at ICARM Kickoff meeting and CIFSQ conference. 8. Visited National Key Laboratory in Meat Safety in Xiamen, affiliated farm and Fudan Univ. in Shanghai, discussed collaboration efforts. 9. Conducted expert interview with Drs. Stuart Levy (Tufts University) and Tara Smith (Iowa University) on AR, as part of the video production targeted for public broadcasting; 10. Served as advisory board member for OSU China Gateway initiative providing expert input on food safety; 11. Accompanied OSU and CFAES leadership (President & Dean) visited China in July 2010, arranged visits and food safety collaboration discussions with CAAS, CAS, Nestle. 12. Conducted seafood AR research project collaboration with South China University of Science and Technology; 13. Presented a seminar at USDA-NIFA to program officers on the Impact of Antibiotic Resistance on Global Food Safety. 14. Presented series of food safety seminars during the trips in China. Eight active funded projects from USDA-ISE, USDA-NIFSI, USDA-NNF, CAPPS, CIFT, OARDC/OSUE, OARDC, Battelle Endowment with total funding of $604,229. Jiyang Lee: 1. Ozonation as a Clean Technology for Environment and Fresh Produce Industry: Disinfection Efficiency and Wastewater Quality (PI: J Lee, co-PI: A. Yousef and Z. Bohrerova, funded by Ohio Water Development Authority). We found that ozonation of fresh produce is more beneficial in disinfecting spores and the wastewater quality was improved by using ozone when compared with chlorine. An abstract was submitted to National Environmental Health Association Annual meeting (June 2011) and a manuscript is under preparation.2. Impact of climate change on Salmonella internalization in fresh produce (PI: Lee, co-Investigators: J. Li, M. Kleinhenz, D. Gardner, funded by Food Innovation Center). We found that Salmonella were more internalized when the plants (lettuce and green onion) underwent extreme weather event stresses. An abstract was submitted to IFT meeting (July 2011) and we will submit a paper in February 2011. PARTICIPANTS: PI: Hua Wang co-PI: Jiyang Lee. The project provided training opportunities for students and post-doctoral fellow Xiaojing Li, Xinhui Li, Lu Zhang, Yingli Li. TARGET AUDIENCES: Academic, industry, government and consumers, both domestic and international. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

    Impacts
    The groundbreaking research from Wang's laboratory since 2004 is changing the picture of the scientific understanding regading antibiotic resistance origination, dissemination, persistance throughoutthe food chain, enviroment and hosts, and its impact on policy and effective mitigation strategies worldwide is anticipated. As the project organizer and co-chair of the scientific organization committee of the 2008 USDA-sponsored project "A food Safety and Public Health Frontier: Minimizing Antibiotic Resistance Transmission through the Food Chain", she summerized and submitted the expert input resulting from the conference to key government agencies, and the content has been directlyy incorporated into USDA-ARS 5-year strategic plan, USDA-NIFSI RFA, and the concept has been reflected in WHO/FAO document as well. The document is now officially published by American Society for Microbiology in 2010, provided scientific directions for academic, government and industry on this highly important food safety and public health topic. In collaboration with the US Dairy industry, Dr. Wang's lab further identified critical control points for antibiotic resistance mitigation in dairy fermentation, directly contributed to the significant reduction of of antibiotic resistant bacteria in fermented dairy products within just 4 years. This sets a great example of effective collaboration among academic and industry for effective AR mitigation. It is anticipated the efforts in global food safey and AR mitigation will contribute to the building of capacity in food safety research, teaching and extension in China and US, enhance global collaboration, and help with effective food industry operation at the global level. USDA, OARDC, as well as other industry and academic fundings supported the study. Dr. Lee conducted the study on Ozonation as a Clean Technology for Environment and Fresh Produce Industry: Disinfection Efficiency and Wastewater Quality (PI: J Lee, co-PI: A. Yousef and Z. Bohrerova, funded by Ohio Water Development Authority). Her group found that ozonation of fresh produce is more beneficial in disinfecting spores and the wastewater quality was improved by using ozone when compared with chlorine. An abstract was submitted to National Environmental Health Association Annual meeting (June 2011) and a manuscript is under preparation. A second project she worked on is Impact of climate change on Salmonella internalization in fresh produce (PI: Lee, co-Investigators: J. Li, M. Kleinhenz, D. Gardner, funded by Food Innovation Center). The group found that Salmonella were more internalized when the plants (lettuce and green onion) underwent extreme weather event stresses. An abstract was submitted to IFT meeting (July 2011) and we will submit a paper in February 2011.

    Publications

    • Peer reviewed papers: 1. Li XJ, Wang HH. 2010. Profiles of antibiotic resistant commensal bacteria from representative ready-to-consume deli and restaurant foods. J Food Prot. 73(10): 1841-1848.
    • 2.Wang HH. 2010. Antibiotic Resistance Mitigation: a Complicated Issue Begging for Targeted Investigation. Microbe.
    • 3.Doyle et al, 2010. Global Food Safety-Keeping Food Safe from Farm to Table. American Academy for Microbiology (AAM) colloquium report.
    • 4.Bi SL, Yan H, Chen M, Zhang Z, Shi L, Wang H. 2010 New variant Salmonella Genomic Island 1-U in Proteus mirabilis clinical and food isolates from South China. J Antimicrob Chemother. Accepted. (JAC-2010-1459.R2).
    • Abstracts and conference presentations: 5.Li XJ, Sun K, Zhang L, Li YL, Wang HH. 2010. The Involvement of Animal Host in the Enrichment of Antibiotic Resistance. IFT annual meeting. #037-45.
    • 6. Zhang L, Li XJ, Wang HH. 2010. The prevalence of antibiotic resistant bacteria in aquaculture products from Hawaii and South China. IFT annual meeting #036-18.
    • 7.Li XH, Alvarez V, Harper WJ, Wang H. 2010. Characterization of an antibiotic resistant Enterococcus faecium strain from fermented dairy product. IFT annual meeting #037-41.
    • 8.Li XJ, Wang HH. 2010. Profiles of Antibiotic Resistant Commensal Bacteria from Representative Ready-to-Consume Deli and Restaurant Foods. 2010 OARDC annual research meeting graduate student research competition poster presentation (2nd place winner); Ohio Branch ASM annual meeting graduate student research competition oral presentation finalist.
    • 9.Zhang L, Li XH, Li XJ, Wang HH 2010. Prevalence of Antibiotic Resistant Bacteria in Seafood Products and Its Impact on Global Food Safety. OARDC annual research meeting poster presentation; OVIFT annual meeting poster presentation.
    • 10. Wang HH. 2010. The impact of the food chain on antibiotic resistance transmission to humans. Chinese Society for Microbiology Annual Meeting Plenary Session speaker.
    • 11. Wang HH. 2010. Organism and society: both sides of the microbial world. Symposium speaker at Chinese Academy of Sciences Institute of Microbiology;
    • 12. Wang HH. 2010. Antibiotic Resistance: How much do we know and where to go from here. Microbial Pathogenesis symposium speaker at Tsinghua University Public Health Forum.
    • 13.Wang HH. 2010. Antibiotic resistance: how much do we know and where to go from here. International Consortium on Antibiotic Resistance Mitigation and Food Safety (ICARM) kickoff meeting, Tianjin, China.
    • 14.Wang HH. 2010. Antibiotic resistance: how much do we know and where to go from here Fudan University College of Biological Sciences. Shanghai, China.
    • 15.Wang HH. 2010. Food Safety: How much do we know and where to go from here. Ohio Agriculture Research and Development Center (OARDC) annual research meeting keynote speaker; Wooster, OH.
    • 16.Wang HH. 2010. Combating Antibiotic Resistance Transmission through the Food Chain-Impact and How to Get There. ASM Ohio Branch annual meeting keynote lecture speaker.Mason, OH.


    Progress 01/01/09 to 12/31/09

    Outputs
    OUTPUTS: 1.Dr. Wang served as the organizer, scientific organization committee co-chair, session moderator and speaker for the USDA NIFSI and OSU co-sponsored Conference on Food Safety and Public Health Frontier: Minimizing Antibiotic Resistance (AR) Transmission through the Food Chain, in April 2009 (Crystal City, VA). More than 80 expert attendees from government, industry and academia re-evaluated the impact of the food chain on AR dissemination for targeted mitigation. Conference brochure including program, compiled abstracts for presentations, and a list of attendees was delivered to attendees. An expert report was later developed and submitted to USDA-NIFA food safety program officers and ARS national food safety program leader to guide future activities. 2. Sponsored by USDA-ISE, She has established research collaboration with South China University of Technology in Guangzhou, China. Two OSU students visited Guangzhou, collected samples and conducted genetic assessment for AR. Dr. Wang provided technical training and guided related research activities there. She visited Hawaii, and collected samples from remote geographic locations for preliminary baseline data assessment. 3. She accompanied FAES leaders Moser, Martin and Krygier visited Beijing and Tianjin, helped with discussions on strategic collaboration with multiple units there. 4. She visited East China University of Science and Technology and Shanghai Jiaotong University, discussed potential collaboration and gave seminar to scientists. 5. She served as session moderator and speaker at the 2009 China International Food Safety & Quality Conference + Expo, delivered the message to help China become a global food safety leader through strategic partnership with US institutions, a strategic movement already well received among US institutions, USDA NIFA program officers, OSU leadership, and FAO leader. 6. She provided food safety leadership by serving as Chair-elect, Food Microbiology Division, the American Society for Microbiology (ASM); Chair, Biotechnology Division, IFT; and panel member for USDA NIFA, NIH, OARDC seed programs, provided professional input to the professional organizations and programs. 7. She served as panel member for AAM Colloquium "Global food safety from farm to table', provided expert input to the colloquium report. 8. As ASM branch lecturership speaker she delivered keynote speech at ASM Northern California Branch and North Central Branch (MN, WI, IA) annual meetings. 9. She served as invited symposium speaker on AR at 2009 IAFP annual meeting. 10. She initiated the communication with Senator Brown's office, provided scientific input for his draft bill on AR, and is planning an AR forum through PHPID. 11. As the winner of the 2009 Battelle Endowment Award for Technology and Human Affairs, she is producing a video on AR. She has already recorded the interviews with close to 10 experts. 12. Two MS students graduated from her laboratory. 13. She taught food fermentation and advanced food microbiology, and disseminated food safety and public health advancements to students. 14. She established new collaboration with Lincoln zoo and Microcide, Inc. on related topics. PARTICIPANTS: Dr. Hua Wang, PI; Dr. Yingli Li, post-doctoral research associate, Xinhui Li, Lu Zhang, Linlin Xiao, Andrew Wassinger, Ph.D. students, Xiaojing Li, Monchaya Rattanaprasert, MS students, all from Wang's lab. Collaborating units: South China University of Technology, Microcide Inc, Lincoln Zoo, Nan Kai University and more Chinese institutions. The project provided training opportunities for the above student participants, as well as for collaborators from SCUT and future collaborators from other Chinese institutions. TARGET AUDIENCES: government agencies, policy makers, industry and medical practitioners, consumers. PROJECT MODIFICATIONS: N/A

    Impacts
    Our activities have already had global impact on both food safety and public health related policy and research activities. As a result of the AR conference and expert report, for the first time USDA NIFSI listed AR and commensal bacteria in the 2010 RFA as special emphasis area with allocation for up to $2 million/each for up to two proposals. This is the first action of its kind from federal agencies in decades. Furthermore, this broadened scope of AR was reflected in the Join FAO/WHO Food Standards Programmer Codex Alimentarius Commission Ad Hoc Intergovernmental Task Force report, published in October 2009, ( http://www.codexalimentarius.net/download/report/730/al33_42e.pdf). Our scope was further incorporated into the ARS 5-year strategic plan. We anticipate FDA NARMS will adopt our concept, and major adjustment on both research activities and industrial practices for targeted mitigation will be followed down the road. The AR video targeted for public broadcasting will have further impact on consumers, medical practitioners, industry, government agencies and policy makers for behavior and practices changes. Furthermore, data from this ongoing project will reveal insights of antibiotic resistance prevalence and potential contributing factors, for future targeted mitigation. The strategic collaboration between US and China in research and education will contribute to the long-term food safety and public health worldwide.

    Publications

    • Li XH, Li YL, Alvarez V, Harper WJ, Wang HH. 2009. Assessment of antibiotic resistance in the dairy fermentation environment. Conference on Food Safety and Public Health Frontier: Minimizing Antibiotic Resistance through the Food Chain. Abstract #P2, abstract book p15.
    • Li YL, Lavin SR, Wang HH. 2009. Prevalence of antibiotic resistance in zoo animal foods. Conference on Food Safety and Public Health Frontier: Minimizing Antibiotic Resistance through the Food Chain. Abstract #P6, abstract book p17.
    • Zhang L, Kinkelaar DF, Wang HH, 2009. Tetracycline resistance in infant digestive tract. Conference on Food Safety and Public Health Frontier: Minimizing Antibiotic Resistance through the Food Chain. Abstract #P7, abstract book p18.
    • Wassinger A, Zhang L, Wang HH. 2009. Revealing Listeria monocytogenes Scott A Biofilm Attributes by Expression Analysis. IFT annual meeting poster presentation.152-36.
    • Xiao L, Wang HH. 2009. Critical issues in assessing live Listeria monocytogenes cells by real-time reverse transcription-PCR. IFT annual meeting poster presentation.120-30.
    • Li XH, Li YL, Alvarez V, Harper WJ, Wang HH. 2009. Critical Control Points to Minimize Antibiotic Resistance Contamination in Cheese Products. IFT annual meeting poster presentation. 200-12.
    • Wang HH. 2009. Antibiotic Resistant Commensal Bacteria in Foods and Hosts. IAFP annual meeting (invited speaker).
    • Jaykus L, Wang HH, Schlesinger L. (eds). 2009. Foodborne Microbes: Shaping the Host Ecosystems. ASM Press.
    • Wang HH. 2009. Commensal bacteria, microbial ecosystems and horizontal gene transmission: adjusting our focus for strategic breakthroughs against antibiotic resistance. In Foodborne Microbes: Shaping the Host Ecosystems (Jaykus L, Wang HH, Schlesinger L., eds). p. 267-281. ASM Press.
    • Wang HH. 2009. Antibiotic resistance in processed foods and human digestive tract. Conference on Food Safety and Public Health Frontier: Minimizing Antibiotic Resistance through the Food Chain. Abstract #S3, abstract book p7.


    Progress 01/01/08 to 12/31/08

    Outputs
    OUTPUTS: We have previously discovered the real magnitude of antibiotic resistant bacteria in the food chain. Our objective for this proposed 6-year study is to continue investigating the microbial ecology associated with the food chain and to reveal the importance of microbial progression to food safety and quality, as well as the contribution of commensal organisms to ecosystem development, disease progression, and resistance to antimicrobial agents. We have examined the antibiotic resistant bacterial flora in commercial dairy products, as well as samples from each steps during cheese making, at both OSU dairy pilot plant and a commercial cheese manufacture plant. We have also examined the prevalence of antibiotic resistant bacteria in retail deli and restaurant foods, as well as assessed the efficacy of home cooking methods on inactivating antibiotic resistant bacteria in shrimps. We have further examined the antibiotic resistant bacteria in animal foods for zoo animals. The results were shared with US dairy industry through Dairy Management Inc. (DMI) and starter culture companies. We organized a symposium, "Foodborne Microbes: Shaping the Host Ecosystems" at the 2008 American Society for Microbiology, to re-evaluate the significance of food chain, particularly foodborne commensal bacteria, in transmitting antibiotic resistance to humans. The research results were also shared with international colleagues through an education and training workshop at the 2008 Chinese Food Safety and Inspection Conference in Beijing, China, 2008 International Association of Food Protection (IAFP) annual meeting, OARDC research conference, Center for Microbial Interface Biology (CMIB) annual retreat, and presentations during an 8-day visit to several Chinese universities and government agencies via a USDA-China Ministry of Agriculture scholar exchange program. I have also given invited talks at Miami University, Ohio Antibiotic Resistance and Animal Agriculture Working Group, CMIB annual retreat, Ohio IAFP branch meeting, went to Senator Brown's office with OSU congressional representative discussed with his staff members regarding the significance of the work and urgent need for effective management strategies. Besides publications, this lines of research also led to several external funded projects including $177,682 from DMI (2007-2009), $25,000 from CIFT (with Lincoln Zoo, 2008), $50,000 from USDA-NIFSI, $99,979 from USDA-ISE (2008-2010). We have also examined the efficacy of culture-independent methods in rapid detection of live microbial cells. This line of study resulted a poster presentation at IAFP annual meeting and funding of $59,750 from CAPPS industry consortium. PARTICIPANTS: Xiaojing Li, graduate student. Yingli Li, Post-doctoral visiting scientist Linlin Xiao, graduate student Lu Zhang, graduate student Xinhui Li, graduate student Dan Kindalaar, Graduate student Valente Alveraz, collaborator Jim Harper, collaborator Shana Larvin, collaborator (Lincoln Zoo) TARGET AUDIENCES: Dairy industry, food and agriculture business. See impact statement. PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    We have communicated our discoveries with the starter culture companies. A common probiotic strain supplemented to all yogurt products was then found to contain tetracycline resistance gene, and believed removed from the market. The prevalence of antibiotic resistant bacteria in name-brand cheeses was reduced significantly in the past couple of years. Through working with smaller cheese manufacturer, we identified several critical control points in cheese fermentation to minimize antibiotic resistant bacterial contamination. We anticipate DMI will recommend the new processing control procedures to the dairy industry. Currently we are leading an international initiative to re-evaluate the impact of the food chain on transmitting antibiotic resistance to humans. This can potentially lead to major changes in public health practices (such as antibiotic prescription) and food industry and agriculture operations. Our discoveries on RNA stability from the rapid detection study will also have major impact on the mainstream data interpretation of RNA related studies.

    Publications

    • 17.Wang HH. 2008. International Collaboration on Food Safety and Public Health: Can We Make a Difference China Agriculture University. USDA-China Ministry of Agriculture exchange program US team 9 food safety visit. Beijing, China. (09/12/08)
    • 18.Wang HH. 2008. International Collaboration on Food Safety and Public Health: Can We Make a Difference South China University of Science and Technology. US team 9 food safety visit. Guangzhou, China. 09/16/08.
    • 19.Wang HH. 2008. International Collaboration on Food Safety and Public Health: Can We Make a Difference Guangdong Center for Disease Control. US team 9 food safety visit. Guangzhou, China. 09/16/08.
    • 20.Wang HH. 2008. International Collaboration on Food Safety and Public Health: Can We Make a Difference East China University of Science and Technology. US team 9 food safety visit. Shanghai, China. 09/17/08.
    • 13.Wang HH. 2008. What are we eating every day Ohio Antibiotic Resistance and Animal Agriculture Working Group. Reynoldsburg OH. (08/11/08)
    • 14.Wang HH. 2008. What are we eating every day Miami University, Oxford, OH.( Invited Seminar Speaker, 08/23/08)
    • 15.Wang HH. 2008. What Are We Eating Every Day OSU Center for Microbial Interface Biology Annual Retreat (Invited Symposium Speaker). Columbus, OH. (09/04/08).
    • 16.Wang HH. 2008. Can we make a difference-food safety and public health frontier: history, challenges and opportunities. East China University of Science and Technology, Shanghai, China. (09/08/08).
    • 21.Wang HH. 2008. International Collaboration on Food Safety and Public Health: Can We Make a Difference Bright Dairy, Inc. US team 9 food safety visit. Shanghai, China. 09/17/08.
    • 22.Wang HH. 2008. Frontiers of Food Safety and Public Health Research. China Center for Disease Control Institute of Infectious Diseases. Beijing, China. 09/22/08.
    • 23.Wang HH. 2008. Frontiers of Food Safety and Public Health Research. China Center for Disease Control Institute of Food Science and Nutrition. Beijing, China. 09/23/08.
    • 24.Wang HH. 2008. Frontiers of Food Safety and Public Health Research. Nan Kai University. Tianjing, China. 09/26/08.
    • 9.Wang HH. 2008. What are we eating everyday ASM general meeting (Symposium speaker). session #119P. Final Program p. 116. 06/03/2008. Boston, MA.
    • 10.Li YL, Lavin SR and Wang HH. 2008. Prevalence of tetracycline and doxycycline resistance in animal foods. CMIB annual retreat.
    • 11.Wang HH. 2008. Antimicrobial resistance (ar) and food safety: what the consumer and industry need to know China International Food Safety & Quality Conference& Expo 2008. (Symposium speaker).
    • 12.Wang HH. 2008. Foodborne microbial biofilms and antibiotic resistance. Ohio IAFP branch meeting, Columbus, OH. (3/17/08)
    • 1.Wang HH. 2008. Commensal bacteria, microbial ecosystems and horizontal gene transmission: adjusting our focus for strategic breakthroughs against antibiotic resistance. In Foodborne Microbes: Shaping the Host Ecosystems (Jaykus L, Wang HH, Schlesinger L, eds). ASM Press. (In press)
    • 2.Li XH and Wang HH. 2008. Prevalence and persistence of antibiotic resistance in food products. OARDC research conference.
    • 3.Li XH, Li YL, Alvarez V, Harper WJ, Wang HH. 2008. Stability of antibiotic resistant Enterococcus sp. in the dairy fermentation environment. International Association for Food Protection (IAFP) annual meeting. #P2-18, program book p36. 08/04/2008.
    • 4.Lu Z, Wang HH. 2008. Assessing the efficacy of home-style cooking methods on reducing tetracycline resistant bacteria in shrimp samples. IAFP annual meeting. P05-55, Program book p54, 08/06/2008.
    • 5.Kinkelarr D, Wang HH. 2008. Profiles of tetracycline resistance bacteria in human microflora associated with infant digestive system. IAFP annual meeting. P05-69, Program book p55, 08/06/2008.
    • 6.Li XJ, Wang HH. 2008. Prevalence of antibiotic-resistant bacteria in deli and restaurant foods. IAFP annual meeting. P5-01, program book p.52, 08/06/2008.
    • 7.Xiao L, Tong W, Wang HH. 2008. Development of a real-time, NASBA-Molecular Beacon system for rapid and specific detection of live microbes in juice products. IAFP annual meeting. P4-28, program book p. 48; 08/05/2008.
    • 8.Li XH,Wang HH. 2008. Prevalence of antibiotic resistant commensal bacteria in seafoods and cheese. ASM general meeting. P-136, program book p. 190, 06/04/2008.


    Progress 01/01/07 to 12/31/07

    Outputs
    OUTPUTS: Given the talk "Antibiotic Resistance in Foodborne Microorganisms" at the FDA public hearing, Tuesday, April 10, 2007, to approximately 50 participants including FDA panel for science review, and representatives from other government agencies, industry, academia and consumers. Provided first-hand scientific evidence to FDA, CDC, USDA regarding the magnitude and spectrum of antibiotic resistance contamination in the food chain, particularly those associated with foodborne commensal bacteria, and urged for policy adjustment to take care of this emerging issue threatening food safety and public health. Organized and delivered two symposia "Impact of the Food Chain on the Emergence of Antibiotic Resistance in Humans" at the 2007 American Society for Microbiology (ASM) General Meeting in Toronto, Canada, and "Implications of Antimicrobial Resistance to Food Processors" at the 2007 Institute of Food Technologists (IFT) Annual Meeting in Chicago, the two largest gatherings for microbiologists and food scientists worldwide to disseminate the important scientific findings to the related scientific community, and urged for new coordinated efforts from food processors to combat the AR problem. The groundbreaking discovery was further disseminated by ASM through ASM Press Conference and Press Release on site in Toronto Canada at the General Meeting (ASM official site http://www.eurekalert.org/pub_releases/2007-05/asfm-rgi051707.php), OSU official site http://www.ag.ohio-state.edu/~news/story.phpid=4129) and major public media around the world such as USA Today (http://www.healthscout.com/news/68/8016275/main.html), UK Channel 4 (http://www.channel4.com/news/articles/society/health/food+may+be+spr eading+superbugs/533442), UK Telegraph (http://www.telegraph.co.uk/news/main.jhtmlxml=/news/2007/05/24/nhea lth24.xml), Europe (France) http://www.foodproductiondaily.com/news-by product/news.aspid=76838&k=antibiotic-resistance-bacteria-food-produ ction. Served as organizer and co-editor for the book "Foodborne Microbes: Shaping the Host Ecosystems", by ASM Press, to re-define the roles of foodborne microbes in public health. Published peer-reviewed research papers and abstracts, and given talks at industry and academic settings. PARTICIPANTS: The following people were involved in conducting the work: Hua Wang (PI) Linlin Xiao, Xinhui Li, Hanna Cortado, Lu Zhang, Andrew Wassinger, xiaojing Li, Yingli Li, Dan Kinkelaar (students) Sophia Kathariou (NCSU) collaborator TARGET AUDIENCES: Acadmedic, governemnt, industry, consumers PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    The rapid emergence of antibiotic resistant pathogens is threatening public health and significantly increased the cost for medical care. Identification of the major routes in antibiotic resistance gene dissemination is critical for establishing effective counter strategies to combat this problem. We have revealed for the first time the prevalence of antibiotic resistant commensal bacteria in various ready-to-eat retail food products. The finding of high magnitude (up to 107 cfu per gram of food) of resistant bacteria in many popular food items strongly supports the notion that food chain might have played an important role in directly disseminating antibiotic resistance genes to the general public, independent from the clinical route. We have also discovered that certain commensal bacteria can serve as intermediate facilitating antibiotic resistance gene transmission more than 10,000 times. This is a breakthrough in understanding the major routes leading to antibiotic resistance in humans and the role of commensal bacteria in the spread of these resistance genes, and most likely will lead to major changes in agriculture and food industry practices, as well as in strategic approaches to minimize antibiotic resistance in clinical therapy. The news was covered by ASM Press Conference and Press Release and other major public media such as USA Today. I am currently organizing an international conference through USDA INFSI to gather renowned experts from government, academia and food industry to develop targeted strategies to combat the dissemination of antibiotic resistance through the food chain. I am also in the process of setting up collaboration with foreign institutes and organizing an international food safety conference in Asia through USDA international program for a global effort to combat this world food safety and public health challenge. The social and financial impacts are tremendous. We have received a total of over 1 million dollars from USDA, DMI, CAPPS and OARDC to support our work. Wang, H. , Alvarez, V. B., Harper, W. J. "Limiting antibiotic resistance microflora in dairy fermentation by processing management. " OARDC $49,900 2007-2008 (Grant); Wang, H. , Alvarez, V. B., Harper, W. J. "Maintaining genotype of starter cultures in dairy fermentation." Dairy Management Inc. $177,682 2007-2009 (Grant); Wang, H. "Rapid detection of live microbes in foods" CAPPS $64,900 2006-2007 (Grant); Lee, K. , Yousef, A. E., Wang, H. "Doctoral training in emerging food safety issues 2006-04318" USDA CSREES National Needs $229,500 2006-2011 (Grant) ; Wang, H. "Minimizing Pseudomonas spp. Biofilms in the Food Environment Using Cleaning-in-Place (CIP) System " MAFMA $22,714 2006-2008 (Grant); Wang, H. "Identification of Listeria monocytogenes biofilm determinants. " CIFT $25,000 2007-2008 (Grant) Kathariou S, Wang, H. "Unique genetic and physiological attributes of Listeria monocytogenes implicated in recent outbreaks of listeriosis in the United States. " USDA-NRI $395,703 2006-2008 (Grant);

    Publications

    • Tong, W., Xiao, L., and Wang, H. 2007. Rapid and specific detection of live spoilage yeasts using a real-time NASBA-molecular beacon detection system. OARDC annual research conference. (4/19/2007). Peer Reviewed.
    • Other scholarly presentations Wang, H. 2007. Rapid detection of live microorganisms in foods. Oral presentation to CAPPS industry board.
    • Wang, H. 2007. Developing biofilm interfering formula for industrial applications. Oral presentation to CAPPS industry board.
    • Wang, H. 2007. Antibiotic resistant commensals in the food chain and potential impacts. Multi-State Food Safety Project SDC-313 annual meeting. San Antonio, TX (11/8/2007).
    • Wang, H. 2007. Biofilms and antibiotic resistance gene transmission in the food environment. (8/10/07, invited seminar speaker, UCLA School of Dentistry).
    • Wang, H. 2007. Methodologies for genetic study and modification of bacteria: gram-positive bacteria. In Encyclopedia of Biotechnology in Agriculture and Food (Bridges A, Hoove DG, Wheeler MB, eds). Marcel Dekker. Peer Reviewed.
    • Jaykus, L., Wang, H., and Schlesinger, L. 2008. Foodborne Microbes: Shaping the Host Ecosystems. Peer Reviewed. ASM Press (near completion).
    • Reviews and Abstracts Wang, H. 2007. Antibiotic resistant commensals in the food chain and potential impacts. 2007 IFT annual meeting symposium presentation. Peer Reviewed.
    • Lehman, M., Li, X., and Wang, H. 2007. Prevalence of antibiotic resistant commensal bacteria in seafood and ready-to-eat salads. The Center for Microbial Interface Biology Retreat. Abstract #44. (6/16/2007). Peer Reviewed.
    • Wang, H. 2007. resistant commensals in the food chain and potential impacts. 107th ASM general meeting symposium presentation. Peer Reviewed.
    • Kinkelarr, D., and Wang, H. 2007. Antibiotic resistance development in human oral and gut ecosystems. The Center for Microbial Interface Biology Retreat. Abstract #45. (6/16/2007). Peer Reviewed.


    Progress 01/01/06 to 12/31/06

    Outputs
    The contamination of pathogens or spoilage microorganisms in foods is an important health threat and causes significant financial loss to both the society and food industry. We use cutting-edge technologies to develop rapid detection methods to improve food safety, quality, and security. Biofilms formed by microorganisms attached to are important sources of secondary contamination, key mechanisms for pathogenic and spoilage organisms persistent in the environment, and major threats to human health. Our goals are to understand the molecular events involved in biofilm formation from initiation, maturation, to detachment, and to develop effective strategies to control and remove biofilms. Risks associated with biofilms in the food environment, besides pathogen virulence, also need to be examined. The rapid emergence of antibiotic resistant (ART) pathogens is a major threat to public health. Understanding the main pathways leading to the evolution and transmission of ART bacteria is essential to combat this problem. We are evaluating the potential role of food chain as an important avenue in the evolution and transmission of ART bacteria to human, besides the clinical settings. Using the rapid detection platform established in our group, we have successfully assessed Listeria monocytogenes gene expression in conditions simulate processing environment by real-time RT-PCR. We have found that both temperature and acids found in food processing and storage environment can significantly affect L. monocytogenes virulence gene expression. Because conventional plating methods cannot properly detect injured cells, and the medium and culturing conditions are limited, we have developed real-time PCR method to properly assess the total antibiotic gene pool in the food chain. Using the developed Taqman real-time PCR method, we have found that the tet(S) (for tetracycline resistance) gene pool in retail cheese samples ranged from 10*4-10*8 copies per gram of food. We have further found that 100% of the ready-to-eat salad samples (both retail and from fast-food-chain restaurants) are contaminated with antibiotic resistant bacteria, and the resistance genes from the salad isolates are transmissible to and functional in human residential bacterium. These data strongly supported the notion that the food chain has emerged as an important avenue in transmitting the antibiotic resistant bacteria and resistance encoding genes to human. For the first time, the American Society for Microbiology (ASM) will have a symposium dedicated to evaluate the impact of the food chain in transmitting antibiotic resistance to human at the 2007 annual meeting and I was selected as the organizer and key speaker for the event. I am also serving as one of the three editors for ASM Press working on the book "Foodborne Microbes: Shaping the Host Ecosystem". The ASM Press book committee and external expert reviewers commented it as "truly groundbreaking" in the field. Our group has now been chosen by the US dairy industry to receive the funding to improve processing procedures to combat the contamination of AR in fermented dairy products.

    Impacts
    Our results revealed for the first time that our retail foods, including many ready-to-eat, generally considered "healthy" items, are heavily contaminated with antibiotic resistant bacteria and these bacteria are transmitted to human through daily food intake. Commensal organisms in the food chain not only can serve as a resistance gene reservoir but an enhancer for the transmitting of resistance genes. Our results illustrated a potentially very important avenue in the dissemination of antibiotic resistance genes in the general public, and likely will have enormous impact on food safety and public health. Besides the contribution to scientific understanding, this research also has significant impact on food industry. In the US alone, this research affects the annual production of $20 million pounds of fermented dairy products business. Worldwide, the number is much larger. Its impact on the even larger fresh produce business is yet to be evaluated.

    Publications

    • Lehman M, Wang HH. 2006. Prevalence of antibiotic resistant bacteria in ready-to-eat salad. IFT annual meeting. Abstract#020B-05.
    • Wang HH, McKay LL. 2006. Horizontal gene transfer as an important mechanism facilitating biofilm ecosystem development by Lactococcus lactis. ASM streptococcus genetics meeting. Abstract#236.
    • Wang HH, Manuzon MY, Lehman M, Wan K, Luo H, Wittum TE, Yousef AE, Bakaletz LO. 2006. Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes. FEMS Micrbiol Lett. 254: 226-231. Erratum in: FEMS Microbiol Lett. 2006 255:328.
    • Lu L, Tomita A, Wang H, Buchholz DR, Shi YB. 2006. Transcriptional regulation of the xenopus laevis stromelysin-3 gene by thyroid hormone is mediated by a DNA element in the first intron. J. Biol. Chem. 281:16870-16878.
    • Hanna SE, Wang HH. 2006. Environmental factors affect the expression of Listeria monocytogenes virulence factor inlA. J Food Protect. 69(11):2754-2757.
    • Hanna ES, Wang HH. 2006. Biofilm formation by Listeria monocytogenes. In Biofilms in the Food Environment. (Balschek HP, Wang HH, Agle M, eds.). Blackwell Publishing Co.
    • Manuzon MY, Wang HH. 2006. Mixed Culture Ecosystem. In Biofilms in the Food Environment (Balschek HP, Wang HH, Agle M, eds.). Blackwell Publishing Co.
    • Blaschek PH, Wang HH, Agle M. (eds.) 2006. Biofilms in the Food Environment. Blackwell Publishing Co.
    • Manuzon MY, Hanna SE, Luo H, Wang HH. 2006. Development of a real-time PCR method to quantify antibiotic resistance gene pool in retail dairy products. ASM annual meeting. Abstract#P065.


    Progress 01/01/05 to 12/31/05

    Outputs
    Our group made three important discoveries regarding biofilm formation and antibiotic resistance gene transfer mechanisms in 2005. First, we have demonstrated that conjugation is an independent biofilm forming mechanism in Lactococcus lactis. This is the first illustration in Gram-positive bacteria, that these organisms can use conjugation to spread biofilm-forming attributes within the community to enhance ecosystem development. We are continuing the study to reveal the regulatory mechanisms involved in microbial biofilm development. Second, we have found that a high frequency conjugation system can serve as an internal enhancer facilitating antibiotic resistance gene transfer (more than 10,000 timers higher) in L. lactis. The role of commensal food-borne organisms in facilitating antibiotic resistance gene transfer within mixed culture ecosystems is currently being investigated. Third, we have also found that up to 100,000,000 CFU per serving of antibiotic resistant bacteria are present in retail foods, including many ready-to-eat items such as cheese, lunchmeat, shrimp and salad. Dairy starter cultures including Streptococcus thermophilus are found to be important host for antibiotic resistance genes in cheese fermentation. This matched with our finding in the lab setting, that commensal bacteria might serve as enhancer facilitated the transfer of antibiotic resistance genes. Our results, for the first time, illustrated not only the prevalence and magnitude of the antibiotic resistance problem in the food chain, but foodborne microbes as intermediates potentially facilitated the dissemination of antibiotic resistance genes to human microbiota. The results strongly support the notion that food chain might have emerged as an important avenue in the transmission and evolution of antibiotic resistant bacteria in human. It opens a new area in food safety research, and is expected to have major impact on food safety and public health. In addition, we have also wrapped up the study developing a rapid detection system for microorganisms in foods. I co-edited the book "Biofilms in the Food Environment", to be published by Blackwell Publishing Professional. So far 1 edited book, 2 book chapters have been submitted, 1 book chapter, 5 peer-reviewed papers and 14 national conference papers and other presentations have resulted from these lines of research. Three graduate students trained in these field(1 Ph.D. and 2 MS) graduated in 2005.

    Impacts
    Our results revealed for the first time that our retail foods, including many ready-to-eat, generally considered "healthy" items, are heavily contaminated with antibiotic resistant bacteria and these bacteria are transmitted to human through daily food intake. Commensal organisms in the food chain not only can serve as a resistance gene reservoir but an enhancer for the transmitting of resistance genes. Our results illustrated a potentially very important avenue in the dissemination of antibiotic resistance genes in the general public, and likely will have enormous impact on food safety and public health. Both the studies on biofilm mechanisms and antibiotics will lead to the development of practical strategies to combat these major challenges in food safety and public health.

    Publications

    • Pettipher GL, Jay JM, Wang H. 2005. Microbiological Techniques. In Encyclopedia of Analytical Science, Second Edition (Worsfold PJ, Alan Townshend A, Poole CF, eds.), Elsevier Science, Oxford, UK. Vol. 6, pp. 16-24.
    • Luo H, Wan K, Wang HH. 2005. A high frequency conjugation system facilitated biofilm formation and pAMbeta1 transmission in Lactococcus lactis. Appl. Environ. Microbiol. 71:2970-2978.
    • Wang HH, Manuzon MY, Lehman M, Wan K, Luo H, Wittum TE, Yousef AE, Bakaletz LO. 2005. Food commensals as a potentially important avenue in transmitting antibiotic resistance genes. FEMS Micrbiol Lett. (In press).
    • Wang HH. 2005. Biofilm formation and gene transfer by foodborne microorganisms: a major challenge to human health. ASM conference: Beneficial Bacteria. p. 54.
    • Wan K, Wang HH. 2005. Horizontal transfer of the broad host range drug-resistance plasmid pAMbeta1 from Lactococcus lactis to foodborne pathogen Listeria monocytogenes. ASM annual meeting. Abstract#P-004, p. 438.
    • Hanna S, Wang HH. 2005. Upregulated expression of Listeria monocytogenes virulence factor inlA in acidic environment. ASM annual meeting. Abstract#P-048, p. 447.
    • Hanna S, Wang HH. 2005. Influence of environment conditions on Listeria monocytogenes virulence factor expression. IFT annual meeting. Abstract#36B-12.
    • Luo H, Wan K, Wang HH. 2005. Bacterial intrinsic mechanism facilitated horizontal gene transfer:an illustration by Lactococcus lactis. 3rd Conference on functional genomics of Gram-positive microorganisms. Abstract#P86.
    • Wang H. 2005. Biofilms and antibiotic resistance gene transfer by foodborne bacteria. Department of Biochemistry, OSU, invited group seminar speaker.
    • Luo H, Wan K, Wang HH. 2005. A high frequency conjugation system facilitated biofilm formation and pAMbeta1 transmission in Lactococcus lactis. OARDC annual conference.
    • Kai W, Wang HH. 2005. Horizontal Transfer of the broad-host-range drug-resistance plasmid pAMbeta1 from Lactococcus lactis to foodborne pathogen Listeria monocytogenes. OARDC annual conference.
    • Lehman M, Wang HH. 2005. The prevalence of antibiotic resistant bacteria in retail foods. OARDC annual conference.
    • Hanna S, Wang HH. 2005. The impact of environmental conditions on the expression of Listeria monocytogenes virulence factor internalin A. OARDC annual conference.
    • Hanna SE, Connor CJ, Wang HH. 2005. Real-time polymerase chain reaction for the food microbiologist: technologies, applications, and limitations. J Food Sci. 70:49-53.
    • Wan K, Yousef AE, Schwartz S, Wang HH. 2005. Rapid, specific and sensitive detection of spoilage molds in orange juice using a real-time Taqman PCR assay. Journal of Food Protection. (In press).
    • Manuzon MY, Luo H, Wang HH. 2005. Natural genetic transformation of tetracycline resistance genes from Lactococcus spp. food isolates to Streptococcus mutans. OARDC annual conference.
    • Wang, HH. 2005. Biofilms and antibiotic resistance gene transfer in the food environment. Center for Microbial Interface Biology work in progress, OSU, seminar speaker.
    • Wang HH. 2005. Food microbiota: detection, biofilms, and antibiotic resistance gene transfer. Department of Food Science and Technology, OSU, seminar speaker, FD SC&TE 810.01.
    • Wang HH. 2005. Rapid detection of microorganisms in foods. Nestle R&D center, Marysville, OH. Invited speaker.


    Progress 01/01/04 to 12/31/04

    Outputs
    The contamination of pathogens or spoilage microorganisms in foods is a major health threat and causes significant financial loss to food industry. Conventional methods take days to weeks to detect the presence of these agents therefore are incompatible with demands from the food industry. We used cutting-edge real-time PCR technology and developed the very first OSU rapid detection system for rapid detection of microorganisms in foods. So far the system includes five components to detect the spoilage Alicyclobacillus spp., multiple thermophilic microorganisms with spoilage potential, the food-borne pathogen Listeria monocytogenes, spoilage yeasts, and molds. Our system has high specificity against target organisms, and can detect the presence of these organisms directly from selected foods within 3-6 hours. This is a significant improvement compare to current mainstream industrial practices. In the past 3 years, we have 1 pending patent, 3 provisional patents, 1 book chapter, 3 peer-reviewed papers, 10 national conference papers and other presentations in this area. Four graduate students and 1 faculty member received training in this field from our program. The system is subject to commercialization. Biofilms formed by microorganisms attached to surfaces are much more resistant to antibiotic treatments and sanitation procedures. They are important sources of secondary contamination, key mechanisms for pathogenic and spoilage organisms persistent in the environment, and major threats to human health. Our goals are to understand the molecular events involved in biofilm formation from initiation, maturation, to detachment, and to develop effective strategies to control and remove biofilms. Risks associated with biofilms in the food environment, besides pathogen virulence, also need to be examined. A system to investigate various biofilms by pathogenic organisms and commensals was established. The L. monocytogenes "honeycomb" biofilm structure was first described. L. monocytogenes virulence strains were found varying in their abilities to form biofilms, and mixed culture biofilm formation was a key mechanism for strain persistence in the environment. In addition, we have made two major discoveries regarding biofilm formation and antibiotic resistance gene transfer mechanisms. First, we have demonstrated that conjugation is an independent biofilm forming mechanism in Lactococcus lactis. This is the first illustration in Gram-positive bacteria, that these organisms can use conjugation to spread biofilm-forming attributes within the community to enhance ecosystem development. Second, we have found that a high frequency conjugation system can serve as an internal "enhancer" facilitating antibiotic resistance gene transfer (more than 10,000 timers higher) in L. lactis. The role of commensal food-borne organisms in facilitating antibiotic resistance gene transfer within mixed culture ecosystems is currently being investigated. During the past 3 years, 2 peer-reviewed papers and 6 national conference papers and other presentations have resulted from this line of research.

    Impacts
    Results from this study will have major impact on human health and food safety. 1) Implementation of the rapid detection system can reduce the time required for microbial quality control test from 2-4 days to within 3-6 hours. This will significantly benefit the food industry and make near-on-line detection possible. 2) The new discovery on biofilm forming mechanism opens the door for developing novel strategies to remove biofilms (OSU will file a provisional patent), which will have major impact on food safety and human health. 3) The discovery of the major role of foodborne commensal organisms in antibiotic resistance gene dissemination will completely change the understanding on the major pathways that lead to antibiotic resistance in human pathogens. Its impact on food safety, public health and medical therapeutic treatment is going to be enormous.

    Publications

    • Luo H, Yousef A, Wang HH. 2004. Rapid detection of Alicyclobacillus species in pure culture and apple juice using real-time PCR. Lett. Appl. Microbiol. 39:376-382.
    • Connor C, Luo H, McSpadden-Gardener BB, Wang HH. 2005. Development of a second rapid detection system for Alicyclobacillus spp. using real-time PCR. Int. J. Food Microbiol. (In press).
    • Hanna S, Connor C, Wang HH. 2005. Real-time polymerase chain reaction for the food microbiologist: technologies, applications and limitations-a review. J. Food Sci. (Accepted)
    • Luo H, Wan K, Wang HH. 2005. A high frequency conjugation system facilitated biofilm formation and pAMb1 transmission in Lactococcus lactis. Appl. Environ. Microbiol. (Accepted)
    • Wan K, Luo H, Yousef A, Wang H. 2004. Rapid and specific detection of spoilage fungi using a real-time Taqman PCR-based system. ASM-Ohio annual meeting. The Ohio J. Sci. 104 (1): A-11.
    • Wan K, Wang H. 2004. Rapid and specific detection of spoilage molds in juice products using a real-time Taqman PCR-based system. IFT annual meeting. Abstract #99A-29, p.253.
    • Luo H, Wang H. 2004. Mixed culture ecosystem: a possible surviving mechanism for non-biofilm formers? IFT annual meeting. Abstract #99D-30, p.268.
    • Wang H, Yousef A, Schwartz S. 2004. Molecular approaches to improve food safety and quality: development and application of the OSU CleanPlant detection system. IFT annual meeting (symposium speaker). Abstract #20-2, p.52.
    • Wang H. 2004. Biofilm development by Listeria monocytogenes and the potential risks. IFT annual meeting (symposium speaker). Abstract #51-3, p.143.
    • Wang H. 2004. Microbial contamination: detection and biofilms. The Procter & Gamble Company, Cincinnati, OH (06/24/2004, invited speaker).
    • Wang H. 2004. Biofilms in the food environment. Department of Oral Biology, OSU (09/30/2004, invited seminar speaker).


    Progress 01/01/03 to 12/31/03

    Outputs
    One of the adaptive living mechanisms for microorganisms is to form biofilms. Biofilm cells are more resistant to adverse environment including antimicrobial treatment and various stress. The presence of food-borne pathogens and spoilage microorganisms in the processing environment is considered to be a serious threat to food safety and quality. Biofilms formed by L. monocytogenes and other natural microflora in sanitation dead corners can become the reservoirs for recurring contamination in the food processing plant environment. However, despite of an increased research interests in the past ten year, our knowledge regarding mechanisms involved in biofilm formation is still very limited; additional risks associated with biofilms in the food environment have not been explored; effective biofilm control and treatment procedures are not available. Understanding the mechanisms involved in biofilm formation and resistance development would allow us to further find counter strategies to deal with this food safety challenge. We have successfully established a biofilm cultivation and observation system. The development of a Listeria monocytogenes biofilm has been documented and a characteristic "honeycomb" structure has been described. We are currently in the process of identifying the relationship between expression of certain cell surface proteins and biofilm formation.

    Impacts
    Biofilm formation is a major problem in food industry. Establishing a basic understanding towards molecular events involved in biofilm development from initiation, maturation, to detachment would enable us to eventually develop effective strategies to control or prevent this problem. It will have significant impact on food safety and quality.

    Publications

    • Abstract: N. Vurma, and H. Wang. 2002. Determination of Listeria attachment using a polystyrene culture tube method. IAFP annual meeting.
    • E. Marsh, H. Luo, and H. Wang. 2003. Biofilm development in Listeria monocytogenes. IFT annual meeting.
    • Peer-reviewed paper: Marsh EJ, Luo H, Wang H. 2003. A three-tiered approach to differentiate Listeria monocytogenes biofilm-forming abilities. FEMS Microbiol Lett. 2003 Nov 21; 228(2): 203-10.


    Progress 01/01/02 to 12/31/02

    Outputs
    We have established the research focus of our group: molecular detection and microbial networking in foodborne microorganisms. We are using the cutting-edge genomic and proteomic approaches to build up our understanding regarding to molecular events and components involved in coordinated microbiological activities such as biofilm formation, development and detachment. We received a seed grant award from OARDC in 2002 to initiate the research. We have made significant progress in the past year and half including presented one poster at 2002 IAFP annual meeting and have one poster submitted for 2003 annual meeting and a manuscript in preparation on that subject. An USDA NRI proposal was submitted on this subject. A framework has been set up to conduct biofilm related research. Essential elements including basic techniques (such as microscopic screening techniques, genetic manipulation techniques in L. monocytogenes, etc.), equipment (biofilm fermentor, proteomic 2-D protein electrophoresis set up), student training have been or are getting in place. Based upon the understanding of physiology and genetics of the microorganisms, we have also conducted application research to fulfill industry needs. We have developed molecular rapid systems to detect foodborne pathogen Listeria monocytogenes and spoilage Alicyclobacillus spp. As less as 10-100 cells can be detected from contaminated foods within a few hours using our systems. A provisionary patent has been filed. We have one poster presented at 2002 IFT meeting, one oral presentation at 2002 OCAPA annual meeting, 4 posters submitted for 2003 IFT annual meeting, a couple manuscripts in preparation. Now we have a total of three extramural funded projects working on related topics. We plan to explore the detection systems and work with nano scientists and food industry to make a commercially deliverable product.

    Impacts
    We expect the development of the rapid detection system can really benefit the industry by cutting lab testing cost and time, minimizing recall, protecting brand image and protecting human health. Fruitful results from biofilm study can further contribute to the basic understanding of metabolic coordination and pathogenicity in Listeria monocytogenes and developing practical biofilm inhibitory approaches to combat this food safety challenge.

    Publications

    • 1. Wang, H., H. Luo, A. Yousef, and S. Schwarts. 2002. Oligonucleotide primer and probes for specific detecting of Alicyclobacillus spp. (Provisionary patent).
    • 2. Wang, H. 2002. Applying biotechnology to improve food safety and quality. OCAPA annual meeting.
    • 3. Kim, M., H. Luo, and H. Wang. 2003. Detecting Listeria monocytoegenes in artificially contaminated meat using a real-time PCR system. 2003 IFT annual meeting (submitted).
    • 4. H. Luo, A. Yousef, and H. Wang. 2003. Rapid detection of Alicyclobacillus species in pure culture and apple juice using real-time PCR. 2003 IFT annual meeting (submitted).
    • 5. Marsh, E. and H. Wang. 2003. Differential biofilm development in Listeria monocytogenes. 2003 IFT annual meeting (submitted).
    • 6. Connor, C., H. Luo, B. B. McSpadden-Gardener, and H. Wang. 2003. Development of a second rapid detection system for Alicyclobacillus spp. using real-time PCR (submitted)


    Progress 09/01/01 to 01/15/02

    Outputs
    Our objectives are to study the molecular coordinating events in microorganisms found in food and their impacts on food safety. We have proposed to: 1). Identifying elements in food processing that may trigger stress responses; Creating a pool of bacteria strains with variability in metabolic coordination using techniques such as mutagenesis, conjugation, etc. 2). Using molecular biology approaches such as 2-dimentional gel electrophoresis, RT-PCR and micro-array to identify proteins synthesized, genes expressed and virulence regulation in these events. 3). Developing protocols for rapid microbial detection, food processing and emerging technology safety evaluation to meet industry needs. To accomplish our goals, we have targeted a microbial community often formed under hostile environment called biofilm. Biofilms have been found associating with raw foods and food processing environment and are believed to be a source of secondary contamination for processed foods. It is documented that biofilm formation triggers microbial gene expression shift. We further propose that metabolic coordination in biofilm is closely related to what might be found in stress responses. We choose to study two Gram-positive food microorganisms, Lactococcus lactis and Listeria monocytogenes to conduct our research. We have established effective methods to detect microbial attachment on abiotic surfaces for both L. lactis and L. monocytogenes. For L. lactis, we have developed a method using 24-well microtiter plates to compare biofilm formation under steady state (Wang, unpublished data). Using this method, visible biofilm by clumping lactococcal strain (for strain details, refer to Wang et al., 1994, J. Dairy Sci., 77:375-384) has been detected after three days of incubation. A modified method has also been established in our laboratory to compare attachment capability of L. monocytogenes starins (Kocaoglu-Vurma and Wang, 2002). These methods will be used to further screening for biofilm forming- or defective- strains in the mutagenesis studies. A L. monocytogenes specific real-time PCR detection system targeting the virulence factor Hly (listeriolysin O) is also developed in our laboratory (Luo and Wang, 2002). Using a specific real-time PCR, we are able to detect the presence of representative L. monocytogenes strains without cross-reacting with strains from L. innocua, L. lactis, P. putida and E. coli. Currently we are exploring potential new virulence factors related to biofilm formation and more probe- and primer-developments are underway. These probe- and primer- pairs will be used to monitor listeria virulence expression under various environmental conditions including in biofilms. Currently, using various mutagenesis approaches, we are in the process of creating a biofilm-defective mutant pool to further identify possible signaling compounds, cell-surface components, and metabolic coordination machinery that are involved in the initiation, development, maturation and detachment of biofilms.

    Impacts
    Our study on identification of biofilm promoting and detachment factors will lead to enhanced scientific understanding of biofilm mechanisms in general and implementing strategies to control food contamination. Revealing novel mechanisms in enhancing listerial virulence expression in the food system will promote risk evaluation and prevention to ensure food safety. The listeria specific real-time PCR system can serve as a prototype for industrial rapid detection of L. monocytogenes based on its virulence while the laboratory handling time is decreased from days or over a week to within a few hours. This work might contribute to establishing a more accurate, early alarming system for listeriosis.

    Publications

    • Kocaoglu-Vurma, N., and H. Wang, 2002. Determination of Listeria attachment using a polystyrene culture tube method. IAFP annual meeting, submitted.
    • Luo, H., A. Yousef, and H. Wang. 2002. Developing a rapid detection system for Listeria monocytogenes using real-time PCR. IFT annual meeting, submitted.