CONTROL OF FOOD-BORNE PATHOGENS IN PRE- AND POST-HARVEST ENVIRONMENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0220951
• Multistate No.: S-1033
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2012
• Program Code: [(N/A)]- (N/A)

Project Director
Mustapha, AZ.

Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211

Performing Department
Food Systems & Bioengineering

Non Technical Summary
Despite the availability of a variety of antimicrobial decontamination methods, food contamination and foodborne diseases continue to occur. The need for effective antimicrobials as well as food safety education and training is ever present. Furthermore, widely used cultural-based pathogen detection methods are time consuming, and often lack sensitivity and selectivity. The use of molecular based methods can overcome many of the limitations of cultural-based methods, in particular, sensitivity, selectivity and speed. A limitation of PCR-based molecular detection methods is the inability to differential between dead or live cells. Our work with the use of ethidium monoazide coupled with PCR will allow for the detection of only live pathogens. The outcomes of this project would contribute to improving food safety by way of the use of highly effective novel antimicrobial compounds and the ability to detect live pathogens in a rapid, sensitive and selctive manner.

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
712	1430	1100	12%
712	1451	1100	11%
712	1452	1100	11%
712	1460	1100	11%
712	3260	1170	11%
712	3320	1170	11%
712	4010	1170	11%
712	3270	1170	11%
712	1461	1170	11%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1460 - Tomato; 3320 - Meat, beef cattle; 1430 - Greens and leafy vegetables; 3260 - Poultry meat; 1461 - Peppers; 1451 - Onion, garlic, leek, shallot; 4010 - Bacteria; 3270 - Eggs; 1452 - Carrot;

Field Of Science
1100 - Bacteriology; 1170 - Epidemiology;

Keywords

antimicrobials

salmonella

e. coli o157:h7

l. monocytogenes

pcr

live and dead cells

Goals / Objectives
1. Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments. 2. Develop rapid, molecular-based detection methods for pathogens in pre-and post-harvest environments. 3. Investigate factors leading to the emergence, persistence and elimination of antimicrobial resistance in food processing and animal production environments. Outputs: Validated decontamination methods that can be used by the fruit, vegetable, seafood, meat and poultry industry to enhance the safety of their finished product Outreach/extension education and training materials for regulatory personnel, producers, processors, consumers

Project Methods
Novel antimicrobial compounds will be tested to determine their effectiveness against pathogens. Natural and synthetic compounds to be tested will include organic acids, bacteriocins, zinc and magnesium oxide nanoparticles. Pathogens, including Escherichia coli, Salmonella and Listeria monocytogenes will be the target organisms. The antimicrobial compounds will be tested in various food matrices. PCR- and novel biosensor-based techniques will be improved upon as techniques for rapid and simultaneous detection of foodborne pathogens, including E. coli O157:H7, Salmonella and L. monocytogenes. DNA and RNA sequences that would make suitable primers for simultaneous detection of target pathogens will be continually searched in the literature. Other biosensor-based rapid methods, such as the use of quantum dots and other nanomaterials, will also be explored to achieve the objectives. Artificially and naturally contaminated food samples will be tested to optimize PCR-based methods, specifically by removing food inhibitors that typically limit the sensitivity of the technique. Foodborne pathogens can be induced to the viable-but-nonculturable (VBNC) state when in the presence of commonly used antimicrobial preservatives or food processes. We will investigate the VBNC phenomenon in E. coli O157:H7, Salmonella and L. monocytogenes in meat products and meat processing equipment following routine cleaning and sanitizing protocols and subsequent storage in various packaging and refrigeration conditions. The meat products will be artificially inoculated with varying concentrations of the pathogens and exposed to different intervention steps, while equipment will be artificially contaminated with the pathogens and the product processed as routinely done, followed by typical cleaning and sanitizing steps. VBNC cells will be monitored by the use of ethidium monoazide (EMA), a stain that can selectively penetrate dead cells because of their compromised membrane/cell wall systems and bind to the intracellular DNA. We will use EMA coupled with PCR to monitor for the presence and recovery of only viable and VBNC cells. The data obtained from this project will be shared with stakeholders via publications, news outlets and classroom activities.

Progress 10/01/09 to 09/30/12

Outputs
Target Audience: Academia, food industry, consumers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Six graduate students were trained and completed. How have the results been disseminated to communities of interest? By scientific publications and presentations at scientific meetings. Some of the research also was publicized by mass media (radio, tv, websites). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have successfully developed a real-time PCR detection method coupled with a staining step to detect live E. coli O157:H7 and Salmonella in foods. We also developed a SERS (Surface enhanced Raman Spectroscopy) method to detect food and waterborne viruses and bacteria. We successfully determined inhibitory activities of ZnO nanoparticles against E. colli O157:H7. A novel argon plasma brush was determined to be effective against Bacillus spores. A patent on microencapsulated probiotics to prevent fecal shedding of E. coli O157:H7 in cattle was filed.

Publications

• Type: Journal Articles Status: Published Year Published: 2009 Citation: Liu, Y., L. He, A. Mustapha, H. Li, Z. Hu, and M. Lin. 2009. Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157:H7. J. Appl. Microbiol. 107:1193-1201
• Type: Journal Articles Status: Published Year Published: 2009 Citation: Wang, L. Y. Li, and A. Mustapha. 2009. Detection of viable Escherichia coli O157:H7 by ethidium monoazide-multiplex-real-time PCR. J. Appl. Microbiol. 107:1719-1728.
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Wang, L. and A. Mustapha. 2010. EMA-real-time PCR as a reliable method for detection of viable Salmonella in chicken and eggs. J. Food Sci. 75(3):M134-M139.
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Fan, C., Z. Hu, L. K. Riley, G. Purdy, A. Mustapha, and M. Lin. 2010. Detecting food- and water-borne viruses by surface enhanced Raman spectroscopy. J. Food Sci. 75(5):M302-M307.
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Yang, B., J. Chen, Q. Yu, M. Lin, A. Mustapha, M. Chen. 2010. Inactivation of Bacillus spores using a low-temperature atmospheric plasma brush. IEEE Transactions on Plasma Science 38(7):1624-1631.
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Fan, C., Z. Hu, A. Mustapha, and M. Lin. 2011. Rapid detection of food-and waterborne pathogens using surface enhanced Raman spectroscopy coupled with silver nanosubstrates. Appl. Microbiol. Biotechnol. 92:1053-1061.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Wang, L., C.-S. Wu, X. Fan and A. Mustapha. 2012. Detection of Escherichia coli O157:H7 and Salmonella in ground beef by a bead-free quantum dot-facilitated method Int. J. Food Microbiol. 156:83-87.

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

Outputs
OUTPUTS: A multiplex PCR reaction coupled to ethidium monoazide (EMA) staining was developed and optimized for detecting Escherichia coli O157:H7 in ground beef and Salmonella in chicken rinses and eggs. Staining cells with 10 mg/mL EMA for 5 min, followed by incubation on ice for 1 min and exposure to bright visible light for 10 min were found to be the optimum conditions to successfully bind dead cell DNA prior to a subsequent real-time PCR. The real-time PCR assay that follows include an internal amplification control (IAC), consisting of 0.25 pg of plasmid pUC19 in order to prevent false negative results. In the presence of 108 CFU/mL dead E. coli O157:H7 cells, the detection limit for live E. coli O157:H7 was 104 CFU/g in ground beef. In the presence of 106 CFU/mL dead Salmonella serotypes Typhimurium and Enteritidis cells, the detection limit for live Salmonella in chicken rinse and egg broth, respectively, was 105 CFU/g. With the incorporation of a 12-h enrichment step, the detection limit for all three pathogens was lowered to 10 CFU/g. ZnO nanoparticles (NPs) at concentrations of 0-12 mmol/L were tested against E. coli O157:H7 and S. Typhimurium by a 24-h exposure in tryptic soy broth, peptone water, distilled water, and yam starch solution. The effect of the different concentrations of ZnO on both pathogens appeared to be bacteriostatic in TSB with numbers approaching those of the control at the end of 12 h. A 7-log reduction was observed after a 3-h incubation of S. Typhimurium in peptone water, distilled water, and yam starch solution containing 3 mmol/L or higher concentrations of ZnO NP suspension. A similar growth decrease of E. coli O157:H7 was observed after 10 h, depending on the growth medium. SEM and TEM results indicate that ZnO NPs attached to the cell surfaces, causing visible damage and disruption of the cell membrane, which potentially led to the death of the cells. Results indicated that availability of nutrients and incubation times were proposed, for the first time, to be important parameters that can influence the antimicrobial effect of ZnO NPs. PARTICIPANTS: Azlin Mustapha, PI Mengshi Lin, Co-PI Luxin Wang, graduate student, Ruoyu Li, graduate student TARGET AUDIENCES: The results of the research would be valuable to food scientists and researchers in the food industry, academia and related government agencies, as far as contributing to the knowledge base for improving food safety via a novel molecular method that detects only viable pathogens and via the use of novel inorganic nanoparticles. PROJECT MODIFICATIONS: None

Impacts
The use of EMA with a DNA-based real-time PCR can successfully prevent false positive results from dead cell detection and represents a simple, yet accurate detection tool for enhancing the safety of food. Inorganic engineered nanoparticles, such as ZnO, are being used more and more in the medical, pharmaceutical and food industries. Our results show that ZnO NPs do have application for inhibiting pathogens that are important in foods.

Publications

• Wang, L. and A. Mustapha. 2010. EMA-real-time PCR as a reliable method for detection of viable Salmonella in chicken and eggs. J. Food Sci. 7(3):M134-M139.
• Wang, L. Y. Li, and A. Mustapha. 2009. Detection of viable Escherichia coli O157:H7 by ethidium monoazide-multiplex-real-time PCR. J. Appl. Microbiol. 107:1719-1728.
• Liu, Y., L. He, A. Mustapha, H. Li, Z. Hu, and M. Lin. 2009. Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157:H7. J. Appl. Microbiol. 107:1193-1201.