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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL FOOD SAFETY OF FRESH AND FRESH-CUT PRODUCE
2009 Annual Report


1a.Objectives (from AD-416)
(1) Develop rapid and sensitive methods for detection of enteric human pathogens and spoilage bacteria from conventional and organically grown produce, (2) Develop effective postharvest sanitizing procedures providing improved antimicrobial activity while maintaining produce quality and shelf-life, and (3) Understand the ecology and mechanisms that allow specific human and spoilage microorganisms to persist on fresh produce and develop control agents to reduce food safety risks.


1b.Approach (from AD-416)
We will design experiments to optimize rapid, real-time PCR-based microbial detection methods for enteric human pathogens and also assess the application of similar methods for spoilage bacteria on fresh and fresh-cut produce. Because of the high organic load in produce wash water, sanitizer applications approved for fresh and fresh-cut preparation often do not provide effective control of human pathogens or may cause adverse effects on produce quality and shelf-life. Sanitation and wash procedures that are more effective in the presence of a high organic load would be developed. We will identify gene families from human pathogens involved in resistance to sanitation agents as well as stress-tolerance which could be used as ‘targets’ to design novel food sanitation agents. We will develop effective Biocontrol agents such as bacteriophages, yeasts and lactic acid bacteria to enhance food safety by limiting human pathogens on fresh-cut produce without affecting its quality or shelf-life.


3.Progress Report
A mucuous-like substance (biofilm) produced by Salmonella enterica serovar Typhimurium strains that allows for survival under low nutrient conditions such as in vegetable wash waters was discovered. In addition, the small molecular weight sugar compound in Salmonella spp. that is essential for biofilm formation was identified. Genetic mutation in the corresponding gene rendered Salmonella strains incapable of forming biofilms. The utility of using several software programs to interpret the genetic information obtained from several genome sequence projects was demonstrated. Genetic analysis revealed new pathways in several Shigella spp. that could aid the pathogen to overcome the human gastric barrier. DNA-based analysis, in combination with antibiotic resistance profiles, were used to classify different Salmonella isolates obtained from retail foods. DNA-finger print patterns of eight isolates matched with the CDC/FDA data base of previous outbreak and clinical isolates indicating their potential to cause disease. With the exception of isolates obtained from alligator meat (tetracycline resistant) and orange juice (chloramphenicol and sulfisoxazole resistant), the remainder of the isolates were susceptible to the panel of 15 antimicrobials tested. The characterization of Salmonella isolates with the proposed scheme enabled us to determine the potential risk associated with individual strains. Studies indicate that Escherichia coli O157:H7 can be transferred from contaminated coring knives to the edible portions of lettuce during lettuce coring-in-field harvesting, and that E. coli O157:H7 can grow significantly on lettuce cored areas during product field-holding and hauling. Risk mitigation approaches were identified, including frequent sanitation of the coring knives, separating coring from cutting, and rapid cooling postcoring/harvesting. Studies were conducted to determine the microbial profile of fresh tomatoes, to investigate pathogen internalization pathways, and to assess the practicality and efficacy of consumer stem scar removal in reducing food safety risks. The vast majority of microorganisms are located on the stem-scars and in the underlying vascular bundle tissues within 15 mm of the stem-scar. The vascular bundles connecting the stem-scar and internal tissues play a critical role in pathogen internalization. Washing tomatoes with chlorinated water effectively inactivates surface attached microorganisms, but not internalized pathogens. Physical removal of stem-scar and underlying tissues, along with surface washing, significantly reduces both surface attached and internalized microorganisms.


4.Accomplishments
1. Identification of critical control points for microbial food safety in harvesting lettuce. Lettuce coring-in-field is a new practice developed by the industry to improve process efficiency and reduce shipping cost, and is widely used for harvesting lettuce destined for fresh-cut processing; however, its potential impact on food safety has not been fully examined. Our studies found that Escherichia coli O157:H7 can be transferred from contaminated coring knives to the edible portions of lettuce during lettuce coring-in-field harvesting, and E. coli O157:H7 can grow significantly on lettuce cored areas during product field-holding and hauling. We further identified risk mitigation approaches including frequent sanitation of the coring knives, separating coring from cutting, and rapid cooling postcoring/harvesting. This study raised the awareness of the potential food safety risks associated with lettuce coring-in-field harvesting and identified an important new research area.

2. Food-borne illness outbreaks associated with consumption of Salmonella spp. contaminated tomatoes are a significant, on-going problem. These outbreaks have negatively impacted public health, consumer confidence in tomatoes, and the U.S. economy. The need to improve tomato food safety is both urgent and critical. USDA-ARS scientists determined the microbial profile of fresh tomatoes, investigated pathogen internalization pathways, and assessed the practicality and efficacy of consumer stem scar removal in reducing food safety risks. The vast majority of microorganisms are located on the stem-scars and in the underlying vascular bundle tissues within 15 mm of the stem-scar. The vascular bundles connecting the stem-scar and internal tissues play a critical role in pathogen internalization. Washing tomatoes with chlorinated water effectively inactivates surface attached microorganisms, but not internalized pathogens. Physical removal of stem-scar and underlying tissues, along with surface washing, significantly reduces both surface attached and internalized microorganisms. This research enables tomato growers and packers to develop reliable strategies and practices to reduce pathogen internalization in post-harvest handling, and by restaurants, harvesters, and processors. This research received a scientific award from the produce industry. FDA scientists found the results useful for future tomato related outbreak investigation.

3. Characterization of genes from Salmonella required for growth in leafy-green vegetable wash-waters. The ability to survive under low nutrient conditions in the environment enables Salmonella spp. to successfully enter the food chain. We discovered that Salmonella enterica serovar Typhimurium strains form a mucuous-like substance (biofilm) under poor growth conditions such as in vegetable wash waters. Survival of human pathogens in biofilms is a persistent cause of food borne infections. We have identified a small molecular weight sugar compound in Salmonella spp. which is essential for biofilm formation. Genetic mutation in the corresponding gene rendered Salmonella strains incapable of forming biofilms. Identification of genes essential for biofilm formation is crucial in designing specific sanitation agents which will be more effective in removing biofilms. The development of better cleaning agents to eliminate Salmonella spp. in food would be extremely beneficial to the food processing industry.

4. Methods and tools for comparative genomics of food-borne pathogens. Outbreaks of enteric human pathogens such as E. coli, Salmonella and Shigella spp. have been reported and the isolated outbreak stains have shown a wide range of genetic differences. Understanding the molecular basis of genetic diversity is important to analyze the risk associated with a particular pathogenic strain. We demonstrated the utility of several software programs to interpret the genetic information obtained from several genome sequence projects. Genetic analysis revealed new pathways in several Shigella spp. that could aid the pathogen to overcome the human gastric barrier. Whole genome sequence analysis tools will strengthen food-borne pathogen surveillance and subsequent risk assessment by various regulatory agencies to make the U.S. food supply safer.

5. Genetic and phenotypic characterization of Salmonella isolates from retail foods. Salmonella enterica serovar strains are important food-borne pathogens and the ability of the pathogen to survive environmental stress conditions plays a crucial role in the spread of almonelosis. The severity of the illness also varies among various strains of this pathogen. Only limited information exists related to growth, biochemical and genetic characteristics of S. enterica serovar strains isolated from retail foods. We used DNA-based analysis in combination with antibiotic resistance profiles to classify different Salmonella isolates obtained from retail foods. DNA-finger print pattern of eight isolates matched with the CDC/FDA data base of previous outbreak and clinical isolates indicating their potential to cause disease. With the exception of isolates obtained from alligator meat (tetracycline resistant) and orange juice (chloramphenicol and sulfisoxazole resistant), the remainder of the isolates were susceptible to the panel of 15 antimicrobials tested. The characterization of Salmonella isolates with the proposed scheme enabled us to determine the potential risk associated with individual strains. Both the food-preparation industry and consumers will benefit from the results of this research.


6.Technology Transfer

Number of New CRADAS1

Review Publications
Bhagwat, A.A., Jun, W., Liu, L., Porteen, K., Dharne, M., Pheh, B., Tall, B.D., Kothary, M.H., Gross, K.C., Angle, S., Jianghong, M., Smith, A.D. 2009. Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice. Microbiology. 155:229-237.

Liu, L., Tan, S., Jun, W., Smith, A.D., Meng, J., Bhagwat, A.A. 2008. Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash-waters and colonization in mice. FEMS Microbiology Letters. 292:13-20.

Xia, X., Zhao, S., Smith, A.D., Mcevoy, J.L., Jianghong, M., Bhagwat, A.A. 2009. Characterization of Salmonella isolates from retail foods based on serotyping, pulse field gel electrophoresis, antibiotic resistance and other phenotypic properties. International Journal of Food Microbiology. 129:93-98.

Chua, T., Bhagwat, A.A. 2009. A rapid and simple DNA extraction procedure to detect Salmonella spp. and Listeria monocytogenes from fresh produce using real-time PCR. Journal of Food Analytical Methods. 2:96-101.

Fratamico, P.M., Bhagwat, A.A., Injaian, L., Cray, P.J. 2008. Characterization of shiga toxin-producing escherichia coli strains isolated from swine feces. Journal of Food Protection. 5(6):827-838.

Bhagwat, A.A., Bhagwat, M. 2008. Methods and tools for comparative genomics of food-borne pathogens. Foodborne Pathogens and Disease. 5(4):487-497.

Wang, H., Feng, H., Liang, W., Luo, Y., Malyarchuk, V. 2009. Effect of surface roughness on retention and removal of Escherichia coli O157:H7 on surfaces of selected fruits. Journal of Food Science. 74(1):E8-E15.

Last Modified: 7/30/2014
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