Adhesion and Control of Human Pathogens to and On Surfaces

Adhesion and Control of Human Pathogens to and on Surfaces (Part B: Produce)

Mandrell RE, Charkowski AO, Cooley MB, Friedman M, Gorski L, Kint S, Miller WG

Agricultural Research Service, Food Safety and Health Unit, WRRC, Albany, CA.
National Program 108
Food Safety Progress Report 2001 (web version)
Section 10: Produce and Animal Manure

CRIS: 5325-42000-022

Summary Project Aims:

Bacterial contamination of fruit and vegetables is an increasing problem in the United States, and recognition of this problem by the American public is gaining. However, very little is known about how pathogenic bacteria exist in food environments. Both basic and applied research is needed to increase our understanding of how these pathogens attach to and survive in environments related to food, including the soil, water, air, plant roots and leaves, and processing environments. Our work has concentrated on the development of: (a) new detection methods for the identification of pathogens on foods; (b) attachment models to better understand the attachment process of several pathogens to food surfaces, including sprouts, lettuce, cilantro and fresh-cut products;(c) screening for natural antimicrobials that may be added to foods; and (d) the development of new strategies to prevent or minimize bacterial contamination during growth and harvesting of produce.

Summary Accomplishments During Entire Project:

We have developed reagents for the detection of Campylobacter in foods; stable fluorescent Salmonella, Campylobacter, E. coli O157:H7, Listeria monocytogenes and plant bacterial strains for studies of the bacterial ecology and biology in complex environments and biofilms; and attachment assays to study pathogen interactions on surfaces. These assays will provide a quantitative means to measure pathogen reduction on produce, and will enable the characterization of specific gene products that can be targeted for anti-adherence strategies. In addition, our work on biofilms should lead to a more thorough understanding of the natural attachment process of pathogens on a variety of produce items. We anticipate that these studies will lead to the identification of non-toxic methods to decrease pathogens in real food environments.

Summary 2000 Accomplishments:

We demonstrated that some of the chemicals used for sanitizing seeds for sprouting are ineffective against the bacteria and/or they damaged the seeds leading to lack of sprouting. We demonstrated that wrinkled and damaged seeds carry a higher level of bacteria than undamaged seeds and that these seeds are more difficult to sanitize. This work has important implications for the sprout industry and points to potential interventions to minimize pathogens in sprouts. New methods for testing sprout irrigation water are needed to decrease the time for results and the cost. We tested commercially available immunomagnetic beads and determined that they can be used to recover several different S. enterica strains, all of which were originally isolated from alfalfa seeds, from dilute bacterial suspensions. Initial studies of the ecology of Salmonella on sprouts, E. coli on lettuce, Salmonella on cilantro, Listeria on a variety of cut produce items.

Projected Research Accomplishments During Next 3 Years:

Identify multiple genes related to E. coli attachment to lettuce, Salmonella and E. coli attachment to sprouts, and Salmonella attachment to cilantro leaves. Characterize Campylobacter signaling pathways and relationship to biofilm formation and attachment. Develop assays for assessing chemical and structural basis for attachment of pathogens to plants. Identify plant antimicrobials useful for minimizing pathogens in produce samples. Develop sorting method for testing sprout seed lots. Develop fast method for identifying pathogen in sprout irrigation water. Determine whether pathogen contamination pre-harvest increases resistance of pathogen to interventions; identify some plant genes important in pathogen attachment. Develop improved methods for isolating multiple pathogens from naturally contaminated produce using new reagents and knowledge gained from basic studies. Determine factors crucial to human pathogen fitness and survival on produce. Conduct preliminary studies of pathogen interactions with soil organisms and pathogen fitness. Develop a comprehensive model for how a human pathogen in manure, water, and/or soil colonizes a plant leaf. Establish methods for identification of enteroviruses present in manure, water and on produce. Develop prototype wash solution compatible for multiple produce items.

Technology Transfer:

Technology transfer is an active part of the current CRIS project. Information and technology will be transferred to industry, government regulators, and to the scientific community via publications, collaborations, CRADA's, and Trust Agreements with industry. We continue to seek partners for licensing anti-pathogen antibodies produced in our labs.

PUBLICATIONS:

Kimura, R., Mandrell, R.E., Galland, J.C., Hyatt, D., Riley, L.W. Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157:H7 strains in environmental samples. Applied Environmental Microbiology 2000; v. 66(6). p.2513-9.

Alfano, J. R., Charkowski A. O., Deng W. L., Badel J. L.,Petnicki-Ocwieja, T.,van Dijk, K.,Collmer, A. The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants. Proceedings of the National Academy of Science USA. 2000. v. 97(9). p.4856-61.

Collmer, A., Badel J.L., Charkowski, A. O., Deng, W. L., Fouts, D. E., Ramos, A. R., Rehm, A. H., Anderson, D. M., Schneewind, O., van Dijk, K. Pseudomonas syringae Hrp type III secretion system and effector proteins. Proc Nat Acad Sci USA 2000.v. 97(16).p.8770-8777.

Licking, E., Gorski, L., Kaiser, D. A common step for changing cell shape in fruiting body and starvation-independent sporulation of Myxococcus xanthus. J. Bacteriology; v. 182. p.3553-3558.

Gorski, L., Gornewold, T., Kaiser, D. A sigma-54 activator protein necessary for spore differentiation within the fruiting body of Myxococcus xanthus. J. Bacteriology. 2000. v.182. p.2438-2444.

Cooley, M. B., Pathirana, S., Wu, H. J., Kachroo, P., Klessig, D. F. Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens. Plant Cell. 2000. v.12. p.663-676.

Miller, W.G., Bates, A.H., T, H.S., Brandl, M.T., Wachtel, M.R., and Mandrell, R.E. 2000. Detection of Campylobacter jejuni cells transformed with new gfp, yfp and cfp marker plasmids on surfaces and in Caco-2 cells Applied and Environmental Microbioogy. V.66 (12). In press.

PROCEEDINGS\ABSTRACTS:

Brandl, M.T., and Mandrell, R.E. Ecological studies ofSalmonella serotype Thompson on cilantro plants support its role in recent epidemics. Ann. Meeting of American Society for Microbiology. Los Angeles, CA. 2000.

Brandl, M.T., and Mandrell, R.E. 2000. Use of confocal microscopy and the green fluorescent protein in ecological studies on Salmonella on plant surfaces. Scanning 2000. 22p. 83.

Harden, L.A., Lieberman, A., Mandrell, R., and Haddon, W.F. A spreadsheet approach to bacterial identification based on MALDI-TOF spectra of whole cells. American Society Mass Spectrometry, Long Beach, CA. 2000.

Mandrell, R.E., Harden, L., Horn, S.T., Haddon, W.F., and Miller, W.G. Analysis of E. coli environmental and diarrheal isolates by MALDI-TOF mass spectrometry: Identification of potential biomarkers ions and a mutation in a gene encoding a biomarker ion. Ann. Meeting of American Society for Microbiology. Los Angeles, CA. 2000.

Wood, D.F., Mandrell, R., Bates, A.H., and Yu, P.C. 2000. Immunolocalization of surface antigens on Campylobacter jejuni using FESEM and a backscatter electron detector Scanning 2000. 22p. 79-80.