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United States Department of Agriculture

Agricultural Research Service


Location: Produce Safety and Microbiology Research

2009 Annual Report

1a.Objectives (from AD-416)
Objective 1: Genome sequencing, annotation, and gene-indexing, of Campylobacter species, Salmonella Enteritidis (SE) and pathogenic E. coli to identify targets for rapid detection and differentiation, and fitness and virulence factors. Objective 2: Develop DNA microarrays, and sequence-based typing methods to detect and analyze multiple critical food-borne pathogens; validate assays with food samples. Objective 3: Develop new and/or improved multi-locus sequence typing (MLST) and multi-locus variable tandem repreat analysis (MLVA) methods for human pathogens with emphasis on enterohemorrhagic E. coli. Combine MLST, MLVA and microarray analysis to identify markers associated with pathogen source and fitness, and relate to epidemiology and culture method bias. Objective 4: Develop specific capture and mass spectrometry (MS) methods to detect and fingerprint foodborne pathogens and threat agents. Objective 5: Evaluate methods for inactivating protein toxins. Problem to be Addressed: Through the use of genomics and proteomics develop multiplex assays to detect, identify and differentiate foodborne pathogens on fresh produce (leafy vegetables) to derive fundamental data to increase the safety and security of this commodity. FY07 Objectives of Research: Genome sequencing, annotation, and gene-indexing, of pathogenic E. coli to identify targets for rapid detection and differentiation, and fitness and virulence factors, with special emphasis on E. coli in the environment of produce production. Use fundamental genomic and proteomic information produced to develop microarray or other multiplex immunoreagent methods to identify and analyze genera, species and strains of critical food-borne pathogens. Identify single nucleotide polymorphism hot-spots in "clonal" pathogens for high resolution fingerprinting. Characterize E. coli O157:H7 strains associated with outbreaks and to identify potential virulence factors and other factors that may enhance fitness in produce production environments (plants, animal hosts, environment).

1b.Approach (from AD-416)
Our objectives address fundamental research to develop high-resolution genotyping methods for characterizing and tracking multiple pathogens related to food. Multiple approaches to methods development are described as contingencies to ensure success. The recent sequence data we have collaborated in producing for Campylobacter and Arcobacter species and collaborations on S. enterica, Ec O157:H7 and Lm genotyping will be invaluable for this work. Recent PSMRU involvement in two outbreak investigations of pre-harvest produce and tree-nuts contaminated with Ec O157:H7 (letter, J. Farrar) and S. Enteritidis (letter, J. Adams), respectively, has confirmed the need for improved methods for tracking pathogens in complex environments, determining their relatedness, and the relevance of these studies also to addressing potential intentional contamination events. The objectives we describe have been organized with the following strategy in mind: (i) Emphasize Campylobacter species, especially emerging species, because they remain underappreciated as food pathogens and causes of serious illness. Recent progress in sequencing and MS analysis facilitate comparative genomics and proteomics, and the expertise gained will be beneficial for development of similar approaches for other pathogens; (ii) Develop microarrays specific for genotyping, to learn as much as possible about virulence factors and fitness characteristics that might be beneficial to interventions during production or processing; (iii) Expand, as appropriate, the microarray approach to more comprehensive DNA microarrays for detection of many pathogens simultaneously; (iv) Develop methods useful for addressing objectives of PSMRU CRIS-040 (“Biology and Control of Human Pathogens on Fresh produce”) to leverage methods and discoveries and increase productivity; (v) Collaborate with other groups who have access to productions systems and/or strains for assessing the robustness of genotyping or protein typing; (vi) Use the novel methods developed to address whether culture bias is affecting the ability to obtain meaningful data about reservoirs, food sources and epidemiology. Replacing 5325-42000-041-00D (4/06).

3.Progress Report
Bioinformatics software for the rapid analysis of foodborne pathogens. A "top-down" proteomics approach for identification of protein biomarkers of foodborne pathogens, viruses and toxins was developed. Strains of E. coli O157:H7 as well as MLVA primer sets were supplied to IBIS Biosciences for genotyping analysis using a novel PCR/mass spectrometry-based platform for foodborne pathogen identification. A collaborative project with Bruker Daltonics to develop benchtop MALDI-TOF bacterial identification was completed.

Plant non-toxic antimicrobials. Edible apple antimicrobial films were provided and used to wrap raw chicken breasts inoculated with S. enterica and E. coli O157:H7; they induced multilog decreases in each pathogen without changes in smell or quality. Additional antimicrobial activities demonstrated were: apple skin and tea powders facilitating thermal destruction of E. coli O157:H7 in ground beef; carvacrol inactivating antibiotic-resistant strains of C. jejuni; and safe plant compounds inactivating antibiotic resistant Salmonella enterica on celery and oysters, E. coli O157:H7 and Salmonella strains in cooked ground beef. Carvacrol both inhibited E. coli O157:H7 and the formation of potentially carcinogenic heterocyclic amines in grilled beef patties. Campylobacter and Arcobacter genomics. We assisted in the annotation and analysis of Campylobacter genome data (5 species) provided by JCVI and participated in a JCVI project to sequence three new oral Campylobacters. We characterized >300 C. jejuni strains by multilocus DNA sequence typing (MLST). MLST methods for C. concisus/C. curvus and Arcobacter strains were developed and used to type human clinical strains of human clinical, food, animal or environmental origin. C. jejuni LOS biosynthesis loci were characterized and revealed rapid changes in LOS structure related to potential virulence.

DNA and oligonucleotide microarrays for genomic analysis of pathogens. DNA oligonucleotide microarrays were expanded to identify the presence of pathogens in environmental samples. Microarray-based indexing of E. coli, S. Typhimurium, C. jejuni, C. coli, C. lari, A. butzleri and Enterobacter sakazakii was completed. These arrays will assist additional studies of virulence and strain relatedness of pathogens.

Cell-based method for Shiga toxin (Stx) detection. A novel, low cost, cell-based fluorescence method was developed to detect Stx and inhibitors of Stx activity expressed by bacterial pathogens. A novel monkey cell line expressing green fluorescent protein allowed extremely sensitive detection of active Stx (pg/ml), and also anti-toxin activities. We screened hundreds of E. coli isolates and identified Stx+ isolates recovered from California and Mexico produce production environments.

Provisional and a utility patents were filed for software developed in our laboratory for rapid identification of proteins and their associated pathogens using mass spectrometry proteomics and DNA sequence data. Bacteriophage capsid proteins are being analyzed for sensitive detection of associated pathogens by mass spectrometry. We continue to develop "bottom-up" proteomic approaches.

1. Environmental investigation of dairies associated with Campylobacter jejuni. ARS scientists in Albany, CA worked with the California Department of Public Health on environmental studies of dairies linked to raw milk outbreaks of Campylobacter jejuni and E. coli O157:H7. Isolates were screened by a new method developed in our lab and we identified human outbreak strains. Identification of outbreak-related strains from both dairies providing crucial epidemiological information for explaining the outbreaks. The results obtained in one of the studies is critical in guidance by the State of California on rules of acceptable levels of fecal contamination of raw milk for human consumption.

2. Mass spectrometry-based bioinformatics software for the rapid identification of foodborne pathogens, viruses and protein toxins. Rapid identification of pathogens and toxins in food are needed, especially for test-and-hold foods like raw produce. Computer software has been developed by ARS scientists in Albany, CA, for rapid identification of markers of foodborne pathogens, viruses and toxins by mass spectrometry analysis. Ions that fragment from intact proteins can be compared against a database of ions from thousands of bacterial, viral and toxin proteins in < 1 minute. This software could shorten the time to identification of pathogens in food. A patent application is currently being drafted, and discussions have been conducted with potential licensees of the software. This software could provide rapid identification of pathogens or toxins in a complex food or processing sample.

3. Sequencing of Arcobacter butzleri and Campylobacter lari genomes. Arcobacter butzleri and Campylobacter lari can cause human bacterial gastroenteritis and can be transmitted via contaminated food, water or shellfish. ARS scientists in Albany, CA completed the genomes of human clinical strains of A. butzleri and C. lari and deposited the data in a national public database. Novel genes, metabolic pathways, and potential pathogenicity genes were identified and facilitated production of DNA chips in our lab. These chip arrays were used to compare genomes of many strains and led to development of a novel Arcobacter DNA typing method for analysis rapidly hundreds of additional strains. These assays will be used for tracking sources of pathogens.

4. Detection and genotyping of E. coli present in environmental samples. Shiga toxin-positive E. coli (STECs) are an emerging issue in food safety. Identification of the potential pathogenic STEC strains requires detection of many genes for each strain. DNA chips were produced by ARS scientists in Albany, CA to detect the presence of STECs in complex samples and as a model for detection of multiple pathogens simultaneously. DNA products from environmental samples were used to detect E. coli virulence genes revealing rapid detection of pathogenic E. coli from a variety of agricultural sources. This method is critical for assessment of risk factors in regions of leafy vegetable production and will provide guidance to the produce industry about risk factors in the production environment.

5. Detection of active Shiga toxin from E. coli. Shiga toxin-positive E. coli (STECs) are an emerging issue in food safety. It is critical to determine whether bacteria that express the toxin genes actually make active toxin. A simple cell-based fluorescence assay was developed by ARS scientists in Albany, CA to assess the activity of Shiga toxin 2 variants from E. coli strains. These variants may be associated with increased virulence of pathogenic E. coli. This non-radioisotope assay is cheap, high-throughput, and more sensitive than standard immunological assays for toxin activity, and provides important information about the virulence of pathogens in food.

6. Rapid and cost-effective method for pathogen detection by DNA microarrays. A major limitation in novel/modern pathogen detection is the expensive and non-portable scanners and labile expensive fluorescence reagents. An innovative, simple, rapid, commercial microarray detection method was evaluated by ARS scientists in Albany, CA for pathogen identification. The method involves a portable scanner suitable for routine testing in a field setting. Pathogenic strains of E. coli could be detected in 2-4 hours, a vast improvement in speed compared to fluorescence detection methods requiring 3-4 days for identification of potential pathogenic strains. This method is a reasonable approach for regulatory lab surveys of food samples for pathogens.

7. Pyocin killing of E. coli O157:H7 strains. Methods for killing E. coli O157:H7 in humans and/or food are needed. ARS scientists in Albany, CA collaborated with a small startup company (AvidBiotics) to test a protein produced by an engineered bacteriophage for it‘s ability to kill E. coli strains. We demonstrated it is specific for E. coli O157:H7 strains and it is highly active. More importantly, it does not stimulate the pathogen to produce more toxin in contrast to what occurs with conventional antibiotics, thus serving as a potential therapeutic for humans to prevent serious kidney disease following severe diarrheal illness. This protein is a novel potential method for killing or minimizing this pathogen in food and a potential therapeutic for humans.

6.Technology Transfer

Number of New CRADAS1
Number of New Patent Applications Filed1

Review Publications
Quinones, B., Miller, W.G., Bates, A.H., Mandrell, R.E. 2009. Autoinducer-2 production in Campylobacter jejuni contributes to chicken colonization. Applied and Environmental Microbiology. 75/1:281-285

Houliston, R.S., Bernatchez, S., Karwaski, M.F., Mandrell, R.E., Jarrell, H.C., Wakarchuk, W.W., Gilbert, M. 2009. Complete chemoenzymatic synthesis of the Forssman antigen using novel glycosyltransferases identified in Campylobacter jejuni and Pasteurella multocida. Glycobiology. 19:153-159.

Kozukue, N., Yoon, K., Byun, G., Misoo, S., Levin, C.E., Friedman, M. 2008. Distribution of Glycoalkaloids in Potato Tubers of 59 Accessions of Two Wild and Five Cultivated Solanum Species. Journal of Agricultural and Food Chemistry. 56(24):11920-11928.

Miller, W.G., Wang, G., Binnewies, T.T., Parker, C. 2008. The complete genome sequence and analysis of the human pathogen Campylobacter lari. Foodborne Pathogens and Disease. 5(4):371-386.

Fagerquist, C.K., Garbus, B.R., Williams, K., Bates, A.H., Boyle, S., Harden, L.A. 2009. Web-based software for rapid "top-down" proteomic identification of protein biomarkers with implications for bacterial identification. Applied and Environmental Microbiology. 75(13):4341-4353.

Friedman, M., Zhu, L., Fienstein, Y., Ravishankar, S. 2009. Carvacrol facilates heat induced inactivation of Escherichia coli 0157:H7 and inhibits formation of heterocylic amines in grilled ground beef patties. Journal of Agricultural and Food Chemistry. 57:1848-1853.

Oh, D., Pan, Y., Berry, E.D., Cooley, M.B., Mandrell, R.E., Breidt, F. 2009. Escherichia coli O157:H7 strains isolated from environmental sources differ significantly in acid resistance compared to human outbreak strains. Journal of Food Protection. 72(3):503-509.

Lee, S., Lee, J., Choi, S., Lee, J., Ohnishi-Kameyama, M., Kozukue, N., Levin, C.E., Friedman, M. 2008. Flavonoid Content in Fresh, Home Processed, and Light-Exposed Onions and in Dehydrated Commercial Onion Products. Journal of Agricultural and Food Chemistry. 56(18):8541-8548.

Sirk, T.W., Brown, E.F., Sum, A.K., Friedman, M. 2008. Molecular Dynamics Study on the Biophysical Interactions of Seven Green Tea Catechins with Cell Membranes. Journal of Agricultural and Food Chemistry. 56:7750-7758.

Wong, Y.Y., Grant, I., Friedman, M., Elliot, C.T., Situ, C. 2008. Antibacterial Activities of Naturally occurring Compounds against Mycobacterium avium subspecies paratuberculosis. Applied and Environmental Microbiology. 74:5986-5990.

Finotti, E., Bersani, E., Vivanti, V., Friedman, M. 2009. Application of a Functional Mathematical Index to the Evaluation of the Nutritional Quality of Potatoes. In: Yee, N;Busseul, W. (Editors) Potato IV, FOOD 3 (Special Issue 2); United Kingdom: Global Scientific Books. p. 30-36.

Friedman, M., Levin, C.E. 2008. Review of methods for the reduction of dietary content & toxicity of acrylamide. Journal of Agricultural and Food Chemistry. 56:6113-6140.

Friedman, M., Levin, C.E., Lee, S., Hyung-Jeong, K., Lee, I., Byun, J., Kosukue, N. 2009. Tomatine-Containing Green Tomato Extracts Inhibit Growth of Human Breast, Colon, Liver, and Stomach Cancer Cells. Journal of Agricultural and Food Chemistry. 57:5727-5733.

Flanagan, R.M., Neal-Mckinney, J.M., Dhillon, A.S., Miller, W.G., Konkle, M.E. 2009. Examination of Campylobacter jejuni putative adhesins leads to the identification of a new protein, designated FlpA, required for chicken colonization. Infection and Immunity. 77:2399-2407

Gaasbeck, E.J., Wagenaar, J., Guilhabert, M., Wosten, M.M., Van Putten, J.P., Parker, C., Van Der Wal, F.J. 2009 A DNase encoded by integrated element CJIE1 inhibits natural transformation of Campylobacter jejuni. Journal of Bacteriology. 191:2296-2306.

Scholl, D., Cooley, M.B., Williams, S.R., Gebhart, D., Martin, D., Bates, A.H., Mandrell, R.E. 2009. An Engineered R-Type Pyocin Is a Highly Specific and Sensitive Bactericidal Agent for the Food-Borne Pathogen Escherichia coli O157:H7. Antimicrobial Agents and Chemotherapy. 53(7):3074-3080.

Habib, I., Miller, W.G., Uyttendaele, M., Houf, K., Dezutter, L. 2009. Clonal population structure and antimicrobial resistance of Campylobacter jejuni from chicken meat in Belgium. Applied and Environmental Microbiology. 75(13):4264-4272.

Habib, I., Louwen, R., Uytendaele, M., Houf, K., Vandenberg, O., Nieuwenhuis, E.E., Miller, W.G., Vanbelkum, A., Dezutter, L. 2009. Correlation between genotypic diversity, lipooligosaccharide gene locus class variation and Caco-2 invasion potential of Campylobacter jejuni from human and chicken meat origin: a contribution to virulotyping. Applied and Environmental Microbiology. 75(13):4277-4288.

Friedman, M., Levin, C.E., Lee, S., Kozukue, N. 2009. Stability of Green Tea Catechins in Commercial Tea Leaves during Storage for 6 Months. Journal of Food Science. 74(2):47-51.

Quinones, B., Masey, S., Friedman, M., Swimley, M.S, Teter, K. 2009. A Novel Cell-Based Method to Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity. Applied and Environmental Microbiology. 75(5):1410-1416.

Friedman, M., Levin, C.E., Choi, S., Lee, S., Kozukue, N. 2009. Changes in the Composition of Raw Tea Leaves from the Korean Yabukida Plant during High-temperature Processing to Pan-Fried Kamairi-cha Green Tea. Journal of Food Science. 74(5):406-412.

Last Modified: 4/20/2014
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