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United States Department of Agriculture

Agricultural Research Service

2008 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 (T-5000) for foodborne pathogen identification. NP108 Action Plan Component 1.2, and sections 1.1.1: Methodology; 1.2.1: Detection/Validation; 1.2.5: Omics.

Plant non-toxic antimicrobials. Edible antimicrobial apple films on raw chicken breasts inoculated with S. enterica and E. coli O157:H7 induced multilog reductions of the pathogens without organoleptic problems. Apple skin and tea powders facilitated thermal destruction of E. coli O157:H7 in ground beef; carvacrol inactivated antibiotic-resistant strains of C. jejuni; plant compounds inactivated three strains of Mycobacterium avium subspecies paratuberculosis (Map) isolated from cattle, raw milk, and humans. NP108 Action Plan Component 1.2, and section 1.1.5: Antibiotic Resistance.

Campylobacter and Arcobacter genomics. We assisted in the annotation and analysis of the JCVI Campylobacter genomes: C. jejuni subsp. doylei, C. fetus subsp. fetus, C. concisus, C. curvus and C. hominis; participated in JCVI oral Campylobacter genome sequencing project to sequence C. rectus, C. showae and C. gracilis strains; and characterized 300+ C. jejuni strains, by multilocus sequence typing (MLST). We developed MLST methods to type C. concisus/C. curvus and Arcobacter strains and typed ~75 human clinical C. concisus isolates and 400+ Arcobacter strains of human, food, animal or environmental origin. NP108 Action Plan Component 1.2, and section 1.1.1: Methodology; 1.1.2: Epidemiology; 1.2.5: Omics.

DNA and oligonucleotide microarrays for genomic analysis of Campylobacter, E. coli. Microarray-based comparative gene indexing (CGI) of seven different species of pathogenic bacteria (E. coli, S. Typhimurium, C. jejuni, C. coli, C. lari, A. butzleri and Enterobacter sakazakii) has been completed. Nineteen classes of lipooligosaccharide (LOS) loci from different C. jejuni strains were mapped, and mechanisms linked to changes in LOS structures were identified, including indels, gene cassettes, and variable gene inactivation by the deletion or insertion of bases. The role of the bile acid, deoxycholate, in triggering the virulence potential of C. jejuni was determined. NP108 Action Plan Component 1.2, and section 1.2.5: Omics, 1.2.8: Pathogenicity. Cell-based method for shiga toxin (Stx) detection. A novel, low cost, cell-based method for the detection of Stx and inhibitors of Stx activity was developed. A Vero cell line was generated to express a destabilized variant of the enhanced green fluorescent protein. Validation experiments demonstrate that this Vero cell- based assay can detect picogram/ml quantities of active toxin. The Vero cell assay facilitated identification of an anti-toxin activity in grape seed and pomace extracts. NP108 Action Plan Component 1.2, and section 1.1.5: Antibiotic Resistance.

1. Environmental investigation of dairies associated with Campylobacter jejuni. The second largest outbreak of Campylobacter illness in US history occurred as a result of contaminated milk processed at a California correctional facility dairy and a small outbreak of C. jejuni illness occurred due to raw milk from a small organic dairy. A study of both dairies by ARS scientists in the Produce Safety abd Microbiology Research Unit in Albany, CA, in coordination with the California Department of Public Health, involved sampling of cattle feces, water, milk and other samples, and isolation of C. jejuni from some of them. Isolates were screened by a novel method and suspect outbreak-related strains were genotyped and compared to genotypes of the human outbreak strains. Successful isolation by PSMRU of outbreak related strains from both dairies provided crucial epidemiological information for explaining the outbreaks. The results obtained in one of the studies is critical in ongoing negotiations between one dairy and representatives of the State of California on rules of acceptable levels of coliforms in raw milk for human consumption. Addresses NP108, Component 1, Problem Statement a2.

2. Mass spectrometry-based bioinformatics software for the rapid identification of foodborne pathogens, viruses and protein toxins. The research addresses the need for fundamental data regarding critical foodborne pathogens. Mass spectrometry-based bioinformatics software has been developed by ARS scientists in the Produce Safety abd Microbiology Research Unit in Albany, CA for rapid identification of protein biomarkers of foodborne pathogens, viruses and toxins by "top-down" proteomics analysis. Sequence-specific ions fragmented from intact proteins are rapidly compared against a database of in silico fragment ions from thousands of bacterial, viral and toxin proteins. This research will yield fundamental information for rapid identification of pathogens and virulence factors. NP108, Component 1, Problem Statements a1 and b1.

3. Sequencing of Arcobacter butzleri and Campylobacter lari genomes. There is a need to obtain more fundamental data regarding critical food-borne pathogens. Arcobacter butzleri and Campylobacter lari are known agents of human bacterial gastroenteritis: A. butzleri is transmitted putatively via contaminated food and water and C. lari is associated with water and shellfish. ARS scientists in the Produce Safety abd Microbiology Research Unit in Albany, CA closed and completed the genomes of the human clinical strains A. butzleri RM4018 and C. lari RM2100. Both genomes were fully annotated, with the sequence and annotation data deposited in the NCBI GenBank database. Analysis of the two genomes identified novel genes and pathways, putative niche-specific genes and potential pathogenicity genes. Completion of the genomes assisted in development of novel A. butzleri and C. lari DNA microarrays. Additionally, the A. butzleri genome sequence is critical for current development of novel Arcobacter typing methods (e.g. MLST). This accomplishment addresses NP108, Component 1, Problem Statement b5.

4. Detection and genotyping of Escherichia coli present in environmental samples. Methods are needed to rapidly detect bacterial pathogens present in agricultural and food samples in order to obtain more fundamental data regarding critical foodborne pathogens. ARS scientists in the Produce Safety abd Microbiology Research Unit in Albany, CA developed DNA oligonucleotide microarrays to detect the presence of Escherichia coli from enrichment cultures following multiplex PCR. These data assist in identifying bacterial species and Shiga toxin producing E. coli. Multiplex PCR products from environmental sample enrichment cultures were hybridized to the array and revealed that the method can detect and identify rapidly pathogenic E. coli from many agricultural sources. This accomplishment addresses NP108, Component 1, Problem Statements a1, a2, b1, and b5.

5. Detection and inactivation of Shiga toxin from E. coli O157:H7. Improved assays are needed to detect E. coli O157:H7 which produces Shiga toxins (Stx1 and Stx2). A novel cell-based assay has been developed to detect Stxs and inhibitors of Stx activity. A Vero cell line enhanced green fluorescent protein as a reporter of activity was used to monitor the toxin-induced inhibition of protein synthesis with a plate reader. Toxin dose response curves demonstrated a picogram/ml threshold for toxin detection. To establish our Vero cell assay as a useful tool for the identification of toxin inhibitors, ARS scientists in the Produce Safety abd Microbiology Research Unit in Albany, CA screened a panel of plant compounds for anti-toxin activities. Fluorescent signals were maintained when Vero cells with reporter were exposed to Stx1/2-containing medium in the presence of either grape seed or grape pomace extract. These results indicate that the Vero cell fluorescence assay is an accurate and sensitive method to detect Stx activity and inhibitors of toxin activity. This addresses NP108, Component 1, Problem statements b1 and b8.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of the New MTAs (providing only)2
Number of Newspaper Articles and Other Presentations for Non-Science Audiences1

Review Publications
Kaakoush, N.O., Sterzenbach, T., Miller, W.G., Suerbaum, S., Mendz, G.L. 2007. Identification of disulphide reductases in campylobacterales. Antonie Van Leeuwenhoek. 92(4):429-441

Fagerquist, C.K., Yee, E., Miller, W.G. 2007. Composite sequence proteomic analysis of protein biomarkers of Campylobacter coli, C. lari and C. consisus for bacterial identification. The Analyst. 132(10):1010-1023.

Miller, W.G., Parker, C., Rubenfield, M., Mendz, G.L., Wosten, M.M., Ussery, D.W., Stolz, J.F., Binnewies, T.T., Hallin, P.F., Wang, G., Malek, J.A., Rogosin, A., Stanker, L.H., Mandrell, R.E. 2007. The complete genome sequence and analysis of the Epsilonproteobacterium Arcobacter butzleri. PLoS One. 2(12):e1358.

Hannis, J., Manalili, S., Hall, T., Ranken, R., White, N., Sampath, R., Blyn, L., Ecker, D., Mandrell, R.E., Fagerquist, C.K., Bates, A.H., Miller, W.G., Hofstadler, S. 2008. High Resolution Genotyping of Campylobacter Using PCR and High-Throughput Mass Spectrometry. Journal of Clinical Microbiology.46(4) 1220-1225.

Quinones, B., Guilhabert, M.R., Miller, W.G., Mandrell, R.E., Lastovica, A.J., Parker, C. 2008. Comparative genomic analysis of clinical strains of Campylobacter jejuni from South Africa. PLoS One. 3(4)e2015_1-e2015_10

Ravishankar, S., Zhu, L., Law, B., Joens, L., Friedman, M. 2008 Plant derived compounds inactivate antibiotic resistant Campylobacter jejuni strains. Journal of Food Protection.71(6)1145-1149(5)

Im, H., Suh, B., Lee, S., Kozukue, N., Ohnisi-Kameyama, M., Levin, C.E., Friedman, M. 2008. Analysis of Phenolic Compounds by HPLC and LC/MS in Potato Plants, Flowers, Leaves, Stems, and Tubers and in Home Processed Potatoes. Journal of Agriculture and Food Chemistry. 56, 3341-3349

Du, W., Olsen, C.W., Avena-Bustillos, R.D., Mc Hugh, T.H., Levin, C.E., Friedman, M. 2008. Storage Stability and Antibacterial Activity against Echerichia coli O157:H7 of Carvacrol in Edible Apple Films made by Two Different Casting Methods. Journal of Agricultural and Food Chemistry. 56:3082-2088.

Kaakoush, N.O., Miller, W.G., De Reuse, H., Mendz, G.L. 2007. Oxygen requirement and tolerance of Campylobacter jejuni. Research in Microbiology. 158(8-9):644-650.

Friedman, M., Levin, C.E., Lee, S., Lee, J., Ohnisi-Kameyama, M., Kozukue, N. 2008. Analysis by HPLC and LC/MS of Pungent Piperamides in Commercial Black,White, Green, and Red Whole and Ground Peppercorns.. Journal of Agricultural and Food Chemistry. 56(9):3028-3036.

Gilbert, M., Parker, C., Moran, A.P. 2008. Campylobacter jejuni Lipooligosaccharides: Structures and Biosynthesis. In: Nachamkin, I, Szymanski, C.M., Blaser, M.J., editors. Campylobacter. 3rd Edition. Washington, DC: ASM Press. p. 483-504.

Last Modified: 7/29/2015
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