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United States Department of Agriculture

Agricultural Research Service

2011 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 repeat 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
This is the final report for project 5325-42000-045-00D terminated in May 2011. The new project 5325-42000-047-00D has objectives that extend from progress made in terminated project. A) Camplylobacter/Arcobacter genomics. We sequenced 22 Campylobacter strains to draft level (30 accepted taxa within the genus Campylobacter now completed), including two novel species, plusan additional 27 C. fetus strains and 7 Arcobacter species. These data provide unprecedented comparative genomics for understanding pathogencity and will be extremely useful to the food safety and infectious disease communities. B) Sequencing of Enteropathogenic (EPEC) E. coli 055:H7. EPECs are leading causes of infant mortality and morbidity in developing countries and a progenitor to enterohemorrhagic E. coli 0157:H7 strains. In collaboration with Life Technologies, we completed the genome sequence of EPEC 055:H7 from California, yielding insight into the evolution and emergence of the deadly 0157:H7 serotype. C) Gene expression E.coli 0157:H7 variants. E. coli 0157:H7 has caused at least 27 outbreaks linked to leafy greens between 1995 and 2008. Camparing global gene expression of E. coli 0157:H7 strains from the 2006 spinach outbreak, revealed differences in strains' resistance to stressful conditions and increase ability to scavenge nutrients. D) Gene expression of C. jejuni during osmotic stress. In collaboration with the University of British Columbia, we identified genes critical for mounting a stress reponse, their presence in a subset of cells in a population, and salt-sensitive phenotypes that may aid in other adaptations. These findings have relevance in different environments. E) Rapid top-down mass spectrometry (MS) identification of pathogenic E. coli virulence factors. Software was developed to construct protein databases to analyze "top-down" MS data, specifically, to identify E. coli Shiga Toxins (STx), amyloid (curli), and acid stress-related proteins and proteins in E. coli that may be associated with chronic illness (Chrons disease). MS analysis provides a useful approach for rapid and molecular characterization of pathogens linked to many different food safety problems, especially identification of gene products related to virulence. F) Antimicrobial activities of non-toxic compounds. Apple juice, apple skin extract, black tea, tomato, and olive (hydoxytyrosol) compounds, have been analyzed for their antimicrobials were developed and effectively killed pathogens on media, ham and chicken. These studies suggest novel approaches for minimizin pathogen contamination. G) Toxicity of pure STx in animal models. Mice fed purified STx2 had damaged kidney, spleen, and thymus tissues. The overall impact of our reseach is to provide insight into the fundamental biological mechanisms of pathogen contamination, virulence, approached for decreasing contamination, and led to new objectives for our 5-year plan initiated in FY2011.

1. Campylobacter genomics. Campylobacter species cause more gastrointestinal (GI) illness than any other foodborne bacteria, but minimal data are available for some species. ARS researchers in Albany, CA, completed extensive sequencing of >60 strains representing all species and taxa to identify critical factors for assessing virulence and typing. In collaboration with Utrecht University, we sequenced the genomes of 23 strains of different subspecies of C. fetus associated with livestock and/or human illness. The identification of novel genes associated with human disease reveal differences that might correspond to the clinical outcomes.

2. Sequencing of Escherichia coli O55:H7 genome. Enteropathogenic E. coli O55:H7 (EPEC O55:H7) is a cause of infant mortality and morbidity in developing countries, and a recent precursor to enterohemorrhagic E. coli serotype O157:H7. In collaboration with Life Technologies Inc., ARS scientists completed the genome sequence of an EPEC O55:H7 from California. Comparative genomics identified bacteriophage insertions intermediate between the previously sequenced O157:H7 and O55:H7 strains. These data provide insight into the evolution and emergence of the deadly O157:H7 serotype and for fundamental analysis of functional differences.

3. Campylobacter jejuni is a serious food pathogen. Global gene expression of E. coli O157:H7 variants. Infections of E. coli O157:H7 accounted for 27 outbreaks linked to leafy produce between 1995 and 2008. ARS scientists in Albany, CA, compared the transcriptional profiles of E. coli O157:H7 strains linked to the 2006 E. coli O157:H7 spinach outbreak. Global gene expression analysis demonstrated that clinical strains and some spinach bag strains exhibited decreased expression of many stress related genes, correlating with the decreased resistance to acid, osmotic, and oxidative stresses. These changes were the result of regulatory mutations among the clinical strains that resulted in an increased ability to scavenge nutrients at the expense of stress tolerance. These findings suggest that stress sensitive E. coli O157:H7 should not be presumed to be less pathogenic.

4. Global gene expression of Campylobacter jejuni during osmotic stress. ARS scientists in Albany, CA, collaborated with the University of British Columbia dissecting the observed genetic variation and global gene expression patterns of the food-borne pathogen C. jejuni during osmotic stress. Microarray expression profiling revealed genes and operons critical for mounting a stress response. Populations of cells were identified that varied in function. For example, a fraction of the population can be salt sensitive and be associated with other adaptations. This variability occurs in other pathogens and is an important emerging area for understanding virulence and fitness in complex environments.

5. Development of rapid and low-cost assays for pathogen detection and genotyping. Shiga-toxin producing E.coli (STEC) have emerged as an important cause of human illness and thus new detection methods are needed. ARS scientists in Albany, CA, developed low-density and cost-effective DNA microarray using a colorimetric method (ampliPHOX) to identify rapidly multiple virulence factors of STEC recovered as part of an intensive survey of a major leafy greens production region in California. These methods will facilitate risk assessments of the food and environmental reservoirs of STECs that causes sporadic- and outbreak-related human illness.

6. Development of mass spectrometry (MS) methods. Rapid methods are needed to identify pathogens and differences related to virulence during outbreak and environmental investigations. Software and a new calibrant were developed to analyze MS data, specifically, to identify E. coli Shiga toxins, amyloids (curli), acid stress-related proteins and E. coli associated with chronic illness (e.g. Crohn’s disease). The new clibrant (alkylated thioredoxin) was developed to increase mass accuracy for top-down proteomic analysis. MS provides new approaches for characterization of pathogens linked to many different food safety problems.

Review Publications
Fagerquist, C.K. 2011. Proteomics of foodborne bacterial pathogens. In: Wiedman, M.; Zhang, W., editors of: Genomics of foodborne bacterial pathogens. New York, NY: Springer. p. 343-402.

Fagerquist, C.K., Sultan, O. 2011. Induction and identification of disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics. Analyst.136:1739-1746. DOI: 10.1039/C0AN00909A.

Sloane, J.Y., Leatherbarrow, A.J., Winstanley, C., Bennett, M., Hart, C.A., Miller, W.G., Williams, N. 2010. A comparison of Arcobacter isolation methods and the diversity of Arcobacter spp. in Cheshire, UK. Applied and Environmental Microbiology. 10.1128/AEM.01964-10.

Wright, S., Wilson, S., Miller, W.G., Mandrell, R.E., Siletsky, R.M., Kathariou, S. 2010. Methylation at GATC sites in genomic DNA of Campylobacter coli from turkeys and swine. Applied and Environmental Microbiology. 76(21):7314-7317.

Behringer, M., Miller, W.G., Oyarzabal, O.A. 2010. flaA-PCR, MLST, PFGE and Rep-PCR Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat. Journal of Microbiological Methods. 84(2011) 194-201.

Fagerquist, C.K., Sultan, O. 2010. Top-down proteomic identification of furin-cleaved alpha-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS. Journal of Biomedicine and Biotechnology. DOI:10.1155/2010/123460.

Rayburn, J.R., Friedman, M. 2010. L-cysteine, N-acetyl-L-cysteine, and glutathione protect xenopus laevis embryos against acrylamide-induced malformations and mortality in the frog embryo teratogenesis assay (FETAX). Journal of Agricultural and Food Chemistry. 58:11/172-11/178.

Lugo-Melchor, Y., Quinones, B., Amezquita-Lopez, B.A., Leon-Felix, J., Garcia-Estrada, R., Chaidez, C. 2010. Characterization of tetracycline resistance in Salmonella enterica strains recovered from irrigation water in the Culiacan Valley, Mexico. Microbial Drug Resistance. 6(3):185-190.

Sirk, T.W., Friedman, M., Brown, E.F. 2011. Molecular binding of black tea theaflavins to biological membranes: relationship to bioactivities. Journal of Agricultural and Food Chemistry. 59:3780-3787.

Friedman, M., Levin, C.E. 2011. Nutritional and medicinal aspects of D-amino acids. Amino Acids. 10.100/s00726-011-0915-1.

Piscatelli, H., Kotkar, S.A., Mcbee, M.E., Muthupalani, S., Schauer, D.B., Mandrell, R.E., Leong, J.M., Chen, J., Zhou, D. 2011. The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation. PLoS One. 6:e19331.

Kondo, S., Hoar, B.R., Mandrell, R.E., Atwill, E.R. 2010. Longitudinal prevalence and molecular typing of Escherichia coli O157:H7 using multiple-locus variable-number tandem-repeats analysis and pulsed field gel electrophoresis in a range cattle herd in California. American Journal of Veterinary Research. 71(11):1339-1347.

Houliston, R.S., Vinogradov, E., Li, J., Dzieciatkowska, M., St.Michael, F., Karwaski, M., Brochu, D., Jarrell, H.C., Parker, C., Yuki, N., Mandrell, R.E., Gilbert, M.E. 2011. Beyond gangliosides: Multiple forms of glycan mimicry exhibited by Campylobacter jejuni in its lipooligosaccharide (LOS). Journal of Biological Chemistry. 286:12361-12370.

Mild, R.M., Joens, L., Friedman, M., Olsen, C.W., Mchugh, T.H., Ravishankar, S. 2011. Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast. Journal of Food Science. 76: M163–M168. doi: 10.1111/j.1750-3841.2011.02065.x.

Choi, S., Kim, S., Kang, M., Nam, S., Friedman, M. 2010. Protective effects of black rice bran against chemically-induced inflammation of mouse skin. Journal of Agriculture and Food Chemistry. 58:10007-10015.

Choi, S.H., Kim, H.J., Lee, I.S., Kozukue, N., Levin, C.E., Friedman, M. 2010. Changes in free amino acid, chlorophyll, carotenoid, phenolic, and glycoalkaloid content in tomatoes during 11 stages of growth, and inhibition of cervical, lung, and lymphoma human cancer cells by green tomato extracts. Journal of Agricultural and Food Chemistry. 58:7547-7556.

Tsuchida, H., Kozuke, N., Han Gyeong, P., Choisuk, H., Levin, C.E., Friedman, M. 2010. Low-temperature Storage of Cucumbers Induces Changes in the Organic Acid Content and in Citrate Synthase Activity. Postharvest Biology and Technology. 58:129-134.

Kim, S., Kang, M., Lee, I., Park, J., Nam, S., Friedman, M. 2011. Composition of liquid rice hull smoke and anti-inflamatory effects in mice. Journal of Agricultural and Food Chemistry. 59:4570-4581.

Choi, S., Ahn, J., Kozukue, N., Levin, C.E., Friedman, M. 2011. Distribution of free amino acids, flavonoids, total phenolics, and antioxidative activities of jujube (Ziziphus jujuba) fruits and seeds harvested from plants grown in Korea. Journal of Agricultural and Food Chemistry. 59:6594-6604.

Last Modified: 11/25/2015
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