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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology of Human Pathogens Associated with Food

Location: Produce Safety and Microbiology Research

2012 Annual Report

1a.Objectives (from AD-416):
Objective 1: Comparative genomic analyses of Campylobacter, Arcobacter, E. coli and S. enterica to identify novel genetic elements or polymorphisms that are associated with virulence, niche specialization or other adaptive traits. Objective 2: Develop sequence-based typing methods to detect and analyze multiple critical food-borne pathogens from multiple sources. Objective 3: Generate gene expression profiles for E. coli, S. enterica, and Campylobacter from varying sources and in response to various environmental stresses to identify factors contributing to virulence and survival in diverse habitats. Objective 4: Develop rapid, simple and inexpensive multiplex assays for pathogen detection and virulence characterization using novel technology for use in surveillance and outbreak epidemiology. Objective 5: Establish proteomic approaches for detecting and typing foodborne pathogens and toxins, and measure pathogen response to environmental stresses by mass spectrometry methods. Objective 6: Investigate mechanisms of bacterial toxicity and evaluate novel methods for inactivating Shiga toxins by developing cell-based assays for assessing toxicity and comparing the relative toxicity of Shiga toxin 1 and 2 variants.

1b.Approach (from AD-416):
Our objectives address fundamental research for comparative genomic analyses of Campylobacter and Arcobacter species, and pathogenic Escherichia coli and Salmonella enterica serovars to identify novel genetic elements or polymorphisms associated within virulence, niche specialization or other adaptive traits. We will analyze the genomic data to develop sequence-based typing methods to detect and analyze multiple critical food-borne pathogens from multiple sources for purposes of improved source tracking to identify reservoirs in food production environments. The genomic data will allow us to develop methods for gene expression profiling of pathogens from different sources and their responses to various environmental stresses to identify factors contributing to virulence and survival in diverse habitats; and develop rapid, simple and inexpensive multiplex assays for pathogen detection and virulence characterization using novel technology for use in surveillance and outbreak epidemiology. The genomic data facilitate developing “top-down” and “bottom-up” mass spectrometry approaches for proteomic analysis for detecting and typing foodborne pathogens and toxins, and measuring pathogen response to environmental stresses. Non-O157 Shiga toxin-producing E. coli (STEC) strains have emerged as a source of more human illness than appreciated previously. Data for isolating and detecting them are a critical need and will be provided through this research. We also will be researching mechanisms of bacterial toxicity to develop and evaluate novel methods for inactivating Shiga toxins by developing cell-based assays for assessing toxicity and comparing the relative toxicity of Shiga toxin 1 and 2 variants. In summary, we will use the latest analytical tools to obtain data for identifying foodborne pathogens, differences in strains related to fitness and virulence, novel interventions related to toxins and, ultimately, approaches for minimizing illness associated with food.

3.Progress Report:
Significant progress was achieved in sequencing, analysis and closure of multiple genomes of Campylobacter and Arcobacter species, including genomes of at least six new species, and multiple Shiga toxin-producing E. coli (STEC) and Salmonella enterica strains associated with multiple outbreaks. These data provide a framework for comparative genomic analyses, identification of novel genetic elements and development of sequence-based typing methods that are major objectives of our 5-year research plan. Comparison of strains from different sources and humans facilitates identification of potential markers of fitness and virulence. We achieved major progress in Campylobacter genomics by sequencing at least one strain of each of the 27 Campylobacter species and identifying multiple new species. Analysis revealed species clustering based on strain source and identification of potential virulence factors. Progress in Campylobacter genomics is timely considering emerging interest in species associated with non-diarrheal human illness; this has led to multiple international collaborations. Software has been patented for constructing a database facilitating identification, as examples, of Shiga toxin variants, acid resistance proteins and Crohn's disease-related E. coli proteins. This discovery will increase accuracy of analysis of bacteria and their protein toxins. Complete genomes facilitated development of rapid and inexpensive chip-based and proteomic methods for STEC O-antigen and virulence identification, detection of toxins and stress-related responses. Similar chip assays for Salmonella and Norovirus characterization are being produced. The STEC chip is being used currently for analysis of thousands of STEC strains isolated as part of large leafy greens production environment and watershed surveys. Identifying the most virulent STEC is critical for determining the risk of STECs in food production. These surveys of central California coast leafy greens production environments spanning seven years, supported partially by extramural grants from USDA-NIFA and FDA, involved frequent testing of livestock, wildlife, leafy greens, soil and water on >50 farms and ranches plus 30 additional watershed sites. Microbial source tracking has identified matching strains from different sources, times and places facilitating identification of genetic and epigenetic factors related to functional differences. Applied research involving surveys of a critical food production region combined with genomics and proteomics approaches for characterizing, in depth, pathogenic strains isolated from surveys will yield important information and reagents for years of future research relevant to intensive food production ecosystems.

1. Sequencing and analysis of Campylobacter genomes. Some Campylobacter species cause human bacterial gastroenteritis and/or disease in food animals. Although most cases of Campylobacter associated food-borne illness are currently attributed to Campylobacter jejuni, emerging Campylobacter species have become linked increasingly to human illness. We have completed the genome sequences of 27 Campylobacter species, subspecies and biovars. Additionally, the genome sequences of multiple novel species, isolated from California agricultural regions, were also completed. Campylobacters colonize a wide variety of mammalian and avian hosts and can be isolated from meat, water, milk and shellfish. Comparative genomics of these completed Campylobacter genomes will provide further insights into the genetic basis of host association, pathogenicity and survival in the environment.

2. Sequencing of salmonella associated with multiple outbreaks. In 2009, shredded lettuce at restaurants seemed the likely source of an outbreak of Salmonella Typhimurium. This was followed in 2010-2011 by a Salmonella outbreak that occurred among students and workers in laboratories. To help elucidate the exact genetic relationship of these outbreak strains, we performed genome sequencing of five outbreak-associated clinical strains that were isolated across the United States between 2009 and 2010. Comparison of these sequences with the seven completed S. Typhimurium genomes revealed that all of these strains are nearly identical to the strain of S. Typhimurium used in clinical and research labs for over two decades. These results have raised concerns about.
1)the 2009 shredded lettuce outbreak source,.
2)the security of laboratory strains, and.
3)persistence of S. Typhimurium strains from “field to fork”.

3. Development of rapid and inexpensive multiplex assay for STEC. The recent STEC outbreaks have emphasized the importance of developing rapid methods for genotyping virulent bacterial strains. An inexpensive colorimetric DNA microarray-based method was developed for simultaneous identification of multiple virulence genes in E. coli O157 and non-O157. This facilitated detection and characterization of STEC isolates recovered from multiple animal sources and environmental samples in agricultural regions in California. The efficiency of this method for STEC has led to collaboration in a major project on human noroviruses, which is the leading cause of human gastroenteritis worldwide.

4. Rapid top-down proteomic identification of shiga toxin variants. STEC are increasingly linked to severe outbreaks of foodborne illness in the US (spinach in 2006) and elsewhere (e.g. fenugreek seeds in 2011 in Germany and France). Toxicity of Stx vary by differences in primary amino acid sequences. We developed a rapid mass spectrometry-based top-down proteomic method to identify sequence-specific Stx variants. The method is simple and fast and can distinguish between highly similar sequence variants. An Stx variant can be identified within 1 to 2 hours after bacterial culturing using this technique and has potential as a rapid identification tool during an outbreak of foodborne illness as well as a powerful research tool.

5. Edible food compounds inactivate virulent bacterial and plant toxins. The increasing number of outbreaks worldwide has raised concern. Stx is the primary virulence factor of STEC, so decreasing active Stx in contaminated food is a strategy for minimizing illness. Stx virulence is based on the 3-D conformation of the protein. In collaborative studies, we discovered that (a) orally ingested Stx damages kidney, spleen, and thymus tissues in mice; (b) freshly prepared juice from Red Delicious apples inhibited the biological activity of Stx; (c) 4-hydroxytyrosol from olives inactivated Staphylococcus aureus and it’s enterotoxin; (d) olive powder inhibited multiple foodborne pathogens; and (e) reconstituted milk inhibited activity of ricin toxin (isolated from castor beans), a toxin similar in activity to Stx. These findings suggest that safe food-compatible compounds can inactivate toxins. The proposed strategies to overcome the virulence of toxins will benefit microbial food safety, animal and human health, food biosecurity, and the economy.

6. Incidence of pathogens in the environment. Thousands of strains of STEC, including O157:H7, Salmonella, Listeria monocytogenes and Campylobacter species have been isolated from > 15,000 environmental samples, and characterized for serotype, genotype and virulence factors. The epidemiology and ecology of pathogens in a major food production region has stimulated multiple interactions with regulatory agencies and industry. In addition to the microbial source tracking information provided for comparison to the field data obtained, these strains provide an unprecedented resource for addressing fundamental biology objectives related to comparative genomics, analysis of foodborne pathogens from multiple sources, and gene expression profiling of related strains from different sources.

Review Publications
Parker, C., Kyle, J.L., Huynh, S., Carter, M.Q., Brandl, M., Mandrell, R.E. 2012. Distinct transcriptional profiles and phenotypes exhibited by Escherichia coli O157:H7 isolates related to the 2006 spinach-associated outbreak. Applied and Environmental Microbiology. 78:455-463.

Fagerquist, C.K., Sultan, O. 2012. A new calibrant for MALDI-TOF-TOF-PSD-MS/MS of non-digested proteins for top-down proteomic analysis. Rapid Communications in Mass Spectrometry. 1241:1248.

Jay-Russell, M.J., Bates, A.H., Harden, L.A., Miller, W.G., Mandrell, R.E. 2012. Isolation of Campylobacter from feral swine (Sus scrofa) on the ranch associated with the 2006 Escherichia coli O157:H7 spinach outbreak investigation in California. Zoonoses and Public Health. 10.1111/j.1863-2378.2012.01465.x.

Rounds, L., Havens, C.M., Feinstein, Y., Friedman, M., Ravishankar, S. 2012. Plant extracts, spices, and essential oils inactivate E. coli O157:H7 pathogens and reduce formation of potentially carcinogenic heterocyclic amines in grilled beef patties. Journal of Agricultural and Food Chemistry.

Kyle, J.L., Cummings, C.A., Parker, C., Quinones, B., Vatta, P., Newton, E., Huynh, S., Swimley, M.S., Degoricija, L., Barker, M., Fontanoz, S., Nguyen, K.M., Patel, R.N., Fang, R., Tebbs, R., Petrauskene, O., Furtado, M., Mandrell, R.E. 2012. E. coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage. Journal of Bacteriology. 194:1885-1896.

Miller, W.G., Chapman, M.H., Yee, E., On, S.L., Mcnulty, D.K., Lastovica, A.J., Carroll, A.M., Mcnamara, E.B., Duffy, G., Mandrell, R.E. 2012. Multilocus sequence typing (MLST) methods for the emerging Campylobacter species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus and C. curvus.. Frontiers in Cellular and Infection Microbiology. 10:3389/fcimb.2012.00045.

Scholl, D., Gebhart, D., Williams, S., Bates, A.H., Mandrell, R.E. 2012. Genome sequence of E. coli O104:H4 leads to rapid development of a targeted antimicrobial agent against this emerging pathogen. PLoS One. 7(3):e33637.doi:10.1371/journal.pone.0033637.

Choi, S., Ahn, J., Kozukue, N., Kim, H., Nishitani, Y., Mizuno, M., Levin, C.E., Friedman, M. 2012. Structure-activity relationships of a-, ß1-, and d-Tomatines and Tomatidine Against Human Breast (MDA-MB-231), Gastric (KATO-III), and Prostate (PC3) Cancer Cells. Journal of Agricultural and Food Chemistry.

Kim, S., Kang, M., Nam, S., Friedman, M. 2012. Dietary rice component ¿-Oryzanol inhibits tumor growth in tumor-bearing Mice. Molecular Nutrition and Food Research. DOI 10.1002/mnfr.201200057.

Fagerquist, C.K., Sultan, O. 2012. Top-down proteomic identification of protein biomarkers of food-borne pathogens using MALDI-TOF-TOF-MS/MS. Lee, M.S. Editor. Mass Spectrometry Handbook. New Jersey:John Wiley & Sons. p. 559-573.

Finotti, E., Bersani, E., Del Prete, E., Friedman, M. 2012. A Functional mathematical index for predicting effects of food processing on eight sweet potato(Ipomoea batatas)cultivars. Journal of Food Composition and Analysis. doi:10.1016/j.jfca.2012.04.001.

Friedman, M., Levin, C.E. 2012. Nutritional value of D-amino acids, D-peptides, and amino acid derivatives in mice. Polleioni & Servi, S., editors. Unnatural Amino Acids Methods and Protocols. New York Dordrecht Heidelberg London. Humana Press. Vol. 794, p. 337-356.

Yang, J., Moon, E., Nam, S., Friedman, M. 2012. Anti-diabetic effects of rice hull smoke extract on glucose-regulating mechanism in type 2 diabetic mice. Journal of Agricultural and Food Chemistry. doi:10.1021/jf3017749.

Rounds, L., Havens, C.M., Feinstein, Y., Friedman, M., Ravishankar, S. 2012. Concentration-dependent inhibition of Escherichia coli O157:H7 and potentially carcinogenic heterocyclic amines in heated ground beef patties by apple,olive,and onion powders and clove bud oil. Journal of Agricultural and Food Chemistry. 60:3792-3799. doi: 10.1021/jf204062p.

Kim, S., Moon, E., Nam, S., Friedman, M. 2012. Hericium erinaceus mushroom extracts protect infected mice against Salmonella typhimurium induced liver damage and mortality by activation of innate immune cells. Journal of Agricultural and Food Chemistry. 60:5590-5596. doi: 10.1021/jf300897w.

Yang, J., Kang, M., Nam, S., Friedman, M. 2012. Anti-diabetic effects of rice hull smoke extract in alloxan-induced diabetic mice. Journal of Agricultural and Food Chemistry. 60:87-94. doi: 10.1021/jf2035077.

Friedman, M., Rasooly, R., Do, P.M., Henika, P.R. 2011. The olive compound 4-hydroxytyrosol inactivates Staphyloccoccus aureus bacteria and Staphylococcal enterotoxin A (SEA). Journal of Food Science. 76:M558-M558. doi:10.1111/j.1750-3841.2001.02365.x.

Kim, S., Kang, M., Park, J., Nam, S., Friedman, M. 2011. Rice hull smoke extract inactivates Salmonella Typhimurium in laboratory media and protects infected mice against mortality. Journal of Agricultural and Food Chemistry. 77:M80-M85. doi: 10.1111/j.750-3841.2011.02478.x.

Pryjma, M., Apel, D., Parker, C., Huynh, S., Gaynor, E.C. 2012. FdhTU-modulated formate dehydrogenase expression and electron donor availability enhance recovery of Campylobacter jejuni following host cell infection. Journal of Bacteriology. 194:3803-3813.

Lu, X., Huang, Q., Miller, W.G., Aston, D., Xu, J., Xue, F., Zhang, H., Rasco, B., Wang, S., Konkel, M.E. 2012. Comprehensive detection and discrimination of campylobacter species using confocal micro-raman spectroscopy and multilocus sequence typing. Journal of Clinical Microbiology. doi:10.1128/JCM.01144-12.

Last Modified: 4/16/2014
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