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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

2013 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:
We made significant advances towards major objectives of our 5-year research plan: the establishment of a framework for comparative genomic analyses; identification of novel genetic elements; and the development of sequence-based methods that type and detect foodborne pathogens. Closure, analysis, and annotation were performed on the genomes of multiple Campylobacter species, several strains of Shiga toxin-producing E. coli (STEC), and Salmonella enterica strains associated with multiple outbreaks. We achieved major progress in Campylobacter genomics, including the identification of 15 new taxa as part of multiple national and international collaborations. Comparative analysis revealed Campylobacter taxa clustering based on gene content that correlated with the environmental source and phenotypes of these taxa. We developed a multiplex PCR method from the genome sequences of the pathogen C. jejuni to quickly and accurately type strains. The genomes of several serotype O111 STEC strains from a survey of the Salinas Valley and from clinical sources were sequenced and gene content comparisons are proceeding as part of identifying the most virulent STECs from environmental and clinical sources. We completed the assembly and annotation of the first closed genome of S. enterica serovar Thompson, a contaminant of many foods. In collaboration with the USDA Natioan Institute of Food and Agriculture (NIFA) Food Virology Project, a focused microarray was designed and validated in conjunction with rapid and inexpensive colorimetric technology for the specific identification of a subset of norovirus strains that are associated with foodborne illness in humans. Finally, research employing whole genome sequencing is advancing to identify key regulatory genes responsible for increased Shiga toxin (Stx) levels, detected in STEC strains from an agricultural region in California. By using a quantitative cell-based assay, these STEC strains, expressing either the Stx2a or Stx2c variant, were identified to have an enhanced ability to inhibit protein synthesis in mammalian cells. We have made further significant progress in proteomics and the characterization of antibacterial compounds from plants. Top-down proteomic identification was combined with RT-PCR to analyze a clinical E. coli strain that produces two variants Stx2a and Stx2c, which are distinguishable and identifiable by MALDI-TOF-TOF mass spectrometry. Protein biomarkers of three S. enterica serovars (Newport, Kentucky, Thompson) were also identified using MALDI-TOF-TOF mass spectrometry. Enzymatic surface shaving experiments were conducted on E. coli O157:H7 and a strain of Listeria monocytogenes. As part of a collaborative project, a protein from L. monocytogenes, that appears to be the antigenic target for a monoclonal antibody, was identified by top-down proteomic identification. In other collaborative projects, we have demonstrated the ability of specific plant compounds alone and in edible films to inactivate S. enterica. These findings provide an excellent scientific basis for the application of plant compound interventions in food packaging.

4. Accomplishments
1. Sequencing of Campylobacter and Arcobacter genomes. Campylobacter and Arcobacter species can cause human bacterial gastroenteritis and/or disease in food animals. We have completed the genome sequences of 45 validly-described Campylobacter and Arcobacter species, subspecies and biovars. Additionally, the genome sequences of multiple novel species, isolated mainly from California agricultural regions, were also completed. These organisms colonize a wide variety of mammalian and avian hosts and can be isolated from meat, water, milk and shellfish. Although most cases of Campylobacter-associated food-borne illness are currently attributed to Campylobacter jejuni, emerging Campylobacteraceae species have become linked increasingly to human illness. Therefore, comparative genomics of these completed genomes will provide further insights into the genetic basis of host association, pathogenicity, and survival in the environment.

2. The complete genome sequence of Salmonella Thompson. Annually, over 40,000 cases of salmonellosis are reported in the United States with an estimated 800,000 unreported cases. There are many different kinds (serovars) of nontyphoidal Salmonella enterica that cause foodborne illness and these are associated with many raw and ready-to-eat foods. S. enterica serovar Thompson has been associated with several outbreaks in the United States including events traced to cilantro and smoked salmon. We have sequenced the first complete genome of S. enterica serovar Thompson. Comparative genomics of S. enterica serovar Thompson with other completed S. enterica genomes will provide insights into possible host association and the design of detection systems for tracking these pathogens.

3. Rapid and inexpensive identification of human noroviruses. Human noroviruses cause up to 21 million cases of foodborne disease in the United States annually and are the most common cause of acute gastroenteritis in industrialized countries. To reduce the burden of foodborne disease associated with viruses, a DNA microarray-based colorimetric method was developed to simultaneously and accurately genotype multiple norovirus strains. This norovirus detection assay has proven to be a sensitive, rapid, and practical method with commercial potential. This method can also be employed for the identification of risk factors to help reduce contamination of noroviruses in pre- and post-harvest environments.

4. Top-down proteomic identification of Shiga toxin 2 variants from Shiga toxin-producing Escherichia coli (STEC) using mass spectrometry. Shiga toxin-producing E. coli (STEC) are increasingly linked to severe outbreaks of foodborne illness and sequence-specific variants of Shiga toxin 2 (Stx2) have been linked to differences in toxicity. Approximately 70 STEC strains were tested for Stx2 production by antibiotic induction. Stx2 variants were identified from un-fractionated bacterial cell lysates by mass spectrometry and top-down proteomic software developed in-house at the USDA. Top-down identification was confirmed by DNA sequencing of the Stx2 genes. This rapid method allows definitive sequence-specific identification of Stx2 variants and thus their likely toxicity. As the method relies upon antibiotic-induced toxin production, STEC response to antibiotic stress is also obtained which has clinical relevance during an outbreak.

5. The antimicrobial effects of cinnamon leaf oil against multi-drug resistant Salmonella Newport on organic leafy greens. Non-typhoidal Salmonella species cause approximately 11% of foodborne illnesses in the United States and 28% of deaths associated with foodborne illness. Salmonella species were responsible for almost 40% of the foodborne outbreaks resulting from produce contamination. There is generally no kill-step when preparing salad vegetables, so there is a risk for foodborne illness outbreaks due to consumption of these vegetables. In a collaborative study with the University of Arizona supported by a USDA grant, we discovered that a 2-min treatment with 0.3% to 0.5% cinnamon oil inactivated the pathogens on four types of organic leafy greens, with no recovery of bacteria by day 3 of storage. Generally Regarded As Safe (GRAS)-listed cinnamon oil has the potential to be used as a treatment option for washing organic baby and mature spinach, and iceberg and romaine lettuces.

Review Publications
Sheppard, S.K., Didelot, X., Jolley, K.A., Darling, A.E., Kelly, D., Colles, F.M., Strachen, N.J., Ogden, I.D., Forbes, K.J., French, N., Carter, P., Miller, W.G. 2012. Progressive genome-wide introgression in agricultural Campylobacter coli. Proceedings of the National Academy of Sciences. 22:1051-1064.

Merga, J.Y., Williams, N.J., Miller, W.G., Leatherbarrow, A.J., Bennett, M., Hall, N., Ashelford, K.E., Winstanley, C. 2013. Exploring the diversity of Arcobacter spp. in cattle in the UK using MLST and whole genome sequencing. PLoS One. 8(2):e55240. doi:10.1371/journal.pone.0055240.

Fagerquist, C.K., Sultan, O., Carter, M.Q. 2012. Possible evidence of amide bond formation between sinapinic acid and lysine-containing bacterial proteins by matrix-assisted laser desorption/ionization (MALDI) at 355 nm. Journal of American Society for Mass Spectrometry. 23(12):2102-2114.

Choi,, S., Ahn, J., Kim, H., Im, N., Kozukue, N., Levin, C.E., Friedman, M. 2012. Changes in free amino acid, protein, flavonoids, and phenolic content in jujube (Ziziphus jujube) fruit during eight stages of growth and antioxidative and cancer cell inhibitory effects by extracts. Journal of Agricultural and Food Chemistry. 60(41):10245-10255. doi:10.1021/jf302848u.

Jay-Russell, M.T., Mandrell, R.E., Yuan, J., Bates, A.H., Manalac, R., Mohle-Boetani, J., Kimura, A., Lidgard, J., Miller, W.G. 2012. Using MOMP typing as an epidemiological tool to investigate outbreaks caused by milkborne Campylobacter jejuni isolates in California. Journal of Clinical Microbiology. 51(1):195-201.

Amezquita-Lopez, B.A., Quinones, B., Cooley, M.B., Leon-Felix, J., Castro-Del Campo, N., Mandrell, R.E., Jimenez, M., Chaidez, C. 2012. Genotypic analyses of Shiga toxin-producing Escherichia coli O157 and non-O157 recovered from feces of domestic animals in rural farms in Mexico. PLoS One. 7(12):e51565. doi:101371/journal.pone.0051565.

Cameron, A., Frirdich, E., Huynh, S., Parker, C., Gaynor, E.C. 2012. The hyperosmotic stress response of Campylobacter jejuni. Journal of Bacteriology. November 2012, 194:(22)6116-6130.

Kim, S., Moon, E., Nam, S., Friedman, M. 2012. Composition of herba pogostemonis water extract and protection of infected mice against salmonella typhimurium-induced liver damage and mortality by stimulation of innate immune cells. Journal of Agricultural and Food Chemistry. 60(49):12122-12130. doi:10.1021/jf304466t.

Sun Phil, C., Sung Phil, K., Seok Hyun, N., Friedman, M. 2012. Antitumor effects of dietary black and brown rice brans in tumor-bearing mice: relationship to composition. Molecular Nutrition and Food Research. 57(3):390-400. doi: 10.1002/mnfr.201200515.

Chen, C.H., Sadhana, R., Marchello, J., Friedman, M. 2013. Antimicrobial activity of plant compounds against Salmonella Typhimurium DT104 in ground pork and the influence of heat and storage on the activity. Journal of Food Protection. 76:1264-1269. doi:10:4315/0362-028X.JFP-12-493.

Fagerquist, C.K. 2013. Top-down proteomic identification of bacterial protein biomarkers and toxins using MALDI-TOF-TOF-MS/MS and post-source decay. Reviews in Analytical Chemistry. 32(2):127-133. doi:10.1515/revac-2012-0035.

Friedman, M., Rasooly, R. 2013. Review of the inhibition of biological activities of selected food-related toxins by natural compounds. Toxins. 5:743-775. doi: 10.3390/toxins50x100x.

Boggess, M.V., Lippolis, J.D., Hurkman II, W.J., Fagerquist, C.K., Briggs, S.P., Gomes, A.V., Righetti, P., Bala, K. 2013. The need for agriculture phenotyping: “Moving from genotype to phenotype”. Journal of Proteomics. doi:

Kim, S., Nam, S., Friedman, M. 2013. Hericium erinaceus (Lion’s Mane) mushroom extracts inhibit metastasis of cancer cells to the lung in CT-26 colon cancer-transplanted mice. Journal of Agricultural and Food Chemistry. 61:4898-1904.

Last Modified: 05/27/2017
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