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United States Department of Agriculture

Agricultural Research Service


Location: Produce Safety and Microbiology Research

2007 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
We develop microarrays (panarrays) for speciating and gene-indexing >20 food pathogen strains in a single experiment. The microarrays provided data for identifying species-specific regions and some virulence determinants of interest, thus discriminating isolates for better epidemiology. The arrays were tested with a model system for identifying Campylobacter species present in poultry package liquid or on poultry carcasses by analyzing for DNA in this complex sample with no, or minimal, pre-processing or culture enrichment. Multi-Campylobacter species microarrays were validated. Full genome sequencing and closure was completed for strains of Campylobacter coli, C. lari and Arcobacter butzleri and facilitated production of full genome DNA microarrays for C. jejuni, C. coli, C. lari and Arcobacter butzleri. These are being used currently for analyzing strains from other parts of the world and sources (hosts, clinical, environmental) to determine whether host- or source-specific genotypes exist. They were critical also in identifying the genomic differences between C. jejuni subspecies jejeuni and subspecies doylei. and Arrays for Salmonella and pathogenic E. coli strains are being produced for analyzing outbreak and environmental strains and for gene-expression studies in collaboration with CRIS project 5325-42000-044-00D. Insertion elements identified during genome sequencing of C. jejuni (e.g. phage, partial phage, plasmid remnants), and added to arrays, facilitated identification of similar or identical insertions present in other strains, thus confirming the prevalence of these elements among clinical and environmental isolates. We developed a cell-based fluorescence assay for measuring shigaliketoxin activity and for measuring the efficacy of non-toxic compounds for inactivation of other protein toxins by chemical modification of sulfhydral groups; this is part of a project in food security. This prototype method must be modified for testing toxins in food samples. These studies will assist in development of methods to detoxify food samples contaminated with toxins. We assisted in development of MultiLocus Variable tandem repeat Analysis (MLVA) of E. coli O157:H7, and “fingerprinting” >600 strains sourced from food, water, animals and humans during environmental and outbreak investigations. A database for source tracking strains during intensive environmental studies has been produced. These genotyping studies have provided data for assessing the potential sources of pathogens in the environment.Conventional (PFGE) and high resolution (SNP arrays) genotyping (fingerprinting) analysis of Salmonella strains causing human illness from consumption of almonds was completed in collaboration with other researchers and confirmed the close relatedness of S. Enteritidis strains even though they had different PFGE profiles and phage types, and were associated with two separate outbreaks.

Detection and genotyping of Campylobacter and Arcobacter present in chicken samples. Methods are needed to detect rapidly multiple pathogens and strains present in food samples. Scientists at PSMRU, Albany, CA developed a prototype microarray method for identifying Campylobacter jejuni, C. coli and Arcobacter butzleri present in naturally contaminated chicken samples. Isolation of strains was achieved from package liquid from whole chicken carcasses by membrane filtration and selective media, without enrichment; enrichment resulted in the isolation of A. butzleri and increased the recovery of C. jejuni. Virulence factors were identified also with the arrays. The DNA microarray detection sensitivity was approximately 10,000 C. jejuni cells. This approachis adaptable for detection simultaneously of multiple foodborne pathogens in food samples. This accomplishment addresses the need to obtain more fundamental data regarding critical foodborne pathogens as noted under NP108 Food Safety Problem Statements 1.1.1: Methodology (Rapid, accurate, and sensitive methodology is needed for identification and quantitation of epizootic pathogens…); 1.1.2: Epidemiology (…to determine the origin and routes of transmission of epizootic pathogens); and 1.2.5: Omics (the development of detection methods, phylogenetic analysis, and elucidation of the biology of the organism at the molecular level under advantageous and disadvantageous environments).

Genetic discrimination of C. jejuni subspecies jejuni and subspecies doylei. C. jejuni subspecies doylei strains are isolated from humans infrequently, but they are associated often with bacteremia, and difficult to distinguish easily from the common C. jejuni subspecies jejuni. A novel multiplex PCR method, based on the nitrate reductase (nap) locus, was developed by ARS scientists at WRRC, Albany, CA to subspeciate C. jejuni subspecies jejuni and subspecies doylei isolates unambiguously. Genomic DNA from >180 strains of these subspecies were tested and resulted in 100% discrimination of C. jejuni subspecies, and facilitated strain identification of the environmental source of illnesses caused by C. doylei. Microarrays were developed and used for measuring the genomic content of C. doylei compared to C. jejuni, and revealed that C. doylei is distinct phylogenetically from C. jejuni. This accomplishment addresses the need to obtain more fundamental data regarding critical foodborne pathogens as noted under NP108 Food Safety Problem Statements 1.1.1: Methodology (Rapid, accurate, and sensitive methodology is needed for identification and quantitation of epizootic pathogens…) and 1.1.2: Epidemiology (…to determine the origin and routes of transmission of epizootic pathogens).

Proteomics identification of foodborne pathogen biomarkers. Mass spectrometry can provide a rapid method to characterize foodborne pathogens and identify biomarkers for development of genetic methods. A method was developed by ARS researchers in Albany, CA for determining the amino acid sequences of foodborne bacteria biomarkers observed in MALDI-TOF spectra, by comparing putative amino acid sequences to similar or identical sequences in fully sequenced strains. Sequences are combined into a composite protein sequence. The identification is made in the absence of any DNA sequence data or de novo MS/MS sequencing. Sequence-specific fragment ions are generated from bacterial protein biomarkers by collision-induced dissociation and laser-induced dissociation on a tandem MALDI time-of-flight instrument (TOF-TOF). Knowledge of the precursor protein ion mass and a short sequence of the protein (sequence tag) allow for definitive identification of the protein and, by inference, the species of the bacteria. This accomplishment addresses the need to obtain more fundamental data regarding critical foodborne pathogens as noted under NP108 Food Safety Problem Statements 1.1.1: Methodology (Rapid, accurate, and sensitive methodology is needed for identification and quantitation of epizootic pathogens…) and 1.2.1: Detection and [Validation] (Detection and quantitation of pathogens, toxins.... Diagnostic tools must be developed for the entire food chain which allow the highest detection capability…).

Antibiotic resistance in C. coli. Some commercial turkey flocks are colonized frequently with C. coli strains that are multidrug resistant (MDR), but the types of strains represented by the MDR C. coli remain poorly characterized. ARS scientists in Albany, CA collaborated with researchers at NCSU (Raleigh, NC), to analyze by multilocus sequence typing (MLST) 59 MDR strains from turkeys. The results revealed that sequence types were consistent with those observed previously for turkey isolates, indication host-associated alleles, and that most of these strains were MDR. MLST data for a larger group of C. coli strains from turkeys were compared to "intervening sequence (IVS) content" in the three 23S rRNA genes and to erythromycin resistance. IVS content and sequence type were good predictors of erythromycin susceptibility of C. coli isolated from turkeys. In other words, if the MLST-based ST or IVS content of a C. coli turkey strain is known, we can predict with confidence whether that particular C. coli strain is susceptible to erythromycin. This accomplishment addresses NP108 Food Safety Problem Statement 1.1.5: Antibiotic Resistance ("The emergence of antimicrobial resistance (AR) among food-borne and commensal bacteria associated with food animal production…" and that …"information regarding the development, prevalence, spread and persistence of AR in food-borne and commensal bacteria is limited…") and the Output of "Combining information on the AR of isolates with genetic or other attributes to obtain specific identification … for isolates."

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of web sites managed1
Number of non-peer reviewed presentations and proceedings5
Number of newspaper articles and other presentations for non-science audiences10

Review Publications
Wosten, M.M., Parker, C., Van Mourik, A., Guilhabert, M.R., Heijmen-Van Dijk, L., Van Putten, J. 2006. The Campylobacter jejuni phos/phor operon represents a non-classical phosphate-sensitive two-component system. Molecular Microbiology.62:1:278-291

Chan, K., Miller, W.G., Mandrell, R.E., Kathariou, S. 2007. Association of erythromycin susceptibility and absence of intervening sequences in 23s ribosomal rna genes of campylobacter coli isolated from turkeys.. Applied and Environmental Microbiology. 73(4)1208-1214

D'Lima, C., Miller, 2007 W.G., Mandrell, R.E., Wright, S.L., Siletsky, R., Carver, D.K., Kathariou, S. Clonal Population Structure and Specific Genotypes of Multi-drug Resistant Campylobacter coli from Turkeys.. Applied and Environmental Microbiology. 73(7) 2156-2164

Friedman, M. 2007 Overview of Antibacterial, Antitoxin, Antiviral, and Antifungal Activities of Tea Flavonoids and Teas. Molecular Nutrition and Food Research. 51(1):116-134

Malik-Kale, P., Rapheal, B., Parker, C., Joens, L., Klena, J., Quinones, B., Keech, A., Konkel, M. 2007. Characterization of genetically-matched isolates of campylobacter jejuni reveals mutations in genes involved in flagellar biosynthesis alter the organism's virulence potential. Applied and Environmental Microbiology. 73(10) 3123-3136

Miller, W.G., Heath, S., Mandrell, R.E. 2007. Cryptic plasmids isolated from Campylobacter strains represent multiple, novel incompatibility groups. Plasmid. 57(2):108-117.

Quinones, B., Parker, C., Janda Jr, J.M., Miller, W.G., Mandrell, R.E. 2007. Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays. Applied and Environmental Microbiology. 73(11)3645-3655

Friedman, M., Henika, P.R., Levin, C.E., Mandrell, R.E. 2007. Recipes for Antimicrobial Wine Marinades against Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica. Journal of Food Science. 72(6):M207-M213.

Stoddard, R.A., Miller, W.G., Foley, J.E., Lawrence, J., Gullard, F.M., Conrad, P.A., Byrne, B.A. 2007. Campylobacter insulaenigrae isolated from northern elephant seals (mirounga angustirostris) in california. Applied and Environmental Microbiology. 7(6)1729-1735

Fagerquist, C.K. 2007. Amino Acid Sequence Determination of Protein Biomarkers of Campylobacter upsaliensis and C. helveticus by "Composite" Sequence Proteomic Analysis. Journal of Proteome Research. 6(7):2539-2549.

Miller, W.G., Parker, C., Heath, S., Lastovica, A. 2007. Identification of genomic differences between campylobacter jejuni subsp. jejuni and C. jejuni subsp. doylei at the nap locus leads to the development of a C. jejuni subspeciation multiplex pcr method. BioMed Central Microbiology. 7:11.

Gilbert, M., Mandrell, R.E., Parker, C., Li, Jianjun, Vinogradov, E. 2007. Structural analysis of the capsular polysaccharide from Campylobacter jejuni RM1221. ChemBioChem. 8(6):625-631.

Last Modified: 7/23/2014
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