Location: Produce Safety and Microbiology Research2016 Annual Report
1a. Objectives (from AD-416):
The overall objective of this project is to develop novel typing methods to identify foodborne pathogens and characterize: bacterial foodborne pathogens through genomics, transcriptomics and proteomics; virulence factors and bacterial toxins; and antibiotic resistance in food production. Specifically, during the next five years we will focus on the following objectives: Objective 1: Develop improved identification technologies for human bacterial and viral pathogens to replace current testing methodologies. Sub-objective 1A: Develop a fast, simple and high throughput array-based method fortyping pathogens. Sub-objective 1B: Validate the array genotyping tool for the identification of viral and bacterial pathogens in samples from agricultural environments. Sub-objective 1C: Develop novel Campylobacteraceae species identification methods. Objective 2: Identify and characterize genetic factors associated with virulence and/or environmental adaptation of human bacterial pathogens using genomic, transcriptional and proteomic analyses. Sub-objective 2A: Identify the transcriptional network patterns of bacterial pathogens during environmental adaptation and modulation of their stress response. Sub-objective 2B: Identify genes involved in host/environmental adaptation and investigate variation in virulence potential through in-depth genome sequencing of selected taxa. Sub-objective 2C: Identify the genetic and epigenetic alterations or factors involved in the environmental adaptation of foodborne pathogens through genomic and methylome analyses. Sub-objective 2D: Quantitative proteomic and transcriptomic analysis of virulence factors of foodborne pathogens can be used to elucidate transcriptional vs. posttranscriptional control of virulence in foodborne pathogens. Sub-objective 2E: Top-down proteomic characterization of bacterial virulence proteins or toxins. Objective 3: Characterize molecular mechanisms contributing to the potency of bacterial toxins. Sub-objective 3A: Identification and characterization of Shiga toxin 2 (Stx2) subtypes in environmental STEC strains. Sub-objective 3B: Characterization of Stx2 expression levels and functional activities in environmental STEC strains. Sub-objective 3C: Characterization of pathogenic mechanisms associated with Stx2 subtypes produced by E. coli strains. Sub-objective 3D: Investigate toxin-inactivation mechanisms by natural plant compounds. Objective 4: Identify antimicrobial resistance gene reservoirs in the food production ecosystem and characterize the fitness and virulence of resistant pathogens. Sub-objective 4A: Complete genomic sequencing and functional metagenomic analyses of antibiotic-resistant Campylobacter. Sub-objective 4B: Characterization of the fitness and virulence of antimicrobial-resistant Campylobacter jejuni and Campylobacter coli.
1b. Approach (from AD-416):
Objective 1: A fast, simple and high throughput array-based method for typing pathogens will be developed. Capture probes will be designed to target norovirus and hepatitis A virus, clinically-important Salmonella serovars and Campylobacter spp. To evaluate probe specificity, viral RNA or bacterial DNA will be extracted from clinical samples or cultured strains. A Cooperative Research and Development Agreement (CRADA) has been established with Arrayit Corporation to develop a fast, simple, and cost-effective test, in conjunction with inexpensive instrumentation. The array genotyping method will be validated using samples from agricultural environments. Also, MALDI-TOF-MS will be assessed as a faster, more accurate and reliable identification of Campylobacteraceae taxa, when compared to current phenotype-based approaches. Objective 2: The transcriptomic patterns that correlate with environmental adaptation and stress modulation for Campylobacter, Salmonella and E. coli will be determined, using RNA-Seq under distinct and relevant environmental conditions. Gene content or alleles that tentatively correlate with niche preference, environmental adaptation or pathogenicity will be identified by sequencing Campylobacter and Arcobacter isolates from a more diverse strain set. Alleles or methylation patterns within a population that correlate with environmental adaptation or pathogenicity will be identified through next-generation genomic analysis. Also, proteomic and transcriptomic analysis will be used to investigate transcriptional/post-transcriptional control of virulence factors and to characterize bacterial toxins and virulence determinants. Objective 3: A genotypic and proteomic screen for identifying and classifying Shiga toxin subtypes, harbored by strains recovered from different sources and locations in a major agricultural region, will be conducted. Using enzyme-linked immunosorbent assay and cell-based assays, the amounts and functional activities of Shiga toxin 2a and 2c subtypes will be determined. Using surface plasmon resonance, the mechanisms contributing to the cytotoxicities associated with the Shiga toxin 2a and 2c subtypes will be characterized, by investigating their role in the inhibition of protein synthesis in mammalian cells, thus providing a better understanding of the toxin’s mode of action. Natural plant compounds, specifically polyphenolics, will be investigated as potential inactivators of bacterial toxins. Objective 4: Genome sequencing of antimicrobial-resistant Campylobacter, isolated from poultry farms, will be performed to identify (potentially novel) antibiotic resistance genes. Metagenomic analysis of bacteria isolated from samples (such as litter, insects and fecal droppings) from these same poultry farms will be performed to identify the pool of ‘available’ antibiotic resistance genes that could potentially be transferred into Campylobacter. The fitness and virulence of resistant Campylobacter will be measured, to determine if increased fitness explains the persistence of resistant strains. Fitness metrics will include survival in insects and on poultry, and fecal colonization.
3. Progress Report:
This is the first report for this new project “Molecular Identification and Characterization of Bacterial and Viral Pathogens”, 2030-42000-051-00D, which continues research from “Molecular Biology of Human Pathogens Associated with Food”, 2030-42000-047-00D. Under Objective 1, progress was obtained on the development of novel typing methods by testing different conditions to improve the detection kinetics using an array-based platform. A combination of methodologies were tested for enhancing target-capture probe binding and signal amplification and resulted in an accurate and sensitive detection of norovirus and hepatitis A virus genotypes. An alternative array-based method, using a semiconductor reporter, is currently being evaluated in collaboration with industry stakeholders to achieve a real-time and sensitive identification of viral and bacterial foodborne pathogens. Within Objective 2, progress was achieved on the response of different strains of Campylobacter jejuni during environmental adaptation. In collaboration with researchers at the Washington State University, we exposed different strains of C. jejuni to human macrophages. The C. jejuni strains were chosen due to their differences in virulence. We examined gene expression differences between the C. jejuni strains using RNAseq. Comparisons between the gene expression profiles should allow us to correlate with virulence expression pathways. Also, under Objective 2, progress was made on the in-depth sequencing of Campylobacter. Draft genomes representing between 5-35 strains from multiple Campylobacter species were generated at Albany or obtained from collaborators. Characterization of this additional genomic data is beginning to reveal both variable gene content within a species and/or gene content unique to a species or species cluster that may be relevant to host colonization or virulence. Progress was achieved under Objective 3 on the characterization of bacterial toxin potencies by subtyping Shiga toxin genes in a collection of STEC strains, recovered from various locations and sources in a major produce-production region in California. Current research is aimed at optimizing methodologies for the quantification of toxin subtype amounts and functional activities in the examined STEC strains. To measure the efficiency of toxin potency in the host, standards, consisting of tagged-toxin subtypes, were successfully constructed and purified for conducting experiments with surface plasmon resonance. To initiate studies that would investigate toxin inactivation mechanisms by plant compounds in an animal model system, chromatographic procedures were optimized for the purification of proanthocyanidins, which are a class of polyphenolic compounds highly enriched in grape extracts. Additionally under Objective 3 on the proteomic screen for identifying and classifying Shiga toxin subtypes, progress was made testing electrically conducting slides as a target surface for matrix-assisted laser desorption/ionization (MALDI) for top-down proteomic identification of Shiga toxin (Stx) and other bacterial protein biomarkers. In addition, a knockout of the B-subunit of stx2a gene of E. coli O157:H7 strain EDL933 was constructed and tested by MALDI-TOF-MS to determine whether artifact peaks observed in the wild-type strain were caused by Stx or another protein. In collaboration with another PSMRU SY (Cooley/050), preliminary experiments were conducted using laser ablation electrospray ionization (LAESI) to analyze single bacterial colonies using high resolution mass spectrometry (Orbitrap). In addition, tests were conducted to determine whether bactericidal lamps and the isolation shell of the LAESI device inactivated and contained bacteria during the LAESI experiment. Initial full genome sequencing of anti-microbial-resistant (AMR) Campylobacter has also commenced under Objective 4. These genomes were obtained from multi-drug-resistant Campylobacter strains obtained from poultry (that is, chickens and turkeys) and AMR strains transmitted by insect vectors at poultry production facilities. These genomic data will be used to identify potential new antibiotic resistance genes and to provide baseline data for downstream fitness/virulence studies.
1. Global gene expression of Campylobacter jejuni in response to changes in oxygen. C. jejuni causes a significant amount of illness in the United States, and is prevalent in chickens. During transit/colonization of a susceptible host, C. jejuni encounters variable oxygen levels. In collaboration with Utrecht University, we examined the alterations in C. jejuni gene expression when the bacteria are grown under different oxygen levels. ARS researchers in Albany, California, utilized the RNAseq technique to measure the transcript levels of all genes within the C. jejuni strain. Our data demonstrate that electron transfer genes are regulated by both oxygen and nitrate. These results will increase our understanding of the physiology of C. jejuni and may provide mechanisms to control this pathogen in foods.
2. Inhibition of bacterial AB5 toxin by polyphenolic compounds. ARS scientists in Albany, California, in collaboration with scientists at the University of Central Florida and the University of Houston, utilized cellular, biochemical and structural studies to provide the first scientific evidence of the specific mechanism of inhibition by purified grape polyphenols against the bacterial AB5-type toxin, cholera toxin. This research identified individual food-compatible inhibitors from grape extracts that blocked cholera toxin binding and activity in human host cells. In addition, these findings have led to a novel formulation of a defined mixture of polyphenolic compounds, generated as waste products by the wine industry. These compounds have proven to be effective inhibitors against toxins expressed by foodborne bacterial pathogens.
5. Significant Activities that Support Special Target Populations:
Crespo, M., Altermann, E., Olson, J., Miller, W.G., Kathariou, S. 2016. Novel plasmid conferring kanamycin and tetracycline resistance in turkey-derived Campylobacter jejuni strain 11601MD. Plasmid Journal. doi: 10.1016/j.plasmid.2016.06.001.
Duarte, A., Seliwiorstow, T., Miller, W.G., De Zutter, L., Uyttendaele, M., Dierick, K., Botteldoorn, N. 2016. Discriminative power of Campylobacter phenotypic and genotypic typing methods. Journal of Microbiological Methods. 125:33-39.