Project : USDA ARS

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Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

2017 Annual Report

Objectives
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.

Approach
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.

Progress Report
Under Objective 1, significant progress was made in the improvement of detection specificity and sensitivity using array-based platform methodologies. These methodologies were tested using enteric foodborne viruses, human norovirus and hepatitis A virus, extracted from clinical specimens or agricultural environments. These methods are now being expanded for the detection of bacterial foodborne pathogens and have set a precedent for the further development of fully-integrated platforms, using simpler and rapid assays, by automating data collection and analysis. In collaboration with a major leafy greens processor in California, methodologies using DNA aptamers are being evaluated to concentrate the targeted foodborne pathogen from samples collected in various postharvest environments. The pathogen recovery efficiency will be determined by molecular amplification of extracted nucleic acids and further detection on an array platform. Within Objective 2, progress has continued on the transcriptomic responses of different strains of Campylobacter jejuni during environmental adaptation using RNA sequencing. In collaboration with researchers at Washington State University, C. jejuni strains with differences in virulence in animal model systems were used to infect human macrophages in tissue culture. Comparisons between the gene expression profiles from the different strains will allow us to correlate these differences with virulence expression pathways. Advances were made in the identification of Campylobacter genes, pathways, or larger genomic suites involved potentially in host/environmental adaptation or virulence. These features were identified through comparative analyses of multiple reference genomes, using the now complete set of Campylobacter genomes and in-depth Illumina sequencing of multiple strains representing several Campylobacter species. For example, sequencing of 76 draft and complete C. lanienae and C. lanienae-related genomes demonstrated that this group of campylobacters cannot utilize the element selenium and thus do not encode a class of respiratory enzymes common to the remainder of the genus. These organisms also encode a unique set of flagellin subunits. Although the role of these features in Campylobacter biology requires further study, these species are only isolated typically from large grazing animals; therefore, it is possible that the novel flagella and the absence of selenium utilization is important for niche association and/or host adaptation in these organisms. Similarly, genes encoding a set of enzymes involved in the utilization of the citrate analog tricarballylate were identified only in Campylobacter strains isolated from hind-gut fermenters (for example, pigs and rabbits), indicating these genes may be critical for host adaptation. Comparative genomics also identified a novel mobile genetic element encoding the zonula occludens toxin. This element, originally described in enteric organisms, such as Salmonella and Yersinia, was also first described in Campylobacter in C. concisus strains. We have identified this element in multiple Campylobacter species, as well as three Arcobacter species. Conservation of key features strongly suggest that these ‘zot islands’ may be a family of pathogenomic elements representing a new type of virulence determinant. In depth sequencing of Campylobacter species has also identified putative subgroups with perhaps differing pathogenic potential. In sequencing performed at Albany and in collaboration with the Institute of Environmental Science and Research (ESR), New Zealand, C. concisus was shown to be divided into two clusters, with one cluster more associated with severe human illness. Additionally, two clusters were further demonstrated to be present in C. upsaliensis. One cluster is almost exclusively associated with human illness, while the other cluster is isolated from clinical samples, cats and canids. Progress was made to identify alleles that exist within a C. jejuni population that correlate with environmental adaptation. In collaboration with researchers at the University of British Columbia, Canada, distinct genetic alleles were determined using next-generation sequencing of C. jejuni colonies that had different shaped cells. C. jejuni are helical shaped cells and their shape is important for initial colonization and host interactions. Passage through the host often results in rod-shaped cells that may be better able to resist phagocytosis and survive in vivo. Sequencing demonstrated variability in genes encoding enzymes for cell wall synthesis. Models are being developed to understand any potential environmental triggers. Under Objective 2, progress was made in the quantitative proteomic and transcriptomic analysis of virulence factors. We constructed single and double gidA and mnmE gene knockouts in Salmonella enterica serovar Typhimurium for further proteomic analysis. Non-radioactive isotopic labeling was also tested for quantitative proteomic analysis on a high resolution Orbitrap mass spectrometer. Progress was made on sub-objective 2E; wherein advanced software (not commercially available) was tested for increased functionality for top-down proteomic analysis using an Orbitrap mass spectrometer. Top-down proteomic analysis was performed on translated as well as a mis-translated sequences of the B-subunit of Shiga toxin 2 from Escherichia coli O157:H7 strain EDL933 using liquid chromatography high resolution Orbitrap mass spectrometry. A number of proteins and their source microorganisms were identified using matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry and top-down proteomic analysis, as part of a collaboration with the ARS Foodborne Toxin Detection and Prevention (FTDP) research unit in Albany, California. The USDA top-down proteomic software was used to identify an important protein biomarker of E. coli as part of a collaboration with University of Wyoming, Department of Chemistry. Continuing progress was achieved under Objective 3 on the characterization of virulence potencies, associated with the expression of Shiga toxin (Stx) subtypes, in a collection of Shiga toxin-producing E. coli (STEC) strains from a major produce-production region in California. Methodologies were optimized for toxin subtype induction, enabling an accurate quantification of toxin functional activities and amounts using cell-based assays and mass spectrometry. To investigate toxin inactivation mechanisms by plant compounds in an animal model system, experimental approaches were developed and validated for assessing the in vivo effect of the bacterial AB-type toxin, cholera toxin. These approaches were then used in preliminary experiments, assessing the in vivo inhibitory effects of polyphenolic compounds, highly enriched in grape extracts, against cholera toxin. Forty-five putative STEC strains from environmental isolates collected in Northern California were screened for Stx production using antibiotic induction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. Stx was over-expressed in these strains by exposure to sub-therapeutic levels of DNA-damaging antibiotics, triggering both expression of bacteriophage-encoded proteins (including Stx) as well as host cell lysis. The latter event avoids the necessity of performing mechanical cell lysis which needlessly contributes to sample complexity. Many of these environmental STEC strains expressed Stx, although the amount generated was strain-dependent. Only Stx2a and Stx2c were unambiguously identified from these strains. Production of Stx1 was not detected in any of these strains, which is consistent with Polymerase Chain Reaction results. Within Objective 4, progress was achieved on the identification of antibiotic resistance genes from metagenomic samples. In collaboration with Kansas State University and North Carolina State University, bacterial samples from a poultry farm in North Carolina were pooled, and DNA from this pool was sheared and cloned. Clones were tested for antibiotic resistance and positive clones were sent to Albany, California, for DNA sequencing. Multiple, different antibiotic resistance genes were identified, including: two ampicillin resistance genes, five gentamicin resistance genes, three streptomycin resistance genes and six tetracycline resistance genes. Furthermore, some antibiotic resistance genes were adjacent to additional genes conferring resistance to the same antibiotic class. Progress was also achieved on the complete genome sequencing of multidrug-resistant Campylobacter. The genomes of two multidrug-resistant strains, resistant to six or seven antibiotics each, a C. jejuni isolated from turkey feces and a C. coli isolated from a housefly on the same turkey farm, were sequenced to completion. A surprising result from the genome sequencing was the identification of a novel mobile genetic element in each strain that confers resistance to gentamicin. The mobile elements from the two strains were essentially identical, indicating that this element can readily move from strain to strain; thus, the presence of this element has important implications for the transmission of antimicrobial resistance within a food production facility.

Accomplishments
1. Detection method for viruses causing food poisoning. Human noroviruses are the leading cause of foodborne illness in the U.S. and are responsible for 19-21 million illnesses and $2 billion in direct and indirect costs annually. Hepatitis A virus causes a lower rate of food poisoning in the U.S. A recent emergence of hepatitis A virus outbreaks have resulted from imported food products. ARS scientists in Albany, California, developed and validated the first molecular detection method for simultaneously identifying virus genetic types, associated frequently with the consumption of various food commodities in the U.S. and Europe. This genetic typing method will be employed by stakeholders from the tech industry as a prototype for the further development of an emerging detection platform, enabling the real time surveillance of foodborne viral pathogens.

2. Short-chain fatty acids modulate expression of Campylobacter jejuni. C. jejuni is a commensal of the intestinal tracts of avian species and other animals and a leading cause of diarrheal disease in humans. The types of cues sensed by C. jejuni to influence responses to promote commensalism or infection are largely lacking. ARS scientists in Albany, California, in collaboration with scientists at the University of Texas Southwestern Medical Center, discovered a set of genes whose expression is modulated by lactate and short-chain fatty acids produced by the microbiota in the intestinal tract. These genes include those encoding catabolic enzymes and transport systems for amino acids that are required by C. jejuni for growth and intestinal colonization of chickens. Gradients of these microbiota-generated fatty acids are cues for spatial discrimination between areas of the chicken intestines so that the C. jejuni can locate niches in the lower intestinal tract for optimal growth. The results provide valuable insights into developing controls of this pathogen within poultry.

3. Identification of a novel hippuricase within Campylobacter. The human pathogen Campylobacter jejuni is a common contaminant of poultry and was initially the only Campylobacter species known to possess hippuricase activity, which is encoded by the hippuricase gene hipO. Consequently, the presence of C. jejuni is routinely determined through a simple colorimetric hippuricase assay. Recently, C. avium, a new Campylobacter species of undetermined pathogenicity, was also isolated from poultry and demonstrated to possess hippuricase activity, and could be potentially confused with C. jejuni in diagnostic tests. ARS scientists in Albany, California, determined the genome sequence of two C. avium strains and showed that hipO is not present within either of the C. avium genomes. Candidates for an alternate C. avium hippuricase gene were screened, and one gene encoding a robust hippuricase was identified and labeled hipA; a gene with moderate similarity to hipA was also found in C. jejuni, but this gene does not encode a functional hippuricase. The hipA and hipO genes possess little sequence similarity; therefore, moving away from assay-based testing towards sequence-based testing would provide more accurate species identification, clarify the prevalence of C. avium on poultry, and yield possible evidence of its presence within human clinical samples.

4. Measuring the effects of bacterial toxins. In collaboration with the University of Central Florida, ARS scientists in Albany, California, measured the lethal effects of cholera toxin from Vibrio cholerae and Shiga toxin from Escherichia coli, main agents of waterborne and foodborne illness, respectively. This research quantified the amounts of bacterial toxins required for complete inhibition of protein synthesis or cell death in the mammalian host cell. Additionally, we found that mammalian host cells can recover from intoxication under certain circumstances and survive the effects of these toxins. These findings have provided fundamental information on the amounts of cholera toxin and Shiga toxin that result in reversible toxin effects and will enable the design of effective intervention strategies for toxin inactivation.

5. Translational errors in the expression of Shiga toxin (Stx) from pathogenic Escherichia coli as measured by mass spectrometry. Stx is an AB5 toxin expressed by foodborne pathogenic Shiga toxin-producing E. coli. The Stx AB5 holotoxin attaches to receptors on the surface of eukaryotic cells via the B-subunits and, after cellular envelopment, the toxin disrupts ribosomal protein synthesis causing cell death. Variations in the amino acid sequence of the five identical B-subunits of the AB5 holotoxin result in significant differences in toxicity due to changes in to eukaryotic cell attachment. ARS scientists in Albany, California, discovered multiple B-subunit sequences for each stx gene (stx1a and stx2a) in E. coli O157:H7 strain EDL933, using advanced mass spectrometry techniques. These additional B-subunit sequences were ascribed to mis-translation of the stx genes during antibiotic-induced stress. Expression of mis-translated sequences from a single stx gene, or multiple stx genes, has implications for the potential diversity of Stx holotoxin conformations and their range of attachment to a variety of surface receptors of eukaryotic cells.

6. Enzymatic “surface shaving”. Pathogenic bacteria interact with their external environment via surface-exposed biomolecules. Enzymatically “shaving” surface-exposed proteins can reveal potential targets for vaccines and other therapies. ARS researchers in Albany, California, performed enzymatic “shaving” on three serotypes of Salmonella enterica enterica: Newport, Kentucky and Thompson. Significantly, components of the flagellar apparatus were detected from Newport samples and Salmonella pathogenicity invasion proteins were detected from all three serotypes, which suggested the triggering of an invasion response within these pathogens. In addition to identifying surface-exposed proteins as potential vaccine targets, enzymatic “shaving” experiments can also elicit unexpected biological responses from a pathogen, leading to a better understanding of how it responds to challenge by proteolytic enzymes that degrade its surface integrity.

U.S. DEPARTMENT OF AGRICULTURE

Produce Safety and Microbiology Research: Albany, CA