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

Location: Produce Safety and Microbiology Research

2018 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
Objective 1: Continued progress was achieved in the development of technologies for the rapid identification of foodborne pathogens. By optimizing methodologies for the recovery of foodborne viruses from agricultural environments, the prevalence of human noroviruses was determined in water samples collected at various sites proximal to leafy green fields in a major production region located on the Central California coast. The research findings revealed that the prevalence of noroviruses varied throughout the seasons. Norovirus detection rates were highest in the fall, followed by spring and winter, with prevalence lowest in the summer. The use of an array-based genotyping tool enabled the accurate identification of norovirus genetic types associated with foodborne illness within these water samples collected from specific agricultural environments. Significant progress was also achieved in the design and validation of improved detection platforms for bacterial pathogens. By optimizing the processes of extraction, concentration, and amplification of nucleic acids, the probe-based method accurately detected Listeria species at a cell concentration below the infectious dose in environmental samples. These findings have set the foundation for the development of commercial food testing applications for real-time monitoring of leafy greens at a processing facility in California. Objective 2: Progress continued in the elucidation using ribonucleic acid (RNA) sequencing of Campylobacter jejuni transcriptional network patterns during environmental adaptation. In collaboration with researchers at Washington State University, C. jejuni strains were cultured within ileal loops, an animal model system. C. jejuni global gene expression was characterized during a time course of bacterial growth in the illeal loops to identify gene expression pathways important to infection. Sub-objective 2B: Advances were made in the identification of Campylobacteraceae genes, pathways or larger genomic suites involved potentially in host/environmental adaptation or virulence. To assist in comparative analyses of the Campylobacteraceae family, the complete genomes of 89 strains sequenced or annotated at Albany, California, (representing the three genera Campylobacter, Arcobacter and the proposed novel genus Avispirillum) were re-annotated. This was accomplished by an all-vs.-all comparison of the approximately 178,000 genes and genomic features present in these 89 genomes with accompanying selective phylogenetic analyses. The annotations of similar genes and genomic features were coordinated across all 89 genomes to construct a consistently-annotated reference source for these three genera. Analysis of these new data uncovered previously-unidentified pathways where new research can be focused. Sub-objective 2C: Advances were made in the identification of genetic alterations involved in the environmental adaptation of foodborne pathogens through genomic analyses. Within the gut of chickens or other vertebrate hosts, C. jejuni is continually exposed to bile, a protective agent against bacterial colonization. C. jejuni quickly adapts to survival in this environment by altering gene expression, causing bile resistance. This gene expression is like a thermostat and can be switched off when bile exposure is reduced; however, some C. jejuni maintain a high bile resistance. In collaboration with researchers at Washington State University, isolates of C. jejuni able to grow in increasing bile were identified and their genomes were sequenced. Genomic comparisons between the original sensitive isolate and the environmentally-adapted isolates identified several mutations possibly involved with bile resistance. However, none of the mutations occurred in genes that exhibit altered gene expression; thus, the cause of bile resistance in these isolates is not obvious. Work is proceeding to both duplicate and verify the role of the mutated gene in bile resistance. Sub-objective 2D: Single and double gene knockout mutants of Salmonella enterica serovar Typhimurium were analyzed by bottom-up proteomic analysis. Gene function in these knockout strains was restored by complementation. Sub-objective 2E: Experiments continued to detect and identify the A-subunit of shiga toxin 2 (Stx2) in wild-type E. coli O157:H7 strain EDL933 using high resolution Orbitrap mass spectrometry. Although the B-subunit was detected and identified, detection of the A-subunit remains elusive, perhaps due to oligomerization of this protein upon dissociation of the holotoxin complex during chromatographic separation. ARS researchers also encountered difficulties in experiments to create a high copy expression plasmid containing the A-subunit due to the size of the gene, about 1100 base pairs, being inserted into the plasmid. Objective 3: Significant progress was achieved on the characterization of virulence potencies, associated with the expression of Stx subtypes, expressed by environmental Shiga toxin-producing E. coli (STEC) strains from agricultural regions in California. Sub-objective 3A and 3B: By employing cell-based assays, mass spectrometry and whole-genome sequencing, a novel plasmid-encoded biomarker in STEC strains was identified from feral pigs and in water expressing high levels of active Stx. Current research is addressing the prevalence of the plasmid-encoded biomarker in STECs from other wildlife and environmental sources. To identify this plasmid-encoded biomarker, an innovative software program was developed to rapidly identify bacterial gene sequences. It is based on proteomic data obtained by tandem mass spectrometry, enabling the discovery of new biomarkers in foodborne pathogens. The combinatorial use of genomic, proteomic and computational analyses also identified cold-shock proteins, CspC, CspE and CsbD, which are highly conserved cold-shock proteins common to both pathogenic and non-pathogenic bacteria, i.e. they are not unique to STECs. Sub-objective 3C: To improve the characterization of pathogenic mechanisms associated with Stx2 subtypes, a cell-based assay was adapted to detect toxin activity by cytofluorometry. This assay enabled a more accurate single-cell analysis of toxin activity. The use of cytofluorometry has contributed to studies documenting cellular recovery from intoxication and to further examine toxin-receptor binding on the host cell. Ongoing studies are aimed at determining if there is a direct correlation between the cellular potency of a Stx2 subtype and the extent of its accumulation in the host cell cytosol. Sub-objective 3D: To investigate toxin-inactivation mechanisms by natural plant compounds, biochemical studies indicated that high molecular-weight polyphenol fractions from grape extract inhibits the cellular activities of bacterial toxins, demonstrating that a defined cocktail of 20 polyphenolic compounds provided substantial cellular resistance to Stx and other AB-type protein toxins. Objective 4: Progress was achieved on the characterization of macrolide resistance (specifically, the antibiotic erythromycin) in Campylobacter. Erythromycin (erm) resistance in Campylobacter is not common but has been steadily increasing. In Campylobacter, erm resistance is conferred usually by either discrete point mutations in the 23S ribosomal RNA (rRNA) gene or erm resistance genes, such as erm(B). Most erm resistance is due to rRNA mutations; however, erm(B) (present on extrachromosomal or mobile elements) has been recently identified in Campylobacter isolated in China, Spain and Turkey, which would be significant since erm resistance genes present on mobile or extrachromosomal elements could lead to a much more rapid dissemination of macrolide resistance. In collaboration with Iowa State University and North Carolina State University, erm-resistant, poultry-associated C. jejuni and C. coli were characterized. In all cases, resistance could be correlated to previously-described 23S rRNA mutations, suggesting that at least in this case and in the U.S. erm(B) has not yet infiltrated into the poultry production chain. In collaboration with Ghent University, Belgium and Mansoura University, Egypt, antibiotic-resistant C. jejuni strains isolated from poultry and human clinical samples were characterized. Here again, erm(B) was not identified. However, a surprising result was that a large percentage of the erm-resistant strains did not contain 23S rRNA mutations, nor mutations in other genes also associated tentatively with macrolide resistance. Thus, the cause of erm resistance in these strains is unknown; work is underway to sequence the genomes of a set of these strains, to determine if a novel mode of erm resistance is present within Campylobacter.

Accomplishments
1. Accurate detection of foodborne viruses in a major agricultural region. Human noroviruses are the leading cause of foodborne illness in the U.S. and are responsible for $2 billion in costs annually. ARS scientists in Albany, California, developed and optimized methods for the efficient recovery and identification of noroviruses from water samples, recovered from public waterways in a major U.S. production region for leafy greens. These methods enabled the rapid and accurate determination of norovirus types predominantly associated with human foodborne illness. This research has characterized the prevalence of relevant types of noroviruses in waterways next to agricultural fields and provides evidence to the leafy green industry of a high prevalence of noroviruses within these waterways. This could potentially lead to the contamination of fresh produce fields from a variety of wildlife sources or floods after heavy rains.

2. Assessing the pathogenic potential in Shiga toxin-producing Escherichia coli (STEC) strains. STEC, responsible for human gastroenteritis with diverse clinical spectra, are a major concern within the food industry since they have been implicated in a wide variety of outbreaks and recalls of various food products. ARS researchers in Albany, California, employed approaches in proteomics and genomics to characterize a key virulence factor, Shiga toxin (Stx), in thirty-eight STEC strains isolated from animal and environmental sources in an important agricultural region along the Central Coast of California. These strains were found to express the clinically-relevant Stx2 subtypes. Eight stx2-positive STEC strains recovered from cattle in the Salinas Valley in California were found by DNA sequencing to have stx2c gene inactivation. Proteomic analysis confirmed stx2c gene inactivation on the basis of very weak detection of the Shiga toxin. In summary, the combinatorial use of genomics and proteomics enabled a better characterization of environmental STEC strains, demonstrating that certain strains are likely less pathogenic based on the expression of clinically-relevant Stx2 subtypes.

3. Antimicrobial resistant (AMR) Campylobacter jejuni from farm to retail. C. jejuni is a commensal organism of the intestinal tracts of avian species and a leading cause of diarrheal disease in humans. Despite a reduction in usage of antibiotics, C. jejuni may encounter multiple antimicrobial agents on the poultry farm where AMR C. jejuni strains are often recovered. Knowledge gaps remain regarding AMR emergence, spread and resilience in C. jejuni populations. ARS scientists in Albany, California, in collaboration with scientists at North Carolina State University, determined the genomic sequences of 10 AMR Campylobacter jejuni strains: six from poultry farms (poultry and flies) and four from retail chicken purchased at grocery stores. All of the strains possessed a tetracycline resistance plasmid (pTet), and some of these pTet plasmids also harbored aminoglycoside resistance genes; such plasmids can be transferred to antimicrobial sensitive strains creating additional AMR C. jejuni. These results provide evidence that AMR of C. jejuni in poultry is multi-dimensional and provides evidence to the poultry industry that AMR C. jejuni persists from farm to retail.

U.S. DEPARTMENT OF AGRICULTURE

Produce Safety and Microbiology Research: Albany, CA