Location: Produce Safety and Microbiology Research
2021 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
This is the final report for project 2030-42000-051-00D, which terminated in February 2021. This plan was replaced by 2030-42000-055-00D. Please see the report for this new project for additional information. Substantial results were realized on all four objectives over the 5 years of the project.
For Objective 1, progress was made on the development of typing methods for foodborne pathogens to replace current testing methodologies. As part of a collaboration with the technology sector in California, a continuous flow-through system was optimized for detecting Listeria species in environmental swab samples. For reliable and highly sensitive detection of Listeria species, a comparative genomic analysis with a dynamic programming algorithm was conducted to evaluate high copy-number sequences and enable the targeted and specific detection of Listeria species. Simulated folding enabled the identification of the best targeted regions for optimal detection, resulting in the development of a nucleic acid-based singleplex assay. Results from inclusivity tests indicated that the singleplex assay had a high level of specificity for detecting various Listeria species when compared to the lack of detected signals for non-targeted bacterial strains. The singleplex assay was finally incorporated in the automated and continuous flow-through system, which employed an adaptation of depth filtering, a processing stage previously used for preventing filter clogging, followed by the use of an aptamer-functionalized column for successfully capturing and concentrating the targeted Listeria cells from the environmental samples. Current efforts are aimed at finalizing the platform as a self-contained, portable, and modular system that is operable with minimal training by non-technical personnel. In addition, the increased sampling size of the method will be optimized to improve the statistical significance of the procedure and to enable a sampling process that is more representative of the entire agricultural field, providing an added value to the food industry.
For Sub-objective 2A, results were made in the elucidation of bacterial gene expression (transcription) patterns during environmental adaptation. Due to their small size, bacteria must respond quickly to changes in their growth environment. These responses are mediated by changes in transcription that alter the production of proteins and enzymes that are encoded by the specific genes. During this project, as part of an international collaboration, the transcriptional patterns of C. jejuni growing in different amounts of oxygen exhibited changes in the expression of putative phospholipid biosynthesis genes. Examination of phospholipids demonstrated previoulsy unknown diversity and dynamic changes in composition. These studies defined a role for certain phospholipids in the motility of C. jejuni. Also, the gene expression of Shiga toxin-producing Escherichia coli (STEC) surviving within protozoa (Tetrahymena sp.) showed that genes involved in the oxidative stress response were activated. With the extensive use of oxidants as sanitizers in the food industry, these findings point to a mechanism of oxidative stress avoidance by STEC and emphasize the importance of exploring the physiology of STEC in environments relevant to food safety.
Under Sub-objective 2B, significant progress was made in the complete and in-depth sequencing of Campylobacter and Arcobacter. During this project, the genomes of all Campylobacter and Arcobacter type strains were sequenced to completion, fully annotated and deposited in GenBank. Additionally, multiple genomes of novel Campylobacter species and multiple draft genomes for several taxa were also generated. Thus, 350+ genomes were sequenced in total; many of the draft genomes have been also deposited with the remainder to be submitted to GenBank upon resumption of duties. Analysis of these genomes has identified putative virulence-associated genes or gene sets. For example, as part of an international collaboration, the chromosomal sequences of several C. showae strains associated with ulcerative colitis and colon cancer were characterized and compared to other C. showae strains. Analysis indicated that the adherent/invasive strains possessed a gene set that was different from the non-adherent/non-invasive strains, suggesting that certain genes are important for the localization, colonization and virulence potential of C. showae. Also, genes associated with Guillain-Barre syndrome (GBS) were identified in six non-C. jejuni campylobacters, including one poultry-associated species (C. hepaticus) and two clinically-relevant species (C. mucosalis and C. ureolyticus), suggesting that multiple Campylobacter species may be capable of eliciting GBS in humans. In another study, an Arcobacter cryaerophilus strain that was isolated from green mussels was found to possess an unusual extrachromosomal element that putatively encodes a secreted toxin.
For Sub-objective 2C, results were obtained for methylome characterization in C. jejuni and STEC. In both bacterial pathogens, diverse strains with distinct restriction-modification systems exhibited various methylation patterns. For one STEC strain, the DNA methylation was shown to affect the transcription of the Shiga-toxin gene and thus may play a major role in virulence.
Under Sub-objective 2D, some progress was made on the analysis of Salmonella enterica enterica (SEE) serovars using bottom-up proteolytic surface-shaving and high-resolution mass spectrometry. These experiments identified effector/invasion proteins (SipA-D). SipB-D are part of the injectosome needle complex of the Type III secretion system, and SipA is an effector protein injected into eukaryotic cells. Our surface-shaving results suggested that the SipB-D needle complex is fully formed for SEE Newport and SEE Thompson strains but was absent in SEE Kentucky. The non-detection of SipA-D in SEE Kentucky would seem to indicate the absence of the needle complex, which would severely impair its virulence and is consistent with previous reports that it is not considered a significant human pathogen.
Under Sub-objective 2E, progress was made on top-down identification of proteins and protein toxins using high- resolution mass spectrometry. The A-subunit of Shiga toxin (Stx) and cytolethal distending toxin-C were detected from two clinical STEC strains. Of particular interest was detection of mistranslated Stx, which was a result of antibiotic stress that had not been reported previously for a fluoroquinolone antibiotic. A new top-down proteomic software (Protein Biomarker Seeker) was developed that was used to identify bacterial stress response proteins, a global regulator of carbon metabolism, and plasmid-encoded immunity proteins of bactericidal enzymes, which provide the bacterial host with competitive survival advantage over other bacteria.
Under Sub-objective 3A, progress was made on the top-down proteomics of Stx in 35 STEC strains of various serotypes collected from an agricultural region in northern California. These environmental STEC strains produced primarily type/subtype Stx2a or Stx2c.
For Objective 3, progress is still ongoing on the characterization of Stx subtypes in presumptive STEC isolates recovered from a major agricultural region for leafy greens in California. Presumptive isolates were recovered based on polymerase chain reaction (PCR)-positive tests for the stx gene; however, results from genotyping assays indicated that these STEC isolates were Shiga toxin-producing Escherichia albertii (STEA), an emerging pathogen misidentified as STEC due to similar phenotypical and biochemical features. The combinatorial use of genome sequencing and cytotoxicity assays will be implemented for the improved identification and assessment of the virulence potential in STEC and STEA.
Within Objective 4, progress was achieved on the characterization of macrolide resistance (erythromycin) in Campylobacter. Erythromycin resistance in Campylobacter is not common but has been increasing over the past 10 years. In Campylobacter, erythromycin resistance is conferred usually by either mutations in the 23S ribosomal RNA (rRNA) gene or the erythromycin resistance gene erm(B). In collaboration with Iowa State University and North Carolina State University, erythromycin-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 United States, 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. erm(B) is often present on extrachromosomal or mobile elements and has been recently identified in Campylobacter isolated in China, Spain and Turkey. Although erm(B) was not identified during this project, mobile elements containing other antimicrobial resistance (AMR) genes were identified and were highly similar to elements found in other non-Campylobacter species. Additionally, some mobile elements were present on conjugative plasmids, indicating two routes by which AMR genes could rapidly disseminate through Campylobacter. Arcobacter genomics also identified a much larger and diverse suite of mobile elements within this genus, with representatives of 13 insertion sequence families and one strain containing approximately 100 complete and degenerate elements. Although many of these mobile elements were simple insertion sequences, some also contained AMR genes, indicating that mobile-element-associated antibiotic resistance may be a common theme within the Epsilonproteobacteria.
Accomplishments
1. Novel detection platform to improve safety of foods. Listeria species are widespread in the environment and, consequently, food production facilities constantly monitor for the presence of this pathogen. To address a request by processors of leafy greens, ARS scientists in Albany, California, collaborated with the technology sector in developing a continuous flow-through system to rapidly detect Listeria species in samples collected from surfaces in food processing facilities. Novel DNA sequences were designed for a Listeria-capturing step using single-stranded DNA sequences (aptamer) and for the development of a nucleic acid-based assay for the detection of multicopy sequences in the Listeria genome. By optimizing the processes of Listeria cell capturing, concentration and lysis, the flow-through method accurately detected Listeria cells in surface samples at low cell concentrations below the infectious dose. These findings have set the foundation for the development of commercialized cartridge-based approaches for pathogen detection in samples from food processing facilities, resulting in food testing applications that enable on-site monitoring that will reduce product hold time.
2. Campylobacter jejuni features associated with developing post infectious neuropathy. Campylobacter jejuni is a major cause of bacterial foodborne gastroenteritis and certain strains that produce sialylated lipooligosaccharides (LOS) can also lead to the post-infectious neuropathy Guillain-Barré syndrome (GBS). Among this group of C. jejuni strains, the risk of GBS following infection with C. jejuni Penner serotype HS:19 is estimated to be at least six times higher than the average risk. To identify novel genomic features within the HS:19 strains that could explain the increased risk for GBS, ARS scientists in Albany, California, in collaboration with scientists at the University of Arizona and Erasmus Medical University in Netherlands, performed comparative genomic analyses. They combined genome alignments, analyzed single nucleotide polymorphism (SNPs) and pan-genome analyses to compare the genome sequences of 36 C. jejuni HS:19 with 874 C. jejuni non-HS:19 genome sequences. These analyses led to the identification of a gene cluster of seven genes involved in modification of biomolecules. Interestingly, these modified biomolecules promote immune responses and may play a role in the development of GBS. The study provides novel insight into possible virulence factors of C. jejuni associated with the HS:19 serotype that may explain an increased risk of GBS.
Review Publications
Foster, G., Baily, J., Howie, F., Brownlow, A., Wagenaar, J.A., Gilbert, M.J., Miller, W.G., Byrne, B.A., Clothier, K.A., Schmitt, T., Patterson, I., Reid, R., Dagleish, M. 2020. Campylobacter pinnipediorum subsp. caledonicus and Campylobacter pinnipediorum subsp. pinnipediorum recovered from abscesses in pinnipeds. Diseases of Aquatic Organisms. 142:41-46. https://doi.org/10.3354/dao03544.
Niedermeyer, J., Miller, W.G., Yee, E., Harris, A., Emanuel, R., Jass, T., Nelson, N., Kathariou, S. 2020. Search for Campylobacter reveals high prevalence and pronounced genetic diversity of Arcobacter butzleri in floodwater samples associated with Hurricane Florence in North Carolina, USA. Applied and Environmental Microbiology. 86:20. https://journals.asm.org/doi/10.1128/AEM.01118-20.
Fan, S., Foster, D., Miller, W.G., Osborne, J., Kathariou, S. 2021. Impact of ceftiufor administration in steers on the prevalence and antimicrobial resistance of Campylobacter spp.. Microorganisms. 9(2):318. https://doi.org/10.3390/microorganisms9020318.
Cao, X., Brouwers, J., Heijmen Van Dijk, L., Van De Lest, C., Parker, C., Huynh, S., Van Putten, J., Kelly, D.J., Wosten, M. 2020. The unique Phospholipidome of the enteric pathogen Campylobacter jejuni: Lysophosholipids are required for motility at low oxygen availability. Journal of Molecular Biology. 432(19):5244-5258. https://doi.org/10.1016/j.jmb.2020.07.012.
Liu, M.M., Coleman, S., Wilkinson, L., Smith, M.L., Hoang, T., Niyah, N., Mukherjee, M., Huynh, S., Parker, C., Kovac, J., Hancock, R., Gaynor, E.C. 2020. Unique inducible filamentous motility identified in pathogenic Bacillus cereus group species. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. 14:2997–3010. https://doi.org/10.1038/s41396-020-0728-x.
Pascoe, B., Schiaffino, F., Murray, S., Bayliss, S.C., Hitchings, M.D., Méric, G., Mourkas, E., Calland, J.K., Burga, R., Penataro-Yori, P., Jolley, K.A., Cooper, K.K., Parker, C., Olortegui, M., Kosek, M.N., Sheppard, S.K. 2020. Genomic epidemiology of Campylobacter jejuni associated with asymptomatic pediatric infection in the Peruvian Amazon. PLoS Pathogens. 14(8). Article e0008533. https://doi.org/10.1371/journal.pntd.0008533.
Negretti, N.M., Ye, Y., Malavasi, L.M., Pokharel, S.M., Huynh, S., Noh, S.M., Klima, C.L., Gourley, C.R., Ragle, C.A., Bose, S., Looft, T.P., Parker, C., Clair, G., Adkins, J.N., Konkel, M.E. 2020. A porcine ligated loop model reveals new insight into the host immune response against Campylobacter jejuni. Gut Microbes. 12(1). Article 1814121. https://doi.org/10.1080/19490976.2020.1814121.
Fagerquist, C.K. 2020. Polypeptide backbone cleavage on the C-terminal side of asparagine residues of metastable protein ions analyzed by MALDI-TOF-TOF-MS/MS and post-source decay. International Journal of Mass Spectrometry. 457:116433. https://doi.org/10.1016/j.ijms.2020.116433.
Cao, X., Brouwers, J., Heijmen Van Dijk, L., Parker, C., Huynh, S., Van Putten, J., Kelly, D.J., Wosten, M.M. 2020. Dataset of the phospholipidome and transcriptome of Campylobacter jejuni under different growth conditions. Data in Brief. 33. Article 106349. https://doi.org/10.1016/j.dib.2020.106349.
Carter, M.Q., Pham, A., Huynh, S., Parker, C., Miller, A., He, X., Hu, B., Chain, P. 2020. DNA Adenine Methylase, not the Pstl restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli. Food Microbiology. 96. Article 103722. https://doi.org/10.1016/j.fm.2020.103722.
