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

Location: Produce Safety and Microbiology Research

2020 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
In support of Objective 1, progress was made on the development of typing methods for foodborne pathogens to replace current testing methodologies. The ongoing collaboration with the technology sector continued to optimize an integrated/continuous flow through system for analyzing samples collected at a leafy greens processing facility. This on-site monitoring system will reduce product hold time, as specifically requested by industry stakeholders. The prototype consisting of a cassette-based system has been optimized in conjunction with a capturing step for the detection of Listeria species with the automated flow through system. Current efforts are aimed at the use of the cassette-based approach for pathogen detection in other types of food commodities. Under Objective 2, progress was made in completion of the DNA sequencing of Arcobacter and Campylobacter type strain genomes. Twenty-seven Arcobacter and 33 Campylobacter type strain genomes were completed, and genes annotated over the course of the project with 27 completed during this fiscal year. All genomes were closed, annotated and deposited into National Center for Biotechnology Information (NCBI) GenBank, with immediate public release. Thus, full type strain genomic data are now publicly available for all current taxa within the Arcobacter and Campylobacter genera. Additionally, 100+ draft genomes, providing a greater sequence depth for select Campylobacter taxa, were also deposited into GenBank. Furthermore, C. jejuni strains with phase variable DNA restriction-modification (R-M) systems were sequenced and genomic methylation patterns determined. The various R-M systems in C. jejuni are involved in site-specific methylation of the pathogen’s genomic DNA and may play a role in global gene expression. These genomes provide a wealth of data, which is critical for the development of novel typing/culturing methods, epidemiology, and research into virulence and the role of gene content in environmental adaptation and host colonization. Under Objective 2, progress was also made in the characterization of mobile elements in Arcobacter. These mobile elements range from simple insertion sequences (elements containing a single gene necessary for movement) to more complex transposable elements, which can also contain antibiotic resistance genes. Mobile elements are common in many bacteria, such as E. coli. However, these elements are rare in the human pathogens Campylobacter and Helicobacter, and where present in these organisms are confined to two or three insertion sequence families. However, the related Arcobacter, recovered from food, water, and human clinical samples, contain a diverse (i.e., representatives of 13 insertion sequence families) and abundant suite of mobile elements, with one strain containing approximately 100 complete and degenerate elements. Several elements were identified in more than one species, suggesting that they can move through the genus via lateral transfer. Additionally, these elements can drive genomic rearrangement and can inactivate genes through element insertion. In one species, these elements have led to a loss of motility due to element-associated gene inactivation and deletion. Therefore, mobile elements likely play a major role in the biology of Arcobacter. Under Objective 2, ARS scientists, in collaboration with researchers at the University of Virginia, determined the genome sequences of several C. jejuni strains isolated from guinea pigs that were genomically distinct from poultry- and cattle-associated strains. These distinctive strains have unique genes that may be host-specific genomic markers in C. jejuni. Such markers will improve source attribution of clinical infections and inform intervention strategies to reduce campylobacteriosis. ARS scientists, in collaboration with researchers at the University of Arizona and Erasmus Medical College in Rotterdam, Netherlands, analyzed genomic differences (e.g., gene content and single nucleotide polymorphisms) of hundreds of C. jejuni strains. Five clusters of genes were identified that were associated with strains of C. jejuni Penner serotype 19 that are associated with Guillain-Barré Syndrome. In support of Objective 2, ARS scientists, in collaboration with researchers at the University of Warwick in Coventry, United Kingdom, analyzed the genomes of Salmonella enterica from a variety of outbreaks. The objective of this collaboration is to identify genomic signatures that allow Salmonella enterica to be distinguished or clustered together. Also in Objective 2, progress was made that resulted in the identification of two plasmid-borne antibacterial (AB) immunity proteins expressed by a pathogenic E. coli strain using antibiotic induction, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic analysis. In addition, a promoter region (SOS box) was discovered upstream of the AB genes on the plasmid that is activated by DNA damage under culturing conditions used in the experiment. Under Objective 3, significant progress was achieved to characterize the potency of Shiga toxin (Stx) subtypes in Shiga toxin-producing Escherichia coli (STEC) strains recovered from a major agricultural region for leafy greens in California. To address research under Sub-objectives 3A and 3B, STEC strains were initially identified based on a typical STEC colony morphology and positive polymerase chain reaction (PCR) tests for the stx gene, but PCR-based genotyping assays showed that these STEC strains were Shiga toxin-producing Escherichia albertii (STEA), an emerging human enteric pathogen misidentified as Escherichia coli, due to similar characteristics. Ongoing research is aimed at the use of genome sequencing and cytotoxicity assays for the improved identification and assessment of the factors contributing to the virulence potential in STEC and STEA. For Sub-objective 3A significant progress was made involving MALDI-TOF-TOF-MS/MS and top-down analysis of B-subunits of Stx2 subtypes. It was discovered that amino acid substitutions within the Stx protein play a significant role in the characteristic pattern of toxin cleavage and the sequence-specific fragment ions that are used for identification. To address Sub-objectives 3C and 3D, continued research characterized the pathogenic mechanisms associated with Stx2 subtypes in cultured mammalian cells. Protocols were optimized for examining the efficiency of receptor binding and delivery of Stx by separating the subpopulations of intoxicated mammalian cells using cytofluorometry. Host cells exposed to Stx2a or Stx2c toxin subtypes exhibited a biphasic response to intoxication in which one subpopulation of cells was unaffected and were resistant to intoxication. To characterize receptor binding and internalization, hybrid toxins were employed to assess the cytotoxicity of the Stx subtypes (Stx1a, Stx2a) in host mammalian cells. Results demonstrated that the identity of the B-pentamer is responsible for the cytotoxic response among the subtypes. Finally, the experimental results provided additional evidence that cell death is not an inevitable outcome of toxin binding, or even toxin activity, and that cells can recover from exposure to low toxin concentrations. Under Objective 4, analysis of results continued for antimicrobial resistant and sensitive Campylobacter isolates and demonstrated the transfer of antimicrobial resistance genes from a resistant strain to a sensitive strain.

Accomplishments
1. Evolution of host specialism of Campylobacter jejuni. ARS scientists in Albany, California, in collaboration with scientists at the University of Bath, United Kingdom, used large Campylobacter jejuni strain collections and comparative genomics techniques, linked to phenotypic studies, to understand the timescale and genomic adaptations associated with the proliferation of the most common foodborne bacterial pathogen within the most prolific agricultural mammal (cattle). The analyses demonstrated genomic adaptation of cattle specialist C. jejuni lineages from an assemblage of host generalist C. jejuni strains that coincided with the dramatic rise in cattle population in the 20th century. C. jejuni adaptation to cattle was associated with horizontal gene transfer and significant gene gain and loss. This genomics-based research illuminates how C. jejuni exploits niches created as a result of increased cattle production and provides clues for reducing the risks posed by this major foodborne pathogen.

2. Top-down proteomic software to identify unknown proteins expressed by foodborne pathogens. Rapid identification of bacterial proteins and toxins is critical to the full characterization of virulence factors expressed by pathogenic microorganisms. It is also critical to exploit proteomic information derived from whole genome sequencing (WGS) of pathogenic strains relevant to food safety. ARS researchers in Albany, California, developed a standalone software program that rapidly identifies proteins and toxins expressed by foodborne pathogens by analysis of mass spectrometry and WGS data. The software can scan hundreds of thousands of full or partial protein sequences in seconds and retrieve a small number or just one candidate protein sequence. The software represents a significant advance in the understanding of protein fragmentation and its use in identifying and characterizing pathogenic bacteria, viruses and fungi important to food safety, public health, outbreak and traceback investigations.

Review Publications
Boukerb, A.M., Penny, C., Serghine, J., Walczak, C., Cauchie, H., Miller, W.G., Losch, S., Ragimbeau, C., Mossong, J., Megraud, F., Lehours, P., Benejat, L., Gourmelon, M. 2019. Campylobacter armoricus sp. nov., a novel member of the Campylobacter lari group isolated from surface water and stools from humans with enteric infection. International Journal of Systematic and Evolutionary Microbiology. 69(12):3969-3979. https://doi.org/10.1099/ijsem.0.003836.
Elhadidy, M., Ali, M.M., El-Shibiny, A., Miller, W.G., Elkhatib, W.F., Botteldoorn, N., Dierick, K. 2020. Antimicrobial resistance patterns and molecular resistance markers of Campylobacter jejuni isolates from human diarrheal cases. PLoS One. 15(1):e0227833. https://doi.org/10.1371/journal.pone.0227833.
Miller, W.G., Yee, E. 2019. Complete genome sequences of the Campylobacter fetus subsp. venerealis, Campylobacter lari subsp. concheus, Campylobacter sputorum bv. sputorum, and Campylobacter volucris Type Strains. Microbiology Resource Announcements. 8(45):e01157-19. https://doi.org/10.1128/MRA.01157-19.
Miller, W.G., Yee, E., Chapman, M.H. 2019. Complete genome sequence of the Arcobacter canalis type strain LMG 29148. Microbiology Resource Announcements. 8(44):e01156-19. https://doi.org/10.1128/MRA.01156-19.
George, A.S., Rehfuss, M.Y., Parker, C., Brandl, M. 2020. The transcriptome of Escherichia coli O157:H7 reveals a role for oxidative stress resistance in its survival from predation by Tetrahymena. FEMS Microbiology Ecology. 96(3). https://doi.org/10.1093/femsec/fiaa014.
On, S.L., Miller, W.G., Kelly, D., Vandamme, P. 2020. An emended description of Arcobacter anaerophilus Sasi Jyothsna et al. 2013: genomic and phenotypic insights. International Journal of Systematic and Evolutionary Microbiology. 70(6):3921-3923. https://doi.org/10.1099/ijsem.0.004214.
Maus, A., Bisha, B., Fagerquist, C.K., Basile, F. 2019. Detection and identification of a protein biomarker in antibiotic resistant Escherichia coli using intact protein LC offline MALDI-MS and MS/MS. Journal of Applied Microbiology. 128(3):697-709. https://doi.org/10.1111/jam.14507.
Bian, X., Garber, J.M., Cooper, K.K., Huynh, S., Jones, J., Mills, M., Rafala, D., Nasarin, D., Kotloff, K.L., Parker, C., Tennant, S.M., Miller, W.G., Szymanski, C.M. 2020. Campylobacter abundance in breastfed infants and identification of a new species in the global enterics multicenter study. mSphere. 5(1):e00735-19. https://doi.org/10.1128/mSphere.00735-19.
Fagerquist, C.K., Zaragoza, W.J., Carter, M.Q. 2019. Top-down proteomic identification of Shiga toxin 1 and 2 from pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry. Microorganisms. 7(488):1-14. https://doi.org/10.3390/microorganisms7110488.
On, S.L., Althus, D., Miller, W.G., Lizamore, D., Wong, S.G., Mathai, A.J., Chelikani, V., Carter, G. 2019. Arcobacter cryaerophilus isolated from New Zealand mussels harbor a putative virulence plasmid. Frontiers in Microbiology. 10:1802. https://doi.org/10.3389/fmicb.2019.01802.
Soto-Beltran, M., Quinones, B., Ibarra Rodriguez, A., Amezquita-Lopez, B.A. 2019. Use of membrane filtration for the recovery of campylobacter from raw chicken carcasses purchased at retail markets in Culiacan, Sinaloa, Mexico. Journal of Biosciences. 7:e698. https://doi.org/10.15741/revbio.07.e698.
Fagerquist, C.K., Zaragoza, W.J. 2019. Top-down and middle-down proteomic analysis of Shiga toxin using MALDI-TOF-TOF mass spectrometry. MethodsX. 6:815-826. https://doi.org/10.1016/j.mex.2019.04.011.
Cherubin, P., Fiddler, D., Quinones, B., Teter, K. 2019. Bimodal response to Shiga toxin 2 subtypes results from relatively weak binding to the target cell. Infection and Immunity. 87:e00428-19. https://doi.org/10.1128/IAI.00428-19.
Mourkas, E., Taylor, A.J., Méric, G., Bayliss, S.C., Pascoe, B., Mageiros, L., Calland, J.K., Ridley, A., Vidal, A., Forbes, K., Strachan, N.J., Parker, C., Parkhill, J., Cody, A., Jolley, K.A., Maiden, M.M., Kelly, D.J., Sheppard, S.K. 2020. Agricultural intensification and the evolution of host specialism in the enteric pathogen Campylobacter jejuni. Proceedings of the National Academy of Sciences. 117(20):11018-11028. https://doi.org/10.1073/pnas.1917168117.
Good, L., Miller, W.G., Niedermeyer, J., Osborne, J., Siletzky, R.M., Carver, D., Kathariou, S. 2019. Strain-specific differences in survival of Campylobacter spp. in naturally-contaminated turkey feces and water. Applied and Environmental Microbiology. 85:e01579-19. https://doi.org/10.1128/AEM.01579-19.
On, S.L., Miller, W.G., Biggs, P., Cornelius, A., Vandamme, P. 2020. A critical rebuttal of the proposed division of the genus Arcobacter into six genera using comparative genomic, phylogenetic, and phenotypic criteria. Systematic and Applied Microbiology. 43(5):126108. https://doi.org/10.1016/j.syapm.2020.126108.
Miller, W.G., Yee, E., Bono, J.L. 2020. Complete genome sequencing of four Arcobacter species reveals a diverse suite of mobile elements. Genome Biology and Evolution. 12(2):3850–3856. https://doi.org/10.1093/gbe/evaa014.
Tran, T.D., Huynh, S., Parker, C., Hnasko, R.M., Gorski, L.A., Khalsa Sat, D., Brown, P., McGarvey, J.A. 2020. Complete genomic sequences of three Salmonella enterica subsp. enterica serovar muenchen strains from an orchard in San Joaquin County, California. Microbiology Resource Announcements. 9(13):e00048-20. https://doi.org/10.1128/MRA.00048-20.