Location: Produce Safety and Microbiology Research2016 Annual Report
1a. Objectives (from AD-416):
1: Elucidate biological factors and molecular mechanisms that enhance or reduce fitness characteristics related to survival and growth of enteric pathogens in the produce production continuum. A: Phenotypic characterization of shiga toxin-producing E. coli (STEC) O111 strains of environmental & clinical origins. B: Characterize traits contributing to fitness of Salmonella strains on pre-harvest lettuce. C: Evaluate formation of STEC persisters & factors contributing to population size in produce production continuum. D: Compare fitness & physiology of different STEC serotypes & associated indigenous microbiota on produce growing in conventional vs organic soil. E: EcO157 biofilm formation under stress conditions in MAP lettuce. F: Identify genes involved in attachment of L. monocytogenes to produce & role of biofilm formation in survival on produce. G: Evaluate interaction of EcO157 with various lettuce accessions. H: Investigate evolution of EcO157 in MAP lettuce. I: Investigate interactions between norovirus & native plant-associated bacteria. 2: Identify environmental factors that affect the persistence and transmission of enteric pathogens in the produce production environment for risk assessment. A: Develop & deploy quantitative assessment of enteric pathogens in surface water in several watersheds in Salinas region to enhance current incidence data supplied to FDA for risk assessment model. B: Determine survival & fitness characteristics of enteric pathogens in water & sediment samples from central California coastal (cCc) produce production regions. C: Determine the impact of indigenous microbial community on environmental persistence of STECs in sediment from watershed locations often positive for STEC strains in Salinas region using metagenomics approach. D: Comparative genomics & transcriptomics applied to characterize environmental Shiga toxin-producing Escherichia coli. E: Investigate survival & persistence of Salmonella & L. monocytogenes in Central California watersheds to measure prevalence, identify subtypes & regional traits. F: Characterize F+ RNA Coliphages in water samples from cCc produce production regions & determine feasibility as indicators for source-tracking enteric pathogens. G: Characterize enteric pathogens transported through aerosols & determine seasonal fluctuations in cCc produce production regions. H: Determine if wild pigs are hosts for human norovirus (HuNoV). 3: Develop methods for the detection and subtyping of enteric bacterial and viral pathogens from produce production environments; to aid epidemiological investigations and to distinguish pathogenic from non-pathogenic strains. A: Develop FT-IR identification libraries for serotype analysis. B: Improve STEC subtyping using sequence of CRISPR & other hypervariable genes to augment existing MLVA method. C: Examine bacterial & produce-associated factors impacting genome & STEC virulence evolution. D: Develop better assay to determine inactivation status of Tulane virus (TV) & HuNoV caused by viral genomic damage. E: Develop accurate, fast, & low-cost multi-amplicon in situ capture qRT-PCR assay determining HuNoV infectivity.
1b. Approach (from AD-416):
Plant-microbe model systems in combination with population studies, ecology, molecular methods, genomics, and microbiology will be used to investigate the interaction of human bacterial and viral pathogens with plants and plant-associated bacteria, as well as to develop improved methods for detection and subtyping of human pathogens on produce.
3. Progress Report:
This is the first report for this new project, which continues research from “Biology and Control of Human Pathogens on Fresh Produce”, 2030-42000-046-00D. The work on many of the sub-objectives in this project is in the early stages of completion. We examined genetic diversity and population structure of environmental STEC E. coli O145 strains and compared their phenotypic traits related to environmental and virulence to several outbreak O145 strains. We found that the core virulence determinants of enterohemorrhagic E. coli were conserved in the environmental STEC O145 strains; however, there was large variation in the expression of virulence traits among the strains that were highly related genotypically. Several cattle isolates exhibited key virulence traits comparable to those of the STEC O145 outbreak strains, emphasizing the emergence of hypervirulent strains in agricultural environments. This work was published in Applied Environmental Microbiology. Environmental and outbreak Shiga toxin producing E. coli (STEC) O111 strains were partially screened for tolerance to sub-lethal osmotic and matric stress in vitro. A PGA-minus mutant of E. coli O157:H7 (EcO157) was constructed and tested for biofilm formation in high and low nutrient media and compared with the wild-type. Lettuce-associated outbreak strain TW14588 was chromosomally marked with antibiotic resistance and a new RpoS-minus mutant of this strain was constructed to match the antibiotic resistance of the parental strain for use in fitness studies. Studies of competitive fitness between wild-type and RpoS mutant on processed lettuce are underway. In our projects to study how strains adapt to growth conditions on plant surfaces we screened 120 isolates of Salmonella from our collection for the mutator phenotype. More strains are being screened, and when done we will select strains for testing on fitness for growth on pre-harvest lettuce. In a separate project we optimized the procedure for quantifying persistence in STEC strains, and determined that the largest population of STEC persisters was induced by growth in water for 1 week at 15oC, followed by growth in spinach lysates for 24 h at 26oC, growth in spinach water for 48 h at 26oC, and finally growth in LB broth at 26oC or 37oC. Using a new quantitative PCR technology (droplet digital PCR, ddPCR) we developed a method to monitor the level of two DNA targets (ehxA, hemolysin and stx2, shiga toxin) to quantify STEC in environmental samples. We determined the ddPCR method can detect at least 50 genome equivalents (GE) of STEC per sample, and have developed similar ddPCR methods to quantitate Salmonella and L. monocytogenes using invA and hlyA, respectively, as targets. This ddPCR method was used to analyze STEC survival during growth of lettuce plants in soils managed as organic, mixed or conventional. Microbial activity was 2-4 times higher than in conventional soils and STEC survival was substantially reduced, both in the rhizosphere and phylosphere of lettuce grown in these soils, compared to mixed and conventionally maintained soils. Biofilm formation, lettuce attachment and growth ability was tested in of monocytogenes strains to select strains for further genomic analysis. We have encountered technical difficulties in our mutant selection process for produce-attachment mutants, and are working with our collaborators at North Carolina State University to resolve these issues. In our Human Nororvirus (HuNoV) work we engineered a strain of E. coli O86:H2 to display GII.4 HuNoV capsid proteins. This strain will be used to search for candidate ligands and/or native plant-associated bacteria that bind to HuNoV. Polyclonal antibodies against GII.4 and GI.1 HuNoV have been made, and we confirmed that type B HBGA was expressed in E. coli O86:H2 and responsible for binding to HuNoV. The E. coli O86:H2 will be used as a positive control for screening plant-associated HuNoV-binding bacteria. Sediment and water samples in Salinas region are routinely collected for STEC isolation. Several locations are often positive; though not all samples at these locations are positive for STEC; leading to the hypothesis that differences in the microbial population of the sediment may control STEC survival. Characterization (by serotype, virulence genes, ompA sequence, and MLVA) of STECs isolated from these samples showed that the two sampling sites demonstrated periods of persistence and lack of persistence of specific STEC genotypes throughout the sampling period. DNA from the corresponding sediment samples, which were saved after initial collection, was extracted and metagenomic analysis in a collaboration with a scientist at Georgia Institute of Technology. Principle component analysis (PCA) indicated significant shift of the microbial populations between the two sample sites and during seasonal changes. Furthermore, one of the sites (with the reduced agricultural impact) showed considerably greater diversity, yet at both sites Proteobacteria are the most dominant phyla. Further PCA analysis is waiting for completion of sequencing of upstream control sediment DNAs. The same sediment and water samples in the Salinas region continue to be assessed for Salmonella and Listeria monocytogenes prevalence and strain subtyping. To date 1557 isolates of Salmonella have been identified, comprising 66 different serovars. The most common serovar (over 350 isolates) is a variation of S. Muenchen [the monophasic 6,8:d:-)]. This serovar showed some spatial localization in the region. Of the 1220 L. monocytogenes isolates approximately 90% were of serotype 4b. Analysis is ongoing. Also ongoing is analysis of E. coli specific coliphages from the same sediment and water samples to predict the sources of fecal contamination in the environment. In our project, to determine if HuNoV can be hosted in wild pigs, we designed qRT-PCR primer/probes to screen porcine norovirus (PoNoV) in wild pig stool samples. Primer sets for sequencing PoNoV also have been designed, and wild pig fecal samples have been collected for future testing. The ability to acquire and incorporate foreign DNA is a major driver of genome and virulence evolution in SETC, as the major virulence determinants of STEC are located on mobile elements. To better understand the conditions that favor acquisition of Shiga toxin (stx) gene and the selective forces to maintain the stx in E. coli, we constructed two stx-donor strains, one is the EcO157 strain linked to the 2006 US spinach-associated outbreak and the other one is the E. coli O104:H4 strain linked to the 2011 large outbreak of HUS in Germany. In each donor strain, the stx gene was replaced with a chloramphenicol acetyltransferase (cat) gene. We will examine the transduction of Shiga toxin-encoding phage from above strains to the E. coli strain belonging to different phylogroups. Strain analysis is ongoing in construction of a Fourier-transform Infrared Spectroscopy (FT-IR) database to serotype Salmonella isolates. We screened over 300 isolates of Salmonella from our strain collection by FT-IR, and have started analysis of patterns in the neural imager software. With the large number (>2400) of serotypes present in the genus Salmonella it may be necessary to construct two databases for comparison of strains in order to determine serotype. To measure viral damage in HuNoV and we are using Tulane Virus (TV) as a surrogate to evaluate for correlations between two cell-based assays (plaque and TCID50 assays) and three molecular genetic qRT-PCR assays. We found that one PFU was roughly-equivalent to 6.7 TCID50 units, 2.8 RNase-treated genomic copies (GCs), 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 Ab-ISC-qRT-PCR GCs. Primer-probe sets for multi-amplicon for TV have been designed and will be tested and validated by cell based assays. Within the last year this project has added a new scientist; therefore, a new sub-objective is under development which will address the ecology of Shiga toxin-producing E. coli (STEC) bacteriophages and their association with bacterial evolution. The new initiative will begin in fiscal year 2017.
1. Virulence potential of environmental Shiga toxin-producing Escherichia coli (STEC) strains. ARS researchers in Albany, California, examined the genetic diversity, population structure, virulence potential, and antimicrobial resistance profiles of environmental STEC O145 strains recovered from a major produce production region in California, and the majority of these environmental STEC O145 belong to sequence type 78 (ST-78), which is a common ST in clinical strains. Diverse stx toxin subtypes were identfied and, and environmental strains were capable of producing enterohemolysin, whereas only a few strains were positive for anaerobic hemolytic activity. Furthermore, nearly half of the strains displayed resistance to at least two different classes of antibiotics. The core virulence determinants of enterohemorrhagic E. coli were conserved in the environmental STEC O145 strains; however, there was large variation in the expression of virulence traits among the strains that were highly related genotypically. Several cattle isolates exhibited key virulence traits comparable to those of the STEC O145 outbreak strains, emphasizing the emergence of hypervirulent strains in agricultural environments. This information regarding the toxicity of environmental STEC strains is important for public health and modelers of produce contamination by enteric pathogens, and the antibiotic resistance information is important for determination of resistance determinants present in the environment.
2. Determination of correlations between cell-based and single-amplicon- qRT-PCR based-assays as methods for measuring infectivity of Tulane Virus. ARS scientists in Albany, California, are using Tulane Virus (TV) as a surrogate for Human Norovirus (HuNoV) to evaluate for correlations between two cell-based assays (plaque and TCID50 assays) and three molecular qPCR-based assays. The researchers found the mathematical correlations between the Plaque Forming Unit in the cell-based assays and the Genome Copies measured in the three molecular based assays. These results will be used as the basis to study more correlations between cell-based and molecular-based assays for TV and HuNoV. This data was published in the Journal of Virological Methods, and will be of interest to risk assessors to determine the viability of HuNoV when attached to produce surfaces.
5. Significant Activities that Support Special Target Populations:
Tian, P., Lei, S., Wang, D., Yang, D.H. 2016. Comparison of in vivo and in vitro assays as methods for measuring infectivity of tulane virus. Journal of Virological Methods. doi: 10.1016.