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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Research Project #438551

Research Project: Human Pathogens within the Produce Production Continuum; their Detection, Mechanisms for Persistence, and Ecology

Location: Produce Safety and Microbiology Research

2022 Annual Report

Objective 1: Elucidate microbial and plant factors and molecular mechanisms that affect fitness characteristics related to survival and growth of enteric pathogens in the produce production continuum. 1.A: Screen lettuce cultivars for genotypes exhibiting a wound response inhibitory to EcO157. 1.B: Assess the role of enzymatic browning in the survival of EcO157 on fresh-cut lettuce. 1.C: Determine whether chemoattractants in lettuce leaves induce the ingression of EcO157 into cut tissue. 1.D: Identify and characterize genetic determinants and physiological traits contributing to colonization of crop plants by STEC and Salmonella enterica. 1.E: Determine the fitness of various genomic subtypes of L. monocytogenes in the colonization of produce. 1.F: Compare fitness and physiology of different STEC serotypes, Salmonella, and L. monocytogenes on produce growing in conventional vs organic soil. 1.G: Isolation and characterization of HuNoV and TV ligands from mammalian cells and bacteria for the development and validation of viral ligand-based assays for determination of viral infectivity. Objective 2: Identify environmental and bacterial factors that affect the persistence and transmission of enteric pathogens in the produce production environment for risk assessment. 2.A: Perform an epidemiological investigation of STEC from the Salinas, CA region with whole genome sequencing. 2.B: Monitor the persistence and distribution of Salmonella and L. monocytogenes genomic types in the Central California Coast agricultural region. 2.C: Determine the survival of EcO157 in water-sediment microcosms from public access watershed sites near a leafy greens-growing region in Central Coastal California. 2.D: Identify environmental reservoirs of enteric pathogen strains exhibiting enhanced pathogenicity potential and environmental persistence by comparative genomics and functional genomics. 2.E: Monitor the transport of pathogenic bacteria in various Salinas watersheds using ddPCR and wgMLST. Objective 3: Develop methods to detect, subtype, and distinguish bacterial pathogen strains from produce production environments to aid surveillance and epidemiological investigations. 3.A: Develop improved media for equivalent enrichment of Salmonella serotypes, and CRISPR-SeroSeq for rapid serotype detection. 3.B: Develop and validate viral ligand-based assays for infective HuNoV and surrogate TV and to determine viral infectivity.

The project will address mechanisms for the detection, persistence, and ecology of human pathogens within the produce production continuum. Objective 1 will address microbial and plant factors that affect the fitness of human pathogens to survive and grow on plants. Objective 2 will focus on the ecology of human pathogens in the plant pre-harvest environment by studying factors that allow the pathogens to survive and be transported through the Central California Coast region. Finally, Objective 3 will concentrate on methods for effective detection of human pathogens for use in surveillance laboratories. Under Objective 1, lettuce cultivars will be screened for genotypes exhibiting a wound response to Escherichia coli O157:H7 (EcO157) to identify cultivars that would reduce potential EcO157 contamination of lettuce in modified atmosphere packaging. The role of browning on fresh cut lettuce regarding EcO157 survival will be measured. Chemoattractants in lettuce leaves will be assessed to determine if they induce the ingression of EcO157 into cut lettuce tissue. EcO157 and two serovars of Salmonella enterica will be studied to determine the genetic determinants that contribute to those human pathogens colonizing spinach, romaine lettuce, cucumbers, tomatoes, cantaloupe, and watermelon. The fitness of Listeria monocytogenes subtype ST382, which has been responsible for several produce-related outbreaks, will be compared with other subtypes on lettuce, cantaloupe, and sprouts. The fitness of various Shiga toxin producing E. coli (STEC) strains, Salmonella, and L. monocytogenes on plants growing in organic and conventional soils will be measured. For Human Norovirus (HuNoV) studies, ligands that bind the virus will be isolated from mammalian cells and bacteria to develop better assays for HuNoV detection. Under Objective 2, an epidemiological investigation of STEC from the Central California Coastal region will be done to track movement and determine contamination routes. The persistence of Salmonella in the same region will be assessed using strains isolated from waters in the area. The ability of EcO157 to survive in soil/water microcosms will be studied using sediment/water samples from the California Central Coast. EcO157 survival and water chemistry will be monitored in these microcosms for up to 3 months. Environmental reservoirs of STEC and Salmonella will be studied by comparing genomic information and epidemiological history of outbreak and environmentally isolated strains. Phenotypic traits that contribute to environmental persistence will be examined, and environmental reservoirs of hypervirulent STEC strains will be identified. Transport of STEC, Salmonella, and L. monocytogenes in Central California watersheds will be monitored using genomic subtyping and detection of virulence genes. Under Objective 3, improved enrichment culture media will be developed to equally enrich various Salmonella serovars for improved Salmonella surveillance. Whole genome methods for detection of relevant serovars will be developed. Assays to detect infectious HuNoV will be developed and validated using ligands for HuNoV isolated under Objective 1.

Progress Report
In support of Sub-objective 1.A, 35 different lettuce cultivars were characterized for phenotypes related to the plant wound response in fresh-cut products stored at 4C and for their inhibition of Escherichia coli O157:H7 (EcO157). Correlation analysis identified chemical and enzymatic traits in lettuce that are significant factors in the inhibition of EcO157 on cold-stored cut lettuce. Regarding the identification of genetic and physiological traits that contribute to the colonization of crop plants by EcO157 and Salmonella, representative strains of EcO157, S. enterica Newport, and S. enterica Javiana were selected for the study. The selections were made based on genomic and phenotypic features of the bacterial strains. After comparing various approaches for generating mutants and DNA sequencing, the random bar code transposon-site sequence (RB-TnSeq) developed at Lawrence Berkeley National Laboratory was selected to use in the project. RB-TnSeq increases the throughput of determining reproductive success (fitness) of mutants by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing to assay mutant fitness. The Tn5 RN TnSeq delivery vectors will be provided by a collaborator, and an MTA is being processed for the vectors. This work is in support of Sub-objective 1.D. Strain selection was made to begin studying the fitness of various L. monocytogenes sequence types (STs) and clonal complexes (CCs) in the colonization of plant tissue in support of Sub-objective 1.E. Strains of ST382, CC1, CC639, and CC217 were selected. Multiple detection protocols were assessed, and ultimately, it was decided to screen individual colonies of L. monocytogenes recovered after inoculation onto plant material via quantitative real time polymerase chain reaction (qPCR). Previously published PCR systems were assessed, and we selected the set published by the Health Protection Agency in the United Kingdom. Primers and probes were designed and ordered. In characterizing ligands that bind to Human Norovirus (HuNoV), a soluble extracellular polymetric substance (SEPS) was isolated from Sphingobacterium strain SC015. This bacterial strain was isolated as a natural resident of lettuce, and the bacterium also binds HuNoV. The extracted SEPS substance has a strong ability to bind HuNoV and Tulane Virus (TV, the commonly used surrogate of HuNoV). Because of this binding ability, the substance has the potential to be used as a ligand to capture both viruses. Furthermore, the SEPS enhances the survival and persistence of HuNoV and TV attached to lettuce and protects the viruses from inactivation. In addition, cleavable viral capsid expressing bacterial systems were constructed for genotypes GI and GII of HuNoV. Construction of a bacterial system for TV is ongoing. This work is in support of Sub-objective 1.G. In the project to investigate the epidemiology of Shiga toxin-producing E. coli (STEC) that exist in the Central California coastal agricultural region, we investigated strains from our previous surveys with whole genome Multi Locus Sequence Typing (wgMLST). This work is related to Sub-objective 2.A. Previous work in subtyping this collection with Multi Locus Variable Tandem Repeat Analysis (MLVA) revealed that hundreds of phylogenetic clusters were present in our STEC strain collection. Several of these clusters were re-examined by wgMLST and evidence of STEC transport in the Salinas region was confirmed, though in a very restricted number of clusters. A collaboration was established with the U.S. Food and Drug Administration (FDA) to facilitate the genome sequencing and wgMLST analysis of several thousand isolates from our entire STEC Salinas collection (4800 strains), in preparation for an extensive epidemiology analysis of STEC transport. To date, over 3200 strains (68%) have been sent to FDA, and their genomes are sequenced and typed. In the project to describe clonal complexes and sequence types of L. monocytogenes present in the Central California coast agricultural region, genome sequencing and core genome MLST (cgMLST) was completed on 1,248 individual isolates from surface waters in the region in support of Sub-objective 2.B. The most common clonal complexes in the region are CC639, CC183, and CC1. Strain clusters in many different sequence types that are persistent in the region were identified. Additionally, the virulence gene complement of the strains was determined. This data was published in Applied and Environmental Microbiology. For environmental sampling of freshwater environments, we purchased a hydrometer with a pH sensor, an oxygen sensor, and a conductivity and temperature sensor for sampling these microcosms. However, due to maximized telework, the sampling time was rescheduled to September 2022. This work relates to Sub-objective 2.C. For Sub-objective 2.D, more than 300 E. coli virulence genes that were deposited into the Virulence Factor DataBase. Additionally, 10 genomic islands (GIs) known to contribute to pathogenicity and fitness traits in STEC were examined systematically in a set of environmental and clinical STEC strains to assess the pathogenicity potential in STEC environmental isolates. Comparative genomic analyses were also performed between clinical and environmental Shiga toxin-producing Escherichia albertii (STEA) strains. The pathogenicity potential of environmental STEA strains were further evaluated by vero-cell based cytotoxicity assays. Birds including Oregon junco, crow, brown headed cow bird, and white-breasted nuthatch were identified as important reservoirs or carriers for these human pathogens. Transport of pathogens in watersheds is dependent on various biotic and abiotic factors, which varies substantially in the Central California coastal region. Several accessible sites, representing four streams, were selected for droplet digital PCR (ddPCR) detection and wgMLST analysis of pathogens (STEC, Salmonella, and L. monocytogenes). Stream velocity, pH, conductivity, salinity, temperature, and oxidation/reduction potential was monitored at these sites and samples for pathogen isolation and ddPCR were collected. This work supports Sub-objective 2.E. In research to develop culture media to equally enrich various serovars of Salmonella, 16 strains of serovars Typhimurium, Kentucky, Enteritidis, and Give were selected in support of Sub-objective 3.A. All strains were analyzed by the Biolog Omnilog system in phenotypic microarrays to assess their nutritional needs. Different nutritional characteristics were noted between the serovars. Notably, strains of serovar Typhimurium and Enteritidis utilized some sugars that strains of serovar Kentucky did not. Additional nutrient differences were noted, and we are awaiting back-ordered reagents to begin experiments in supplementing growth media and assessing fitness in enrichment media. Growth curves of the strains were done in Buffered Peptone Water (BPW), modified Buffered Peptone Water (mPBW), Trypticase Soy Broth (TSB), and one-tenth strength TSB. Growth differences were noted, especially a die-off of Enteritidis strains in BPW. The genomes of the 16 strains were sequenced and are being analyzed for detection targets for screening of culture media inoculated with combinations of serovars. In the development of an assay to detect infective HuNoV and TV, primers and probes for ddPCR were designed. Cell cultures for TV were developed. This work supports Sub-objective 3.B.

1. Seasonality of E. coli O157:H7 survival and microbiome in cold-stored fresh-cut lettuce. Outbreaks of Escherichia coli O157:H7 (EcO157) linked to romaine lettuce grown on the Central West Coast of California are more prevalent in the fall, and the cause of these seasonal trends is unknown. These outbreaks have greatly impacted the California lettuce industry, which is valued at over $2 billion annually. ARS researchers in Albany and Salinas, California, in collaboration with scientists at the Food and Drug Administration, discovered that EcO157 survives better on cold-stored fresh-cut romaine harvested in the fall than in the spring, and that the lettuce microbiome also has seasonality. This points to seasonal properties of lettuce and/or its microbiome, or interaction thereof, as factors in the seasonal behavior of EcO157 in this commodity. The results open a new branch of inquiry for the identification of plant traits and microbiome components that may be used in plant breeding or manipulated in order to suppress enhanced EcO157 survival in fall fresh-cut romaine. These findings led to a scientific publication, a press release picked up by numerous national and international outlets including Associated Press, and consultations by, and talks to, the leafy greens industry.

2. Virulence gene patterns in Listeria monocytogenes strains native to Central California coastal waters. Over half of the fatalities resulting from bacterial foodborne illness are caused by Listeria monocytogenes and increasing numbers of outbreaks and product recalls are due to contaminated produce. In a collaboration between ARS researchers in Albany, California, and Food and Drug Administration (FDA) researchers in College Park, Maryland, the genomes of over 1,200 isolates of L. monocytogenes isolated from surface waters along the Central California coastal region were sequenced and characterized. All the isolates contained core L. monocytogenes virulence genes in Listeria Pathogenicity Island 1 (LIPI-1), which allows the strains to cause listeriosis. Pathogenicity islands LIPI-3 and LIPI-4, which are found in strains that cause neurological illness and higher mortality, were intact in 73% and 63%, respectively, of the isolates. These findings have led to continued collaborations between ARS and FDA to define risk factors for environmental strains of L. monocytogenes. This information is needed for risk assessments and modelers of foodborne pathogens in the vicinity of agricultural production.

3. Identification of Salmonella serovars present in Central California coast surface waters. Salmonella is one of the leading causes of foodborne illness world-wide and produce-related outbreaks have been sourced to tomatoes, cucumbers, peppers, onions, and leafy greens. ARS researchers in Albany, California, undertook a five-year survey of surface waters along the Central California coastal agricultural region and determined a 56% prevalence of Salmonella in lakes, ponds, streams, and rivers. Sixteen of the 24 most common serovars detected in the region are among the Top 20 serovars reported to cause the most human salmonellosis in the United States, and some serovars had location and seasonal bias. These findings have resulted in multiple collaborations with government and academic institutions to describe types of Salmonella present in water and in the environment. This information is needed for risk assessments and modelers of foodborne pathogens in the vicinity of agricultural production.

4. Identification of novel molecular targets for detection of hypervirulent STEC strains. Shiga toxin producing Escherichia coli (STEC) is estimated to cause 2.8 million cases of acute illnesses globally each year. Strains of STEC vary greatly in pathogenicity; thus, genetic markers of hyper-virulent strains are critically needed for rapid detection and risk assessment. ARS researchers in Albany, California, systematically examined over 300 E. coli virulence genes in a large number of STEC strains isolated from various environmental samples collected in a major leafy greens production region in California and identified a list of genes that are important for STEC pathogenesis. New molecular targets were discovered to assess virulence potential of STEC isolates. The study provided additional molecular targets to develop a systematic approach for STEC detection, which improves the sensitivity of detection and the accuracy in developing risk assessment models.

5. A rapid and sensitive method to remove assay inhibitors in elutes from produce. Fruit and vegetable-related human norovirus outbreaks are becoming prevalent worldwide. Detection of human noroviruses in food samples has been a challenge due to the assay inhibitors present in washings from fruits and vegetables. ARS researchers in Albany, California, developed an easy method to remove these inhibitors using bentonite-coated activated carbon. The method was applied for detection of human noroviruses as well as surrogates spiked onto fruits and vegetables. The average detection limits of these viruses were reduced by approximately 10 to 100 fold. The method developed is a rapid, sensitive method to remove inhibitors and has the potential to be used to detect low numbers of food-borne viruses in produce.

Review Publications
Leonard, S., Simko, I., Mammel, M., Richter, T., Brandl, M. 2021. Seasonality, shelf life and storage atmosphere are main drivers of the microbiome and E. coli O157:H7 colonization of post-harvest lettuce cultivated in a major production area in California. Environmental Microbiome. 16. Article 25.
Gorski, L.A., Cooley, M.B., Oryang, D., Carychao, D.K., Nguyen, K., Luo, Y., Weinstein, L., Brown, E., Allard, M., Mandrell, R., Chen, Y. 2022. Prevalence and clonal diversity of over 1,200 Listeria monocytogenes isolates collected from public access waters near produce production areas on the central California coast during 2011 to 2016. Applied and Environmental Microbiology. 88(8). Article e00357.22.
Carter, M.Q., Laniohan, N.S., Lo, C., Chain, P.G. 2022. Comparative genomics applied to systematically assess pathogenicity potential in Shiga toxin-producing Escherichia coli O145:H28. Microorganisms. 10(5). Article 866.
Wang, N., Pan, G., Guan, S., Rong, S., Wang, D., Gao, Z., Tian, P., Li, Q. 2022. A broad-range disposable electrochemical biosensor based on screen-printed carbon electrodes for detection of human noroviruses. Frontiers in Bioengineering and Biotechnology. 10. Article 845660.
Zhang, Z., Liu, D., Zhang, Z., Tian, P., Li, S., Wu, Q., Wang, D., Tian, Z. 2021. Complete genome sequence of GII.9 norovirus. Archives of Virology. 167:249-253.
Gorski, L.A., Liang, A.S., Walker, S., Carychao, D.K., Aviles Noriega, A., Mandrell, R., Cooley, M.B. 2022. Salmonella enterica serovar diversity, distribution, and prevalence in public-access waters from a central California coastal leafy green-growing region from 2011 to 2016. Applied and Environmental Microbiology. 88(3). Article e01834-21.
George, A.S., Brandl, M. 2021. Plant bioactive compounds as an intrinsic and sustainable tool to enhance the microbial safety of crops. Microorganisms. 9(12). Article 2485.