Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

2018 Annual Report

Objectives
Objective 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. Sub-objective 1A-1I (Refer to uploaded Project Plan) Objective 2: Identify environmental factors that affect the persistence and transmission of enteric pathogens in the produce production environment for risk assessment. Sub-objective 2A-2H (Refer to uploaded Project Plan) Objective 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. Sub-objective 3A-3E (Refer to uploaded Project Plan) Objective 4: Study the ecology of Shiga toxin-producing E. coli (STEC) bacteriophages and its association with bacterial hosts. Objective 5: Development of immuno-, bacteriophage-, and mass spectrometry-based methods for rapid detection of foodborne pathogens.

Approach
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 on produce. Pathogenic E. coli is a foodborne pathogen that has been linked to numerous outbreaks of foodborne illnesses, and the illnesses are primarily attributed to the ingestion of Shiga toxin-producing E. coli (STEC). Previous research has indicated the virulence markers such as stx genes, of STEC strains are conferred to stx-encoding bacteriophages and can be transduced into the susceptible bacterial hosts. In order to understand the interplay between STEC-specific phages and their bacterial hosts in the environment to enhance the safety of food products and the prevention of new emerging foodborne pathogens, the initial focuses of the phage research are to isolate, collect and characterize STEC phages and to understand the relationship between phages and their hosts in the environment. Efficient methods for isolation of STEC bacteriophages will be utilized. Characterization of STEC bacteriophages will be established using genomic sequencing and proteomic analyses. The association of fecal contamination with the population of STEC bacteriophages in the environment will be determined. Environmental factors that influence the geographical distribution of STEC bacteriophages will be identified. This will establish a foundation to study biological interactions between phages and their hosts and the association of phages with bacterial evolution as well as to utilize collected phages to develop biosensors and pre-harvest biological controls for STEC to improve the microbiological food safety of the food supplies.

Progress Report
Objective 1: Studies of fitness and survival of enteric pathogens in produce production environments were continued. In vitro studies of Shiga toxin producing Escherichia coli (STEC) O111 strains revealed that differences in aggregative behavior dictated the comparative ability of strains to form biofilms in media with various osmolyte levels. Specifically, the gene ribonucleic acid (RNA) Polymerase, sigma S (RpoS) function affected the ability of strains to aggregate in vitro, which was inversely correlated with biofilm formation. RpoS is a central regulator of the general stress response and not only allows the cell to survive environmental challenges, but also prepares the cell for subsequent stresses. An inverse correlation was also seen with the aggregative protein Cah. Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. STEC may benefit from aggregative behavior when seeking protection in dense cell aggregates, but aggregation may hamper the relocation of free cells to an attached state. Sub-objective 1A: An article about cah, and its ecological role was published. Persister cells of STEC tolerate stress and likely enhance survival on produce. The formation of persister populations in 4 STEC outbreak strains was tested in field water, spinach leaf wash water, spinach lysates, and on lettuce plants. The proportion of EcO157 persister cells increased in the lettuce phyllosphere population under conditions of carrying capacity, multiplication, and decline caused by low water availability. Sub-objective 1C: The number of persister cells per leaf varied across a collection of leaf samples, indicating the persistence of EcO157 fluctuated within a lettuce plant and across plants. This work was submitted to the journal, Applied and Environmental Microbiology. Sub-objective 1D: ARS researchers in Albany, California, in collaboration with University of California, Davis, and Russell Ranch, monitored the survival of STEC on plants grown in organic, mixed or conventional soils with different irrigation methods (furrow vs drip). Indigenous microbes in organic soil affected the survival of STECs. Soils were collected twice during the growing season (cover crop to cash crop) from a series of farm plots that had specific amendments. Indigenous microbial activity was 2-4 times higher in organic soils, and STEC survival varied depending on soil type and irrigation method. Metagenomic analysis of the soils is ongoing. Additional experiments are being conducted with commercial soil amendments. Sub-objective 1G: Lettuce accessions were evaluated for their expression of basal plant defense genes and colonization by STEC following cutting and storage under Modified Atmosphere Conditions (MAP). A lettuce accession that had greater resistance to plant diseases, and on which EcO157 had lower survival, also displayed greater expression of genes involved in plant hormone signaling; whereas expression of genes in alternate plant defense pathways were repressed. Sub-objective 1F: Biofilms were measured to assess the attachment and growth of L. Monocytogenes to produce on other surfaces. Twenty strains showed differential attachment to plastic, glass, stainless steel, and lettuce. ARS scientists in Albany, California, in collaboration with scientists at North Carolina State, are identifying genes important for attachment to produce via targeted whole genome sequencing (WGS). Sub-objective 1I: With collaborators at Shanghai Jiao Tong University and Shanghai Institute of Technology, a novel system to study human norovirus (HuNoV) attachment to produce-associated bacteria were developed. Cleavable HuNoV P-Proteins were displayed on the surface of transformed bacteria. The bacteria-P-Protein-complex (BPC) was used to purify HuNoV P-proteins, identify binding of HuNoV proteins to lettuce and bacteria, and isolate ligands for HuNoV binding to lettuce, bacteria, and MK2 cells. The system was also used to characterize conditions for BPC binding to and removal from lettuce. Three manuscripts describing this work were published, and one is in preparation. Sub-objective 2C: Metagenomic analysis of sediment samples from the Salinas, California, area is proceeding to determine if indigenous microbial communities affect the persistence of STECs. Previous work identified 2 sampling sites with periods of presence or absence of specific STEC genotypes in an extended sampling period. Statistical analysis indicated a significant shift of the microbial populations between the 2 sample sites. ARS collaborators at the Georgia Institute of Technology could not detect STEC in the sequence data due to the complexity of the samples. However, a high abundance of antibiotic resistance genes was found, indicating that sediment from the region serves as a reservoir of antibiotic resistance. A manuscript is in preparation. Sub-objective 2D: Genome sequencing was done on 12 environmental STEC O145 strains that vary either in genotype (ST) or in the expression of virulence traits. All 12 genomes are annotated, and comparative genomic analyses with clinical strains for the discovery of new fitness and virulence traits is ongoing. Characterization of environmental STEC O121 strains revealed that a clonal population of ST-655, a common ST in clinical strains, was the predominant genotype. Carbon utilization profiles revealed a highly divergent structure within this clonal STEC O121 population. While carbon utilization profile-based clustering failed to differentiate the environmental from the clinical strains, it did exhibit better discriminating power than Multi Locus Sequence Typing (MLST) as strains linked to the same outbreak were all clustered together. A manuscript is under preparation. Sub-objective 2E: Subtyping 1693 Salmonella and 1253 Listeria monocytogenes isolates acquired through watershed sampling of the Salinas area continued. For Salmonella, 75 percent of the strains have been serotyped, and 73 serotypes were identified. Some serotypes show geographical bias. All L. monocytogenes isolates are undergoing WGS by ARS collaborators at the U.S. Food and Drug Administration in Maryland. In a study that included 25 of these L. monocytogenes strains, collaborators at North Carolina State University showed that 2 are of hypervirulent Clonal Complex 4, and 8 are ST-382, a genotype responsible for a 2014 stone fruit outbreak. ST-382 had a significant association with water. The genotype data was published in mBio, and the other work is being prepared for publication. Sub-objective 2H: To determine if wild pigs could be hosts for HuNoV, 70 feral pig stool samples were collected from various regions of California. Of these, 44 percent were positive for Type G1 HuNoV, and 17 percent were positive for GII HuNoV, as measured by our quantitative Reverse Transcriptase PCR (RT-qPCR) method. Porcine Norovirus was not detected. The virus obtained from the positive samples is being sequenced and a manuscript describing this work is being prepared. Sub-objective 3A: A model to use Fourier Transform Infrared Spectroscopy to serotype Salmonella based on the Top 20 Salmonella serovars related to U.S. illness was developed. Some serotypes identified well, but others were problematic in our model. A manuscript on this project was submitted. Sub-objective 3B: DNA sequence analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and hypervariable regions was not sufficient to differentiate STEC strains from the Salinas, California, area. The method was switched to Whole Genome MLST (wgMLST), which resolved 64 different STEC strains isolated from a variety of locations in the region, an improvement over our previous method. Data indicate that long-range transport of STEC strains may occur primarily through domestic and wild animals. Sub-objective 3C: Continuing studies into whether plants help maintain core virulence genes in STEC strains, through WGS additional strains were identified, including RM9872, cattle isolate from Salinas, California, that carries a prophage encoding the Stx2a toxin, that exhibited high cytotoxicity. A test to determine if the stxa genes in these strains could be donors for horizontal gene transfer both in vitro and when placed on plant surfaces is under development. The recipients for these experiments will be 3 environmental EcO145 strains that lack stxa genes. Sub-objectives 3D and 3E: A new Multi-Amplicon quantitative PCR method as a more accurate assay for infectious HuNoV and its surrogate Tulane Virus (TV) was validated. The results were variable with each primer and probe set, however, and were not consistent with results with TV in tissue culture. Therefore, a novel DNA-aptamer-based assay was developed in collaboration with scientists at Shanghai Jiao Tong University, to determine the infectivity of HuNoV. Two aptamers were selected to incorporate into the current RT-qPCR methods to detect HuNoV. A manuscript describing this work was submitted for publication. Objective 4: Using environmental samples taken from 50 miles south of San Jose, California, ARS researchers improved their original phage isolation procedure to identify phage and STEC bacteria. Lytic phage specific to STEC serogroups O26, O45, O103, O111, O121, O145, and O157 were isolated, but no STEC bacteria were found in these samples. WGS was done for genomic characterization, and to facilitate proteomic studies, of the phages specific to O45, O103, O111, O121, O145, and O157 isolated from non-fecal compost. No STEC virulence genes were encoded in these phages, which is important if they are to be used in biocontrol interventions. Phages in the Siphoviridae (O45, O111, and O145) were most dominant in number, followed by Myoviridae (O121 and O157), and Podoviridae (O103) in samples.

Accomplishments
1. A box liner with a slow release sulfur dioxide pad enhances the killing of foodborne pathogens on table grapes. California produces 99 percent of the commercial table grapes in the U.S. ARS researchers in Albany, California, examined the survival of three common foodborne pathogens, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Thompson, inoculated on commercially packed table grapes under simulated refrigerated transit conditions. Results showed that a box liner in the shipping container enhances the bactericidal effect of a sulfur dioxide (SO2) pad in a pathogen-dependent manner. The use of slow release SO2-generating pad combined with box liner is effective in reducing foodborne pathogens L. monocytogenes and S. enterica Thompson, while the use of SO2-generating pad alone was more effective in killing E. coli O157:H7. The results were communicated to growers in California through the California Table Grape Commission, who requested this research.

2. Creation of a pathogen quantification method using droplet digital polymerase chain reaction (PCR). Foodborne pathogens pose a risk to fresh produce, and while simple detection of pathogens in a produce environment can be done, absolute quantification of pathogens in those environments is extremely difficult and time consuming. This severely limits accurate prediction of pathogen levels and rate of transport in the environment. ARS researchers in Albany, California, developed a new method using droplet digital PCR (ddPCR) to determine the actual number of cells of Escherichia coli, Salmonella, and Listeria monocytogenes in contaminated environmental samples. The ddPCR method was shown to be significantly less sensitive to PCR inhibitors commonly found in soil and sediment that adversely affect other quantification methods. During method development the researchers also developed a robust DNA extraction and purification procedure to recover DNA from several different types of bacteria with equal efficiency, which allows for better comparisons of levels of each type of pathogen that might be present. The data from this ddPCR method supplements pathogen incidence data previously done in collaboration with the Food and Drug Administration, who is using the information provided by this work in the development of risk assessment models.

3. An effective method to dry and decontaminate wet whole almonds. California produces 80 percent of the world’s almonds with a value of over $5.33 billion. Contamination of almonds with Salmonella has caused several large and expensive recalls by the industry and outbreaks of human illness. The occurrence of rain during the harvest season may cause the complete loss of an almond crop due to increased risk of microbial contamination and lack of adequate drying technology. ARS scientists in Albany, California, developed an effective and energy-saving new technology based on sequential infrared heat and hot air to simultaneously dry and decontaminate wet whole almonds. The published results were provided to the industry and contributed to ARS scientists receiving the 2018 Research and Development Award by the Institute of Food Technologists.

4. Isolation of bacteriophages that kill Shiga toxin-producing Escherichia coli related to produce outbreaks. Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually. ARS researchers in Albany, California, isolated and characterized several novel bacteriophages or phages (viruses that infect bacteria) specific to STEC subtypes that have caused outbreaks related to produce, including O26, O45, O103, O111, O121, O145 and O157. The phages were isolated from local non-fecal composts. None of the cognate STEC bacteria were isolated from these samples, and analysis indicates this is due to the phages eliminating these STEC subtypes. These phages provide a new tool for biocontrol interventions, replacing chemicals and antibiotics during pre-harvest and post-harvest management. The phage biocontrol intervention provides a valuable new tool to protect growers, farmers, and the industry from foodborne contamination losses and to improve food safety.

5. Escherichia coli O157:H7 utilizes plant-derived choline for survival of osmotic stress in lettuce wounds. E. coli O157:H7 is the most common bacterial agent causing outbreaks from fresh-cut packaged lettuce. Plant damage is inherent to the production of fresh-cut lettuce and enteric pathogens multiply to high densities in lettuce wounds. Through transcriptomics, chemical analyses, and bacterial competitive fitness studies on lettuce plants and processed lettuce, ARS scientists in Albany, California, uncovered that E. coli O157:H7 experiences osmotic stress in cut lettuce leaves but benefits from its import of plant choline at wound sites to synthesize glycine betaine, a bacterial osmoprotectant. These published results provide important information to the fresh cut industry. They integrate hurdle technologies into mitigation steps based on the physiology of enteric pathogens at various stages of fresh-cut lettuce production.

6. Development of assays to measure the infectivity of Human Norovirus. Human Norovirus (HuNoV) is extremely difficult to grow in culture, which makes it difficult to measure how much active, infectious virus might be present when searching food or environmental samples for HuNoV infection. ARS researchers in Albany, California, developed a new simple and sensitive assay called in situ Capture RT-qPCR based on the capture of active, infectious virus particles, to estimate the number of infective HuNoV particles present in environmental and oyster samples. An additional novel assay was developed using DNA aptamers to capture active virus particles. These results provide important information to the industry for the development of assays to detect infectious HuNoV from contaminated foods before they are sent to market.

7. Shiga toxin-producing Escherichia coli (STEC) makes several adhesins involved in bacterial attachment and host colonization. ARS researchers in Albany, California, and Ames, Iowa, in collaboration with scientists from Pennsylvania State University, demonstrated that the autotransporter protein Cah confers on E. coli cells the ability to autoaggregate, which is inversely correlated with its ability to form biofilms and plays a strain-specifc role in plant and animal colonization by STEC. Although the cah gene is widespread in the STEC population, a loss-of-function mutation in cah is common. Therefore, cah mutants might be selected in ecological niches relevant to the produce production environment in which autoaggregation could be detrimental to bacterial populations, such as during the initiation of a new biofilm or disassembly of a mature biofilm on plants or plant processing equipment or the need to seek nutrient sources, e.g. plant surfaces or produce processing surfaces. This study provides evidence of environmental pressures in selection of STEC variants with improved fitness at diverse ecological niches that STEC colonize, providing risk assessment modelers technologies for reducing pathogen occurrence in produce.

8. Human Norovirus can bind to proteins on the surfaces of bacteria and lettuce. Human Norovirus (HuNoV) is the most common cause of foodborne illness due to produce contamination, but the natural receptors for HuNoV in humans are the Human Blood Group Antigens (HBGA). ARS researchers in Albany, California, in collaboration with scientists at Shanghai Jiao Tong University and Shanghai Institute of Technology, showed that bacteria engineered to carry the HuNoV binding protein (the P protein) could bind to lettuce and lettuce extract. Further work showed that HuNoV could bind to factors other than HBGA, which would allow for natural contamination of produce surfaces directly (via carbohydrates and proteins present on lettuce cells), or by binding to bacteria naturally present on lettuce surfaces. Bound HuNoV could be removed from lettuce surfaces by low or high pH washes, high ionic strength rinses, or with mild detergents. Additionally, virus attached to bacteria was still capable of being deactivated by heat. These results provide important information to the industry for improved mitigation technologies based on the physiology of HuNoV and could be used as intervention strategies to remove virus from contaminated produce.

9. Salmonella enterica interaction with plants does not proceed via type 3 secretion as in plant pathogens. Salmonella is an important causal agent of illness associated with produce. Bacterial plant pathogens commonly interact with plant cells by injection of effectors (proteins and other molecules) via a specific type of system called the Type 3 Secretion System (T3SS), and this helps them escape the plant immune response to survive in the plant tissue. There is significant effort into the study of the Salmonella T3SS to see if it affects survival of Salmonella in plants. ARS researchers in Albany, California, in collaboration with researchers at The Volcani Center and the University of Tel Aviv, Israel, demonstrated that, unlike bacterial plant pathogens, Salmonella does not have the ability to translocate effectors into plant cells via its type 3 secretion apparatus. These published results provide critical guidance to current efforts by governmental and academic research programs in developing intervention strategies for the prevention of Salmonella contamination of produce using plant genetics based on this type of interaction.

Review Publications
Carter, M.Q., Brandl, M., Kudva, I.T., Katani, R., Moreau, M.R., Kapur, V. 2017. Conditional function of autoaggregative protein cah and common cah mutations in Shiga toxin-producing Escherichia coli. Applied and Environmental Microbiology. 84(1):e01739-17. https://doi.org/10.1128/AEM.01739-17.
Cox, C.E., Brandl, M., De Moraes, M.H., Gunasekera, S.P., Teplitski, M. 2018. Production of the plant hormone auxin by Salmonella and its role in the interactions with plants and animals. Frontiers in Microbiology. 8:2668. https://doi.org/10.3389/fmicb.2017.02668.
Tadepalli, S., Bridges, D.F., Driver, R., Wu, V.C. 2018. Effectiveness of different antimicrobial washes combined with freezing against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes inoculated on blueberries. Food Microbiology. 74:34-39. https://doi.org/10.1016/j.fm.2018.02.018.
Carter, M.Q., Feng, D., Chapman, M.H., Gabler, F. 2017. Survival of foodborne pathogens on commercially packed table grapes under simulated refrigerated transit conditions. Food Microbiology. 72:199-205. https://doi.org/10.1016/j.fm.2017.12.004.
Liao, Y., Quintela, I., Nguyen, K.M., Salvador, A., Cooley, M.B., Wu, V.C. 2018. Investigation of prevalence of free Shiga toxin-producing Escherichia coli (STEC)-specific bacteriophages and the correlation with STEC bacterial hosts in produce-growing area in Salinas, California. PLoS One. 13(1):e0190534. https://doi.org/10.1371/journal.pone.0190534.
Bridges, D.F., Rane, B., Wu, V.C. 2018. The effectiveness of closed-circulation gaseous chlorine dioxide or ozone treatment against bacterial pathogens on produce food control. Food Control. 91(2018):261-267. https://doi.org/10.1016/j.foodcont.2018.04.004.
George, A., Cox, C., Desai, P., Porwolik, S., Chu, W., De Moraes, M.H., McClelland, M., Brandl, M., Teplitski, M. 2017. Interactions of Salmonella enterica Serovar Typhimurium and Pectobacterium carotovorum within a tomato soft rot. Applied and Environmental Microbiology. 84:e01913-17. https://doi.org/10.1128/AEM.01913-17.
Chalupowicz, L., Nissan, G., Brandl, M., McClelland, M., Sessa, G. Popov G., Barash, I., Manulis-Sasson, S. 2018. Assessing the ability of Salmonella enterica to translocate Type III effectors into plant cells. Molecular Plant-Microbe Interactions. 31(2):233-239. https://doi.org/10.1094/MPMI-07-17-0166-R.
Cooley, M.B., Carychao, D.K., Gorski, L.A. 2018. Optimized co-extraction and quantification of DNA from enteric pathogens in surface water samples near produce fields in California. Frontiers in Microbiology. 9:448. https://doi.org/10.3389/fmicb.2018.00448.
Scott, R.A., Thilmony, R.L., Harden, L.A., Zhou, Y., Brandl, M. 2017. Escherichia coli O157:H7 converts plant-derived choline to glycine betaine for osmoprotection during pre- and post-harvest colonization of injured lettuce leaves. Frontiers in Microbiology. 8:2436. https://doi.org/10.3389/fmicb.2017.02436.
Wang, M., Rong, S., Tian, P., Zhou, Y., Guan, S., Li, Q., Wang, D. 2017. Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to HBGA-like molecules in romaine lettuce. Frontiers in Microbiology. 8:251. https://doi.org/10.3389/fmicb.2017.00251.
Tian, P., Yang, D., Lei, S., Wang, D., Li, Q., Gorski, L.A., Lee, B.G., Quinones, B., Cooley, M.B. 2017. Concurrent detection of human norovirus and bacterial pathogens in water samples from an agricultural region in Central California Coast. Frontiers in Microbiology. 8:1560. https://doi.org/10.3389/fmicb.2017.01560.
Zhou, Z., Tian, Z., Li, Q., Tian, P., Wu, Q., Wang, D., Shi, X. 2017. In situ capture RT-qPCR: a new simple and sensitive method to detect human norovirus in oysters. Frontiers in Microbiology. 8:554. https://doi.org/10.3389/fmicb.2017.00554.
Liao, Y., Liu, F., Sun, X., Li, R.W., Wu, V.C. 2018. Complete genome sequence of Escherichia coli Phage vB_EcoS Sa179lw isolated from surface water in a produce-growing area in northern California. Genome Announcements. 6(27):e00337-18. https://doi.org/10.1128/genomeA.00337-18.
Gorski, L.A., Huynh, S., Cooper, K.K., Parker, C. 2017. Complete genomic sequences of two salmonella enterica subsp. enterica serogroup C2 (O:6,8) strains from central California. Genome Announcements. 5:e01234-17. https://doi.org/10.1128/genomeA.01234-17.