Location: Produce Safety and Microbiology Research
2024 Annual Report
Objectives
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.
Approach
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
This report documents progress for project 2030-42000-052-000D, titled, “Understanding Ecological, Hydrological, and Erosion Processes in the Semiarid Southwest to Improve Watershed Management”, which started in December 2020.
As part of the work under Objective 1, lettuce cultivars that were part of the screen for Escherichia coli O157:H7 (EcO157) inhibition in cut cold-stored leaves in Sub-objective 1A, were assessed for multiplication of the pathogen in the latex secreted from cut lettuce stems during harvest. Phenolic production and polyphenol oxidase enzyme (PPO) and peroxidase (POD) activity, all of which are associated with the plant wound response, were also measured in the latex of each cultivar. Five cultivars of interest for the behavior of EcO157 with latex on the cut stems were identified. One cultivar repressed EcO157 growth, two were highly inhibitory more than others, and two promoted high multiplication of the pathogen. These five cultivars were planted in the field in Salinas in the summer of 2024 in order to confirm the results obtained in the laboratory. Partial progress was made in Sub-objective 1B, and cultivars characterized with high and low PPO activity when grown under laboratory conditions were planted in the field in the summer of 2024. They will be tested for PPO activity in cut lettuce and EcO157 colonization. No progress was made in Sub-objective 1C due to a critical vacancy in a laboratory technician position.
In support of Sub-objective 1D, three strains each of clinical Salmonella enterica serotype Newport and serotype Javiana linked to fresh produce-associated outbreaks were selected for whole genome sequencing in collaboration with scientists at ARS in Clay Center, Nebraska, and the Center for Diseases Control and Prevention (CDC) in Atlanta, Georgia. The complete genome sequences of the six strains were determined and deposited in GenBank. Those strains will be used to construct Tn-5 mutant libraries. Conditions to examine the attachment proficiency of Shiga toxin-producing E. coli (STEC) on romaine lettuce and of S. enterica Newport and S. enterica Javiana on cantaloups, cucumbers, and tomatoes were optimized.
For work under Sub-objective 1E, screening for competitive fitness of Listeria monocytogenes strains of Sequence Type (ST) 382 in direct competition with serotypes 1/2a and 4b strains on lettuce was completed. Strains were inoculated at a 1:1:1 ratio onto green leaf lettuce and allowed to grow at 4- and 25-degrees C. Screening of colonies and serotype determination was done using multiplex real time polymerase chain reaction (PCR). In testing of 12 strains, ST 382 showed comparable fitness to strains of serotypes 1/2a and 4b on cut lettuce incubated at 25- or 4-degrees C. However, the percentage recovery of ST 382 strains was statistically better from lettuce incubated at 4-degrees C than at 25-degrees C. A manuscript on this work was submitted for publication in the Journal of Food Protection. Work on this sub-objective continues with screening cantaloupe rinds and flesh to determine if the trend is the same on different produce types.
In work supporting Sub-objective 1G, a mining platform to search for ligands that bind human norovirus (HuNoV) was constructed in collaboration with a scientist at Shanghai JiaoTong University. The platform uses a bacterial cell surface display system (BSDS) with E. coli expressing the P protein of HuNoV on the surface of the transformed cell as a cellular vector. The transformed bacteria are then used to fish candidate ligands for HuNoV binding from various sources. The complexes of the P domain and ligand candidates were isolated, released by thrombin and identified by a nanoliter liquid chromatography tandem with a quadrupole orbital trap mass spectrometer. A proteinaceous oyster heat shock protein 70 (oHSP70), was isolated and showed a strong ability to bind HuNoV. Recombinant oHSP70 was constructed and used to concentrate and capture HuNoV. The recombinant oHSP70 showed a higher binding ability to HuNoV than commonly used pig stomach mucin. The BSDS system was used with MK2 cells; however, no new ligands other than well- known Human Blood Group Anitgen ligands in MK2 cells were isolated. A material transfer agreement was initiated with Baylor College of Medicine to obtain HIE cells, and the BSDS system will be used to isolate candidate ligands from HIE cells for HuNoV.
Under work for Objective 2, 43 strains of Salmonella serovar Infantis isolated from the Central California coast surface waters were screened for large plasmids. Eighteen contained plasmids greater than 100 kilobases in size. Plasmid sequence analysis was done with Whole Genome Seqeuencing (WGS) using the Illumina platform and by testing adaptive sampling methods using the Oxford Nanopore system. Additionally, the Infantis strains were screened for antibiotic resistance against the National Antimicrobial Resistance Monitoring System (NARMS) panel of antibiotics. All strains showed some level of antibiotic resistance. The top three antibiotics for antibiotic resistance were sulfisoxazole, azithromycin, and streptomycin. Azithromycin resistance is being studied further. This work supported Sub-objective 2B.
In support of Sub-objective 2C, 30 water samples inoculated with STEC strains under three test conditions were processed for both metagenomic shotgun sequencing and 16S sequencing in collaboration with a scientist at Oklahoma University. Data analyses are in progress. A PCR-based method was developed to quantify the Viable but Not Culturable (VBNC) EcO157 population under the testing conditions. In support of Sub-objective 2D, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential and environmental persistence of Escherichia albertii strains isolated from wild birds in a major agricultural region in California in collaboration with scientists Los Alamos National Laboratory, the CDC, and at ARS, Clay Center, Nebraska. Among the 10 avian-sourced strains examined, two were identified that exhibited both a high level of cytotoxicity and strong biofilm production, implying a potential to emerge into a high-risk pathogen. Application of Whole Genome Sequencing (WGS) in outbreak investigations has facilitated the identification of EcO157 strains that reoccur, emerge, or persist, and cause illnesses over periods of months or years and those strains are defined as REP strains. Comparative genomics and phenotypic assays have been applied to reveal genetic loci as well as phenotypic traits contributing to the emergence of REPEXH01 and REPEXH02 strains.
In support of Objective 3, a study of Salmonella serotypes recovered from naturally contaminated chicken rinsates was done in collaboration with scientists at ARS in Clay Center, Nebraska, and The University of Georgia. In the portion of the work conducted in Albany, California, combinations of strains of serovars Infantis, Kentucky, and Enteritidis were compared in pair-wise inoculations into Tetrathionate Broth (TT) and Rappaport Vassiliadis Soya Peptone Broth (RVS) in concentration ratios of 1:1, 1:10 and 10:1. Salmonella Kentucky strains outcompeted the Infantis and Enteritidis strains when the Kentucky strains were inoculated at 10 times the level of the other serotypes, but not when the inoculation ratios were 1:1 or when the Kentucky strains were inoculated at one-tenth the level. One implication of the work is that the high levels of Salmonella Kentucky isolated in chicken surveillance may be due to high levels of the serotype on the chickens, and not better growth of the Kentucky strains in enrichment culture. This work was published in Food Microbiology.
In support of Sub-objective 3B, two cell culture-based assays to measure HuNoV infectivity were established. The Tissue Culture half Infective Dose (TCID50) was established. Work to correlate infectivity and in vitro detection continues. The standard curves for Droplet Digital (dd) and Reverse Transcriptase (RT) quantitative PCR were established. The project was significantly impacted by renovation of the office and lab, as well as facility issues leading to extremely high laboratory room temperatures, leading to malfunction of equipment.
An additional project in collaboration with a chemist at ARS, Albany, California, was undertaken to determine if self-assembling chemical compounds with biocidal activity would affect the growth of foodborne pathogens. Contact time experiments are ongoing with compounds in distilled water before moving to compounds diluted in tap water.
Accomplishments
1. Differential fitness of Salmonella serotypes in common enrichment media. Detection of virulent serotypes of Salmonella during routine food surveillance is needed to ensure food safety. Routine media used to grow and identify Salmonella shows a bias toward different serotypes. ARS researchers in Albany, California, and Clay Center, Nebraska, in collaboration with a scientist at the University of Georgia, demonstrated that Salmonella serotype Enteritidis was more likely to be isolated from Tetrathionate Broth media, while serotype Infantis was more likely to be isolated using Rappaport Vassiliadis Soya Peptone Broth. Additionally, the research showed that the lesser virulent serotype Kentucky was able to outperform the other serotypes in both media when initially present at 10 times higher levels, but not when inoculated at equivalent levels. This observation was critical for USDA-FSIS since it indicated that high recovery of serotype Kentucky from some FSIS samples may be due to high levels of Kentucky on the product and not from better performance of that serotype in standard enrichments.
2. Wild birds carry STEC strains with increased potential to cause severe human diseases. Current knowledge about the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) is largely limited to strains of several predominant groups such as serotype O157:H7. Recently non-O157:H7 serotypes associated with foodborne outbreaks and human infections have increased, thus information about virulence and pathogenesis of diverse STEC strains is needed for the development of a One Health mitigation strategy. ARS researchers in Albany, California, characterized the pathogenicity potential of STEC strains isolated from wild birds in a major agriculture region in California and revealed novel virulence determinants in the diverse non-O157:H7 avian STEC strains. Such information is important for risk assessment in preharvest environments and contributes to the development of effective control strategies to ensure the safety of food from farm to fork.
3. Wild birds carry Escherichia albertii strains exhibiting comparable cytotoxicity with STEC outbreak strains. Escherichia albertii is an emerging foodborne pathogen and the cause sporadic outbreaks worldwide; however, pathogenesis and environmental reservoirs of this foodborne pathogen are not well understood. With rapid development of Next Generation DNA Sequencing technology, genomic characterization is needed for improved detection and to understand the pathogenesis of this organism. ARS researchers in Albany, California, performed comparative pathogenomic analyses of ten E. albertii strains, isolated from wild birds in an agricultural region in central California, with the clinical strains collected by the CDC from 1954-2014 in the U.S. to reveal the virulence genes repertoire of E. albertii bird strains. The study revealed both novel virulence genes in E. albertii bird strains and identified two Shiga toxin-producing E. albertii (STEA) bird strains exhibiting significantly higher cytotoxicity than several STEC outbreak strains. Such information is valuable for elucidating the pathogenesis and disease ecology of E. albertii as well as for risk assessment in the preharvest environment.
Review Publications
Liao, N., Chen, X., Tang, M., Tian, P., Liu, C., Ruan, L., Pan, H., Shu, M., Zhong, C., Wu, G. 2024. Grapefruit essential oil combined with UV-C treatment: A technology to improve the microbial safety of fresh produce. Innovative Food Science and Emerging Technologies. 93. Article 103639. https://doi.org/10.1016/j.ifset.2024.103639.
Carter, M.Q., Quinones, B., He, X., Pham, A.C., Carychao, D.K., Cooley, M., Lo, C., Chain, P.S., Lindsey, R.L., Bono, J.L. 2023. Genomic and phenotypic characterization of Shiga toxin-producing Escherichia albertii strains isolated from wild birds in a major agricultural region in California. Microorganisms. 11(11). Article 2803. https://doi.org/10.3390/microorganisms11112803.
Lyu, C., Luo, G., An, R., Tian, P., Wang, D. 2023. Development of recombinant oyster heat shock protein 70 mediated in situ capture RT-qPCR to detect human norovirus and Tulane virus. Food Control. 158. Article 110245. https://doi.org/10.1016/j.foodcont.2023.110245.
Gorski, L.A., Shariat, N., Richards, A.K., Siceloff, A.T., Aviles Noriega, A., Harhay, D.M. 2023. Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media. Food Microbiology. 119. Article 104431. https://doi.org/10.1016/j.fm.2023.104431.
George, A.S., Simko, I., Brandl, M. 2024. Identification and characterization of lettuce cultivars with high inhibitory activity against the human pathogen Escherichia coli O157:H7: Toward a plant-intrinsic hurdle approach to microbial safety. Postharvest Biology and Technology. 211. Article 112816. https://doi.org/10.1016/j.postharvbio.2024.112816.