Project : USDA ARS

ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Research Project #430338

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

Location: Produce Safety and Microbiology Research

2021 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
This is the final report for project 2030-42000-050-00D, which was replaced by project 2030-42000-052-00D. Significant progress was made on all objectives regarding the fitness, ecology, and detection of foodborne pathogens as they relate to produce contamination. Several Shiga-toxin producing Escherichia coli (Ec, STEC) and Salmonella subtypes have caused outbreaks and/or recalls on leafy greens. Comparisons of EcO111 strains revealed varying levels of expression of RpoS, a mediator of the bacterial stress response. Subsequent construction of mutants lacking rpoS revealed the mutants assimilated a wider array of nutrients than parent strains, but not preferred substrates common in lettuce tissue. Studies of EcO111 and EcO157 on lettuce in Modified Atmosphere Packaging (MAP) showed that rpoS-deficient mutants have lower competitive fitness, suggesting that RpoS promotes leaf colonization. RpoS controlled the autoaggregation of EcO111 via high expression of curli fimbriae, a phenotype displayed by environmental and outbreak isolates, but not those causing sporadic illness. Curli fimbriae confers EcO157 with a competitive trait during biofilm formation with spinach leaf-associated microflora, likely via mediating the interaction between EcO157 cells and spinach bacteria that are more proficient in surface attachment. In EcO157, the Cah protein caused autoaggregation, and deletion of cah enhanced EcO157 attachment to spinach leaves. Comparison of cah sequences in multiple E. coli serovars indicates this gene may be a target of adaptive mutation in response to environmental niches. Autoaggregation may benefit E. coli when seeking protection in dense aggregates but hamper free cells when relocating to an attached state (Sub-objectives 1A and 1H). Transcriptomics showed that EcO157 is exposed to osmotic stress in the cut wounds of ready-to-eat lettuce. High-performance liquid chromatography mass spectrometry (HPLC-MS) and biosynthetic mutants revealed that EcO157 uses choline derived from cut plant tissue to synthesize the osmoprotectant glycine betaine (Sub-objective 1H). Study of EcO157-lettuce interactions in ready-to-eat MAP products revealed that fall-harvested lettuce and cultivars with a poor shelf life grown in California support greater survival of EcO157 and a microbiome enriched in Erwiniaceae during cold storage. Under temperature abuse, EcO157 multiplication in MAP lettuce was independent of shelf life but highly correlated with CO2 levels in the packaging (Sub-objective 1G). This new knowledge of EcO157 behavior on fresh-cut lettuce is applicable to hurdle technologies and lettuce breeding to improve food safety. In basil and lettuce plants, Salmonella strains of serovar Senftenberg outcompeted those of Typhimurium during multiplication in the leaf apoplast and phyllosphere, likely due to differences in nitrogen utilization between the serovars. Factors involved in the formation of antibiotic and stress-resistant persister cells were examined in STEC strains. High STEC persister levels were detected in environments relevant to lettuce production, such as field water and on the leaves under dry conditions (Sub-objective 1C). A three-year study of organic and conventional soils indicated significant suppression of six STEC serotypes on lettuce grown in organic vs conventional soils. Metagenomic analysis showed that species more abundant in organic soils are closely related to the pathogens, hence, promoting competition with the pathogens (Sub-objective 1D). Growth and survival of Listeria monocytogenes populations attached to lettuce and stainless-steel surfaces were studied in multiple serotypes and sequence types that originated from clinical, food, and environmental sources. Strains showed similar attachment kinetics, but variable growth of those attached cells on romaine lettuce pieces. Comparisons indicated that some strains that grew well on cantaloupe were poor performers on lettuce, indicating different factors needed for the two produce types (Sub-objective 1F). Factors that allow Human Norovirus (HuNoV) to bind to romaine lettuce and to lettuce epiphytic bacteria were identified. The lettuce binding target was a chimera molecule with aspects of Human Blood Group Antigen (HBGA) type A and Lewis A antigen. HBGA-like molecules were identified on the surfaces of some bacteria that were isolated from lettuce surfaces (Sub-objective 1I). Among projects to study foodborne pathogens in the environment, a digital droplet polymerase chain reaction (ddPCR) method was developed to quantify E. coli, Salmonella, and L. monocytogenes in water samples collected from the Central California Coast, and this new method correlated well with traditional microbial analysis (Sub-objective 2A). In a collaborative study with Georgia Institute of Technology, metagenomic analysis of sediment samples from the same area revealed significantly different metagenomes from six different sediment samples. Metagenomes were moderately correlated with rainfall and contained large numbers of antibiotic production and resistance genes (Sub-objective 2C). EcO145 genomics was studied with 20 environmental isolates. Key virulence genes were conserved in O145:H28 strains, but not in O145:H11 or O145:H34 strains. Transcriptomic studies revealed that the function of a novel type II DNA modification methylase (PstI-RM system) encoded by a Stx2- converting prophage in E. coli was to regulate transporters and adhesins. However, it also contributes to maximize Shiga toxin production (Sub-objective 2D). Analysis of a collection of >2000 Salmonella and 1,326 L. monocytogenes isolates from public access waters of the Central California Coast was completed. Whole genome analysis of 1231 L. monocytogenes strains revealed 75 different sequence types, and the most common were CC639, CC183, and CC1 making up 27%, 19%, and 13%, respectively. All isolates contained core virulence genes, and pathogenicity islands LIPI-3 and LIPI-4 identified in 73% and 63%, respectively, of the sequenced isolates. The most common Salmonella serovars included Muenchen, Give, and Typhimurium. Salmonella prevalence was not affected by season; however, L. monoctogenes was isolated more often in the winter and spring months and positively correlated with large rain events (Sub-objective 2E). In studies to assess the prevalence of HuNoV and Porcine NoV in the wild pig population of California, 168 feral pig stool samples were collected over 3 years. Using real-time quantitative reverse transcription PCR (qRT-PCR), clear signals for HuNoV genogroups I and II were detected in 2.4% and 7.1%, respectively, of the samples, indicating a potential reservoir of HuNoV (Sub-objective 2H). In projects to aid in subtyping and detection of foodborne pathogens, Fourier-Transform Infrared Spectroscopy was able to speciate Listeria and to differentiate between serotypes 1/2a, 1/2b, and 4b of L. monocytogenes (Sub- objective 3A). Whole Genome Multi Locus Sequence Typing (wgMLST) was used to subtype 120 STEC strains isolated from the Central California Coastal region, and pathogen transport in the area was confirmed. Deeper analysis indicated that transport of pathogens in the region occurs primarily through domestic and wild animals (Sub-objective 3B). In collaboration with Georgia Institute of Technology, imGLAD, a metagenomic based pathogen detection pipeline was developed. The STEC limit of detection of STEC with imGLAD was 100 cells/100-g of field-grown baby spinach leaves. Due to sequencing depth issues, imGLAD was not able to detect STEC via shiga toxin (stx) genes in culture-positive samples of sediments. In vitro transduction of stx genes due to exposure to common DNA-damage reagents was demonstrated in two E. coli produce outbreak strains. Genetic loci contributing to high level shiga toxin 2 (Stx2) were identified (Sub-objective 3C). Projects to develop assays for capture and amplification of infectious HuNoV were investigated. After investigating several methods, three were shown successful in detecting three different subtypes of HuNoV. These assays rely on HBGA-based target capture and RT-qPCR, an aptamer-based assay, and an immunochromatographic assay (Sub-objectives 3D and 3E). Over 50 lytic bacteriophages that are specific to the EcO157 and six other STEC serotypes were isolated and described (Objective 4). The genomes of >20 were sequenced to determine that they did not carry stx genes and could be used to develop intervention technologies. Phages were characterized to determine host range, stress stability, plating efficiency, and growth characteristics. The phages belonged to several different families including Myoviridae, Podoviridae, Siphoviridae, and Autographiviridae. One T4-like phage, Sa45lw, was able to infect both STEC O45 and STEC O157. A bacteriophage-based, electrochemical biosensor STEC detection system was developed to detect viable STEC cells (Objective 5). This low-cost, portable, wireless system was designed to be used on-site for regulators, inspectors, producers, or processing plant operators to evaluate product safety without transporting samples to testing laboratories. A patent was filed and published. Bacteriophage-based liquid reagent was converted into pre-made dry format by freeze-drying or air-drying. Both systems resulted in a comparable level of reagent bioactivity. Storage temperatures and sugar preservatives were analyzed, and refrigeration temperatures preserve the reagent for >2 months, while both trehalose and pullulan preserve the reagent similarly. Another method developed is a prototype of a closed-enrichment system that can simultaneously enrich and colorimetrically detect low levels of foodborne pathogen contaminants in food samples. The leak-proof container allows the addition of colorimetric reagents to detect foodborne pathogens and a biocide to minimize contamination and infection risks for end-users in the field.

Accomplishments
1. Shelf life and season are main drivers of E. coli O157:H7 survival on cut lettuce. Escherichia coli (E. coli) O157:H7 infections from contaminated lettuce continue to impact public health and the U.S. lettuce industry, which is valued annually at nearly $2 billion. Outbreaks linked to products grown in California occur predominantly from fall-harvested lettuce, and the reason for this seasonality is unknown. Using fresh-cut lettuce stored in modified atmosphere packaging in the cold, ARS researchers at Albany, California, in collaboration with scientists at the Food and Drug Administration Center for Food Safety and Nutrition, identified the fall season and lettuce cultivars with poor shelf life as the main drivers of E. coli O157:H7 survival and microbiome structure. These results open new fields for inquiry into the seasonal aspects of the physiology of fresh-cut lettuce and its microbiome that may prevent the seasonal occurrence of E. coli O157:H7 infections. Likewise, the identification of shelf life as an important lettuce trait in E. coli O157:H7 survival implies that genetic breeding for improved lettuce shelf life must be part of a successful strategy to enhance produce safety.

2. Detecting and distinguishing among intact Shiga toxins in complex samples. Shiga toxin-producing Escherichia coli (STEC) produce a variety of Shiga toxins that can cause serious food poisoning. Preventing food poisoning requires the rapid identification of the type and subtype of Shiga toxin produced by the STEC. ARS scientists in Albany, California, developed a mass spectrometry-based means of detecting both the binding and toxic portions of the known Shiga toxins. The approach permits researchers to rapidly identify the type and subtype of intact Shiga toxins in serum and bacterial growth media without isolating the STEC cells. This is a rapid method that can replace cell- and animal-based assays.

3. Commercialization of novel rapid detection technology for gluten in foods. American consumers and food producers need effective gluten testing methods to meet demand for product labeling and gluten-free food choices. ARS researchers in Albany, California, developed and optimized novel technologies suitable for both producers and consumers to achieve rapid and cost-effective detection of gluten from food products. These technologies have been patented and commercially licensed to provide robust gluten detection solutions throughout the food supply chain. These rapid technologies will be useful for consumers who eat a gluten-free diet, especially those suffering from celiac disease and other ulcerative stomach illness.

4. Bacteriophages as alternative methods to control STEC on contaminated mung bean seeds. Sprouting seeds contaminated with foodborne pathogens, such as Shiga toxin-producing Escherichia coli (STEC), are a concern for sprout growers in the United States. Methods to prevent foodborne pathogen contamination rely heavily on chemicals and antibiotics, which can result in antimicrobial resistance. ARS researchers in Albany, California, developed an effective biocontrol method for STEC contamination of mung bean sprouts using bacteriophages that specifically kill STEC. These bacteriophages have resistance to adverse temperatures and pH, and they lack any STEC toxin genes, making them usable for intervention techniques. The bacteriophages killed STEC of serotypes O45 and O157, two different types of dangerous STEC bacteria, on mung bean seeds within 6 hours of treatment. The bacteriophages provide a cost-effective solution to the produce industry to control STEC contamination that minimizes the potential for antimicrobial resistance.

5. A molecular marker for hypervirulent Shiga toxin producing Escherichia coli strains. Shiga toxin-producing Escherichia coli (STEC) is one of the major bacterial pathogens linked to fresh-produce-associated outbreaks and causes life-threatening illnesses in humans. Production of Shiga toxin (Stx), especially the subtype 2a (Stx2a), is associated with STEC virulence. ARS researchers at Albany, California, revealed that induction of Stx2a is dependent on several factors that are related to bacteriophages integrated into the genome (also called “prophages”) of STEC cells. These factors are the type of prophage carrying the stx gene, a specific DNA sequence in front of the stx gene, and the presence of the Q993 gene, which encodes a specific protein called “antiterminator Protein Q993.” Types of STEC that express the antiterminator Protein type known as Q933 produce the highest levels of Stx2a; therefore, the Q933 protein could serve as a biomarker for hypervirulent STEC strains that may be contaminating foods.

6. Regulation of stress resistance and virulence in E. coli. Consumption of contaminated produce accounts for an estimated 46% of the over 9 million cases of foodborne illness in the United States each year. Shiga toxin-producing Escherichia coli (STEC), one of the major bacterial pathogens linked to fresh-produce-associated outbreaks, can cause life-threatening diseases in humans. However, factors impacting survival and persistence of STEC in fresh-produce production environments are not fully understood. ARS researchers in Albany, California, uncovered that a protein called DNA adenine methylase (Dam) plays a central role in regulating the expression of genes involved in virulence, stress resistance, and energy metabolism in STEC. Dam also represses the expression of Shiga toxin, a toxin which causes diarrhea in humans. Deletion of the Dam gene (or deficiency of the Dam protein) in STEC resulted in the killing of STEC cells, due to activation of dormant phages in the STEC genomes. The critical role of Dam in STEC virulence and stress resistance sheds new insight into the development of effective intervention strategies.

7. Evaluation of community DNA sequencing to detect foodborne pathogens. Current agricultural practices contribute to contamination in the environment and the spread of food- and water-borne disease and antibiotic resistance. Traditionally, the level of pollution and risk to public health from fecal pollution are assessed by culture-based tests; however, the accuracy of these traditional methods, and particularly their suitability for sediments are inadequate. The use of DNA sequencing as a method for environmental detection of Shiga toxin-producing Escherichia coli (STEC), is not fully developed. ARS researchers in Albany, California, collected sediments from one of the most highly productive agricultural regions in the United States to assess how agricultural runoff affects native, sediment microbial communities, and if STEC could be detected directly in sediments by DNA sequencing. Metagenomic analysis of these samples discovered a large number of antibiotic resistance genes, suggesting substantial contamination from domestic animal feces, but did not discover sequences from STEC, indicating the need to substantially improve sequencing depth for use of this tool as a direct assessment of public health risk of agricultural sediments.

U.S. DEPARTMENT OF AGRICULTURE

Produce Safety and Microbiology Research: Albany, CA