2010 Annual Report 1a.Objectives (from AD-416) To characterize the ecology of enteric pathogens on produce, the ecology and epidemiology of E. coli O157:H7 in the produce production environment and develop improved methods for the detection of bacterial and viral enteric pathogens on produce. FY06 objective: Develop and validate receptor based methods for capturing ricin and related plant toxins from food(s). Problem to be Addressed:. 1)Identify mechanisms critical to the attachment, growth, and/or survival of human pathogens, as well as to their interaction with the natural microflora on fresh produce - particularly in relation to biofilm formation. . 2)Develop rapid methods for the concentration, detection, quantification and characterization of enteric pathogens found in and on produce. FY07 Objectives of Research: Identify bacterial genes that are involved in the attachment, colonization and survival of enteric pathogens on produce. Identify plant genes that mediate the attachment, colonization and survival of enteric pathogens on plants. Determine the biotic and molecular factors that drive the interaction of enteric pathogens with the bacteria on produce, and that are conducive to mixed biofilm formation. Develop methods and biosensors for the concentration and detection of enteric pathogens from produce and soil, development of improved methods for isolation and detection of enterohemorrhagic E. coli and use of these methods for studying the sources of E. coli O157:H7 contamination of the produce production environment, development of methods for identifying Noroviruses in water and produce production environment. 1b.Approach (from AD-416) Plant-microbe model systems in combination with population studies, molecular methods, genomics, proteomics, and microscopy, will be used to investigate the interaction of human pathogens with plants and with plant-associated bacteria, as well as to develop improved methods for the detection of human pathogens on produce. Formerly 5325-42000-040-00D (11/05). FY06 Program Increase. 3.Progress Report Significant progress was made in identifying plant factors that affect the colonization of lettuce by E. coli O157:H7 (EcO157). Specific plant compounds were identified that are involved in the differential growth of EcO157 in the leaf lesions of various lettuce cultivars. Additionally, we determined that recombinant Norwalk virus viral-like particles (rNVLP) applied to the surface of Romaine lettuce and cilantro leaves localized as large clusters primarily on the leaf veins, whereas its distribution was widespread on the leaf surface of iceberg lettuce. Immunoassays showed that extracts from Romaine lettuce leaves bound rNVLP particles in a dose-dependent manner and that non-HBGA molecules in the extract bind human norovirus in human tissues and similar molecules in shellfish that bind noroviruses, bind rNVLP by a binding site(s) different from the defined binding pocket on the virion. Extracts from cilantro, iceberg lettuce, spinach and celery also bound rNVLP. A manuscript about Norovirus attachment to produce has been submitted to the Journal of Applied and Environmental Microbiology. [Sections 1.2.3 (Production and Processing Ecology) and 1.2.7 (Risk Assessment).] Several outbreaks of salmonellosis have been linked to raw almonds and other nuts and have cost millions of dollars to the nut production industry. Our investigation of Salmonella with other microbes present in nut orchards revealed that the pathogen forms complex biofilms with Aspergillus niger. This association requires cellulose production by Salmonella and leads to increased internalization into humancell lines. The production of such mixed biofilms may facilitate the high persistence of Salmonella in almond orchards and provides insight into its ecology in nonhostenvironments. The results of this study are described in a manuscript submitted to PNAS. [Section 1.2.3 (Production and Processing Ecology).] We have determined that variants of curli, adhesive fimbriae that are involved in surface attachment, cell aggregation, biofilm formation, and interaction with host cells, are widely distributed in EcO157 strains. Most importantly, curli variants from a colonal population display distinct survival fitness. Curli-producing (C+) variants grow much better under nutrient-limiting conditions than curli-deficient (C-) variants whereas C- variants are much more resistant to acidic pH than C+ variants. DNA sequence analysis of the EcO157 gene encoding RpoS, a master regulator of curli production, revealed several mutations in the C+ variants. Our data suggest that maintaining curli variants in an EcO157 population provides a survival strategy in which C+ variants may be selected in a nutrient-limiting environment whereas C- variants may have a selective advantage during the initial interaction of the pathogen with a human host. These results provide insight into the evolution of the pathogen and, the epidemiology EcO157 infection. A manuscript describing our results has been submitted to the Journal of Applied and Environmental Microbiology. [Section 1.2.7 (Risk Assessment).] 4.Accomplishments 1. New insight into the physiology of EcO157 in lettuce wounds helps explain its association with processed lettuce. Fresh-cut lettuce is the food vehicle associated with most outbreaks of EcO157 infection. Microarray transcriptional profiling of EcO157 in lettuce lysates and real time-qPCR analysis in shredded lettuce was used to identify determinants involved in survival of the pathogen in the cut lesions of lettuce. ARS researchers in Albany, CA, found that EcO157 adapted rapidly to an array of chemical stresses in cut lettuce, including oxidative stress, and had increased resistance to chlorine sanitizers in that environment. This enhanced tolerance to chemical stress in leaf lesions, combined with its rapid growth as we reported previously, provides risk assessment data to explain the strong epidemiological link of processed leafy greens with EcO157 infections. Data also provide new information for EcO157 stress-responsive pathways while in lettuce lesions that can be targeted in technologies for the reduction of microbial contamination of processed produce. 2. Salmonella enrichment culture conditions optimized. There is an increasing concern about the association of Salmonella with produce and recalls and withholding of leafy greens due to contamination with Salmonella are on the rise. More sensitive methods for detection and isolation of the pathogen from produce are needed. ARS researchers in Albany, CA, have shown that molecular detection of Salmonella from produce was up to 1000-fold more sensitive when enrichment culture was done in Tryptic Soy Broth (TSB) than in two other media commonly used for isolation of Salmonella from food, and that common immunomagnetic techniques do not enhance detection from TSB. We also characterized the plant microflora that multiplies when produce is analyzed for contamination into these standard enrichment media. This information is of use to public health agencies, private laboratories that provide quality testing to the produce industry, and the industry that develops molecular detection kits and equipment. 3. Specificity and sensitivity of histoblood group antigen (HBGA)-conjugated magnetic beads to concentrate and detect multiple strains of norovirus (NoV). NoV is considered to be the most common etiologic agent of food-borne disease linked to produce, but sensitive methods for its detection still need to be developed. To develop an improved method for detection of NoV in produce, the effect of pH and ionic strength on the binding kinetics of NoV to HBGA, a candidate receptor for NoV was characterized. ARS Researchers in Albany, CA, demonstrated that NoV was captured quickly and effectively under low pH and that heat release of NoV RNA resulted in a better recovery rate from the beads compared to extraction with a conventional reagent. Pig mucin, which contains HBGA, can be conjugated to magnetic beads and thus concentrate all eight GI strains from 28- to 1502-fold and 11 of 13 GII strains from 30- to 1014-fold. This information is of use to public health agencies and private laboratories that provide quality testing to the produce industry, and to the biotech industry that develops equipment and molecular detection kits for NoV. 4. Stress alters fingerprint profiles of Eco157. The evolution of EcO157 in the environment is still poorly understood. ARS researchers in Albany, CA, have determined that short-term exposure in the laboratory to high temperature; UV radiation and starvation alter the profile of hypervariable tandem repeats (TRs) in the EcO157 that are exploited as an important method of differentiating/fingerprinting different strains. These mutations indicate that O157 strains previously subjected to harsh environments are more closely related due to the increased rate of mutation. Although studies that correlate the similarity of strains by the number of different TRs in multiple TR locations in the genome remain valuable during outbreak investigations, environmental stress-related changes should be considered in this type of analysis. Strain fingerprinting and similarity analysis is necessary to assess the movement of EcO157 in the animal and crop production environments. 5. Shiga toxin-producing E. coli prevalence in produce growing areas in the Salinas Valley. The initial source of the contamination in outbreaks of EcO157 linked to leafy vegetables produced in the Salinas Valley in California remains unknown. Therefore, there is a great need to establish a baseline for the environmental prevalence of EcO157 and non-O157 E. coli in this important agricultural region. In collaboration with the University of California at Davis and the California USDA APHIS Wildlife Services, ARS researchers at Albany, CA, have isolated over 3000 strains of E. coli O157 and non-O157:H7 shigatoxin-positive E. coli from 12,000 samples from water, animals and their feces, crops and soil, and have determined their MLVA and ompA type, and virulence gene profile. We also have submitted several O157 PFGE profiles to CDC PulseNet. O157 and non-O157 E. coli prevalence varies considerably among sources, with the highest prevalence associated with cattle feces (7% and 33%, respectively), but that other animal species also are a significant source of the pathogens. This information provides the industry and public health agencies with the first epidemiological data for E. coli in this important agricultural region of the US and will be used to develop good agricultural practice for produce production. 6. Salmonella is more able to survive in humans after passage through protozoan. The number of Salmonella cells contaminating produce necessary to make humans sick remains unclear. Salmonella remains undigested in the food vacuoles of the common protist, Tetrahymena, and is released as viable cells in its fecal pellets. Microarray gene expression profiling of S. Typhimurium cells in Tetrahymena revealed that many genes required for survival and replication within human cells also had increased expression in Tetrahymena. Furthermore, ARS researchers in Albany, CA, showed that the pathogen is more acid resistant in Tetrahymena fecal pellets than as free cells. Our results indicate that passage through this protozoan confers on Salmonella physiological advantages relevant to its contamination cycle and may lower the number of cells that can make a human sick. Review Publications Barak, J., Gorski, L.A., Liang, A.S., Narm, K. 2009. Previously uncharacterized Salmonella enterica genes required for swarming play a role in seedling colonization. Microbiology. 155:3701-3709 Kyle, J.L., Parker, C., Goudeau, D.M., Brandl, M. 2010. Transcriptome Analysis of Escherichia coli O157:H7 Exposed to Lysates of Lettuce Leaves. Applied and Environmental Microbiology. 76(5):1375-1387 Gorski, L.A., Liang, A.S. 2010. Effect of Enrichment Medium on Real-time Detection of Salmonella enterica from Lettuce and Tomato Enrichment Cultures. Journal of Food Protection. 73(6):1047-1056 Cooley, M.B., Carychao, D.K., Nguyen, K.M., Whitehand, L.C., Mandrell, R.E. 2010. Effects of Environmental Stress on Stability of Tandem Repeat in Escherichia coli O157:H7. Applied and Environmental Microbiology. 76(10)3398-3400 Pan Y, Breidt F, Gorski LA. 2010. Synergistic effects of sodium chloride, Glucose, and temperature on biofilm formation by Listeria monocytogenes serotype 1/2a and 4b strains. Appl Environ Microbiol. 76(5):1433-1441.