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United States Department of Agriculture

Agricultural Research Service

Research Project: Biology and Control of Human Pathogens on Fresh Produce

Location: Produce Safety and Microbiology Research

2012 Annual Report

1a. Objectives (from AD-416):
Objective 1: Identify and characterize the microbial genes that are involved in the attachment, colonization and survival of enteric pathogens on produce. Objective 2: Determine the genetic and biochemical factors in plants that effect the attachment, growth and survival of human pathogens in/on plants. Objective 3: Assess the role of other microflora and aerosols in survival and transmission of enteric pathogens in agricultural environments. Objective 4: Develop methods for the detection of enteric viral and bacterial pathogens from produce and soil.

1b. Approach (from AD-416):
Plant-microbe model systems in combination population studies, molecular methods, genomics, and microscopy, will be used to investigate the interaction of human pathogens with plants and plant-associated bacteria, as well as to develop improved methods for detection of human pathogens on produce.

3. Progress Report:
Analysis is proceeding on results from the 2.5 year survey of water, domestic and wildlife samples, soil and produce from the Salinas Valley region and is enhanced by data from an improved STEC typing method that was applied retroactively to our entire STEC collection. Another survey of the Salinas watershed for a variety of pathogens is now being conducted in collaboration with FDA and NASA. Incidence of Salmonella and Listeria monocytogenes in the Salinas Valley watershed was assessed with over 500 samples. A new non-selective enrichment method for L. monocytogenes was developed for this project and is being compared with common selective enrichment media. Preliminary results indicate a high incidence of the latter pathogens, and serotypes and subtyping of isolates is ongoing. Also, 86 Salmonella isolates from a survey of wild amphibians and reptiles in California were characterized for antibiotic resistance and serotyped. Methods for phenotypic screening of a large number of nonO157-STEC strains isolated from various environmental samples in the Salinas Valley are being developed and variants of the German outbreak STEC O104:H4 were tested phenotypically and on sprouts. To improve our understanding of STEC epidemiology, acid resistance proteins were characterized in various STEC strains and comparative transcriptomics of E. coli O157:H7 curli variants derived from the 1993 hamburger- and 2006 spinach-linked outbreak strains was performed. The genome of six E. coli O145:H28 outbreak strains, including one associated with lettuce in the US was sequenced, with one genome fully closed. The basal defense response of leaves to injury, such as by cutting during leafy greens processing, was investigated for its role in colonization by E. coli O157:H7 using lettuce, and Arabidopsis thaliana parental and mutant lines. A new method for gene expression profiling in E. coli O157:H7 using RNAseq is being developed to acquire data on the physiology of the pathogen in packaged shredded lettuce. We have identified a new surrogate virus for human norovirus to enable us to investigate the biology of the pathogen on produce. A rapid and sensitive method based on receptor-binding capture and magnetic sequestration was developed and applied to the detection of human norovirus in fresh produce and environmental samples. To assess the potential for produce contamination in the field by aerosolized enteric pathogens, we monitored culturable bacteria in aerosols using high volume cyclonic samplers and characterized bacterial communities by 16S rRNA gene sequencing. Seasonal fluctuations in culturable bacteria correlated with atmospheric temperature, humidity and wind velocities. Relative consumption of E. coli O157:H7 strain variants by predatory protozoa native to dairy wastewater and their survival to passage through the protists were measured in order to better understand the extended survival of the pathogen in animal rearing operations.

4. Accomplishments
1. Selective enrichment media bias for Salmonella enterica strains isolated from mixed strain cultures. During food-borne outbreak investigations it is critical to isolate the relevant strain from food and/or environmental sources, but if a sample is contaminated with multiple strains of the pathogen, the relevant strain might be missed. ARS researchers in Albany, California, determined that 1) some Salmonella strains were more likely to be isolated than others when we tested nineteen Salmonella enterica strains in mixed Salmonella pure cultures, and in cattle fecal enrichments using different Salmonella enrichment media, and 2) different enrichment media affected the distribution of the strains recovered. Strains of serogroup B, which included serotypes often linked to outbreaks such as serovars Typhimurium, Saint- Paul, and Schwarzengrund were less likely to emerge as dominant strains. This provides information to public health agencies, as well as to the industry, that during investigations of food and/or other environmental samples, multiple enrichment protocols should be used to ensure isolation of target strains of Salmonella. Our findings were published in PLoS One.

2. Salmonella habitat conditions overlap in produce soft-rot lesions and in the animal intestine. The incidence of Salmonella contamination of retail produce has been positively correlated with the presence of soft-rot, a common post-harvest disease that affects all fruit and vegetables. ARS researchers in Albany, California, have determined through gene expression profiling that Salmonella experiences in soft-rot lesions, conditions that are required also for its colonization and pathogenicity in the animal host intestine. This high adaptation of the human pathogen to degraded plant tissue has implications for safe shelf-life standards set by the produce and food industry, and means that the presence of soft-rot should be included in microbial risk assessment models for produce. Manuscript submitted for publication to Applied Environmental Microbiology.

3. E. coli O157:H7 forms mixed biofilm with spinach-associated microorganisms. The dynamics of mixed biofilm formation that include E. coli O157:H7 leading to its persistence in produce processing environments are unknown. ARS researchers in Albany, Califronia, have observed that E. coli O157:H7 produces thick biofilms on stainless steel surfaces in the presence of spinach lysates. Using physiological and metagenomic approaches, they determined that the early success of E. coli O157:H7 in these mixed biofilms was associated with its efficient utilization of spinach nutrients, whereas its population decline at later stages of the biofilm was due to its poor competition for macronutrients. This study provides risk assessment information for investigation of factors that may compound the occurrence of outbreaks linked to produce. Our results were published in the Plos One journal.

4. Prevalence of enteric pathogens in produce growing areas in the Salinas Valley, California. Several outbreaks of E. coli O157:H7 infection have been linked to leafy vegetables and numerous recalls of produce have been caused by contamination with Listeria monocytogenes and Salmonella enterica. ARS researchers in Albany, California, in collaboration with FDA-CFSAN and NASA-GSFC have conducted a survey of the Salinas watershed for the presence of E. coli O157 and non-O157:H7 shigatoxin-positive E. coli, Salmonella, Listeria, and Campylobacter. This survey has provided data for a predictive geospatial risk assessment model (PGRAM). Data collected to date from more than 500 samples indicate substantial differences in the prevalence of the different pathogens with a definite correlation to sampling region and date. This information provides the industry and public health agencies with valuable epidemiological data for risk assessment of these pathogens from this important agricultural region of the US.

5. Characterization of bacteria transported through aerosols. The role of enteric pathogen aerosolization in the contamination of produce remains unclear. ARS scientists in Albany, California, evaluated a flow cytometry-linked culture method to monitor and characterize live bacteria from airborne particles by collecting them in liquid rather than with filters, which decreases bacterial viability. The successful detection of culturable pathogens from airborne particles with our method indicates its potential for monitoring the environmental transport of pathogens and complements current molecular-based methods to detect live enteric pathogens in aerosols transported to produce grown in proximity of dairies and feedlots. Our method has applicability in surveillance by public health, and for research agencies to improve our understanding of the epidemiology of enteric pathogens in agricultural areas where animal and crop production are geographically intertwined.

6. Optimization of method for detection of human norovirus from produce and from sewage. Norovirus is considered the most common etiologic agent of outbreaks linked to produce, and water is one of the most important routes of transmission. However, effective methods for its concentration from produce in order to detect it and determine it's infectivity are lacking. ARS researchers in Albany, California, have developed a rapid and sensitive method to detect human norovirus in fresh produce and sewage using a receptor-binding capture and magnetic sequestration (RBCMS) method. Compared with other commonly used approaches, the RBCMS method is more sensitive, has better concentration power, and enriches for the infectious encapsulated virus. This information, which was published in the Food Microbiology and Applied Environmental Microbiology Journals, is of use to public health agencies, private laboratories that perform testing for the industry, and the biotech industry that develops equipment and molecular biology kits for detection of norovirus.

Review Publications
Carter, M.Q., Louie, J.W., Fagerquist, C.K., Sultan, O., Miller, W.G., Mandrell, R.E. 2012. Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7. Applied and Environmental Microbiology. 78:1004-1014.

Pan, L., Zhang, Q., Li, X., Tian, P. 2012. Detection of human Norovirus in cherry tomatoes, blueberries and vegetable salad by using a receptor binding based capture and magnetic sequestration(RBCMS) method. Food Microbiology. 30:420-426.

Brandl, M., Carter, M.Q., Parker, C., Chapman, M.R., Huynh, S., Zhou, Y. 2011. Salmonella biofilm formation on Aspergillus niger involves cellulose - chitin interactions. PloS ONE 6(10):e25553.

Smith, C.D., Berk, S.G., Brandl, M., Riley, L.W. 2012. Survival characteristics of diarrheagenic Escherichia coli pathotypes and Helicobacter pylori during passage through the free-living ciliate, Tetrahymena sp. FEMS Microbiology Ecology. 82:574-583.

Ravva, S.V., Hernlem, B.J., Sarreal, C.Z., Mandrell, R.E. 2012. Bacterial communities in urban aerosols collected with wetted-wall cyclonic samplers and seasonal fluctuations of live and culturable airborne bacteria. Journal of Environmental Monitoring. 14(2):473-481. doi: 10.1039/clem10573d.

Gorski, L.A. 2012. Selective enrichment media bias the types of salmonella enterica strains isolated from mixed strain cultures and complex enrichment broths. PLoS One.7:e34722

Tian, P., Yang, D., Pan, L., Mandrell, R.E. 2012. Application of a receptor-binding-capture qRTPCR assay to concentrate human norovirus from sewage and to study the distribution and stability of the virus. Applied and Environmental Microbiology. 78(2):429-36 doi:10.1128/AEM.06875-11.

Carter, M.Q., Xue, K., Brandl, M., Liu, F., Wu, L., Louie, J.W., Mandrell, R.E., Zhou, J. 2012. Functional metagenomics of Escherichia coli O157:H7 interactions with spinach indigenous microorganisms during biofilm formation. PLoS One. 9:e44186.

Carter, M.Q., Parker, C., Louie, J.W., Huynh, S., Fagerquist, C.K., Mandrell, R.E. 2012. RcsB contributes to the distinct stress fitness between Escherichia coli O157:H7 curli variants of 1993 hamburger-associated outbreak strains. Applied and Environmental Microbiology. doi:10.0028/AEM.02157-12.

Last Modified: 06/25/2017
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