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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

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: 10/19/2017
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