Location: Produce Safety and Microbiology Research2013 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:
Objective 1: A Salmonella Typhimurium isolated in our survey was shown to be fitter on lettuce than two other serovars also isolated in the Salinas Valley watershed. In collaboration with scientists at the ARS, Ames, Iowa, and at Pennsylvania State University, we comparatively characterized E. coli O157:H7 (EcO157) supershedder strains and their mutants for attachment to leafy greens and biofilm production in leaf lysates. RNAseq was used to characterize the transcriptome of EcO157 in lettuce under modified atmosphere packaging conditions and to characterize the role of methylation in the biology of E. coli O145. Environmental and clinical shigatoxin-producing O111 strains were characterized for aggregative behavior and its role in attachment to plants. Objective 2: Colonization of EcO157 in wounded leaf tissue was assessed in Arabidopsis mutants defective in basal defense pathways. Other planned work under this objective was redirected to the pathogen survey in California and genetic characterization of isolates, under objective 4 below. Objective 3: Testing of Salmonella and EcO157 strains for survival to grazing by protozoa is ongoing. Results on EcO157-protozoa interactions in dairy waste water were published. Approaches to study attachment of norovirus to plant microflora failed and new methodology is being applied to achieve this objective. Objective 4: A survey of the Salinas Valley watershed for the presence of Salmonella, Listeria monocytogenes, EcO157and shigatoxin-producing E. coli (STEC) in collaboration with the FDA and NASA was conducted into its second year. Pulse field gel electrophoresis analysis was carried out on all Salmonella isolates. Multi Locus Sequence Typing and Multi Locus Tandem Variable Repeat Analysis of Salmonella were assessed and deemed unsuitable; a Luminex-based serotyping is being investigated. Improved detection methods were applied to quantify STEC levels at high prevalence sites. Our new STEC typing method is being tested on these new isolates, indicating greater than 1000 different types, including all the clinically important serotypes. Location data for STECs from the Salinas Valley watershed were formulated into matrices along with our existing typing data and partially analyzed to map the movement of various STECs in that environment. The genome sequence of two STEC O111 animal isolates from the Salinas Valley was obtained and compared with that of sequences of clinical strains. Genetic and phenotypic differences in 500 strains of EcO157 were compared to evaluate the fitness of pathogenic E. coli in animals, humans, and soil. The virulence profile and genetic diversity of over 100 E. coli O145 strains from various sources including from the Salinas Valley were characterized. Coliphages specific to E. coli from animals and humans were characterized to source-track pathogenic E. coli in California produce production regions. Using Tulane virus as a surrogate for human norovirus, our new detection method was developed and validated for distinguishing between infectious and inactivated viruses, and applied to demonstrate inactivation conditions for human norovirus.
Goudeau, D.M., Parker, C., Zhou, Y., Sela, S., Kroupitski, Y., Brandl, M. 2013. The Salmonella transcriptome in lettuce and cilantro soft rot reveals a niche overlap with the animal host intestine. Applied and Environmental Microbiology. 79:250-262.