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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

2013 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.

1. High prevalence of Salmonella among wild reptiles and amphibians with access to production fields in California. Recalls of leafy greens due to Salmonella contamination have economically impacted produce growers in California. Cold-blooded vertebrates could be a source of microbial contamination of produce since they are known carriers of Salmonella. ARS Researchers in Albany, California, in collaboration with scientists at University of California (UC), Davis, sampled 460 amphibians and reptiles and associated waters for the presence of Salmonella in a major produce growing region of California, and observed an incidence of 59% in snakes, 15% in lizards, 5% in toads, 1% in frogs, and 18% in water samples. Most of the animal isolates and a small subset of the water isolates were S. enterica subsp. Diarizonae, and 62% of the animal isolates were resistant to one or more antibiotic. The findings, which were published in Foodborne Pathogens and Disease, provide the industry and public health agencies with valuable epidemiological data for risk assessment for this important agricultural region of the US.

2. Identification of Salmonella genes with a role in attachment and persistence on plant surfaces. Salmonella contamination of leafy greens and tomato fruit has caused numerous recalls of produce and outbreaks of illness, with serious impact on public health and the produce industry. In collaboration with scientists from the Illinois Institute of Technology and from the Volcani Center in Israel, ARS scientists in Albany, California, identified several new genes in Salmonella that have a role in biofilm formation and attachment to plant surfaces. Some of these genes also affected tolerance to chlorine treatment or persistence on lettuce under cold storage conditions, or had a known role in interactions with animal hosts. These results, which were published in the Phytopathology and PLoS ONE journals, enhance knowledge of the biology of this human pathogen on plants that can be used to develop more effective interventions to mitigate contamination of produce.

3. Genome sequencing reveals signatures of E. coli O145:H28. Non-O157:H7 Shiga toxin-producing E. coli (STEC) are emerging food-borne pathogens and have caused outbreaks of infections associated with produce. ARS Researchers in Albany, California, have determined the complete genome sequences of four Escherichia coli O145 strains (EcO145), two of which were linked to the 2010 U.S. lettuce-associated outbreak and two to the 2007 Belgium ice-cream-associated outbreak. Comparative genomic analyses suggest that, unlike any other known non-O157 STEC strain, EcO145 ascended from a mutual lineage with E. coli O157:H7 (EcO157), and evolved to a unique enterohemorrhagic E. coli (EHEC) group by acquisition of the core set of EHEC virulence factors. This study provides new genomic information about an important STEC serotype and will facilitate the discovery of STEC signature genes in order to improve the detection of STEC in food.

4. Occurrence of natural genetic variants of rcsB and rpoS in E. coli O157:H7 population. Genetically diverse populations of E. coli O157:H7 exist in agricultural environments, but the genetic factors involved in this diversity and their role in the biology of this human pathogen are unknown. ARS researchers in Albany, California, have discovered a high prevalence of mutations in the rcsB and rpoS genes of curli-producing variants of 1993 hamburger-associated outbreak strains and of 2006 U.S. spinach-associated outbreak strains, respectively. These mutations lead to distinct stress tolerance phenotypes in E. coli O157:H7 curli variants and may impart overall fitness and virulence to E. coli O157:H7 populations throughout the contamination cycle of the pathogen. The results of this study were published in Applied and Environmental Microbiology and provide public health agencies with valuable knowledge about the epidemiology of this important food-borne pathogen.

5. Development of methods for isolation of both E. coli O157 and non-O157 STEC from agricultural environments. Surveillance for the presence of Shiga toxin-producing E. coli (STEC), outbreak investigations, and quality control by the industry and inspection agencies require effective methods for STEC recovery from environmental samples, but such methods had not yet been developed. The abundance of these pathogens is very low and the harsh environment often restricts growth, making recovery difficult. ARS scientists at Albany, California, developed a very robust method for recovery of E. coli O157 from wildlife, soil, sediment and water, leading to isolation of the outbreak strain from numerous environmental sources during the 2006 spinach outbreak investigation. Subsequent improvements in the method to include all STEC and its application to a survey of thousands of samples from the Salinas region provides valuable data for risk assessment relevant to the microbial safety of produce grown in this important agricultural environment.

6. Growth inhibition of environmental protozoa extends the survival of E. coli O157:H7. On-site control of E. coli O157:H7 is essential to prevent contamination of produce grown in proximity to feedlots and dairies. ARS researchers at Albany, California, observed that monensin, a commonly used feed supplement, inhibits certain protozoa, thereby extending the survival of E. coli O157:H7 in dairy wastewater. This antibiotic feed supplement also altered the community structure of both protozoa and bacteria in wastewater. These data suggest prudent use of antibiotic dietary supplements is warranted as such treatment enhances the persistence of E. coli O157:H7 in the agricultural environment. The findings, which were published in PLoS ONE, identify for the industry and public health agencies, a risk factor in the contamination of produce in areas where animal and crop production are geographically intertwined.

7. Optimized method for detection of human norovirus also reveals its infectivity. Norovirus (HuNov) is considered the most common etiologic agent of outbreaks linked to produce, but common norovirus detection methods do not differentiate between infectious and nonviable particles. ARS researchers in Albany, California, have developed a new in situ capture qRT-PCR (ISC-qRT-PCR) method to concentrate HuNoV from food and environmental samples that distinguishes infectious and inactivated viruses. Using this approach, they also demonstrated inactivation conditions for HuNov and observed that HuNov is more heat- and ethanol-resistant, but more sensitive to chlorine treatment relative to its commonly used surrogates. This innovative technology resulted in a patent application because of its high value to public health agencies and private laboratories that perform testing for the industry, and to the biotechnology industry that develops equipment and molecular biology kits for detection of norovirus.

Review Publications
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.

Salazar, J., Deng, K., Tortorello, M., Brandl, M., Wang, H., Zhang, W. 2013. Genes ycfR, sirA and yigG contribute to the surface attachment of Salmonella enterica Typhimurium and Saintpaul to fresh produce. PLoS One. 8(2):e57272. doi:10.1371/journal.pone0057272.

Brandl, M., Cox, C.E., Teplitski, M. 2013. Salmonella interactions with plants and their associated microbiota. Phytopathology. 103:316-325.

Brandl, M., Sundin, G.W. 2013. Focus on food safety: Human pathogens on plants. Phytopathology. 103:304-305.

Gorski, L.A., Jay-Russell, M., Liang, A.S., Walker, S., Bengson, Y., Govoni, J.A., Mandrell, R.E. 2013. Diversity of pulsed field gel electrophoresis pulsotypes, serovars and antibiotic resistance among Salmonella isolates from wild amphibians and reptiles in the California central coast. Foodborne Pathogens and Disease. 10:540-548.

Yang, J., Pan, Z., Takeoka, G.R., Mackey, B.E., Bingol, G., Brandl, M., Garcin, K., Mchugh, T.H., Wang, H. 2012. Shelf-life of infrared dry-roasted almonds. Journal of Food Chemistry. 138(1):671-678. DOI: 10.1016/j.foodchem.2012.09.142.

Benjamin, L., Atwill, E.R., Jay-Russell, M., Cooley, M.B., Carychao, D.K., Gorski, L.A., Mandrell, R.E. 2013. Occurrence of generic E. coli, E. coli O157:H7 and Salmonella spp. in water and sediment from leafy green produce farms and streams on the Central California coast. International Journal of Food Microbiology. 10.1016/j.ijfoodmicro.2013.04.003 165(1)65-76.

Ravva, S.V., Sarreal, C.Z., Mandrell, R.E. 2013. Protozoan and bacterial community shifts during enhanced survival of Escherichia coli O157:H7 in monensin-treated dairy wastewater. PLoS One. 8(1):e54782.doi:10.1371/journal.pone.0054782.

Friedman, M., Henika, P.R., Levin, C.E. 2013. Bactericidal activities of health-promoting,food-derived powders against the foodborne pathogens Escherichia coli,listeria monocytogenes, salmonella enterica,and staphylococcus aureus. Journal of Food Science. doi:10.1111/1750-3841.12021 78:M270-M275.

Rounds, L., Havens, C.M., Feinstein, Y., Friedman, M., Ravishankar, S. 2013. Concentration-dependent inhibition of Escherichia coli O157:H7 and heterocyclic amines in heated ground beef patties by apple, olive, and onion powders and clove bud oil. Journal of Meat Science. doi: 2010.1016/j.meatsci.2013.03.010..

Cooley, M.B., Jay-Russell, M., Atwill, E.R., Carychao, D.K., Nguyen, K.M., Quinones, B., Patel, R., Walker, S., Swimley, M., Pierre-Jerome, E., Gordus, A., Mandrell, R.E. 2013. Development of a robust methos for isolation of shiga toxin positive Escherichia coli (STEC) from fecal, plant, soil and water samples from leafy greens production region in California. PLoS One. 8(6):e65716. doi:10.1371/journal.pone.0065716.

Kroupitski, Y., Brandl, M., Pinto, R., Belausov, E., Tamir-Ariel, D., Burdman, S., Sela, S. 2013. Identification of Salmonella enterica genes with a role in persistence on lettuce leaves during cold storage by recombinase-based in vivo expression technology. Phytopathology. 103:362-372.

Last Modified: 4/18/2014
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