Biology and Control of Human Pathogens on Fresh Produce

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1212	2000	12%
712	1420	1000	16%
712	1430	1100	29%
712	1460	2000	21%
712	1122	1000	10%
712	1123	1100	12%

Progress 12/28/10 to 12/27/15

Outputs
Progress Report 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. 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. This is the final report for project 2030-42000-046-00D. Significant progress was made on all of the objectives, and some sub-objectives were redirected to other projects. Using clinical and environmental strains in several molecular genetic analyses we showed that curli fimbriae (cell- surface adhesive filaments) play important roles in shiga-toxin producing Escherichia coli O111 (STEC O111) and E. coli O157:H7 (EcO157) biofilm formation and interaction with plant-associated microbiota, EcO157 surface attachment, and Shiga-toxin E. coli (STEC) O111 attachment to spinach leaves. STEC O111 strains of environmental and outbreak origin were highly aggregative; whereas sporadic clinical strains were not. This aggregative behavior depended on curli production variation by the strains. Both inter- and intra-strain variations in curli production are widespread in EcO157 with the relative proportions of curli-producing (C+) and curli-deficient variants (C-) in an EcO157 population depending on the growth conditions. Furthermore, curli variants derived from the same strain displayed differences in survival fitness, with C+ variants growing better than C- in nutrient-limited conditions, and C- variants being significantly more acid resistant. These differences were due to natural mutations in the genes for the alternative sigma factor rpoS and the response regulator of the RcsCDB signal transduction system, and we discovered these mutations were found in EcO157 strains linked to spinach and hamburger outbreaks. Our transcriptome analysis suggested that STEC genomes are highly flexible, and capable of generating variant strains with different phenotypic traits. The heterogeneous population resulting from this flexibility would result in the fittest variant being amplified under different environmental conditions. Several new Salmonella genes that have roles in biofilm formation, attachment to and persistence on produce under cold storage were identified; and several had a known role also in interactions with animal hosts. We also demonstrated that different strains of Salmonella showed variable fitness in growth on pre-harvest baby lettuce, with a S. Typhimurium strain isolated originally from the Salinas Valley having the greatest fitness among a panel of strains tested. Disease lesions in a lettuce line resistant to downy mildew supported less EcO157 multiplication than those of a highly susceptible line, which lower expression of the plant basal immunity parker PR-1. Transcriptomics using RNAseq revealed that EcO157 experiences osmotic stress on cut lettuce. EcO157 used plant choline released in lettuce wounds to make the osmoprotectant glycine betaine and alleviate its solute stress in wounded tissue. We demonstrated that factors in plants affect the attachment of Human Norovirus (HuNoV) to plant surfaces. HuNoV attach to Human Blood Group Antigen (HBGA) proteins in order to infect human cells, and we demonstrated that HBGA-like glycoproteins are involved in the attachment of HuNoV to lettuce. This binding could lead to pre-harvest contamination of lettuce with HuNoV. Capsid proteins of subtype GII.4 HuNoV can bind to the surface of romaine lettuce leaves EcO157 is not a strong biofilm former in standard culture media, but we showed it forms extensive biofilms in unsterile field water, in spinach lysates, and as part of a population of spinach-associated microbes. Spinach-associated microbes enhanced initial attachment of EcO157, and also decreased the population of EcO157 in biofilms on stainless steel. The success of EcO157 in the mixed biofilm is likely associated with its metabolic potential in utilizing spinach nutrients, and successful competition with indigenous spinach bacterial biofilm species. In association with diseased plant tissue we showed that EcO157 cells were a 1,000-fold more numerous in wet downy mildew lesions than on healthy lettuce leaf tissue, and persisted in greater numbers on dry leaves when the disease was present. Gene expression profiling showed that Salmonella experiences in soft-rot lesions physicochemical conditions similar to those during colonization of the animal host intestine. This high adaptation to degraded plant tissue has implications for shelf-life standards set by the produce industry. We demonstrated that five E. coli pathotypes survived passage as viable cells through Tetrahymena, a protozoan ubiquitous on plants and in agricultural environments. In a different project we demonstrated that EcO157 survival in dairy wastewater lagoons was affected by protozoa by measuring an increase in predatory protozoa concomitant with a decrease in EcO157 numbers. Monensin, a dairy feed supplement that controls the protozoa population, extends the survival of EcO157, suggesting the prudent use of antibiotics is warranted. We demonstrated that application of neem eliminated 90% of EcO157 from dairy manure within 2 days, suggesting this inexpensive supplement might be an effective way to control pathogens. Strains of EcO157 isolated from soil and animal feces survived longer in soil and resist protozoa predation better than clinically isolated strains of EcO157. We demonstrated the longer surviving strains lack production of curli fimbriae. In analysis of aerosols near dairies we determined the bacterial populations were diverse in different areas, but did not reflect the species dominant in manure. This work will be useful in establishing buffer zones between dairies and vegetable crops. We demonstrated that E. coli O86:H2 expresses a Type-B HBGA, which gave us a tool for developing assays for HuNoV detection. Using Tulane Virus (TV) as a surrogate for HuNoV, we developed a new in situ capture (ISC) qRT-PCR method, and confirmed the binding of TV to the surface of E. coli O86:H2. Using this method we captured functional virus particles for stress testing. We further developed a more reliable cell-culture-based infectivity assay using the captured TV, and showed that HBGA-expressing bacteria did not confer protection to TV under heat stress, but binding to HBGA-like ligands on plant surfaces might allow pre-harvest contamination by HuNoV. Surveys of wildlife and watersheds in the leafy green growing region of Central California were continued from the previous project such that our understanding of pathogen prevalence and transport in the Salinas region is based on results from 3 major studies covering more than 10 years and involving samples of water, domestic and wildlife, soil and produce. In the current study of watersheds involving bimonthly sampling of up to 30 sites within a 500 sq. mile region, prevalence of EcO157 and STEC were 7% and 9%, respectively; 17% for Campylobacter; 24% for HuNoV; 43% for L. monocytogenes; and 56% for Salmonella. Presence of Salmonella was statistically similar between watersheds during all seasons, but some variation was seen according to season and location for the other pathogens. In concurrent surveys Salmonella incidence was high among amphibians and reptiles in the same region with the highest incidence (51%) in snakes. These animals harbored mostly Salmonella enterica subtype diarizonae, which are less commonly seen in outbreaks. Furthermore, no correlation was found between watershed samples testing positive for HuNoV and the incidence of generic E. coli or the other enteric pathogens in the study. L. monocytogenes isolates from the region are approximately 90% serotype 4b, and likely to contain intact copies of the virulence gene inlA. We created improved methods were developed to detect and distinguish the infectivity of HuNoV from environmental and food samples to use in this survey. Using these assays determined that 65% of the GI and GII subtypes (those affecting humans) found in the survey were not infectious. Our HuNoV assays have been used to assess HuNoV contamination in oysters from retail markets, and these methods may be applicable to HuNoV detection in oysters. We demonstrated that different serogroups of Salmonella displayed differential fitness in standard enrichment culture protocols with strains of serogroup B (including serovars Typhimurium, Saintpaul) demonstrating less fitness when in competition with other serogroups. Often in outbreaks more than one strain of Salmonella can be present, and the finding of variable fitness indicates decreased efficiency in detection of relevant strains during outbreak investigations. Incorporating more than one enrichment protocol enhances the chances of finding all the subtypes in a given sample. Digital image analysis showed that Tween-80 dispersed large Salmonella aggregates from cilantro into the wash water, suggesting that surfactants may complement other sanitizing approaches during produce processing. A novel technology was developed based on sequential infrared heating and hot air that dried pistachios while reducing the Salmonella surrogate Enterococcus faecium populations on kernels and shells by more than 6- and 5-log cfu/g, respectively. Comparative genomics of four E. coli serotype O145 (EcO145) genomes from four strains (two food and clinical isolates each linked to a U.S. lettuce-associated outbreak and a Belgium ice-cream-associated outbreak) revealed a large core (5,173 genes) and a considerable amount of strain- specific genes. Additionally, the two EcO145 genomes linked to the two outbreaks display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome), implying divergent evolution of EcO145 strains. This is the final report for this project, which expired on December 27, 2015. Please see the report for the new project, �Ecology and Detection of Human Pathogens in the Produce Production Continuum�, 2030-42000-050- 00D, for additional information. Accomplishments 01 Prevalence of E. coli, Salmonella, Campylobacter, and Listeria monocytognes in produce growing areas in the Salinas Valley. ARS researchers at Albany, California, in collaboration with the Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition (CFSAN) and National Aeronautics and Space Administration (NASA) are completing a 5 year survey of the Salinas watershed of California for the presence of E. coli O157 and non-O157:H7 shigatoxin-positive E. coli, Salmonella, Campylobacter, and L. monocytogenes to provide data for a predictive geospatial risk assessment model (PGRAM). Despite the current drought, more than 3000 water samples have been processed to date, of which 7% and 9% were positive for E. coli O157 and STEC, respectively. Incidence of Salmonella, Campylobacter and L. monocytogenes was much higher, ranging from 17% for Campylobacter, 43% for L. monocytogenes, to 56% for Salmonella. Samples are also being collected at higher frequency in selected regions to improve the resolution of E. coli O157. This information will provide valuable data to the industry and public health agencies for risk assessment of these pathogens from this important agricultural region of the U.S. 02 Variability in the aggregation of Shiga toxin-producing E. coli (STEC) O111 is dictated by curli and RpoS. STEC O111 is prevalent in produce growing regions and has caused outbreaks linked to consumption of salad and cabbage. ARS researchers in Albany, California, showed that aggregation is common among environmental and outbreak, but not sporadic case, strains of STEC O111. Aggregation promoted or inhibited STEC O111 biofilm formation, depending on hydrodynamic conditions. Curli was identified as the primary factor in this aggregative behavior. Polymorphism in O111 aggregation was attributed to functional variability in the stress responsive global regulator RpoS, demonstrating the high adaptability of STEC to changing environments throughout food production. These results, which were published in Applied and Environmental Microbiology, inform the industry, and public health and regulatory agencies about the epidemiology of nonO157:H7 STEC in the food production continuum. 03 Serotyping of Listeria monocytogenes by Fourier Transform Infrared Spectroscopy (FT-IR). ARS researchers in Albany, California, used FT- IR in conjunction with artificial neural network software to develop a database for the rapid identification Listeria species and serotyping of L. monocytogenes strains. A spectral library was constructed from 254 strains of Listeria spp to give a biochemical fingerprint from which identification of unknown samples is made. Eleven serotypes of L. monocytogenes were identified with 96.6% accuracy. This database and neural network definition may provide a rapid and inexpensive method for L. monocytogenes subtyping that would be useful for public health laboratories. This work was published in Public Library of Science ONE. 04 Coliphages as surrogates for source-tracking pathogenic E. coli in produce production environment. Source-tracking of pathogens from fork to farm is critical for development of pathogen control measures. ARS researchers in Albany, California, determined that E. coli specific coliphages survive differently based on their genetic differences, and under different environmental conditions. Thus, the methods they have developed to detect phages can be used to detect both those resulting from recent fecal contamination, and those that persist longer as a result of cooler temperatures and increased presence of host bacteria. Data on the prevalence and fate of coliphages may be invaluable in predicting the sources of fecal contamination and aid in developing methods to prevent enteric pathogen contamination from likely sources during produce production. 05 Development of improved methods to detect and distinguish infectivity of Human Norovirus (HuNoV) in environmental and food samples. ARS researchers in Albany, California, developed new, improved protocols for the detection of HuNoV from environmental water samples and to detect the infectious potential of those HuNoV particles. Using these new assays, which include the incorporation of Tulane Virus (TV) as an internal control, and an in situ capture qRT-PCR (ISC-qRT-PCR) it was determined that at least 65% to 71% samples exhibiting HuNoV signals in qRT-PCR were unlikely to be infectious. The ISC-qRT-PCR method was also utilized by collaborators to detect HuNoV in oyster samples from retail markets in Shanghai, China, and found subtypes GI and GII of HuNoV in 33% and 3% of the oyster samples, respectively. The results, which were published in Food Microbiology show promise for ISC-RT-qPCR as a method for detecting HuNoV in oyster samples. 06 Development of a simultaneous drying and decontamination technology for pistachios. The U.S. is the second leading producer and exporter of pistachios, providing 24 percent of the world total production. In 2014, the U.S. produced 257,000 tons of pistachios valued at $1.6 billion. Pistachios have been recalled due to contamination with Salmonella and have caused outbreaks of salmonellosis, impacting public health and the pistachio industry. ARS researchers in Albany, California, have developed a novel technology based on sequential infrared heating and hot air that dries pistachios with energy savings of 34% compared with traditional hot air drying alone, and effects an approximate one million times reduction in population sizes of the Salmonella surrogate Enterococcus faecium on pistachio kernels and shells, respectively. This technology provides the industry with an efficient and effective approach to produce high quality safe pistachios and reduce the incidence of foodborne illness. 07 Curli fimbriae enhance the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel. Curli fimbriae are surface features that are conditionally required for surface attachment and biofilm formation in many bacteria. ARS researchers at Albany, California, reported that curli significantly enhanced the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel surfaces by 5-fold. These fimbriae were also required for E. coli O157:H7 biofilm formation on stainless steel, and enhanced 19-27 fold the biofilm production on glass in some culture media. Furthermore, curli played an essential role in the formation of mixed biofilm by E. coli O157:H7 and plant-associated microorganisms. These studies, which were published in Food Microbiology, provide new information about the contribution of an important adhesion, curli fimbriae, to survival fitness of enteric pathogens in both host and nonhost environments, and could help contribute to science-based interventions to aid pre-harvest food safety based on the pathogen physiology in the agricultural environment. 08 Identification of Human Blood Group Antigen (HBGA)-like ligands in lettuce involved in Human Norovirus (HuNoV) binding. Norovirus bind to HBGA proteins in humans as part of the infection process, but HuNoV will also bind to lettuce surfaces. ARS researchers in Albany, California, engineered a bacterial strain to display the subtype GII.4 capsid protein of HuNoV, and demonstrated that the HuNoV capsid proteins could bind to Romaine lettuce leaves. They further demonstrated that HBGA-like glycoproteins on the lettuce leaves were involved in the binding to HuNoV. This information, which was published in Frontiers in Microbiology, is important in showing that HuNoV may be a persistent contaminator of pre-harvest lettuce. 09 Listeria monocytogenes isolates from produce growing areas in the Salinas Valley are more likely to carry intact inlA genes and potentially be virulent. The inlA gene, encoding Internalin A, is necessary for L. monocytogenes virulence, but this gene is found to be mutated in 40-50% of foodborne and food processing plant isolates of L. monocytogenes. ARS researchers in Albany, California, assessed the inlA gene sequence of 112 strains of L. monocytogenes isolated from a survey of Salinas Valley watersheds. Over 90% of the isolates carried intact inlA genes. This work, which has been submitted to Public Library of Science ONE, indicates that environmental isolates that would be available for pre-harvest contamination of produce growing in the region are more likely to carry intact copies of this essential virulence gene. This information is vital for risk assessment and modeling of foodborne contamination and illness. 10 Transport studies of E. coli O157 and non-O157 STEC in Monterey and San Benito Counties. ARS researchers in Albany, California, processed more than 13,000 samples from domestic animals, wildlife, water, soil, and produce collected between April 2008 and October 2010 that yielded 363 and 1904 E. coli O157 and STEC types, respectively. Many of these subtypes (or closely related subtypes) were found at several locations and/or sample dates, indicating the pathogens are transported within the environment. Location data has been formulated into matrices and these matrices were analyzed, with additional geospatial data from the region, to monitor risk-associated parameters of closely related pathogens in both counties. The completed analysis will provide valuable data for risk assessment of the proximity of domestic or wildlife to produce.

Impacts
(N/A)

Publications

Romanolo, K.F., Gorski, L.A., Wang, S., Lauzon, C. 2015. Rapid identification and classification of Listeria spp. and serotype assignment of Listeria monocytogenes using fourier transform-infrared spectroscopy and artificial neural network analysis. Public Library of Science for Pathogens. PLoS ONE 10(11): e0143425. doi:10.1371/journal.pone.014325.
Diodati, M.E., Bates, A.H., Miller, W.G., Carter, M.Q., Zhou, Y., Brandl, M. 2016. The polymorphic aggregative phenotype of Shiga toxin-producing Escherichia coli O111 depends on rpoS and curli. Applied and Environmental Microbiology. 82:1475�1485. doi: 10.1128/AEM.03935-15.
Carter, M.Q., Quinones, B., He, X., Louie, J.W., Zhong, W.W., Lee, B.G., Yambao, J.C., Mandrell, R.E., Cooley, M.B. 2015. An environmental shiga toxin-producing Escherichia coli O145 clonal population exhibits high- level phenotypic variation that includes virulence traits. Applied and Environmental Microbiology. doi: 10.1128/AEM.03172-15.

Progress 10/01/14 to 09/30/15

Outputs
Progress Report 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. 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. A collection of Shiga toxin producing E. coli (STEC) serovar O111 strains from environmental and clinical sources was screened for phenotypic characteristics relevant to multicellular behavior on plants, and the strong biofilm-forming phenotype depended on curli production. Comparative genotypic analysis revealed that certain outbreak strains had high phylogenetic similarity to several strains isolated from environmental sources in a major produce growing region of California, and that some strains circulate over time and geographically in this area. These results were published in Foodborne Pathogens and Disease. E. coli O157:H7 supershedder strains and their mutants were characterized for attachment to leafy greens and biofilm production in leaf lysates in collaboration with scientists at the ARS, Ames, Iowa, and at Pennsylvania State University. In collaboration with scientists from the Illinois Institute of Technology and from the Volcani Center in Israel, several Salmonella genes involved in biofilm formation, attachment to plant surfaces, adaptation to chlorine treatment and cold storage conditions were identified. Digital image analysis showed that Salmonella aggregates on cilantro plants were dispersed into smaller ones and single cells in the wash water by addition of Tween-80. The Salmonella studies were published in Phytopathology, PLoS ONE and Applied Environmental Microbiology. Natural curli variants of E. coli O157:H7 linked to several outbreaks as well as their isogenic curli-deletion mutants were examined for surface attachment, biofilm formation, and adherence to animal host cells. Curli plays a role in initial attachment of E. coli O157:H7 to plant and abiotic surface and mediate the formation of mixed biofilm by the pathogen and plant-associated microorganisms, but curli contributed minimally to biofilm on abiotic surfaces when cells were grown in spinach lysates. This was presented at American Society for Microbiology (ASM) 2015 and a manuscript is in preparation. In collaboration with Georgia Institute of Technology genome analysis of 1493 E. coli STEC strains was done to compare virulence factors such as Shiga toxin, intimin, the intimin receptor, and the hemolysin toxin, in different phylogroups to determine why some strains of STEC cause severe disease and others cause no disease at all. The STEC virulence genes were detected mostly in phylogroup E and B1. In contrast no phylotype B2 or D strains were positive in selected virulence genes. In collaboration with scientists at Shanghai Jiao Tong University, Shanghai, China, the genetic diversity and virulence of Staphylococcus aureus in raw and processed foods was identified. Fourier-transform Infrared Spectometry (FT-IR) showed promise as a superior method to differentiate between strains of Salmonella than the previously used colony immunoblot method, and will be used to differentiate strains in fitness experiments on pre-harvest lettuce. In collaboration with scientists at North Carolina State, progress was made on screening outbreak and environmental strains of Listeria monocytogenes for attachment and growth on romaine lettuce, biofilm formation on different abiotic surfaces. A protocol was developed for selection of mutants deficient in attachment to cantaloupe using amplicon sequencing and the Illumina MiSeq to determine genes important for plant attachment. This work was presented at the International Association for Food Protection (IAFP) 2015, and a manuscript is in preparation. RNAseq was used to characterize the transcriptome of E. coli O157:H7 in lettuce under Modified Atmosphere Packaging conditions and several candidate genes were investigated that affect the colonization of shredded lettuce. Two manuscripts describing these findings are in preparation. Arabidopsis mutants that are defective in 2 basal defense pathways were tested for colonization by E. coli O157:H7 and differences in survival of the human pathogen were observed. Further studies are ongoing. A study was published in BMC Microbiology showing that downy mildew disease lesions promote elevated growth and survival rates of E. coli O157:H7 on lettuce and a lettuce line expressing high levels of the plant basal defense marker PR-1 support lower densities of E. coli O157:H7 in downy mildew lesions than another line with lower PR-1 expression. In mixed species biofilms, curli-producing E. coli O157:H7 formed large aggregates with spinach-associated microorganisms on various abiotic surfaces; whereas curli-deficient E. coli O157:H7 mostly remained as single cells in the aggregates of plant-associated microorganisms. This work was submitted to Food Microbiology. Transcriptome analysis of Salmonella in macerated cilantro and lettuce tissue infected with the plant pathogen Dickeya dadantii revealed that physicochemical conditions in produce soft-rot lesions overlap with those in the animal intestine, which has implications for safe shelf-life standards and suggests the presence of soft-rot should be included in microbial risk assessment. The results were published in Applied Environmental Microbiology. Predatory protozoa in wastewater play an important role in controlling the populations of E. coli O157:H7 in dairy lagoons. Vorticella microstoma, Platyphora sp and Colpoda aspera from dairy wastewater were capable of consuming E. coli O157:H7 preferentially. Monensin, a dietary supplement for enhanced milk and meat production, inhibits and eliminates protozoa that control E. coli O157:H7. Work on the variability of interactions between enteric pathogen strains and Tetrahymena was begun, but not completed due to a lack of human resources. Aerosols generated on dairies were characterized for bacterial communities using cultural, molecular and flow cytometry methods. The predominant bacteria in aerosols were diverse among locations and did not reflect the predominant species of bacteria present in cow feces. Bacterial isolates with similar light scattering properties share identical 16S rDNA gene sequences. While many DNA sequences of human pathogens were detected, foodborne pathogens of interest were not found. In contrast to genetically indistinguishable clinical isolates, environmental strains of E. coli O157:H7 from soil and animal feces survive longer in soil and resist protozoan predation. Strains that survive longer lack curli. The curli-deficient phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments. Soil exposure altered the expression of stress-response genes linked to fitness of E. coli O157:H7 and mutations were detected in the stress-related sigma factor, rpoS, which may confer a differential expression of genes associated with mechanisms of survival and virulence. Tulane virus was used as a surrogate for Human Norovirus (HuNoV) in experiments to determine the role of plant microbial flora on HuNoV ecology in agricultural environments. Human Blood Group Antigen (HBGA) expressing E coli were tested for attachment of Tulane virus, and showed no difference from control bacteria, suggesting that HuNoV binds to bacteria by mechanisms other than HBGA. A manuscript is in preparation. RNA-seq based transcriptomics was used to understand the role of DNA methylation via the novel type II restriction-modification (R-M) BsuBI/ PstI system found in the E. coli 2010 lettuce-associated outbreak strains. Unlike Dam DNA methylase, the novel system has a much smaller regulon, and many of the genes are related to stress tolerance including iron limitation, osmotic stress, and multidrug resistance. A manuscript is in preparation. In collaboration with the Food and Drug Administration (FDA) and National Aeronautics and Space Administration (NASA), a third year of the Salinas region watershed has been completed and found a much higher incidence with pathogens other than STEC, including Salmonella, L. monocytogenes, and Campylobacter. The highest was Salmonella, which averaged 60% of all water samples, but with differences between sample sites and sample season. Different patterns of variation were seen with the other pathogen types, indicating potentially multiple and varied sources for these pathogens in the Salinas region. However, the incidence of all pathogens was reduced compared to previous years, primarily due to the drought. A survey of these samples for HuNoV was completed, and the overall prevalence was 24.2% with seasonal variation. There was no significant difference in HuNoV detection rates between samples testing positive or negative for the 4 bacterial pathogens, and overall detection levels of generic E. coli and HuNoV showed no correlation. The efficacy of Moore Swab in recovery of HuNoV was also evaluated, and the correlation of viral in vivo assays and in vitro assays was investigated. One manuscript has been submitted and another is in preparation. At the end of this fiscal year we will have completed four years of sampling (approximately 2500 samples). Among L. monocytogenes isolates from food processing plants about 40% have truncated forms of the virulence gene inlA, which renders the strains less virulent. Among inlA genes from 112 L. monocytogenes isolates from 2012 to 2014 from the Salinas Valley and 101 (90%) had intact genes. Only 3 (2.6%) of the strains carried truncated alleles. Truncated alleles did not persist in the region, and were only identified in strains isolated in early 2012. A manuscript is in preparation. In collaboration with California State East Bay, we showed that FT-IR could be used to efficiently differentiate between Listeria species, and between O serogroups of L. monocytogenes. Differentiation among H antigens remains less efficient, but we are screening more strains. A manuscript is in preparation. FT-IR is also being used to differentiate Salmonella subspecies. We have developed several rapid testing methods to concentrate HuNoV from food and environmental samples. Accomplishments 01 Downy mildew disease promotes the multiplication and persistence of E. coli O157:H7 on lettuce. Lettuce is one of the main crop implicated in produce-linked outbreaks of E. coli O157:H7 infections and is frequently infected with the downy mildew pathogen in the Salinas Valley, California, one of most important lettuce growing region in the U.S. ARS scientists in Albany and Salinas, California, determined that E. coli O157:H7 multiplied 1,000-fold more in downy mildew lesions than on healthy lettuce leaf tissue under warm temperature and on wet leaves. On dry lettuce leaves, E. coli O157:H7 persisted in greater numbers when downy mildew disease was present. Downy mildew lesions on a lettuce line that is more resistant to the plant pathogen supported less E. coli O157:H7 multiplication than those of a line that was highly susceptible to the disease and that showed lower expression of the plant basal immunity marker PR-1. The findings suggest that breeding lettuce against downy mildew may be effective also as a defensive line against E. coli O157:H7 colonization. The research was presented to the California Leafy Greens Research Board and published in BMC Microbiology, where the article has been among the top accessed in the journal. 02 Prevalence of E. coli in produce growing areas in the Salinas Valley. ARS researchers at Albany, California, in collaboration with the Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition (CFSAN) and National Aeronautics and Space Administration (NASA) Goddard Space Flight Center (GSFC), are conducting a 5 year survey of the Salinas watershed of California for the presence of E. coli O157 and non-O157:H7 shigatoxin-positive E. coli, Salmonella, Campylobacter and Listeria to provide data for a predictive geospatial risk assessment model. The third year of a survey of the Salinas watershed was completed and a fourth year is being conducted to monitor incidence. Despite the current drought, more than 2000 water samples have been processed to date, of which 7% and 9% were positive for E. coli O157 and Shiga toxin-producing E. coli (STEC), respectively. Incidence of Salmonella, Campylobacter and Listeria monocytogenes was much higher, at 59%, 17%, and 40%, respectively. Samples are being collected at higher frequency in selected regions to improve the resolution of E. coli O157. This information will provide valuable data to the industry and public health agencies for risk assessment of these pathogens from this important agricultural region of the U.S. 03 Transport studies of E. coli O157 and non-O157 Shiga Toxin producing E. coli (STEC) in Monterey and San Benito Counties. ARS researchers in Albany, California, processed more than 13,000 samples (domestic animals, wildlife, water, soil, produce) between April 2008 and October 2010 which yielded 363 and 1904 E. coli O157 and STEC types, respectively. Analysis of the more prevalent STEC types indicated apparent transport over long distances (>20 Km) and subsequent survival for up to 18 months. Often transport of STEC was supported by changes in the environment or the presence of wild-life. Improved strain typing is being developed to verify these findings, and to monitor the risk of closely related pathogens in both counties. The completed analysis will provide valuable data for risk assessment of the proximity of domestic or wildlife to produce. 04 Prevalence of human norovirus (HuNoV) and the correlation between detection rates of HuNoV and bacterial pathogens in water in production fields in California. Human noroviruses are highly contagious viruses and are the main cause of acute gastroenteritis. Contamination of irrigation water could cause a food-borne outbreak. ARS Researchers in Albany, California, tested 626 samples from a leafy greens production region in California, and the overall prevalence was 24%. There was no correlation between samples testing positive for Norovirus and incidence of generic E. coli or other foodborne pathogens. The findings provide the industry and public health agencies with valuable epidemiological data for risk assessment for this important agricultural region of the U.S. 05 Coliphages for source-tracking shiga-toxigenic E. coli. To provide data for traditional trace-back studies from fork to farm it is necessary to determine the environmental sources for shiga-toxigenic E. coli. ARS researchers in Albany, California, developed fluorescent PCR methods to determine the prevalence of E. coli specific coliphages as indicators of fecal contamination in waters from a produce production region of the central coast of California. Most of the coliphages in waters were those sourced to humans and originated from human-impacted sites. However, shiga-toxigenic E. coli also were detected from locations influenced by agriculture, and data on the prevalence of coliphages may be invaluable in predicting the sources of fecal contamination and aid in developing methods to prevent enteric pathogen contamination from likely sources during produce production. 06 Characterization of the role of curli in surface attachment, biofilm formation, and host cell adherence in various E. coli O157:H7 strains. Curli are highly aggregative fimbriae and produced by several species of enteric pathogens. ARS Researchers in Albany, California, investigated the role of curli in surface attachment and biofilm formation in several systems relevant to fresh produce production and processing. Curli enhanced the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel, and contributed to the formation of mixed biofilms by the enteric pathogen and plant-associated bacterial species on abiotic surfaces in spinach-wash water. However, curli contribute minimally to biofilm formation on abiotic surface when cells were grown in spinach lysates. In collaboration with ARS Researchers in Ames, Iowa, ARS researchers in Albany, California, also showed that curli modulate the adherence of E. coli O157:H7 to bovine rectoanal junction squamous epithelial cells. These studies provide new information about the contribution of an important adhesion, curli fimbriae, to survival fitness of enteric pathogen in both host and nonhost environments, and could help contribute to science-based interventions to aid pre-harvest food safety based on the pathogen physiology in the agriculture environment. 07 Development of rapid in vivo and in vitro assays for determination of infectivity of virus. Traditionally the infectivity of a culturable virus was determined traditionally by plaque and/or half tissue culture infectious dose (TCID50) assay, both of which are time-consuming (at least 5 days) and labor-intensive. ARS researchers in Albany, California, have developed a method for rapid determination of infectivity of culturable viruses using a culture mediated amplification assay (CMA-qRT-PCR) combinng a molecular approach with the traditional tissue culture based assay. Cells are infected with samples containing virus for 12-24 hours followed by amplification of viral genomic signals by qRT-PCR. The new assay converts the number of viral genome copy numbers into infectious units. Overall, the CMA-qRT- PCR method takes significantly less time and labor and exhibits high sensitivity in distinguishing the infectivity of Tulane Virus (a surrogate for Human Norovirus). This assay has applications for many types of culturable human and plant viruses, and will speed up detection of human and crop illness by several days. 08 Salmonella can be eliminated rapidly from aerated wastewater from dairy lagoons. Salmonella is the most commonly identified foodborne pathogen in produce, meat and poultry. Cattle are known reservoirs of Salmonella and the pathogen excreted in feces ends up in manure flush lagoons. ARS researchers in Albany, California, demonstrated that Salmonella serovars declined rapidly with decimal reduction times of less than 2 days in aerated microcosms prepared from lagoon equipped with circulating aerators. Populations of Salmonella decreased significantly in aerated microcosms compared to nonaerated waters and in summer compared to winter. Some growers use dairy lagoon water for growth of fodder plants and for watering cows on the dairy farms. The results indicate that holding the wastewater for sufficient decimal reduction cycles in lagoons with these types of aerators yield pathogen-free nutrient-rich water for crop irrigations and fertilization, and is important information for these growers. 09 Neem supplements eliminate E. coli O157:H7 from manure. E. coli O157:H7 shed in cattle manure can survive for extended periods of time and intervention strategies to control this pathogen at the source are critical as produce crops are often grown in proximity to animal raising operations. Neem oil, from Azadirachta indica (neem tree) is used in organic agriculture as pest control. ARS researchers in Albany, California, discovered that neem leaf and bark supplements eliminate 90% of the pathogen from manure in less than 2 days. The active ingredient responsible for the inhibition can be extracted with ethyl acetate. These results indicate that application of inexpensive neem supplements to control pathogens in manure and possibly in produce fields may represent a novel strategy to control the transfer of foodborne pathogens in organic farming from farm to fork. 10 High genotypic and phenotypic similarity among shiga toxin-producing E. coli (STEC) serovar O111 environmental and outbreak strains. E. coli O111 is an emerging non-O157:H7 STEC that has been associated with produce-linked outbreaks in the U.S. ARS scientists in Albany, California, assembled and characterized a collection of environmental and clinical strains of E. coli O111 from diverse sources, and showed by Pulse Field Gel Electrophoresis (PFGE) analysis that several environmental strains from water, cattle and wild life were identical to each other and had high phenotypic and genotypic similarity to outbreak and sporadic illness strains. Additionally, all environmental O111 strains encoded shiga toxin and multiple virulence factors. The findings, which were published in Foodborne Pathogens and Disease, show that STEC O111 similar to known O111 outbreak strains and with the potential to be pathogenic to humans, circulate via water, cattle and wild life around agricultural fields over large distances and over long periods of time. This provides epidemiological data to government agencies for decision making regarding the regulation of non-O157 STEC in plant-derived foods.

Impacts
(N/A)

Publications

Song, M., Yalong, B., Jie, X., Carter, M.Q., Chunlei, S., Shi, X. 2014. Characterization of foodborne Staphylococcus aureus isolates: association of toxin gene profile with genotype and food commodities in Shanghai, China. International Journal of Food Microbiology. doi: 10.1016/j. ijfoodmicro.2014.11.020.
Carter, M.Q., Chapman, M.H., Gabler, F., Brandl, M. 2015. Effect of sulfur dioxide fumigation on survival of foodborne pathogens on table grapes under standard storage temperature. Food Microbiology. 49:189-196. doi: 10. 1016/j.fm.2015.02.002.
Simko, I., Zhou, Y., Brandl, M. 2015. Downy mildew disease promotes the colonization of romaine lettuce by Escherichia coli O157:H7 and Salmonella enterica. BMC Microbiology. 15:19. doi: 10.1186/s12866-015-0360-5.
Diodati, M.E., Bates, A.H., Cooley, M.B., Walker, S., Mandrell, R., Brandl, M. 2015. High genotype and phenotypic similarity among Shiga toxin- producing Escherichia coli O111 environmental and outbreak strains. Foodborne Pathogens and Disease. 12:235-243.
Ravva, S.V., Sarreal, C.Z., Cooley, M.B. 2015. Male-specific coliphages for source tracking fecal contamination in irrigation waters and prevalence of Shiga toxigenic Escherichia coli in a major produce production region of central coast of California. Environmental Science: Processes & Impacts. DOI: 10.1039/C4EM00537F.
Tian, P., Wang, D. 2013. Inactivation Conditions for Human Norovirus Measured by an In Situ Capture-qRT-PCR Method. International Journal of Food Microbiology. 172:76-82.
Xu, S., Wang, D., Yang, D., Tian, P. 2015. Alternative methods to determine infectivity of Tulane virus: a surrogate for human norovirus. Food Microbiology. DOI: 10.1016/j.fm.2014.12.004.
Wang, D., Xu, S., Yang, D., Young, G.M., Tian, P. 2014. A new In Situ Capture-qRT-PCR method uses for an alternative approach to determine inactivation of Tulane virus. Applied and Environmental Microbiology. 80:2120-2124.
Ravva, S.V., Sarreal, C.Z. 2014. Survival of Salmonella enterica in aerated and nonaerated wastewaters from dairy lagoons. International Journal of Environmental Research and Public Health. 11:11249-11260.
Ravva, S.V., Korn, A.M. 2015. Effect of Neem (Azadirachta indica) on the survival of Escherichia coli O157:H7 in dairy manure. International Journal of Environmental Research and Public Health. 12:7794-7803.
Fagerquist, C.K., Zaragoza, W.J., Sultan, O., Woo, N., Quinones, B., Cooley, M.B., Mandrell, R.E. 2014. Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization-Tandem Time of Flight mass spectrometry. Applied and Environmental Microbiology. 8(9):2928-2940.
Lee, Y., Van Nostrand, J.D., Tu, Q., Lu, Z., Cheng, L., Yuan, T., Deng, Y., Carter, M.Q., He, Z., Wu, L., Zhou, J. 2013. The PathoChip, a functional gene array for assessing pathogenic properties of diverse microbial communities. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. doi: 10.1038/ismej.2013.88.
Cote, R., Katani, R., Mathew, M.R., Kudva, I.T., Arthur, T.M., DebRoy, C., Mwangi, M.M., Albert, I., Garay, J.R., Li, L., Brandl, M., Carter, M.Q., Kapur, V. 2015. Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells. PLoS One. 10(2):e0116743. DOI: 10.1371/journal. pone.0116743.
Gorski, L.A. 2014. Serotype assignment by sero-agglutination, ELISA, and PCR.In: Jordan, K., Fox, E.M., Wagner, M., editors. Methods in Molecular Biology. New York, NY: Humana Press. p. 41-61.
Parker, C., Gorski, L.A., Huynh, S. 2013. Complete genome sequence of salmonella enterica subsp. enterica Serovar Thompson Strain RM6836. Genome Announcements. 1(6):e00900-13. doi: 10.1128/genomeA.00900-13.

Progress 10/01/13 to 09/30/14

Outputs
Progress Report 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. 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. Objective 1. Experiments are in progress to assess the fitness of environmental and outbreak isolates of Shiga toxin-producing E. coli (STEC) and Salmonella on leafy vegetables. A broad collection of STEC serovar O111, including numerous isolates from the Salinas Valley, California, survey performed in this project, was characterized phenotypically. Supershedder strains of E. coli O157:H7 obtained from our collaborators at other ARS locations were tested for their attachment to spinach relative to non-supershedder strains. S. Typhimurium is located throughout the Salinas region with serotypes Give and the monophasic 6, 8:d:- being localized near cattle grazing areas; this information will be used for the next rounds of strain selection for the lettuce fitness studies. Natural curli variants of Escherichia coli O157:H7 linked to several outbreaks were examined for production of Shiga toxin under both induced and non-induced conditions. No significant difference in Stx production was observed in any pair of curli variants. Furthermore, no differences were observed in their adherence to bovine rectoanal junction squamous epithelial cells. Objective 2. Significant progress has been made to test E. coli O157:H7 mutants based on the transcriptome data obtained by RNA-seq analysis of the pathogen in Modified Atmosphere Packaged lettuce. Additionally, Arabidopsis thaliana mutants that are defective in two basal defense pathways were tested for colonization of intact and wounded leaf tissue by E. coli O157:H7. Hormone treatments of lettuce to trigger some of these basal defense pathways and test their effect on the colonization by this pathogen are underway. Other planned work under this objective was redirected to the pathogen survey in California and genetic characterization of resulting isolates, under objective 4 below. Objective 3: Indigenous bacteria associated with baby spinach leaves and from Salinas River water collected close to produce growing areas were isolated and characterized by 16S gene sequences. The role of these spinach and water isolates in biofilm formation of E. coli O157:H7 is being investigated. In addition, a flow cytometry method was developed to rapidly detect predation of E. coli O157:H7 by protozoa to aid in monitoring the rates of enteric pathogen destruction by protozoa in produce production environments. Soil and feral pig feces isolates of E. coli O157:H7 survived longer than clinical strains in soil and with certain protozoa isolated from dairy wastewater. Furthermore, curli- positive subpopulations of E. coli O157:H7 decreased after exposure to protozoa and only curli-negative phenotypes survived in soils. Curli negative phenotype appears to be a selective trait for survival and possibly transport of the pathogen in soil and water. In order to assess the role of plant microbial flora on NoroVirus biology on plants, we constructed recombinant human norovirus in a baculovirus system and corresponding polyclonal antibodies from rabbit to use in an ELISA assay and microscopy. Progress was impeded by strong background signal likely due to nonspecific binding of bacteria to the antibodies. An alternative approach to directly measure the captured viral genome via qRT-PCR for Human NoroVirus is being tested. Objective 4: DNA methylation is known to play a role in gene expression, replication, and bacterial virulence. Comparative genomics of E. coli O145 revealed distinct methylomes between US lettuce-associated and Belgian ice cream-associated outbreak strains. We generated a dam deletion mutant and a deletion mutant in the two-gene operon that encodes the type II restriction-modification system in the 2010 lettuce- associated outbreak O145 strain. RNA-seq was applied to investigate the regulon of each methylase. The second year of a survey of the Salinas watershed was completed and a third year is being conducted to monitor incidence levels of various pathogens in collaboration with the Food and Drug Administration (FDA) and the National Aeronautics and Space Administration (NASA). 414 water samples were collected this year, of which 2% and 5% were positive for E. coli O157:H7 and STEC, respectively. Samples are also being collected at higher frequency in select regions to improve the resolution for the risk assessment. Additionally, we are collaborating with Roka BioScience by supplying 134 enrichments from water samples in the Salinas region for testing of their new RNA-based STEC detection system. Surveys of six Salinas Valley watersheds for Salmonella and L. monocytogenes continued with isolation frequencies of the pathogens consistent (62% prevalence for Salmonella and 42% prevalence for L. monocytogenes) with observations over the previous 1.5 years. Serovar analysis was completed on 60% of the 1072 isolated Salmonella strains. Serotype diversity was greatest in the Salinas River (30 different serovars) and least in the Gabilan (9 different serovars). The 87 serovar Typhimurium isolates were found among all 6 watersheds at incidences of 4 � 25%. L. monocytogenes serotype 4b continues to be the dominant serotype in the region, and a subset of all L. monocytogenes strains are being assessed to determine if they carry an intact allele of the virulence factor internalin A (inlA) gene. Salmonella and L. monocytogenes prevalence data were provided to FDA and NASA for risk assessment modeling. Pulsed Field Gel Electrophoresis and serotype analysis continue to be done on all Salmonella isolates. A collaboration was initiated with California State University, East Bay, in Hayward, California, to assess the use of Fourier-transform Infrared Spectroscopy (FT-IR) in the differentiation of L. monocytogenes serotypes and Salmonella subtypes. Improved methods for isolation of Norovirus from produce and environmental samples were developed based on a new In Situ Capture qRT-PCR (ISC-qRT-PCR) method to concentrate Human Norovirus from food and environmental samples. With the new method, we have tested water samples from a produce production region in the Salinas Valley, California. Among 626 samples tested, 107 and 104 samples were positive for G1 and G2 human norovirus, respectively. Among the qRT-PCR positive samples, 62 and 20 samples were confirmed by sequencing for G1 and G2, respectively. G.1.1 was found to be the dominant strain for G1 subtype and all G2 strains were G2.4. Accomplishments 01 High prevalence of Salmonella and L. monocytogenes in a major produce production area of the U.S. Recalls of leafy vegetables due to Salmonella and L. monocytogenes are on the rise and have economically impacted produce growers in California. ARS researchers in Albany, California, in collaboration with the Food and Drug Administration (FDA) and the National Aeronautics and Space Administration (NASA) sampled watersheds near produce production fields in the Salinas Valley, California bimonthly over 2 years and discovered overall prevalence of 62% for Salmonella and 42% for L. monocytogenes. Salmonella was present at equivalent levels in all seasons throughout the region, whereas L. monocytogenes showed spatial bias and was more prevalent in the winter and spring. L. monocytogenes serotype 4b, a type implicated often in foodborne outbreaks, comprised 94% of the isolates. The findings provide valuable epidemiological data to the industry and public health agencies as well as risk assessment data for modeling to the FDA and NASA for this vital agricultural region in the U.S. 02 Enrichment bias for L. monocytogenes serotype 1/2a in standard detection protocol. Large outbreaks of listeriosis associated with produce and numerous recalls of leafy vegetables due to contamination with L. monocytogenes have raised great public health concerns in recent years in the U.S. As part of a watershed survey for L. monocytogenes in the Salinas Valley in California, ARS researchers in Albany, California, compared 304 L. monocytogenes strains isolated from the region in two different enrichment procedures to determine if the methodology influenced the types of strains that were recovered. Strains of serotype 1/2a were more likely to be isolated than serotype 4b strains from a protocol that includes the commonly used Fraser broth as a selective secondary enrichment medium, while a non-selective secondary enrichment medium did not show this bias. The overall high incidence of the common food outbreak serotype 4b, based on both enrichment protocols, indicates that this serotype is dominant ecologically in the region, which is different from environmental surveys of other regions for L. monocytogenes. 03 Transport of E. coli O157 and non-O157 shiga toxin-producting E. coli (STEC) throughout major produce growing regions of the U.S. E. coli O157:H7 and non-O157 shiga toxin-producing E. coli are major zoonotic pathogens that caused large outbreaks associated with produce in the United States. ARS researchers in Albany, California, processed over 13, 000 environmental samples (domestic animals, wildlife, water, soil, produce) obtained in the Monterey and San Benito Counties of California, in the period 2008-2010 and demonstrated the presence of 363 and 1904 E. coli O157:H7 and STEC types, respectively, in these samples. Typing of the isolates revealed that many of these types (or closely related types) were detected at several locations in these regions and/or sample dates, indicating transport of the pathogens in the environment. Location data have been formulated into matrices and these matrices, along with additional geospatial data from the region were analyzed in collaboration with the Food and Drug Administration (FDA) and the National Aeronautics and Space Administration (NASA) to monitor risk- associated parameters of closely-related pathogens in both counties. The analysis provides valuable data to the industry and public health agencies for risk assessment of the proximity of domestic and wildlife animals to produce fields. 04 Prevalence of human norovirus in water in production fields in California. Human noroviruses are the main cause of acute gastroenteritis and the most important causal agent of produce-linked outbreaks in the United States. Irrigation water may contaminate crops with norovirus. ARS researchers in Albany, California, tested 626 samples from irrigation water sampled in an important leafy vegetable production region in California. Seventeen percent and 16 percent of the samples were positive for norovirus genotype 1 and 2, respectively. The findings provide the industry and public health agencies with valuable epidemiological data for risk assessment of norovirus contamination in this important agricultural region of the United States. 05 Development of a functional gene array for assessing pathogenic properties of microbial communities. Methods to rapidly detect the presence of human pathogens in environmental and food samples are direly needed. In collaboration with scientists at the Institute for Environmental Genomics and Department of Microbiology and Plant Biology at the University of Oklahoma, ARS scientists in Albany, California, designed a functional gene array, the PathoChip, to assess the pathogenic potential of the microflora of diverse habitats. The PathoChip contains a total of 3715 probes representing 13 virulence factors including adherence, colonization, motility, invasion, toxin, immune evasion and iron uptake. The PathoChip provides a useful tool to identify virulence genes in microbial populations, examine the dynamics of virulence genes in response to environmental perturbations and determine the pathogenic potential of microbial communities. 06 Development of a rapid assay for determination of infectivity of culturable virus. The infectivity of a culturable virus was determined traditionally by plaque and/or half tissue culture infectious dose (TCID50) assay, both of which are time-consuming (at least 5 days) and labor-intensive. ARS researchers in Albany, California, developed two methods for rapid determination of infectivity of culturable viruses. The culture-based amplification assay (CMA-qRT-PCR) combines a molecular approach with the traditional tissue culture-based assay. Overall, the new method takes significantly less time and labor, and is high sensitive in assessing viral infectivity. These rapid assays provide useful tools for the detection of, and evaluation of the effect of interventions on, culturable food-borne pathogenic viruses. 07 Soil exposure alters the expression of stress-response genes linked to fitness of E. coli O157:H7. Airborne dust from feedlots is a potential mechanism of contamination of nearby vegetable crops with Escherichia coli O157:H7. ARS researchers at Albany, California, observed that E. coli O157:H7 strains from environmental sources survived longer in fine soil of less than 45-�m particle size and lacked expression of curli, a protein associated with attachment and virulence. Soil exposure also altered expression of stress-response genes linked to fitness of E. coli O157:H7 and mutations were detected in the stress-related sigma factor, rpoS. These findings identify for the industry and public health agencies, risk factors associated with mechanisms of survival and/or virulence of pathogens in produce production environments. 08 Comparative genomic analysis of E. coli O145:H28 food and clinical isolates. Shiga toxin-producing E. coli (STEC) O145 was identified as a cause of hemolytic uremic syndrome (HUS) at least two decades ago and is one of the six non-O157 serotypes most frequently associated with human disease in the United States. ARS researchers in Albany, California, performed whole-genome comparison and identified single nucleotide polymorphisms (SNPs) among strains of food and clinical origin associated with the same outbreak. Whereas 18 SNPs were identified in non-repeat regions of the genome of the 2007 Belgian ice cream-linked outbreak strains, no SNPs were observed in such regions among the two strains linked to the 2010 lettuce-linked outbreak in the U.S. A nearly identical methylome was detected among the pair of E. coli O145 strains of food and clinical origin that were associated with each of these outbreaks. This study provides new information about genomic diversity in an important STEC serotype and will facilitate the discovery of STEC signature genes in order to improve the detection of these important pathogens in food, including produce. 09 Sulfur dioxide fumigation is effective against contamination of table grapes with food-borne pathogens. California is the largest producer of table grapes in the U.S. and its annual crop is valued at $1.6B. ARS researchers in Albany, California, examined the persistence of common foodborne pathogens after their inoculation onto table grapes followed by treatment with sulfur dioxide and storage at cold temperature. Sulfur dioxide fumigation regimes were identified that effectively sanitized table grapes under these conditions. This study provides the table grape industry with critical information to develop standard protocols to ensure the microbial safety of table grapes. 10 The surfactant Tween-80 disperses Salmonella aggregates contaminating cilantro leaves. Salmonella has caused numerous outbreaks linked to herbs and leafy vegetables, and Salmonella aggregates/biofilms that form on plant surfaces are a major concern for decontamination of produce because these assemblages are more tolerant to sanitization. ARS scientists at Albany, California, used digital image analysis of a large number of micrographs of Salmonella cells that colonized cilantro plants and were washed off from the leaves with or without the surfactant Tween-80. Their results showed that Tween-80 dispersed large Salmonella cell aggregates into smaller ones and into single cells in the wash water. This study demonstrates that studies at the bacterial cell level have the potential to reveal foodborne pathogen behavior that is of relevance to the development of improved technologies aimed at minimizing microbial contamination of produce. 11 Novel technology to dry and decontaminate pistachio nuts. California is the world�s largest supplier of tree nuts and the annual pistachio crop is valued at more than $1.3B. The California pistachio industry has been economically impacted by one outbreak of salmonellosis linked to pistachios and numerous recalls of pistachios due to contamination with Salmonella. Current drying with hot air technology does not effectively pasteurize pistachio nuts. ARS researchers in Albany, California, developed an infrared�based technology that reduces microbial contamination by over 5-log10 bacterial cells/g nut. This new technology provides the industry with an effective process for both drying and sanitizing pistachios.

Impacts
(N/A)

Publications

Ravva, S.V., Hernlem, B.J., Sarreal, C.Z. 2014. Rapid Detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous tetrahymena by flow cytometry. Frontiers in Cellular and Infection Microbiology. DOI:10.3389/fcimb.2014.000057.
Gorski, L.A., Walker, S., Liang, A.S., Nguyen, K.M., Govoni, J.A., Carychao, D.K., Cooley, M.B., Mandrell, R.E. 2014. Comparison of subtypes of Listeria monocytogenes isolates from naturally contaminated watershed samples using a combination of non-selective and selective enrichment methods. PLoS One. 9(3):e92467. DOI: 10.1371/journal.pone.009246.
Cooley, M.B., Quinones, B., Oryang, D., Mandrell, R.E., Gorski, L.A. 2014. Prevalence of Shiga toxin producing Escherichia coli, Salmonella enterica and Listeria monocytogenes at public access watershed sites in a California central coast agricultural region. Frontiers in Cellular and Infection Microbiology. 4:30.
Ravva, S.V., Cooley, M.B., Sarreal, C.Z., Mandrell, R.E. 2014. Fitness of outbreak and environmental strains of Escherichia coli O157:H7 in aerosolizable soil and association of clonal variation in stress gene regulation. Pathogens. 3:528-548. DOI: 10.3390/pathogens3030528.
Cooper, K.K., Mandrell, R.E., Louie, J.W., Korlach, J., Clark, T.A., Parker, C., Huynh, S., Chain, P.S., Ahmed, S., Carter, M.Q. 2014. Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7. Biomed Central (BMC) Genomics. DOI: 10.1186/1471-2164-15-17.
Carter, M.Q., Louie, J.W., Huynh, S., Parker, C. 2014. Natural rpoS mutations contribute to the phenotypic heterogeneity of clonal populations in Escherichia coli O157:H7. Food Microbiology. 44:108-118.
Cooper, K.K., Mandrell, R.E., Louie, J.W., Korlach, J., Clark, T.A., Parker, C., Huynh, S., Chain, P.S., Ahmed, S., Carter, M.Q. 2014. Complete genome sequences of two Escherichia coli O145:H28 outbreak strains of food origin. Genome Announcements. 2(3):e00482-14. DOI: 10.1128/genomeA.00482- 14..
Brandl, M., Huynh, S. 2014. Effect of the surfactant Tween 80 on the detachment and dispersal of Salmonella enterica Thompson single cells and aggregates from cilantro leaves as revealed by image analysis. Applied and Environmental Microbiology. 80(16):5037-5042. DOI: 10.1128/AEM.00795-14.

Progress 10/01/12 to 09/30/13

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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.

Impacts
(N/A)

Publications

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.
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.
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.
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.

Progress 10/01/11 to 09/30/12

Outputs
Progress Report 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. 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. 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. Accomplishments 01 Selective enrichment media bias for Salmonella enterica strains isolated from mixed strain cultures. During food-borne outbreak investigations i is critical to isolate the relevant strain from food and/or environmenta 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 be isolated than others when we tested nineteen Salmonella enterica strains in mixed Salmonella pure cultures, and in cattle fecal enrichmen 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 t 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 finding were published in PLoS One. 02 Salmonella habitat conditions overlap in produce soft-rot lesions and in the animal intestine. The incidence of Salmonella contamination of reta 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 expressi profiling that Salmonella experiences in soft-rot lesions, conditions th 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 shoul be included in microbial risk assessment models for produce. Manuscript submitted for publication to Applied Environmental Microbiology. 03 E. coli O157:H7 forms mixed biofilm with spinach-associated microorganis The dynamics of mixed biofilm formation that include E. coli O157:H7 leading to its persistence in produce processing environments are unknow ARS researchers in Albany, Califronia, have observed that E. coli O157:H 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 compou the occurrence of outbreaks linked to produce. Our results were publishe in the Plos One journal. 04 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 bee caused by contamination with Listeria monocytogenes and Salmonella enterica. ARS researchers in Albany, California, in collaboration with F 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 f 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. 05 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 cytometr 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 i 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 dairi 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. 06 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 rout 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) meth Compared with other commonly used approaches, the RBCMS method is more sensitive, has better concentration power, and enriches for the infectio encapsulated virus. This information, which was published in the Food Microbiology and Applied Environmental Microbiology Journals, is of use public health agencies, private laboratories that perform testing for th industry, and the biotech industry that develops equipment and molecular biology kits for detection of norovirus.

Impacts
(N/A)

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.
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.
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., 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.
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.
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.
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
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.
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.

Progress 10/01/10 to 09/30/11

Outputs
Progress Report Objectives (from AD-416) Objective 1: Identify and chacterize 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. 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 well as to develop improved methods for detection of human pathogens on produce. Replacing 5325-42000-044-00D 03/2011. In the light of the large outbreak of enteroaggregative E. coli O104:H4 in Europe in 2011, frozen samples from a survey of pathogenic E. coli in the Salinas region, CA, are being re-screened for specific E. coli serotypes (O104 and O55), as well as for enteroaggregative and extra- intestinal pathogenic E. coli. As non-O157:H7 shigatoxin-producing E. coli (STEC) are emerging in the US, we began comparative genomics of STEC O145, a strain of which caused an outbreak linked to lettuce from a major produce-growing region of the US. Additionally, a method was developed for high throughput screening of fitness traits in numerous STEC strains isolated from the Salinas Valley in order to improve our understanding of their epidemiology in agricultural habitats, including produce. We previously demonstrated large inter- and intra-strain variations in the production of curli fimbriae in E. coli O157:H7, and difference in stress tolerance among curli variants of the pathogen. We have shown that this variation is likely due to a regulatory switch, suggesting a molecular mechanism in E. coli O157:H7 that generates variants to ensure the survival of the pathogen population under various stress conditions. The genome instability of E. coli O157:H7 was measured after desiccation and acid treatments for comparison to genomic instability observed with starvation, UV and high temperature. We investigated bacterial communities in biofilms that include E. coli O157:H7 and showed that the pathogen is a strong competitor and biofilm producer on abiotic surfaces and leaf tissue when in field water (collected in the Salinas Valley) containing simple nutrients. Survival of E. coli O157:H7 in naturally contaminated soil was monitored on particles dispersed in a wind tunnel, then applied onto lettuce leaves or seedlings. Additionally, the relationship between the size of particles and the level of natural contamination in soil samples was determined. Also, the survival of outbreak strains was monitored in moist soil microcosms under drying conditions that simulate those leading to aerosolization. Regarding our work with Salmonella, we determined levels of resistance to ultraviolet and oxidative stress in 34 different strains of Salmonella of various serotypes and serogroups originating from meat and produce. Nutrient utilization profiles of Salmonella strains were determined using the Biolog system. Studies on molecular detection of Salmonella has continued with testing of various combinations of real time PCR primers and probes with enrichment cultures in addition to immunological methods of detection of various serogroups of Salmonella when co-cultured under standard enrichment conditions. We have extended a survey for the prevalence of Salmonella in California by testing 1000 samples from various animals, and determined the antibiotic resistance of isolates. Concerning our studies of norovirus biology on produce, we have shown that virus binding to lettuce or raspberries was enhanced under acidic conditions mainly due to the interaction of electric charges, suggesting that acidic wash should be avoided for the removal of norovirus from contaminated produce. Accomplishments 01 Cell individuality in E. coli O157:H7 with relevance to its epidemiology Curli fimbriae are important in host and plant colonization, biofilm formation, and induction of inflammatory response. ARS researchers in Albany, CA, observed large E. coli O157:H7inter- and intra-strain variations in the production of curli fimbriae. Curli variants derived from the same pathogenic strain displayed marked differences in their response to environmental and host-related stresses. Whereas, curli- producing variants were more fit under nutrient limitation, curli- deficient variants were more resistant to acidic pH such as that in the human stomach. DNA sequence analysis indicated that a genetic switch may underlie this phenomenon. These results provide insight into the evoluti of the pathogen and are critical to understanding the survival strategie of the pathogen during its cycling between host and nonhost environments and its epidemiology. 02 Aerosol transport of bacteria from concentrated animal feeding operation Aerosols have been suspected to transport food pathogens and contaminat fruits and vegetables grown in close proximity of concentrated animal feeding operations. However, studies investigating such transport are scarce, and science-based guidelines for the produce industry regarding the safe distance between produce fields and processing plants, and anim operations are lacking. ARS researchers in Albany, CA, used gene sequencing methods to characterize bacterial communities in manure and a samples collected from dairies in Central and Coastal valleys of California. Whereas known enteric pathogens were not detected in aerosol samples, bacterial species originating from cows at the dairy operation the vicinity were identified. Thus, bacteria specific to each dairy may used as tracers for detecting the source of pathogens that are transport to produce or fruit crops from feedlots or dairies. This suggests a new strategy for trace back epidemiological investigations by regulatory agencies and for quality testing by the produce industry.

Impacts
(N/A)

Publications