Progress 10/01/10 to 09/30/15
Outputs
Target Audience:Our primary target audience is other scientists working in the area of food safety and fresh produce. We also are interested in reaching producers and regulators in the same field. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The work carried out in the last year has largely been the work of a single graduate student that graduated with a Masters degree in May of 2015. During the course of his research he learned to carry out microbiological experiments and both classical and next generation sequencing experiments. Much of the data analysis incldung the design of the pipelines that we currently use was done by him in the course of his thesis research. He has presented the results of his work at local and national meetings and is in the process of revising two manuscripts for publication. How have the results been disseminated to communities of interest?Results have been disseminated to the scientific, regulatory and industrial communities through presentations at national meetings that bring these groups together. One manuscript related to this work has been recently published and two more are in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The past 20 years have seen a striking increase in the number of cases of foodborne illness associated with eating fresh plant material. The reasons for this remain unclear, in part because we have limited knowledge of how human pathogenic bacteria associate with plants, whether or not they persist once they are there and how easily they are detected on plants when present. The current project has provided new information concerning the types of bacteria that naturally associate with plants, as well as how those bacteria influence our ability to easily detect human pathogens like Listeria monocytogenes when it is present on plant material. We have used modern DNA sequencing methods to sample the bacterial community present on romaine lettuce more deeply than has been done previously and have shown that it contains hundreds of genera of bacteria. Moreover, the community is quite different in different locations on the plant surface rather than uniformly distributed as is often assumed. We have also tested how this complex plant-associated bacterial community influences a classical set of microbiological assays designed to rapidly screen for the presence of Listeria in food samples. These assays were designed to work primarily with meat and dairy products and in those systems they perform quite well, falsely reporting the presence of Listeria only 5-10% of the time. In contrast, the plant associated bacterial community contains many species that can masquerade as Listeria in these classical assays. In fact, 90-95% of the bacteria that appear to be Listeria are in fact found in other bacterial genera entirely. While these assays are never taken to be definitive by themselves, the very high false positive rate found with lettuce samples greatly reduces the utility of the assays as an initial screen for Listeria. We've extended this research by beginning a detailed characterization of the genome sequences of some of the species that cause problems for these assays. This information will be used to develop more effective assays that can eliminate the false positive results due to these specific species. Specific Objectives Determine what bacterial organisms make up the "community" associated with fresh produce and how frequently this includes human pathogens or their relatives. Develop an understanding of the interactions that take place between the bacteria and the plant. This will involve identifying specific plant genes involved in response as well as genes required by the bacteria for growth and persistence in the plant. Determine if bacteria that are better able to survive and persist on plants are selected in natural environments. This might involve selection of naturally occurring mutations or potentially the horizontal transfer of genes from organisms better adapted to a plant-based lifestyle. For the first objective we have characterized the bacterial community associated with fresh produce in two ways - using next generation metagenomic sequence analysis and by characterizing members of the community that survive classical microbiological enrichment and selection assays designed to select for species in the genus Listeria. The sequencing assays produced two important results. First they demonstrated that the bacterial communities associated with lettuce are very complex, containing species from more than 300 genera of bacteria. While the majority of the species are from 20 or so genera, deep sequencing reveals that there are numerous other genera represented at low levels. The second major result is that the communities found on lettuce leaves are extremely variable. Comparing the community found on small sections of leaf show that even regions of leaf separated by just a few centimeters can contain numerous species that are unique. This is important because it emphasizes how critical it is to develop sampling strategies based on a better understanding of how the communities vary over the surface of the plant and to identify locations that are most likely to harbor human pathogenic bacteria. The classical microbiological assays revealed that there are numerous common bacterial community members that appear to be Listeria following enrichment and selective plating. This greatly reduces the utility of these assays as a preliminary screen, since they result in roughly 80% of the lettuce samples tested appearing to be positive for Listeria. To improve the utility of these assays we have sequenced the genomes of multiple strains from the genus Cellulomonas that generate false positives. We have screened these genomes to identify possible antibiotic targets that could be used to select against these in our Listeria assays. If this effort is successful we can repeat it with the other problem genera to create a selective assay with a much lower false positive rate. We unfortunately did not have the resources (either in personnel or research funding) to successfully pursue the second and third objectives of the proposal. Given the limited resources available, as well as the sources of funding, focusing on the first objective presented the best opportunity to generate valuable new findings.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Bach, C.E. (2015) INFLUENCE AND CHARACTERIZATION OF MICROBIAL CONTAMINANTS ASSOCIATED WITH THE FDA BAM METHOD USED TO DETECT LISTERIA MONOCYTOGENES FROM ROMAINE LETTUCE, M.S. Thesis, Purdue University.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Deering, A.J., D.R. Jack, R.E. Pruitt and L.J. Mauer (2015) Movement of Salmonella serovar Typhimurium and E. coli O157:H7 to Ripe Tomato Fruit Following Various Routes of Contamination. Microorganisms 3, 809-825. doi:10.3390/microorganisms3040809
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Our target audience is academics and research scientists working in the food safety field, as well as scientists working for regulatory agencies that formulate policy with respect to food safety sampling/testing. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? All of the experimental work and much of the data analysis has been conducted by two graduate students, one of whom should graduate with a Masters degree in the next semester. Both students have presented their work at local gatherings of the plant science community and one has presented the results of the Listeria project at the national IFT meeting in New Orleans. How have the results been disseminated to communities of interest? Results have been disseminated through poster presentations and oral presentations at local meetings and workshops, as well as at national meetings related to food safety. Three manuscripts describing the results of this project are in preparation. What do you plan to do during the next reporting period to accomplish the goals? During the next year we will expand the number of Listeria false positive genome sequences we have and complete the analysis of similarities and differences found in those genomes. We will also carry out a complete spatial charcaterization of the lettuce bacterial community on single leaves and attempt to identify consistent patterns in the spatial variation. Lastly we will also carry out experiments to examine how well human pathogenic bacteria can integrate into the existing bacterial community and whether variation in the efficiency of integration can be correlated with the presence or absence of specific bacterial species within the naturally occurring communities.
Impacts
What was accomplished under these goals?
We have continued to expand our bacterial community sequencing data for romaine lettuce to get better coverage of the entire community and identify those rare genera of bacteria that are present in some communities and not others. At the same time we have created sequence datasets for the bacterial communities found on the surface of small leaf disks isolated from different portions of the leaves. These datasets indicate that there can be a large degree of variation in community composition a few centimeters apart on the same leaf, especially in genera that represent relatively small fractions of the total community. We have also expanded our collection of bacteria isolated from romaine lettuce that appear to be Listeria based on selective enrichment and plating assays (Listeria false positives). We now believe we have a fairly complete collection (at least at the level of bacterial genera) and have begun sequencing complete genomes of several of these strains to try and identify common features that allow them to survive the Listeria selective media.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Evaluation of selective media used for the detection of Listeria monocytogenes from Romaine Lettuce. (2014) Christopher E. Bach, Amanda J. Deering, and Robert E. Pruitt. IFT National Meeting, New Orleans, LA.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Our primary target audience is other scientists that are working in the food safety field related to fresh produce. Secondarily we are interested in reaching producers, processors and regulatory officials inolved in the produce industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Virtually all of the experimental work has been carried out by a graduate student, who has received training in microbiology, molecular biology, next generation sequencing and bioinformatics. He has presented his research locally and will present it at a national meeting in the next year. How have the results been disseminated to communities of interest? Results have primarily been distributed to academic and industry professionals through talks and posters at scientific meetings and workshops. Manuscripts describing the results are in preparation. What do you plan to do during the next reporting period to accomplish the goals? Metagenomic analyses of the datasets that have been generated will be carried out to characterize how the bacterial community changes in response to sanitizers and storage as well as to map the variability of the community spatially over the leaf surface. Bacterial species isolated from lettuce that survive standard selective enrichment and plating for Listeria species will be further characterized at the taxonomic and genome sequence levels to try and identify common factors that might be used to select against them in selective isolation experiments. Experiments are also being prepared that will test how well E. coli, Salmonella and Listeria integrate into the naturally occurring bacterial community and whether this varies from one lettuce sample to another. If variation exists we will attempt to identify the bacterial species that are responsible for enhancing or restricting the growth of the human pathogenic bacteria.
Impacts
What was accomplished under these goals?
We have begun to generate a more complete set of metagenomic sequence data describing the microbial community found associated with romaine lettuce. This dataset will focus on distinguishing bacteria associated with the exterior of the leaf from those that are found within the interior portion of the plant. In addition, we have begun to characterize how different genera of bacteria are distributed over the surface of the leaf in order to determine how homogeneous the community is spatially. This information could be critical in determining the best approach to sample fresh produce for contamination with human pathogens. We have also begun to characterize how commonly Listeria monocytogenes is found in association with fresh produce. As a first step we have conducted some simple isolation experiments based on FDA recommended selective enrichment and plating. In these experiments virtually everything that passes these first screening tests is not in the genus Listeria, indicating that the standard primary screens used for this pathogen do not work effectively when applied to fresh produce communities. We are currently characterizing these “false-positive” bacteria further to determine if it would be possible to devise a better selective medium for use with plant derived samples.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Our efforts in this year of the project have been directed at improving our ability to carry out metagenomic analyses of plant associated bacterial communities as well as characterizing the ability of human pathogenic bacteria to invade and persist within those communities. In order to increase the quantity of data we can obtain in a single experiment we have spent a great deal of effort in adapting our 16S metagenomic sequencing tools to utilize Illumina's MiSeq platform (rather than the Roche 454 platform) and develop a pipeline that will analyze the data using either GAST or QIIME software. These tools have been used to characterize the microbial communities associated with lettuce and spinach and how those communities are altered following a commercial sanitization treatment and cold storage. These preliminary data have provided indications of what bacterial genera are important in reducing shelf life of these products as well as how sanitization can alter the growth rate of various genera during refrigerated storage. They have also provided indications that the composition of the lettuce and spinach communities is very different. These experiments have led to a collaborative arrangement with Dole Fresh Vegetables that allows us to obtain matched commercial lettuce and spinach samples as harvested and following commercial processing. In the coming year we plan to develop a fungal ITS based metagenomics system that will allow us to characterize the fungal members of these microbial communities in parallel with the bacterial communities. We have also continued to characterize the ability of E. coli O157:H7 and Salmonella to persist in various commercial tomato cultivars following contamination of seeds, soil or leaves. These experiments demonstrate that enterobacterial pathogens are able to persist in tomato from the seed all the way through fruit production. In the coming year we plan on using next generation sequencing technologies to quantitatively characterize the ability of these pathogens to penetrate and persist within the endogenous bacterial community. Results of this work have primarily been disseminated through scholarly publications and presentations at scientific/industrial conferences. PARTICIPANTS: Amber Furrer, undergraduate intern; Chris Bach, graduate student, Amanda Deering, collaborative research scientist; Lisa Mauer, collaborating faculty. We have a collaborative arrangement with Dole Fresh Vegetables to provide produce samples and limited funding. The project provides training opportunities for graduate and undergraduate students in microbiology, molecular biology, genetics, plant biology and bioinformatics. TARGET AUDIENCES: Other scientists and food industry professionals nationally and globally. Information concerning the ongoing project has primarily been disseminated through presentations at national and international meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Analysis of the data generated from our next generation sequencing experiments provides an important window on how the communities of bacteria found in association with fresh produce change during processing and storage. These changes are likely to be key players in food spoilage and an understanding of how processing affects this may lead to better methods to extend shelf life of fresh produce. Continued outbreaks of foodborne illness associated with fresh produce indicate that human pathogenic bacteria have the ability to enter and persist in bacterial communities found on fresh produce and our characterization of these endogenous communities is a first step in understanding the mechanisms that allow these interactions to take place. Experiments continue to indicate that associations between human pathogens and plants can extend through the life of the plant and these pathogens may be transmitted from one generation to another through seed contamination. Better characterization of the microbiome associated with commercial seeds is necessary to determine whether this constitutes a major source of contamination in outbreaks of foodborne illness.
Publications
- Deering, A.J., R.E. Pruitt, L.J. Mauer and B.L. Reuhs (2012) Examination of the internalization of Salmonella serovar Typhimurium in peanut, Arachis hypogaea, using immunocytochemical techniques. Food Research International, 45: 1037-1043. doi:10.1016/j.foodres.2011.01.061
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Most of our effort in the first year of this project has focused on the first two objectives. Experiments carried out under the first objective has been directed towards developing and improving the efficiency of our ability to generate useful sequence from the bacterial community associated with fresh produce. We can now carry out such experiments in a reasonable time frame (provided adequate funding is available) and have generated useful community data from several lettuce samples purchased at grocery stores and farmer's markets. These preliminary data suggest that there is a great deal of variability in the bacterial species associated with different lettuce samples and that members of the family Enterobacteriaceae are very commonly found associated with lettuce purchased by consumers. DNA sequence data also suggests that enterobacterial human pathogens can be detected in these samples at low frequency. At the same time we have been pursuing contacts within the fresh produce industry in order to obtain funding to carry out larger scale experiments as well as to obtain samples of commercially grown lettuce at various stages of processing to see how the bacterial community changes. Experiments related to the second objective have been directed at examining how readily human pathogenic bacteria get internalized into plant tissues via various routes of contamination. Secondary experiments have examined how far it is possible for bacteria to move in the plant once internalized and for what length of time the bacteria can persist. Results indicate that bacteria are particularly easily internalized if they are present when seeds germinate and that they can persist within the plant throughout the life-cycle. Future experiments will examine what factors in the plant, bacterium and environment influence the success with which the bacteria colonize the plant. Results of this work have primarily been disseminated through scholarly publications and presentations at scientific/industrial conferences. PARTICIPANTS: Tara Stapay, undergraduate intern; Amanda Deering, collaborative research scientist; Lisa Mauer, collaborating faculty; Brad Reuhs, collaborating faculty. The project provides training opportunities for graduate and undergraduate students in microbiology, molecular biology, genetics, plant biology and bioinformatics. TARGET AUDIENCES: Other scientists and food industry professionals nationally and globally. Information concerning the ongoing project has primarily been disseminated through presentations at national and international meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Data generated as part of this project suggest that human bacterial pathogens that come in contact with plants can become internalized relatively easily and persist for long periods of time after an initial contamination event. Given that it is also possible that these bacteria can pass via seed from parent to offspring, these results could have a major impact on food safety considerations for fresh produce. A recent outbreak of pathogenic E. coli in Europe is thought to have arisen from sprouts grown from contaminated seed imported from outside Europe. Based on our studies we would anticipate that there would be no sanitization method currently in use that could have rendered these sprouts safe for human consumption if they were being grown from contaminated seeds. Our sequencing experiments also suggest that human bacterial pathogens and their relatives are found in association with produce purchased by consumers with moderate frequency and that more experiments of this type are warranted to discover what factors influence these associations.
Publications
- Deering, A.J., R.E. Pruitt, L.J. Mauer and B.L. Reuhs (2011) Identification of the cellular location of internalized Escherichia coli O157:H7 in mung bean, Vigna radiata, using immunocytochemical techniques. Journal of Food Protection, 74, 1224-1230. doi: 10.4315/0362-028X.JFP-11-015
- Deering, A.J., L.J. Mauer and R.E. Pruitt (2011) Internalization of E. coli O157:H7 and Salmonella spp. in plants: A review. Food Research International, in press. doi: 10.1016/j.foodres.2011.06.058
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