Summary of Awards to Date

Pyrosequencing and community profiling for risk assessment in leafy greens.

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Sep. 1, 2008 - Aug. 31, 2010

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Andrew K. Benson, Ph.D.
University of Nebraska


Walter, J., Hutkins, R.


The indicator organism concept is based on use of a given microbial species or group of species as an indicator of the relative risk that a sample is contaminated with pathogenic species. Indicator organisms such as generic E. coli have traditionally been used to assess the relative microbiological risk that food or water is contaminated with fecal matter since the mammalian gastrointestinal tract is the primary habitat of this species. One of the problems with this approach, however, is the fact that the species E. coli is only a very minor fraction of the total mass of microorganisms present in the mammalian GI tract, making up only less than 0.1% of the total population. While other more abundant species would certainly provide more sensitivity, most currently accepted microbiological methods rely on culturing of the organisms from the sample and the vast majority of species in the GI tract are difficult, if not impossible to culture routinely. We therefore propose to test the validity of a new non-culture-based approach which combines the powerful PCR-based amplification of target genes from each microbe in a sample, coupled with massively parallel DNA sequencing of hundreds of thousands of these target genes to enumerate the different species that are present. This approach therefore provides a profile of the entire community of microorganisms in a sample, allowing the analyst to determine if the community of microorganisms present is comprised of those that are expected from that type of food or if the community contains species that are characteristic of other environment such as feces or soil. The experiments in this proposal are designed to test two criterion that must be met in order to validate this community profiling concept. First, the microbial community of a given commodity must be predictable and distinguishable from fecal and soil communities. Second, the approach must be able to detect the presence of species from feces when present. These criteria will be evaluated using spinach as an experimental model with plants derived from different cultivars, climates, and growing seasons.