1. Science-based policy for the food industry: Embracing the BEST Science that’s out there Andrew K. Benson Professor, Department of Food Science Director, Printed Microarray Core Facility University of Nebraska Powered by

2. Microbes exist in complex ecosystems within different food matrices

3. Autochonous Allocthonous Uncontaminated sample Contaminated sample

4. Indicator organism concept 1.Easy to culture 2.Present in all mammalian feces 3.Durable in the environment 4.Associated with presence of pathogens *E. coli is the most common indicator used Problems associated with use of indicators 1.Relatively low abundance in feces a.E. coli present at 10 6 /gram b.Others (e.g. Bacteriodes, Clostridia) present at 10 13 /gram 2.Some species are present in environment (problem with coliforms) 3.Correlation with pathogens questionable: a.Instances of “indicator free” foods causing outbreaks b.Poor correlation of indicators with actual pathogens

5. Eckburg et al. Science. 2005. 308:1635-1637 Taxonomic group to which E. coli belongs Deep sequencing surveys confirm that E. coli is a minor member Of the human fecal microbiota

6. Do the math: 1,000 Kg production lot contaminated with 1g of feces Assumptions: E. coli present at 10 6 per gram of feces Bacteroides present at 10 13 per gram If the contamination is homogenously distributed, each gram of the 1,000,000 g (1000 Kg) sample would contain: 1 E. coli 1,000,000 Bacteroides How many grams must be sampled to ensure detection of E. coli? at least 10 grams, 100 grams would be better How many grams must be sampled to ensure detection of Bacteriodes? 10-100 mg

7. Culture-based methods count only the organisms that can be grown in vitro… And we can culture 10% (at best) or species From any environment

8. Non-culture-based methods that allow us to survey composition of the community

9. Extract nucleic acids Specific PCR tests for individual species 1.Effective for specific pathogens 2.Back to the same problem for indicators Community profiling 1.Use of 16S rDNA as a target 2.Profiles composition of entire community 3.Able to identify allocthonous species 4.Does the ecosystem contain fecal flora?

10. Extract nucleic acids PCR amplify 16S rDNA from all bacteria Attach to microspheres PCR amplify on beads Deposit beads into picotitre plates Massively parallel Pyrosequencing: The next generation of DNA sequencing

11. Picotitre plate—single bead per well, ~1 million wells Parallel sequencing of all 1 million wells ***At least 400,000 individual sequences…this number is rapidly growing Will be 1 Million reads per run by 2009

12. 0 10 100 1000 Species Samples 1234....1234.... Relative abundance Looking at the output

13. Uncontaminated samples contaminated samples Allocthonous species or fecal species

14. Indicator organism or community profile? It’s time we step up to where science is headed