P2-115 Development of a New Strategy for Mapping Microbiomes of Food Manufacturing Facilities

Tuesday, July 24, 2012
Exhibit Hall (Rhode Island Convention Center)
Brad Ziebell, ConAgra Foods, Omaha, NE
Stefanie Gilbreth, ConAgra Foods, Omaha, NE
Andrew Benson, University of Nebraska-Lincoln, Lincoln, NE
Kelly Dawson, ConAgra Foods, Omaha, NE
Joseph Nietfeldt, University of Nebraska-Lincoln, Lincoln, NE
Ryan Legge, University of Nebraska-Lincoln, Lincoln, NE
Introduction:   Food manufacturing facilities are collections of microbial ecosystems with microbial members in harborage sites in equipment and the environment converging from diverse origins such as ingredients, air, dust, and employees.   New culture-independent tools such as deep pyrosequencing provide a means to identify and quantify complex communities of microorganisms in a production setting, providing insight into the origins of these communities. 

Purpose:   One of the most significant issues in application of culture-independent PCR-based approaches is determining viability of taxa that are detected. In this study, a method was established and used to identify composition of viable microbes in communities from pre-kill portions of a thermal processing line. 

Methods:   Samples from ingredients, environmental swabs, and finished product were collected from a manufacturing facility.  DNA was extracted directly from one portion (non-enriched) and another portion was inoculated into broth incubated at three different temperatures (enriched).  DNA content from extracts of the “non-enriched” and “enriched” samples was measured by quantitative Real-Time PCR (qPCR) using the 16S ribosomal RNA gene (16S rDNA) as a target.  Growth was measured independently by aerobic plate counts (APC) and by deep pyrosequencing of 16S rDNA amplicons from these same samples.    

Results:   At the optimal enrichment temperature of 35 °C,   samples having enriched/unenriched 16S rDNA qPCR ratios >10 had viable cells as measured by APC while samples with ratios <10 had APCs ranging from undetectable to 100 CFU, establishing 100 CFU as a minimum viable cell threshold. As with APC, deep pyrosequencing of samples with enriched/unenriched qPCR ratios >10 were consistent with viable cells being present, showing large changes in the proportions of taxa detected after enrichment. Compositional analyses of sequences from sequential samples of a production line by linear discriminant analysis were consistent with viable organisms from ingredients and the environment converging to form communities on equipment prior to the thermal process step.  

Significance:   Optimization of this sample preparation and sample differentiation is an important first step in application of deep pyrosequencing to study the microbial ecology of food manufacturing facilities. We have used this method along with deep pyrosequencing to define composition of viable microbial communities in a manufacturing plant.