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P3-58 Evaluation of Enriched Microflora of Raw Milk Cheese Spiked with E. coli O157:H7 and E. coli O103 Using Next-Generation Sequencing Technology

Wednesday, August 3, 2016
America's Center - St. Louis
Tina Pfefer, U.S. Food and Drug Administration, College Park, MD
Julie Kase, U.S. Food and Drug Administration, College Park, MD
Padmini Ramachandran, U.S. Food and Drug Administration, College Park, MD
James White, Resphera Biosciences, LLC, Baltimore, MD
Andrea Ottesen, U.S. Food and Drug Administration, College Park, MD
Introduction: Between 1998 and 2014, there were 38 outbreaks in the U.S. directly linked to unpasteurized cheese resulting in 804 illnesses, 172 hospitalizations, and 3 deaths. Enrichment broths are an essential component of detection methods used to isolate bacterial pathogens from foods implicated in outbreaks. A better understanding of enrichment dynamics can inform efforts to optimize enrichment broths to increase levels of the target microbe while decreasing interfering background microflora.

Purpose: Describe microflora diversity during enrichment of raw milk cheese in order to improve detection and isolation of Escherichia coli from this food matrix. 

Methods: Raw milk cheese was spiked with either Escherichia coli O157 or O103 and enriched overnight using R&F Escherichia coli O157:H7 Enrichment Broth or mBPWp broth. High-throughput 16S rRNA amplicon sequencing resulted in a survey of bacterial species for each treatment.  Reads were preprocessed with QIIME and taxonomic assignment was performed using Resphera Insight. Reads given multiple calls (e.g., identified as E. coli, S. sonnei and S. boydii) were noted as “possible.”

Results: All portions before enrichment were dominated by Lactococcus lactis at 96% and above. After enrichment, L. lactis and total possible E. coli (E. coli and possible E. coli) dominated all samples at 24 to 68% and 16 to 68%, respectively, followed by Enterobacter (4 to 16%) and Shigella (1 to 7%). Next-generation sequencing tools were able to detect and roughly measure E. coli introduced into cheese, with significant differences noted in E. coli as well as total possible E. coli between cheese spiked with E. coli O103 enriched with mBPWp and non-spiked cheese enriched with mBPWp.

Significance: This study leverages NGS technologies to describe microbial diversity of raw milk cheeses before and during enrichment. These data will be used to improve precision of broths that detect and isolate E. coli from this important food matrix.

Back to: Poster Session 3 – Antimicrobials, Laboratory and Detection Methods, Microbial Food Spoilage, Modeling and Risk Assessment, Retail and Food Service Safety and Sanitation
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