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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL ECOLOGY AND SAFETY OF FRESH PRODUCE

Location: Environmental Microbial and Food Safety Laboratory

Title: Recovery of Escherichia coli O157:H7 by immunomagnetic separation techniques and potential for regrowth in finished composts

Authors
item Callahan, Mary Theresa
item Reynnells, Russell
item Roberts, Cheryl
item Handy, Eric
item Ingram, David
item Millner, Patricia
item Sharma, Manan

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 22, 2013
Publication Date: N/A

Technical Abstract: Introduction: Mature, finished compost made from various feedstocks should undergo testing for the presence of Escherichia coli O157:H7 to ensure thermal destruction of the pathogen during composting. Immunomagnetic separation (IMS) –based methods may provide an assay which can be conducted within 24-48 h to determine presence or absence of E. coli O157:H7 in finished compost. Purpose: 1) To compare the ability of two IMS methods – automated recirculating (AR) and direct plating (DP) - to recover E. coli O157:H7 from compost samples containing biosolids, manure, or yardwastes; 2) to determine the ability of compost samples to support regrowth of E. coli O157:H7 in compost. Materials and Methods: Twenty-nine USCC-STA certified compost samples were collected from across the U.S. Aliquots (400g) of each sample were inoculated with stx-2 positive E. coli O157:H7 at 10^1 to 10^2 cfu/g. Homogenized samples were incubated 5 h at 37oC for DP, then serially diluted and incubated 2 h at room temperature, then 42oC for 6 h. For both methods, samples were incubated overnight at 4oC before addition of immunomagnetic beads. Sample recirculation (30 min) and DNA purification from immunomagnetic beads, was followed by real-time PCR targeting the stx2 gene. For DP, immunomagnetic beads were added to diluted samples, mixed, removed, and then plated onto chromagarO157. Results: The recirculation and DP methods both recovered E. coli O157:H7 from 30/ 30 (100%) samples. PCR detection of E. coli O157:H7 was greatly enhanced by the removal of polyphenols and humic acids from DNA samples. Regrowth of E. coli O157:H7 in compost varied based on feedstock. Significance: IMS recovery, coupled with either direct plating or real-time PCR detection, is an efficient method to recover E. coli O157:H7 from finished compost within 24 – 48 h. Regrowth potential of E. coli O157:H7 in finished composts is affected by physicochemical parameters of compost.

Last Modified: 10/22/2014
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