P3-10 Evaluation of Immunomagnetic Separation Techniques for the Recovery and Re-growth Potential of Escherichia coli O157:H7 in Finished Composts from Manure, Biosolids, and Yardwaste Feedstocks

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Mary Theresa Callahan, U.S. Department of Agriculture-ARS, Beltsville, MD
Russell Reynnells, U.S. Department of Agriculture-BARC-EMFSL, Beltsville, MD
Cheryl Roberts, U.S. Department of Agriculture-ARS-EMFSL, Beltsville, MD
David Ingram, U.S. Department of Agriculture-ARS, Beltsville, MD
Patricia Millner, U.S. Department of Agriculture-ARS, Beltsville, MD
Manan Sharma, U.S. Department of Agriculture-ARS, Beltsville, MD
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 techniques may provide assays 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.

Methods: Twenty-nine USCC-STA certified compost samples were collected from across the U.S.  Three aliquots (400 g each) were inoculated with stx-2 positive E. coli O157:H7 at 101-102 CFU/g.  AR homogenized samples were incubated 5 h at 37°C.  DP samples were serially diluted and incubated 2 h at room temperature, then 42°C for 6 h.  All samples were incubated overnight at 4°C before addition of immunomagnetic beads.  AR 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 AR 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.