Recirculating Immunomagnetic Separation and Optimal Enrichment Conditions for Enhanced Detection and Recovery of Low Levels of Escherichia coli O157:H7 from Fresh Leafy Produce and Surface Water

Abstract

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (C(T) values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42 degrees C for 5 h provided better detection than enrichment at 37 degrees C. Extended incubation of surface water for 20 h at 42 degrees C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

Same-Day Detection ofEscherichia coliO157:H7 from Spinach by Using Electrochemiluminescent and Cytometric Bead Array Biosensors

“…Separate duplicate rinse aliquots were processed exactly according to the FDA-BAM procedure, which confirmed that E. coli O157:H7 was absent from control leaves and present in spiked samples. Viable target counts obtained from FDA-BAM enrichments at the 7-h time point were 2 to 3 logs lower than those in SOP enrichments (2.4 ϫ 10 2 to 4.1 ϫ 10 3 CFU/ml), an observation that is consistent with previous reports (23,25). ECL signals and S/N ratios increased over baseline after 3 to 4 h of enrichment and continued to increase over the experimental time course (not shown).…”

“…Enrichment at 42°C without antibiotics served as the standard operating procedure (SOP) for all assays, except where direct analyses according to the FDA-BAM procedure are specified. The SOP conditions were chosen for short-term enrichments based on published reports indicating enhanced E. coli O157:H7 recovery at 42°C compared to that at 37°C (23,25). Aliquots of enrichment cultures were taken at all indicated time points (hourly), plated on CTSMAC, and processed for analysis using the ECL or IMS-cytometric bead array (IMS-CBA) assays.…”

The use of essential oils for postharvest decay control. A review.

“…[43,47,[60][61][62] New methods to make an earlier diagnostics of potential foodborne illness agents, such as E. coli O157 : H7 from fresh leafy produce and surface water, [63] can also help to avoid further consequences.…”

Recovery and Detection of Escherichia coli O157:H7 in Surface Water, Using Ultrafiltration and Real-Time PCR

“…In order to simulate normal environmental conditions, this study focused on the recovery of low organism seed levels from 40-liter surface water samples. Many studies have reported successful recovery of EHEC O157:H7 by the IMS/culture method in surface water, but these studies typically limited the analyses to sample volumes of ϳ100 ml of water and reported limits of detection ranging from 1 to 6 CFU/100 ml (10,11,18). One study in The Netherlands processed 1-liter samples and reported a limit of detection of 4 MPN/liter (7).…”