Laboratory and pilot-scale dead-end ultrafiltration concentration of sanitizer-free and chlorinated lettuce wash water for improved detection of Escherichia coli O157:H7.

An automated dead-end (single pass, no recirculation) ultrafiltration device, the Portable Multi-use Automated Concentration System (PMACS), was evaluated as a means to concentrate Escherichia coli O157:H7 from 40 liters of simulated commercial lettuce wash water. The assessment included generating, sieving, and concentrating sanitizer-free lettuce wash water, either uninoculated or inoculated with green fluorescent protein-transformed E. coli O157:H7 at a high (1.00 log CFU/ml) or low (-1.00 log CFU/ml) concentration. Cells collected within the filters were recovered in approximately 400 ml of buffer to create lettuce wash retentates. The extent of concentration was determined by viable plate counts using a medium selective for the transformed E. coli O157:H7. The samples were qualitatively analyzed for E. coli O157:H7 according to the U. S. Food and Drug Administration Bacteriological Analytical Manual enrichment method and with an electrochemiluminescence immunoassay. This concentration method was then evaluated in a pilot-scale production line at Michigan State University using chlorinated (100, 30, and 10 ppm of available chlorine) lettuce wash water. The total PMACS processing times were 82 ± 6 and 65 ± 5 min for sanitizer-free and chlorinated washes, respectively. Overall, E. coli O157:H7 populations were approximately 2 log higher in retentates than in unconcentrated lettuce wash samples. The higher E. coli O157:H7 levels in the retentates enabled cultural and electrochemiluminescence immunoassay detection in some samples when the corresponding lettuce wash samples were negative. When combined with standard and rapid detection methods, the PMACS concentration method may provide a means to enhance pathogen monitoring of produce wash water.

Automated dead-end ultrafiltration for concentration and recovery of total coliform bacteria and laboratory-spiked Escherichia coli O157:H7 from 50-liter produce washes to enhance detection by an electrochemiluminescence immunoassay.

An automated concentration system (ACS) based on dead-end ultrafiltration was used in this study to concentrate bacteria, including Escherichia coli O157:H7, from 50-liter produce washes (PWs, sieved produce wash). Cells trapped in the filters were recovered in approximately 400 ml of buffer to create PW retentates (PWRs). Extent of concentration was determined by analyzing PWs and PWRs for total coliform bacteria and E. coli O157:H7 using standard methods. In addition, an electrochemiluminescence immunoassay was evaluated for detection of E. coli O157:H7 in spiked PWs and PWRs to demonstrate usefulness of the ACS for same-day detection. The levels of total coliform bacteria and E. coli O157:H7 in PWRs were higher than those in PWs by 1.85 ± 0.41 log most probable number per 100 ml and 1.82 ± 0.24 log CFU/ml, respectively. Electrochemiluminescence detection of E. coli O157:H7 was accomplished within 2 h using ACS concentration of lettuce and spinach wash water artificially spiked with the pathogen at levels as low as 0.36 log CFU/ml and 1.39 log CFU/ml, respectively. Detection of E. coli O157:H7 at -0.93 ± 0.15 log CFU/ml in lettuce wash occurred within approximately 6 h when a 4-h enrichment step was added to the procedure. Use of dead-end ultrafiltration increased bacterial concentrations in PWR and allowed same-day detection of low levels of E. coli O157:H7 in PW. This concentration system could be useful to improve the sensitivity of current rapid methods for detection of low levels of foodborne pathogens in PW water.

Dead-end ultrafiltration concentration and IMS/ATP-bioluminescence detection of Escherichia coli O157:H7 in recreational water and produce wash.

The purpose of this study was to develop a detection method for viable E. coli O157:H7 in fresh produce and recreational water. The method was evaluated using eight samples of produce wash and recreational water with or without spiked E. coli O157:H7 at ≤10(2) CFU·ml(-1) and concentrated using dead-end ultrafiltration (DEUF) to produce primary and secondary retentates. Fifty-four matrix replicates of undiluted secondary retentates or dilutions (1:2 or 1:10 in buffer) were evaluated using an IMS/ATP bioluminescence assay (IMS/ATP). Combining primary and secondary DEUF yielded a 2-4 log(10) increase in E. coli O157:H7 concentrations in spiked samples and resulted in signal-to-noise ratios 2-219 fold higher than controls, depending on the sample type. DEUF increased the concentration of E. coli O157:H7 to within the detectable limits of IMS/ATP. The combined assay provided detection of viable E. coli O157:H7 in produce and recreational water. Accurate detection of microbial pathogens using DEUF and IMS/ATP could reduce disease outbreaks from contaminated water sources and food products.