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

Abstract

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.

Laboratory and Pilot-Scale Dead-End Ultrafiltration Concentration of Sanitizer-Free and Chlorinated Lettuce Wash Water for Improved Detection of Escherichia coli O157:H7

“…Aliquots from each processing stage were reserved for subsequent microbial analysis and E. coli 0157:H7 immunoassay detection (details described below). The detection assay chosen was an ECL immunoassay that was previously evaluated in our laboratory with E. coli 0157:H7-spiked spinach rinsate and produce washes (26,29). Finally, triplicate 40-liter lettuce wash samples containing 10, 30, or 100 ppm of available chorine were evaluated using Michigan State University's pilotscale leafy green processing line.…”

“…The different particle sizes for the homogenized lettuce were not directly determined. However, based on the use of different sizes of sieving pans in our previously published study (29), the particle sizes can be estimated. The 125-pm mesh and 5-pm mesh were used to remove large particulates of >5 pm that would clog the PMACS filter.…”

“…Flow rate and pre-and postfilter pressures were monitored during filtration. The PMACS automated recovery cycle was then initiated to recover the material collected on the filter: 0.01% sodium polyphosphate in 0.1 M sodium phosphate buffer (PB-NaPP) (23,27,29) was pumped into the filter in the direction opposite to the forward flow (back flushed) to generate approximately 400 ml of retentate. The retentate was collected in a sterile polystyrene bottle and measured using a sterile 500-ml graduated cylinder.…”

“…In other studies, 1-to 10-liter aliquots of liquid food matrices were processed using tangential HFUF before analysis (24,32). Previous work conducted in our laboratory demonstrated the effectiveness of dead-end (single-pass, no recirculation) HFUF for concentrating E. coli 0157:H7 from 40-liter aliquots of sieved lettuce and spinach wash water (29).…”

“…Hence, the goal of this study was to evaluate an automated system based on dead-end ultrafil tration, the Portable Multi-use Automated Concentration System (PMACS) (27), for concentrating E. coli 0157:H7 from large-volume samples of inoculated simulated com mercial lettuce wash water containing a 5% organic load (wt/vol; blended iceberg lettuce). The work included establishing the PMACS performance parameters and evaluating its concentration efficacy using standard micro biological procedures and an electrochemiluminescence (ECL) immunoassay (23,29). Following these laboratory characterization studies, the combined concentration and detection method was assessed in pilot-scale tests at Michigan State University using their leafy green processing line.…”