An integrated flow cytometry-based system for real-time, high sensitivity bacterial detection and identification

Abstract

Foodborne illnesses occur in both industrialized and developing countries, and may be increasing due to rapidly evolving food production practices. Yet some primary tools used to assess food safety are decades, if not centuries, old. To improve the time to result for food safety assessment a sensitive flow cytometer based system to detect microbial contamination was developed. By eliminating background fluorescence and improving signal to noise the assays accurately measure bacterial load or specifically identify pathogens. These assays provide results in minutes or, if sensitivity to one cell in a complex matrix is required, after several hours enrichment. Conventional assessments of food safety require 48 to 56 hours. The assays described within are linear over 5 orders of magnitude with results identical to culture plates, and report live and dead microorganisms. This system offers a powerful approach to real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

Figure 1. Photograph of the Vivione Biosciences 9013 Flow Cytometer.

Figure 2. Flow cytometer gate logic for RAPID-B assays.

The flow of information is indicated by the arrows. LS1 and LS2 represent standard flow cytometry scatter channels side and forward scatter, respectively. FL1, FL2, and FL3 represent standard green, yellow, and red fluorescence flow cytometry channels, respectively.

Figure 3. Screen capture from the 9013 flow cytometer showing the flow of information.

The sample is E. coli O157. Information starts at the scatter plot (upper left) and then passes through gates that are a combination of scatter and fluorescence regions to the final target region in the fluorescence plot at the bottom right. The count of events progresses from 7011 in the left upper panel to 6814 in the final counting region. The order of the panels corresponds to the order of gating logic in Figure 2.

Figure 4. Fluorescent excitation and emission spectra of fluorescein isothiocyanate.

Figure 5. Effects of background reduction.

Forward versus side scatter plots (left panels) and Fluorescence (FL3 vs. FL1) intensity plots (right panels) of the RAPID-B Pathogen Specific (PS) assay for E. coli O157:H7 stx1+ and stx2+ (ATCC 43895). A. Sterile spinach sample blank (25 g) B. Spinach sample (25 g) spiked with trace (single digits) of the E. coli O157 ATCC 43888 strain. Both samples were incubated in 75 ml BHI for 6 hr, subjected to phloxine photobleaching, followed by Percoll gradient centrifugation.

Figure 6. Time to results (TTR) for both RAPID-B and FDA BAM pathogen contamination assays.

The sequence of steps in the RAPID-B Total Plate Count Assay and Pathogen-Specific Assay are contrasted to those in the FDA BAM 4a assay.

Figure 7. Representative flow cytometry data for RAPID-B Total Plate Count assay.

Scatter (left panels) and fluorescence intensity (right panels) plots of the RAPID-B Total Plate Count (TPC) assay for Ralstonia picketti. A. Non-stressed Ralstonia sample (111,607 live, 558 injured (0.5%)). B. Heat stressed Ralstonia sample, events above the counting region due to PI penetrating damaged cell walls (82,081 live, 6894 injured (8.4%)).

Figure 8. Representative flow cytometry data for RAPID-B Pathogen Specific assay.

Scatter (left panels) and fluorescence intensity (right panels) plots of the RAPID-B Pathogen Specific (PS) assay for E. coli O157:H7 stx1+ and stx2+ (ATCC 43895); results from a spinach validation study. A. Positive spinach sample (368 live E. coli O157 cells, 48 dead). B. Negative spinach sample (4 events; a threshold of 6 events was used for blanks based on historical data).

Figure 9. Linearity of the RAPID-B E.coli O157 assay.

The linearity of the RAPID-B E.coli O157 assay is demonstrated and compared with 2 different types of culture plates. The bacterial isolate used was Arkansas Department of Health #3000372, EHEC E.coli O157:H7. Each data point for the plate count agar (PCA) and Sorbitol-MacConkey Agar (SMAC) is an average of 6 plate counts.

Figure 10. Growth curve of E.coli O157.

As measured by RAPID-B (real time) and culture plates (historical). Two RAPID-B measurements were averaged at each time point for the RAPID-B curve, and two PCA culture plates are averaged for the culture plate curve.