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

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

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts

Growth curve of <i>E</i>.<i>coli</i> O157.

<p>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.</p

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

<p>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.</p

Linearity of the RAPID-B <i>E.coli</i> O157 assay.

<p>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.</p

Flow cytometer events for a negative control sample carried through the pathogen specific protocol.

<p>The numbers of events in the scatter and fluorescence regions are given for a negative control spinach sample processed through the pathogen specific protocol; note the reduction in both particulate matter and fluorescence interference as the sample moves through each of the steps.</p

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

<p>The sample is <i>E. coli</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094254#pone-0094254-g002" target="_blank">Figure 2</a>.</p

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

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

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

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