Thymol treatment of bacteria prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis aids in identifying certain bacteria at the subspecies level.

RATIONALE: The identification of bacteria based on mass spectra produced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become routine since its introduction in 1996. The major drawback is that bacterial patterns produced by MALDI are dependent on sample preparation prior to analysis. This results in poor reproducibility in identifying bacterial types and between laboratories. The need for a more broadly applicable and useful sample handling procedure is warranted. METHODS: Thymol was added to the suspension solvent of bacteria prior to MALDI analysis. The suspension solvent consisted of ethanol, water and TFA. The bacterium was added to the thymol suspension solvent and heated. An aliquot of the bacterial suspension was mixed directly with the matrix solution at a 9:1 ratio, matrix/bacteria solution, respectively. The mixture was then placed on the MALDI plate and allowed to air dry before MALDI analysis. RESULTS: The thymol method improved the quality of spectra and number of peaks when compared to other sample preparation procedures studied. The bacterium-identifying biomarkers assigned to four strains of E. coli were statistically 95% reproducible analyzed on three separate days. The thymol method successfully differentiated between the four E. coli strains. In addition, the thymol procedure could identify nine out of ten S. enterica serovars over a 3-day period and nine S. Typhimurium strains from the other ten serovars 90% of the time over the same period. CONCLUSIONS: The thymol method can identify certain bacteria at the sub-species level and yield reproducible results over time. It improves the quality of spectra by increasing the number of peaks when compared to the other sample preparation methods assessed in this study. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Photobleaching with phloxine B sensitizer to reduce food matrix interference for detection of Escherichia coli serotype O157:H7 in fresh spinach by flow cytometry.

A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.

Reduction of food matrix interference by a combination of sample preparation and multi-dimensional gating techniques to facilitate rapid, high sensitivity analysis for Escherichia coli serotype O157 by flow cytometry.

Escherichia coli serotype O157 strains, which may be found in foods, often produce enterohemorrhagic toxins. The research goal was to facilitate rapid, sensitive detection in foods of E. coli serotype O157 by flow cytometry. Sample preparation methods were developed for potential use in 15 foods. Combined with multi-dimensional gating, these methods decreased time-to-results (TTR) for determination of low-level contamination. They mitigated the effects of interfering food components, concentrated cells for analysis without growth or, when necessary, used short-term incubation. The results showed qualitative analysis that was equivalent to culture plating in accuracy and superior in sensitivity and speed. Preparation time was 10-30 min per sample and detection took 3-4 min. Culture growth, if required, took an additional 4-6 h. A protocol for raw spinach analysis, using 4 h culture incubation, was 94% correct with one false negative for a low level inoculation. Its projected limit-of-detection (LOD) was 1 viable cell per 25 g of spinach, based on an average of 28 counts detected after growth and an estimated counts-to-threshold (C/T) ratio of 1.3. The results suggested potential uses for regulatory screening and food industry process control.

Rapid Flow Cytometry Detection of a Single Viable Escherichia coli O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique.

Very low cell count detection of Escherichia coli O157:H7 in foods is critical, since an infective dose for this pathogen may be only 10 cells, and fewer still for vulnerable populations. A flow cytometer is able to detect and count individual cells of a target bacterium, in this case E. coli O157:H7. The challenge is to find the single cell in a complex matrix like raw spinach. To find that cell requires growing it as quickly as possible to a number sufficiently in excess of matrix background that identification is certain. The experimental design for this work was that of a U.S. Food and Drug Administration (FDA) In-House Level 3 validation executed in the technology's originating laboratory. Using non-selective enrichment broth, 6.5 h incubation at 42°C, centrifugation for target cell concentration, and a highly selective E. coli O157 fluorescent antibody tag, the cytometry method proved more sensitive than a reference regulatory method (p = 0.01) for detecting a single target cell, one E. coli O157:H7 cell, in 25 g of spinach. It counted that cell's daughters with at least 38× signal-to-noise ratio, analyzing 25 samples in total-time-to-results of 9 h.

Flow-Cytometry-Based Method to Detect Escherichia coli and Shigella Spp. Using 16S rRNA-Based Probe.

Detection of microbial contamination in foods before they go on to the market can help prevent the occurrence of foodborne illness outbreaks. Current methods for the detection of Escherichia coli are limited by time-consuming procedures, which include multiple culture incubation steps, and require several days to get results. This unit describes the development of an improved rapid flow-cytometry-based detection method that has greater sensitivity and specificity. This method requires less time-to-results (TTR) and can detect a small number of E. coli in the presence of large numbers of other bacteria. Clear step-by-step protocols for cell concentration determination, sample preparation, and flow cytometric analysis are provided. © 2017 by John Wiley & Sons, Inc.

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.

Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach.

The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed.