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Efficacy of Propidium Monoazide on Quantitative Real-Time PCR-Based Enumeration of Staphylococcus aureus Live Cells

Hajime Takahashi1, Ryota Kasuga1, Satoko Miya1

  • 1Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan.

Journal of Food Protection
|October 12, 2018
PubMed

Insights

Propidium monoazide quantitative real-time PCR (PMA-qPCR) accurately enumerates live bacteria after treatment with certain sanitizers like ethanol but fails with others. Its effectiveness for live bacterial enumeration depends heavily on the specific sanitizer used.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Quantitative real-time PCR (qPCR) combined with propidium monoazide (PMA) is used to differentiate live from dead bacterial cells by excluding DNA from dead cells.
  • Previous studies have focused on PMA's effectiveness with heat-treated bacteria, with limited research on sanitizer-treated bacteria.

Purpose of the Study:

  • To evaluate the accuracy of PMA-qPCR for enumerating live bacteria (Staphylococcus aureus) treated with various common sanitizers.
  • To compare PMA-qPCR performance across different live/dead cell ratios and sanitizer types.

Main Methods:

  • Preparation of dead Staphylococcus aureus cells using six different sanitizers: ethanol, isopropyl alcohol, benzalkonium chloride, sodium hypochlorite, hydrogen peroxide, and nisin.
  • Mixing treated dead cells with live bacterial cultures at various ratios.
  • Enumeration of live bacteria using PMA-qPCR and comparison with traditional plate counts.

Main Results:

  • PMA-qPCR accurately enumerated live bacteria (<0.5 CFU/500 μL difference from plate counts) for treatments with ethanol, isopropyl alcohol, and nisin.
  • Accurate enumeration was achieved for live/dead ratios from 10/1 to 0.01/1 (ethanol, isopropyl alcohol) and 10/1 to 0.1/1 (nisin).
  • PMA-qPCR failed to accurately enumerate live cells treated with benzalkonium chloride and hydrogen peroxide. qPCR without PMA showed accuracy comparable to plate counts for sodium hypochlorite-treated cells.

Conclusions:

  • The efficacy of PMA-qPCR for enumerating live bacteria is sanitizer-dependent and not universally applicable.
  • Researchers should carefully consider the type of treatment applied to bacterial cells when deciding to use PMA-qPCR for live cell enumeration.
Keywords:
EnumerationEthanolPropidium monoazideQuantitative real-time PCRSanitizerStaphylococcus aureus

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