Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium monoazide

Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique's inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not an appropriate method for discriminating between live and dead cells to enumerate Legionella for regulatory purposes.

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Evaluation of viability of E. coli O157: H7 on chlorine and lactic acid treated spinach leaves using combined propidium monoazide staining and real-time PCR

LWT ◽

10.1016/j.lwt.2020.109259 ◽

2020 ◽

Vol 125 ◽

pp. 109259

Author(s):

Vijay Singh Chhetri ◽

Yu Han ◽

Marlene E. Janes ◽

Achyut Adhikari

Keyword(s):

Lactic Acid ◽

Real Time ◽

Real Time Pcr ◽

Propidium Monoazide ◽

E Coli ◽

Spinach Leaves

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Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay

Plant Disease ◽

10.1094/pdis-11-19-2397-re ◽

2020 ◽

Vol 104 (8) ◽

pp. 2225-2232

Author(s):

A-li Chai ◽

Hai-yan Ben ◽

Wei-tao Guo ◽

Yan-xia Shi ◽

Xue-wen Xie ◽

...

Keyword(s):

Real Time ◽

Pseudomonas Syringae ◽

Real Time Pcr ◽

Light Exposure ◽

Qpcr Assay ◽

Bacterial Suspension ◽

Propidium Monoazide ◽

Chromosomal Dna ◽

Tomato Seed ◽

Viable Cells

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 μmol liter−1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml−1 and 11.86 CFU g−1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g−1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

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