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Published on: February 1, 2014
Enumeration of viable Escherichia coli by real-time PCR with propidium monoazide
1Hachinohe National College of Technology, Tamonoki, Hachinohe, Japan.
Abstract:
A photo-inducible DNA-binding dye, propidium monoazide (PMA), was used to distinguish viable and dead Escherichia coli cells. Microscopic observations using a combination of the dyes 4',6-diamidino-2-phenylindole and PMA indicated that PMA stained only dead cells, with membrane damage, red. Mixtures of viable and heat-treated E. coli cells were subjected to real-time polymerase chain reaction (PCR) with PMA treatment. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells in the mixtures of viable and heat-treated cells, as long as the ratio of dead cells to viable cells was no greater than 10. In the wastewater treatment plants, total, viable and culturable E. coli were enumerated by real-time PCR, real-time PCR coupled with PMA treatment and the most probable number method using EC-MUG medium, respectively. The concentrations of viable E. coli in the wastewater treatment plants were much higher than those of culturable cells. In addition, viable cells were even more chlorine resistant than culturable ones.
Insights
Propidium monoazide (PMA) effectively distinguishes viable and dead Escherichia coli. This method accurately quantifies viable E. coli in wastewater, revealing higher concentrations than culturable cells.
Area of Science:
- Microbiology
- Molecular Biology
- Environmental Science
Background:
- Distinguishing viable from dead bacteria is crucial for environmental monitoring.
- Traditional methods may underestimate bacterial populations in complex samples like wastewater.
Purpose of the Study:
- To evaluate propidium monoazide (PMA) combined with real-time PCR for enumerating viable Escherichia coli.
- To compare viable E. coli concentrations with culturable E. coli in wastewater treatment plants.
Main Methods:
- Utilized propidium monoazide (PMA) and 4',6-diamidino-2-phenylindole (DAPI) for microscopic differentiation.
- Applied PMA-treated real-time PCR to quantify viable E. coli in mixtures and wastewater samples.
- Compared PMA-real-time PCR with the most probable number (MPN) method.
Main Results:
- PMA selectively stained membrane-damaged (dead) E. coli cells.
- A linear relationship was observed between viable cell counts and real-time PCR threshold cycles for PMA-treated cells (dead:viable ratio ≤ 10).
- Viable E. coli concentrations in wastewater were significantly higher than culturable E. coli, and viable cells showed greater chlorine resistance.
Conclusions:
- PMA combined with real-time PCR is a reliable method for quantifying viable E. coli.
- Wastewater contains substantial populations of non-culturable but viable E. coli.
- Viable E. coli poses a potentially underestimated public health risk due to its resilience.
