J Korean Acad Conserv Dent.  2008 Nov;33(6):537-544. 10.5395/JKACD.2008.33.6.537.

Selective detection of viable Enterococcus faecalis using propidium monoazide in combination with real-time PCR

• Kim S
• Lee S
• Kim E
• Seo D
• Song Y
• Jung I

• KMID: 1428464
• DOI: http://doi.org/10.5395/JKACD.2008.33.6.537

Abstract

Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.

Keyword

E. faecalis; real-time PCR; propidium monoazide; chlorhexidine

MeSH Terms

Azides
Bacteria
Cell Count
Chlorhexidine
DNA
Enterococcus
Enterococcus faecalis
Polymerase Chain Reaction
Propidium
Real-Time Polymerase Chain Reaction
Azides
Chlorhexidine
DNA
Propidium

Figure

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  Figure 1 Ct value measured by real-time PCR after serial dilutions of E. faecalis (A. 105 dilution, B. 104 dilution, C. 103 dilution, D. 102 dilution, E. 10 dilution)

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  Figure 2 Effect of PMA on the amplification of different ratios of viable and dead E. faecalis (A. Table showing mixing ratios of viable and dead E. faecalis, B. Ct value of amplified genomic DNA shown as a function of the percentage of viable E. faecalis, C. Relative difference of viable E. faecalis measured by real-time PCR)

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  Figure 3 Correlation between relative difference measured by real-time PCR and log CFU by plate counting (group A. Relative difference of non-PMA treated E. faecalis at each optical density, group B. Relative difference of PMA treated E. faecalis at each optical density, group C. Log CFU by plate counting at each optical density)

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  Figure 4 Monitoring exposure of E. faecalis to increasing time periods of 2% chlorhexidine (A. Increase of Ct values as increasing exposure time of 2% CHX, B. Decrease of relative difference of viable cells as increasing exposure time of 2% CHX)

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