Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide

Abstract

Real-time PCR (qPCR) is a widely used technique in analysing environmental and clinical microbiological samples. However, its main limitation was its inability to discriminate between live and dead cells.Recently, propidium monoazide (PMA) together with qPCR has been used to overcome this problem, with good results for different bacterial species in different types of samples.Our objective was to implement this technique for analysing mortality in multi-species oral biofilms formed in vitro with five oral bacteria: Streptococcus oralis, Streptococcus gordonii, Veillonella parvula, Fusobacterium nucleatum and Prevotella intermedia. We also tested its effectiveness on biofilms treated with an antiseptic solution containing 0.07% w/w cetylpyridinium chloride (CPC).Standardisation of the qPCR-PMA method was performed on pure, heat-killed planktonic cultures of each species, detecting mortality higher than 4 log in S. oralis, S. gordonii and F. nucleatum and higher than 2 for V. parvula and P. intermedia. We obtained similar results for all species when using CPC.When we analysed biofilms with qPCR-PMA, we found that the mortality in the non-CPC treated multi-species biofilms was lower than 1 log for all species. After treatment with CPC, the viability reduction was higher than 4 log in S. oralis and S. gordonii, higher than 3 log in F. nucleatum and P. intermedia and approximately 2 in V. parvula.In short, we standardised the conditions for using qPCR-PMA in 5 oral bacterial species and proved its usefulness for quantification of live and dead cells in multi-species oral biofilms formed in vitro, after use of an antiseptic.

Figure 1

Visualisation of multispecies oral biofilm using confocal microscopy. Staining was performed with Live/Dead BacLight bacterial viability kit. Live and dead cells are visualised in green and red, respectively.

Figure 2

Optimisation of PMA methodology. Pure planktonic cultures were subjected to heat- and CPC-killing conditions and treated with 100 μM PMA. Amount of live cells (qPCR-PMA) was subtracted from the total amount of cells (qPCR) to obtain the number of dead cells (Δlog₁₀ cells). Bars indicate the mean values in the negative control (empty bars), heat-killed cells (striped bars) and CPC-killed cells (grey bars).

Figure 3

Use of PMA on multispecies biofilms. Oral multispecies biofilm comprising by S. oralis, S. gordonii, V. parvula, F. nucleatum and P. intermedia were exposed to CPC and treated with 100 μM PMA. The number of dead cells (Δlog₁₀ cells) was found by calculating the difference between the amount of total cells (qPCR) and the amount of live cells (qPCR-PMA). Bars indicate the mean values for each species in a biofilm not exposed to CPC (empty bars) and a CPC-killed biofilm (grey bars).