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

<jats:title>Abstract</jats:title><jats:p>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.</jats:p><jats:p>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.</jats:p><jats:p>Our objective was to implement this technique for analysing mortality in multi-species oral biofilms formed<jats:italic>in vitro</jats:italic>with five oral bacteria:<jats:italic>Streptococcus oralis</jats:italic>,<jats:italic>Streptococcus gordonii</jats:italic>,<jats:italic>Veillonella parvula</jats:italic>,<jats:italic>Fusobacterium nucleatum</jats:italic>and<jats:italic>Prevotella intermedia</jats:italic>. We also tested its effectiveness on biofilms treated with an antiseptic solution containing 0.07% w/w cetylpyridinium chloride (CPC).</jats:p><jats:p>Standardisation of the qPCR-PMA method was performed on pure, heat-killed planktonic cultures of each species, detecting mortality higher than 4 log in<jats:italic>S. oralis</jats:italic>,<jats:italic>S. gordonii</jats:italic>and<jats:italic>F. nucleatum</jats:italic>and higher than 2 for<jats:italic>V. parvula</jats:italic>and<jats:italic>P. intermedia</jats:italic>. We obtained similar results for all species when using CPC.</jats:p><jats:p>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<jats:italic>S. oralis</jats:italic>and<jats:italic>S. gordonii</jats:italic>, higher than 3 log in<jats:italic>F. nucleatum</jats:italic>and<jats:italic>P. intermedia</jats:italic>and approximately 2 in<jats:italic>V. parvula</jats:italic>.</jats:p><jats:p>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<jats:italic>in vitro</jats:italic>, after use of an antiseptic.</jats:p>