PMA-qPCR to quantify viable cells in multispecies oral biofilm after disinfectant treatments

Abstract

Conventional quantitative real-time PCR (qPCR) amplifies DNA from viable and dead cells, which can lead to an overestimation of live bacteria. Viability qPCR aims to eliminate DNA from membrane-compromised cells through treatment with propidium monoazide (PMA).

Here, we evaluated PMA-qPCR to enumerate viable cells of Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans, and Veillonella dispar. Five-species oral biofilms were grown on hydroxyapatite discs for 64 h. The biofilms were exposed to 0.2 % chlorhexidine (CHX) or 3 % sodium hypochlorite (NaOCl) for 2 min, either once before cell harvest at 64 h or six times during biofilm growth. The total and single species cells were quantified by culture (CFU) and qPCR from samples with and without PMA treatment before DNA extraction. For species-specific qPCR, TaqMan assays were applied. To determine total bacteria counts, a SYBR green qPCR was established using universal degenerative primers for the conserved dnaK gene.

For biofilms treated once with CHX, the addition of PMA led to a 1 to 1.6 log₁₀ reduction in PCR counts. This closely matched CFU and PMA-qPCR counts for total bacteria and all single species, except for F. nucleatum, where PMA-qPCR detected significantly more bacteria than culture. NaOCl treatment directly affected DNA and inhibited subsequent PCR amplification, even in samples without PMA. Single treatment of biofilms with 3 % NaOCl and six-fold exposure of biofilms to disinfectants resulted in no viable cell detection by culture. However, PMA did not completely prevent PCR amplification, indicating that disinfectant efficacy measured by viability PCR could be underestimated.