Illumina Sequencing in Conjunction with Propidium Monoazide to Identify Live Bacteria After Antiseptic Treatment in a Complex Oral Biofilm: A Study Using an Ex Vivo Supragingival Biofilm Model

<b>Background/Objectives</b>: The evaluation of the efficacy of antibacterial treatments in complex oral ecosystems is limited by the inability to differentiate live from dead bacteria using omic techniques. The objective of this study was therefore to assess the ability of the combination of the 16S rRNA Illumina sequencing methodology and the action of propidium monoazide (PMA) to study viable bacterial profiles in oral biofilms after exposure to an antiseptic compound. <b>Methods</b>: Cariogenic supragingival biofilms were developed in an ex vivo model for 96 h, using saliva from healthy volunteers. The biofilms were treated with 0.12% chlorhexidine (CHX) combined with 0.05% cetylpyridinium chloride (CPC), for 60 s, using phosphate buffered saline as a control. After exposure, each biofilm was treated or not with PMA to then extract the bacterial DNA, quantify it by Qubit, quantify the bacterial population using qPCR, and perform the metataxonomic study of the samples using Illumina 16S rRNA sequencing. <b>Results</b>: A significantly lower DNA concentration in the PMA-treated biofilms (<i>p</i> < 0.05 compared with those not exposed to PMA) was observed. The viable bacterial count obtained by qPCR differed significantly from the total bacterial count in the biofilm samples exposed to the antiseptic (<i>p</i> < 0.05). The viable microbiome differed significantly from the total bacterial profile of the samples treated with CHX/CPC after exposure to PMA (<i>p</i> < 0.05 at the α- and β-diversity levels). <b>Conclusions</b>: The combination of Illumina 16S rRNA sequencing and PMA helps solve the inability to evaluate the efficacy of antibacterial treatments in the bacterial profile of complex ecosystems such as oral biofilms.