Evaluation of chlorhexidine substantivity on salivary flora by epifluorescence microscopy

Abstract

Epifluorescence microscopy permits evaluating the antimicrobial activity of CHX on the salivary flora in real-time. Fluorescence assays could be particularly useful to analyse simultaneously the effect of antimicrobials that alter the cytoplasmic membrane integrity on different oral ecosystems.

In situ neutralisation of the antibacterial effect of 0.2% Chlorhexidine on salivary microbiota: Quantification of substantivity

“…It has been stated that this specific solution has a general tendency to artificially increase the levels of non-vital bacteria 28 with regard to the plate cultures and the plate efficiency. In a previous study, 12 it has been shown a correlation between the immediate effect and the plate cultures; however, this correlation was lost as time passed, with the effect of CHX being overestimated by plate cultures, 12 indicating that the bacterial vitality may be underestimated with plate cultures or overestimated with fluorescence techniques, which is in contra to what has been recently stated. 28 In this same way, Hannig et al 29 showed that live/dead techniques were valid when assessing bacterial vitality after the use of antiseptic agents, being the combination of Syto 9/propidium iodide a reliable tool when assessing bacterial vitality in a multispecies biofilm.…”

“…Since epifluorescence microscopy has been considered an observer-dependent technique, 12 it is important to determine the intra-and inter-observer correlations. In the present series, the ICC mean values were !0.85.…”

“…The first, can give information on the mechanism standpoint such as minimum inhibitory concentration or bacterial kill-time curves, but give very limited clinical perspective on a so dynamic ecosystem like the oral cavity. On the other hand, in vivo models vary from methods based on plaque re-growth assessments on short periods of time 17 or others using saliva [8][9][10]12,18,19 and/or dental plaque after a single application. [19][20][21] Fig.…”

“…1 -Scheme of the protocol followed by all volunteers from the randomization to the application of the different tests. In vivo models, the evaluation of the duration of the antibacterial effect of a single CHX mouthwash implies the consideration of two aspects: the repercussion that the immediate antibacterial effect of the CHX has on the bacterial regrowth and the posterior antibacterial effect that the CHX continues to exert due to its slow release (this posterior antibacterial effect is what is known as substantivity [8][9][10]12,18,19 ).…”

“…6 The action of CHX on the membrane structure permits the use of fluorescence staining techniques based on combinations of dyes, which can differentiate vital from non-vital cells due to membrane permeability and integrity. A common combination used in studies on the activity of antimicrobial agents both on dental plaque 7,8 and on salivary microbiota [7][8][9][10][11][12] has been found in the LIVE/DEAD 1 BacLight TM Viability Kit (Molecular Probes, Leiden, The Netherlands), which is composed of Syto 9 and propidium iodide. Their staining principle is simple, as both are based on membrane permeability; Syto 9 penetrates all vital and non-vital cells due to its ability to penetrate intact cells, while propidium iodide only penetrates those cells with higher cell membrane permeability (i.e.…”