Oral Rehabilitation, Faculty of Dentistry,The University of Hong Kong, Hong Kong SAR, ChinaChina
Technological and Higher Education Institute of Hong Kong, Hong Kong SAR, ChinaChina
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To investigate the effectiveness of a delmopinol, sodium chlorite, povidone iodine, and lactoferrin-lysozyme-lactoperoxidase mouthrinse against C. albicans planktonic cells and biofilms. Methods Two-fold dilutions of the mouthrinse solutions were prepared (1:2, 1:4, 1:8, 1:16, 1:32, 1:64), and dispensed into 96 well microtitre plates. Standardized inocula of wild type C. albicans SC5314 was added and the minimum inhibitory concentrations (MICs) were determined in accordance with Clinical and Laboratory Standards Institute (CLSI) Guidelines for the broth microdilution assay. Optically clear wells were cultured for determination of minimum fungicidal concentrations (MFCs). The effect of the mouthrinses against C. albicans mature biofilms was also evaluated. The metabolic activity of the biofilms was semi-quantitatively determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. Results The lowest MICs were exhibited by delmopinol (1:16), followed by sodium chlorite (1:8), povidone iodine (1:4), and lactoferrin lysozyme- lactoperoxidase (1:4). Delmopinol exhibited fungicidal activity at 1:16 dilution, while MFCs were achieved by higher concentrations of povidone iodine (1:4) and sodium chlorite (1:2). Lactoferrin-lysozyme-lactoperoxidase did not exhibit fungicidal activity at all tested concentrations. Among the four agents at two-fold dilution, sodium chlorite and povidone iodine exhibited the greatest reductions (>90%) in C. albicans biofilms (p<0.005). Significantly greater reductions in metabolic activity were achieved with sodium chlorite compared to the other agents at dilutions of 1:4 through to 1:32 (p<0.005). Conclusions Delmopinol exhibited the highest activity against planktonic C. albicans, while sodium chlorite exhibited greater effects against mature biofilms. Clinical Significance Sodium chlorite was shown in this study to exhibit potent antifungal activity against C. albicans biofilms, and products containing this active ingredient deserve consideration for inclusion in oral health promotion measures conducted amongst vulnerable patient groups.
Keywords: Yeast; Mouthrinse; Antifungal activity
Two-fold dilutions of the mouthrinse solutions were prepared (1:2, 1:4, 1:8, 1:16, 1:32, 1:64), and dispensed into 96 well microtitre plates. Standardized inocula of wild type C. albicans SC5314 were determined in accordance with Clinical and Laboratory Standards Institute (CLSI) Guidelines for the broth microdilution assay. Optically clear wells were cultured for determination of minimum fungicidal concentrations (MFCs). The effect of the mouthrinses against C. albicans mature biofilms was also evaluated. The metabolic activity of the biofilms was semi-quantitatively determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay.
The lowest MICs were exhibited by delmopinol (1:16), followed by sodium chlorite (1:8), povidone iodine (1:4), and lactoferrin-lysozyme-lactoperoxidase (1:4). Delmopinol exhibited fungicidal activity at 1:16 dilution, while MFCs were achieved by higher concentrations of povidone iodine (1:4) and sodium chlorite (1:2). Lactoferrin-lysozyme-lactoperoxidase did not exhibit fungicidal activity at all tested concentrations. Among the four agents at two-fold dilution, sodium chlorite and povidone iodine exhibited the greatest reductions (>90%) in C. albicans biofilms (p<0.005). Significantly greater reductions in metabolic activity were achieved with sodium chlorite compared to the other agents at dilutions of 1:4 through to 1:32 (p<0.005).
Delmopinol exhibited the highest activity against planktonic C.
mature biofilms.
Sodium chlorite was shown in this study to exhibit potent antifungal activity against C. albicans biofilms, and products containing this active ingredient deserve consideration for inclusion in oral health promotion measures conducted amongst vulnerable patient groups.
Yeast; Mouthrinse; Antifungal activity.
cc Copyright 2018 by Lam OLT. This is an open-access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0), which allows to copy, redistribute, remix, transform, and reproduce in any medium or format, even commercially, provided the original work is properly cited. was added and the minimum inhibitory concentrations (MICs)
albicans, while sodium chlorite exhibited greater effects against
andida albicans is an opportunistic pathogen commonly isolated from the oral cavity, and is a major etiologic agent of oral C mucosal infections. It is the fourth leading cause of nosocomial bloodstream infections,1 and the association of sepsis with preceding oral colonization has been demonstrated in AIDS patients, haematopoietic stem cell and bone marrow transplant patients, as 2-4 well as low-weight neonates. Candida albicans has also been implicated as an etiological agent of pneumonia in certain medically 5-7 compromised patient groups.5-7 While C. albicans is commonly isolated from various oral sites in healthy individuals, higher prevalence rates have been observed in hospitalized patients, and oral colonization has been associated with factors such as xerostomia, broad spectrum antibiotic therapy, immune status, nutritional deficiencies, and endocrine disorders.8
Although conventional antifungal agents such as polyenes and azoles have been the mainstays of prophylaxis and treatment for systemic and oral candidiasis, resistance to such agents have been increasingly reported in medically compromised patients. There is thus an urgent need for the evaluation of the antifungal activity of existing commercial oral antiseptic agents, which is currently poorly understood.9
The aim of this study was to investigate the efficacy of four existing commercial mouthrinses against C. albicans planktonic cells and biofilms. Tested products included: Betadine® mouthrinse (povidone-iodine 1% w/v, 8.7% ethanol, menthol, methyl salicylate, glycerol, saccharin sodium, purified water), Decapinol® mouthrinse (delmopinol hydrochloride 2mg/ml, aqua, alcohol, aroma, saccharin sodium, sodium hydroxide), TheraBreath® mouthrinse (sodium chlorite, water, PEG-40 hydrogenated castor oil, tetrasodium EDTA, sodium bicarbonate, sodium benzoate, mentha piperita oil, sodium hydroxide), and OralSevenTM mouthrinse (lactoferrin, lysozyme, lactoperoxidase, potassium thiocyanate, calcium lactate, glucose oxidase, aqua, monopropylene glycol, xylitol, hydrogenated starch hydrosylate, poloxamer 407, hydroxyethyl cellulose, sodium benzoate, aloe barbadensis, mentha piperita, benzoic acid, zinc gluconate).
The antifungal activity of mouthrinses was tested against wild type C. albicans strain SC5314, which was cultured as previously described.10 Two-fold dilutions of the mouthrinse solutions were prepared (1:2, 1:4, 1:8, 1:16, 1:32, 1:64) in RPMI, and minimum inhibitory concentrations (MICs) were determined in accordance with Clinical and Laboratory Standards Institute (CLSI) Guidelines for the broth microdilution assay.11 Optically clear wells were cultured on Sadbouraud’s agar for determination of minimum fungicidal concentrations (MFCs). The effect of the mouthrinses against C. albicans mature biofilms was also evaluated using a previously established protocol (Figure 1).10 In brief, standard C.
albicans inocula (2-3x106 cells/ml) were added to 96-well polystyrene microtitre plates (Iwaki) and incubated at 37 oC with agitation.
Following the adhesion phase, supernatant was aspirated, and wells were washed twice with PBS. Plates were incubated for a further o 24 hours at 37 oC. Following aspiration of liquid and washing with
PBS (2x), previously prepared serial dilutions of mouthrinses (1:2, 1:4, 1:8, 1:16) in YNB were added. Plates were incubated for an additional 24 hours, and YNB without mouthrinses were included in positive control wells. The metabolic activity of the biofilms was semi-quantitatively determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. All assays were done in triplicate.
Figure 1. Workflow for The Evaluation of The Effectiveness of Mouthrinses Against C. Al- bicans Mature Biofilms The lowest MICs were exhibited by delmopinol (1:16), followed by sodium chlorite (1:8), povidone iodine (1:4), and lac- toferrin-lysozyme-lactoperoxidase (1:4). Fungicidal activity was achieved at 1:16 dilution for delmopinol, and at higher concentra-
® tions for povidone iodine (1:4 dilution), and sodium chlorite (1:2 dilution). Lactoferrin-lysozyme-lactoperoxidase did not exhibit ® fungicidal activity at all tested concentrations. Among the four agents at two-fold dilution, sodium chlorite and povidone iodine ® exhibited the greatest reductions (>90%) in C. albicans biofilms (p<0.005) (Figure 2). Significantly greater reductions in metabolic activity were achieved with sodium chlorite compared to the other TM agents at dilutions of 1:4 through to 1:16 (p<0.005). Lactoferrin-lysozyme-lactoperoxidase consistently exhibited the weakest antifungal activity, with only a 40% reduction in biofilm metabolic activity at 1:2 dilution.
Prior studies have investigated the effectiveness of a wide range of commercial mouthrinses (eg. chlorhexidine, benzydamine hydrochloride, lawsone methyl ether, povidone iodine, lactoperoxidase-hypothiocyanate, amine fluoride, melaleuca) against oral yeasts in hospitalized and medically compromised patients, as well as their acceptability among both patients and nursing staff in hospital wards.9 Nevertheless, it has been clear that in vitro activity against Candida often does not equate with clinical effectiveness, and there currently remains a lack of evidence supporting the clinical efficacy of chemical agents contained in mouthrinses.
In this study, sodium chlorite demonstrated potent antifungal activity against C. albicans biofilms, and products containing this active ingredient deserve consideration for inclusion in oral health promotion measures conducted amongst vulnerable patient groups.
o High-quality randomized controlled trials are required to evaluate the in vivo effectiveness of this agent as an adjunct to conventional mechanical oral hygiene interventions, as well as its patient acceptability, and cost-effectiveness.
Figure 2. Effect of Four Commercial Mouthrinses on Candida Albicans Biofilms Biofilm Metabolism Following Incubation with Mouthrinses At A) 1:2 Dilution. Kruskal-Wallis Test (P<0.001). Pairwise Mann-Whitney U Tests (A<B<C; P<0.005). B) 1:4 Dilution. Kruskal-Wallis Test (P<0.001). Pairwise Mann-Whitney U Tests (A<B<C; P<0.001). C) 1:8 Dilution. Kruskal-Wallis Test (P<0.001). Pairwise Mann-Whitney U Tests (A<B<C; P≤0.008). D) 1:16 Dilution. Kruskal-Wallis Test (P<0.001). Pairwise Mann-Whitney U Tests (A<B; P<0.001).
The authors declare that they have no conflicts of interest.