Commercial antimicrobials mouthrinses on caries and periodontitis-related biofilm control: a review

Esta revisão tem como objetivo discutir o potencial antimicrobiano de diferentes enxaguatórios bucais em relação ao controle da cárie dentária e doença periodontal. A pesquisa foi realizada usando PubMed e as seguintes palavras-chave: “agente antimicrobiano” ou “agente antiplaca”, “biofilme dental” e “cárie dentária” ou “doença periodontal” ou “gengivite”. Foram selecionados os estudos publicados em inglês, de 2011 a 2015, em revistas com fator de impacto maior que 0,8. Foram encontrados no total 22 artigos, 13 relacionados à cárie dentária e 9 relacionados à doença periodontal. Entre os 13 estudos envolvendo bactérias e/ou biofilme cariogênicos, 6 foram realizados in vitro, 3 in situ e 4 in vivo. Entre os 9 estudos envolvendo doença periodontal, 2 foram in vitro e 7 in vivo. Os principais agentes ativos testados foram: CHX-Clorexidina, CPC-cloreto de cetilpiridínio e OE-óleos essenciais (com álcool ou sem álcool). A CHX foi comparada ao OE em 6 estudos, mostrando superioridade em 3 estudos, similaridade em 1 estudo e inferioridade em 2 estudos. CPC mostrou menor efeito na redução da placa em comparação à CHX e ao OE. Ainda há controvérsias sobre o efeito do álcool, mas alguns estudos têm mostrado superioridade no caso de OE e CHX com álcool sobre biofilmes cariogênicos e periodontopatogênicos, respectivamente, quando comparados à versão sem álcool; para o CPC, não foi encontrada diferença. Mais estudos clínicos são necessários para melhor compreensão sobre mecanismo de ação e as diferenças de desempenho entre os agentes antiplaca. ABsTRACT

T he oral cavity is directly in contact with microorganisms [1].Saliva, gingival fluid and our diet supply nutrients for them, making the environment propitious to microbiota development [2,3] Microbiota can be organized as biofilm that potentially can cause oral diseases as dental caries and periodontitis [4].
Dental caries is one of the most relevant oral chronic diseases caused by microorganisms from different species organized in a supragingival biofilm.The cariogenic microorganisms metabolize sugar, especially sucrose derived from the diet, producing acids that reduce the biofilm pH and cause tooth decay [4,5].The main cariogenic microorganisms present in biofilm are S. mutans, Lactobacillus, bifidobacteria and fungi.S. mutans, in particular, produce insoluble extracellular polysaccharides from sucrose in the biofilm matrix, increasing metabolic efficiency and protecting themselves against host defenses mechanisms [4,6].
The mechanical disorganization of dental biofilm by toothbrushing is extremely important to prevent dental caries [8] and gingivitis [9], but sometimes insufficient for patients who have unfavorable conditions as, for example, xerostomia [10] and using fixed orthodontic appliances.The use of antimicrobials agents may be an alternative for those patients at high risk of dental caries [10] and periodontal disease [11,12] Among the active agents, chlorhexidine digluconate (CHX), Cetylpyridinium Chloride (CPC) and essential oils (EO) [13] are the most used by the population, who applied them for halitosis control [14].CHX is considered a gold-standard antimicrobial agent applied in patients with periodontal diseases.It has been indicated as a temporary coadjutant to regular oral hygiene procedures, as a preoperative and/ or postoperative rinse either [15].
Despite the popularity of the antimicrobial agents often found in supermarkets, there is sparse information about their efficacy on the control of oral diseases.Therefore, the aim of this review was to compile information about the efficacy of the main commercial antimicrobial agents applied to prevent tooth decay and periodontal disease.

RevIeW oF lITeRATuRe
The survey was conducted using PubMed and the following keywords: "antimicrobial agent" or "antiplaque agent", "dental biofilm" and "dental caries" or "periodontal disease" or "gingivitis".Only studies published in English, from 2011 to 2015, in journals with impact factor greater than 0.8, were selected.We found a total of 22 papers, 13 related to dental caries and 9 related to periodontal disease, involving in vitro, in situ and in vivo models.

Commercial Agents and Biofilm/Dental Caries
Chlorhexidine (CHX) is considered a gold standard antimicrobial agent applied in dentistry.Accordingly, most studies testing new antimicrobial agents have included CHX as a positive control.Cetylpyridinium chloride (CPC), also a cationic agent as chlorhexidine, is indicated to combat dental plaque and halitosis [16].Essential oil (EO: eucalyptol, thymol, salicylate and menthol), a non-ionic agent, is other agent popularly applied to control dental plaque [17].The agents are mostly available as mouthrinses.

Different
commercial mouthrinses containing CHX, in concentrations ranged from 0.05 to 0.2%, were compared to EO (formulae with alcohol) and water (negative control) using multispecies biofilms (A.naeslundii, V. dispar, F. nucleatum, S. mutans, S. oralis and C. albicans).The total CFU were determined using Columbia blood agar, and the S. mutans and S. oralis CFUs were counted using Mitis-Salivarius agar.The treatments were done after 16.5, 24.5, 40.5, and 48.5 h of biofilm formation.After a total time of 64.5 h, CFUs were determined for total microorganisms (A.naeslundii, V. dispar, F. nucleatum, S. mutans, S. oralis and C. albicans), S. mutans and S. oralis.The total CFU numbers were not significant different among the mouthrinses.Biofilm formation was reduced in 7 log10 steps by 0.2% CHX (formulae with alcohol), and in 3 log10 steps by EO, 0.05% CHX (formulae with alcohol), and 0.12 and 0.2% CHX (without alcohol) solutions compared to water [18].
Wakamatsu et al. [20] compared the penetration kinetics of four mouthrinses (CHX, EO, CPC and isopropylmethylphenol-IPMP) into S. mutans biofilm.The penetration velocities were determined by monitoring fluorescence loss between 30 s and 5 min exposure.EO showed the best penetration, but within 30 s no mouthrinse had any antiplaque effect.After 30 s, EO induced the highest reduction in CFU number, but not in bacteria detachment compared to the other mouthrinses.
Nanoemulsions prepared with 25% soybean oil, 1% CPC and 10% Triton X-100-TRI (NE) were applied on S. mutans, L. casei, C. albicans and A. viscosus both isolated and in a mixed-culture.S. mutans and L. casei biofilms were stained using a live/dead kit.MIC and MBC were determined for all microorganisms isolated and in a mixed-culture.The time kinetics was also analyzed for all microorganisms (1, 5, 15, 30 and 60 min) using optical density.The adherence of microorganism on glass plates (24 h) and the growth of biofilm (72 h) were analyzed after fixing and staining, using optical density.NE has shown reduced in 83% the viability of S. mutans and L. casei biofilm compared to negative control.MIC and MBC of NE were 9-to 27-fold smaller than those from CHX (positive control).In respect to killing curves, NE had a faster and powerful effect compared to CHX.The level of adhesion on glass surface was reduced by 94.2 to 99.5% in NE treated groups compared to positive (CHX) and negative controls.The antiadherence and anti-biofilm effects of NE suggest a promising anti-caries action [21].
Commercial rinses containing 0.05% CPC, alcohol or free-alcohol, were compared with 0.05% fluoride mouthwash (F) and 0.12% chlorhexidine (positive control-CHX).MIC was firstly determined for each mouthrinse considering 25 microorganism species associated with oral diseases.The second part of the study evaluated the antimicrobial activity using supragingival biofilm collected from 15 subjects, which was exposed ex vivo to the mouthrinses for 5-7 days in anaerobic environment.MIC values were significantly lower for both CPC rinses compared to fluoride rinse especially against gram-negative bacteria (most involved in halitosis etiology), showing a broad-spectrum activity.CHX had the greatest antimicrobial effect.This ex vivo model showed no difference between CPC rinses formulated with alcohol or without alcohol.Both CPC (> 90% killing) and CHX (98% killing) showed higher antimicrobial activity compared to F [22].
The effect of antimicrobial mouthrinses was also studied in adult patients under orthodontic treatment.The patients were treated with 0.1% CHX alcohol-free, essential oil (alcohol/alcohol-free) or negative control (1% hydroalcoholic solution) for 4 days (1x30s/ day).Supragingival biofilm and microorganism on tongue were collected and analyzed for UFC counting (S. mutans).All mouthrinses were similarly able to significantly reduce the number of S. mutans colonies compared to control for both samples (tongue and biofilm) [23].
The antiplaque effect of EO with or without alcohol was compared in vivo.Thirty subjects were divided into two groups (EO with and without alcohol).They rinsed twice a day for 3 days.EO with alcohol showed better plaque inhibitory effect (plaque index of 2.18 in whole mouth) than alcohol-free solution (plaque index of 2.46) [24].
Albertsson et al. [26] evaluated the antimicrobial effect of EO and CHX alcohol-free mouthrinses on S. mutans and Lactobacillus in saliva.Twenty healthy volunteers applied the mouthrinses twice times during 16 days after the regular mechanical oral hygiene.Saliva was collected and analyzed for CFU/ml.Only CHX rinse showed a significant reduction in S. mutans and Lactobacillus counting, while EO did not have antimicrobial effect.Two mouthrinses, EO (0.092% of eucalyptol, 0.042% menthol, 0 .060% of methyl salicylate and 0.064% of thymol) and 0.2% CHX, were tested on biofilm in situ.Bacterial viability, thickness and covering grade were evaluated after 4 days of applying each of the mouthrinses (2 times x 30s/day).CHX showed 13.2% and EO 14.7% of live bacteria.CHX was better in reducing biofilm thickness compared to EO (CHX 6.5 μm vs. EO 10.0 μm) and covering grade (CHX 20.0% vs. EO 54.3%).CHX showed better antiplaque effect compared to EO [27].
The effect of CPC (concentrations of 0, 0.025%, 0.05%, 0.075%, and 0.1%), applied twice day for 1 min, during early (0h to 50h) and mature (48h to 98h) S. mutans biofilm formation, was determined.All CPC concentrations showed complete anti-biofilm activity during early biofilm formation.For old biofilm, the highest CPC concentrations had effect on dry weight, viability and acidogenicity, but they had no effect on water-insoluble extracellular polysaccharides production.Therefore, CPC has inhibitory effect on young S. mutans biofilm only [28].
Hannig et al. [29] compared the effect of fluoride solution (100 ppm F as AmF and 150 ppm F as NaF) to 0.2% CHX (positive control) on biofilm adherence to enamel and dentin in situ after 8 h of 1 min-rinse.The bacterial viability and CFU for total microorganism were determined.In the control group, significantly higher amounts of adherent bacteria were detected on dentin (4.8 x 10 6 ± 5.4 x 10 6 bacteria/cm 2 ) than on enamel (1.2 x 10 6 ± 1.5 x 10 6 bacteria/cm 2 ).Chlorhexidine significantly reduced the amount of adherent bacteria (dentin: 2.8 x 10 5 ± 3.4 x 10 5 bacteria / cm 2 ; enamel: 4.2 x10 5 ± 8.7 x 10 5 bacteria/cm 2 ).Rinses with the fluoride solution also significantly reduced bacterial adherence to dentin (8.1 x 10 5 ± 1.5 x 10 6 bacteria/cm 2 ).The viability was reduced by both chlorhexidine and fluoride.While a significant reduction of bacterial adherence on enamel and dentin was seen for chlorhexidine, F reduced the bacterial adherence on dentin only.
Rabe et al. [30] compared the antimicrobial effect of 0.1% CHX with 0.2% NaF.Enamel discs were mounted on healing abutments in the premolar region of three subjects for 7 days.After this period, the treatment was done for 1 min.Then, the architecture, bacterial viability and total biomass of the biofilm were evaluated using fluorescence methods.The biofilm architecture was similar for both groups, however CHX had effect on the biofilm surface, while F caused cell damage in the middle and deep biofilm layers.Both rinses were able to significantly reduce the bacterial vitality (63% vs. 95% in control) and the total biomass (6.5 x 10 6 arbitrary units/mm2 for control, 0.82 x 10 6 arbitrary units/mm2 for CHX and 0.87 x 10 6 arbitrary units/mm 2 for F).Rinse with CHX has antimicrobial effect in the cell/liquid interface at the top of biofilm.NaF, however, is able to penetrate and exert effect in the middle and deep levels of the biofilm.Some in vitro studies have shown that fluoride reduces the production of lactate and the biomass of S. mutans biofilm when applied in high concentrations [31,32], however, other studies have shown no differences in lactate production, CFU number and pH drop by the application of fluoride compared to control in S. mutans biofilm [33,34].The antimicrobial effect of fluoride is still not a consensus.
Table I summarizes the results found in the above-cited studies.Generally, CHX and EO seem to be the best antimicrobial agents against cariogenic bacteria.Both CHX and EO showed better antimicrobial effect than CPC, while F has limited antimicrobial effect.Few studies have analyzed the impact of these mouthrinses on the prevention of tooth demineralization, which should be the most relevant question to be answered.

Commercial Agents and Biofilm/
Periodontal Disease CHX, as previously shown, has been widely tested as antiplaque agent.In this in vivo study, the authors tested two formulations.The volunteers rinsed twice a day 0.12% CHX (alcohol) or 0.1% CHX (alcohol-free) with 0.1% of Formaldehyde (CHX-F) during 7 days.After the treatment, plaque indexes were recorded.The mean plaque of first group (0.76±0.38) was significantly lower compared to the second group (1.43±0.56),showing that alcohol might have some influence on the antiplaque effect of CHX [35].
A crossover study was done with ten volunteers using an experimental gingivitis model.
Three mouthrinses (0.12% chlorhexidine plus 0.05% CPC; 0.12% CHX pure and 0.12% CHX plus NaF) were tested for the inhibition of oral bacteria related with periodontal diseases (S. oralis, A. naeslundii, V. parvula, F. nucleatum, A. actinomycetemcomitans, P. gingivalis) and on biofilm formed in vitro.The effect of the mouthrinses was analyzed in bacteria under planktonic phase, which were treated for 1 min using the short interval-killing test.The antimicrobial effect was measured as CFU/mL and no difference was found among the rinses.For biofilm formation, the bacteria were grown on sterile ceramic calcium hydroxyapatite (HAP) discs for 12 h at 37 ºC using a bioreactor.The discs were immersed in the mouthwash for 2 min and the biofilm cultivated for 5 days.The viable cells were analyzed using culture methods, scanning electron microscopy (SEM), Live/Dead staining and fluorescence in situ hybridization (CLSM).SEM showed a typical biofilm structure.The fluorescence in situ hybridization technique confirmed the presence of the six bacterial species in biofilms older than 3 days.The live/dead ratio revealed that the majority of cells were alive in 3-, 4-and 5-d biofilms.Cells in biofilms showed more tolerance compared with planktonic cells.In 4-d biofilm, CHX+CPC showed more antimicrobial effect than CHX+NaF and CHX [37].
Both CPC and EO were also compared in an in vivo study, in which 142 subjects wore the mouthrinses for 2 weeks (2x30s/day).The Modified Gingival Index (MGI), Plaque Index (PI) and bleeding Index (BI) were analyzed.EO demonstrated significant reduction in MGI (9.4%), PI (6.6%) and BI (29%) compared to CPC.EO presented clinical superiority compared to CPC in the short-term management of plaque and gingivitis [38].
Commercial mouthrinse with 0.075% CPC (fluoride-free/alcohol-free) was compared with EO (fluoride-free/alcohol) in respect to antiplaque and antigingivitis effects in vivo.Fluoride-free/alcohol-free mouthwash was used as negative control.After 6 weeks, the subjects from CPC, EO and NC groups exhibited reductions in GI of 28.6%, 22.6% and 1.70%, respectively; while PI was reduced in 31%, 28% and 1.4% for CPC, EO and NC, respectively.Both mouthrinses provide a significant reduction in dental plaque and gingivitis [41].
A new rinse with CPC (alcohol-free) was tested to control plaque and gingivitis in 67 adults with moderate gingivitis during 6 months (3x30 s/day).PI, bleeding on marginal probing (BOMP) and stain (S) indexes were applied.The presence of A. actinomycetemcomitans, P. gingivalis, P. intermedia/nigrescens, T. forsythia, P. micra, Capnocytophaga spp., E. corrodens, Eubacterium spp.and F. nucleatum were determined in the biofilm.Significant reduction of the clinical parameters was observed for the tested CPC solution compared to placebo.Among the periodontopathogenic bacteria, P. intermedia showed a clear reduction after 3 and 6 months of CPC treatment.CPC shows ability to reduce biofilm accumulation after 3 and 6 months of use [12] Based mainly on clinical trials as shown in Table II, EO has a superior antiplaque and antigingivitis effects compared to CPC, while EO has a similar efficacy compared to CHX.On the other hand, CHX has often been responsible for inducing undesired effects as tooth discoloration.

CFU counting for periodontal bacteria
In planktonic phase (S. oralis F. nucleatum, P. gingivalis and A. actinomycetemcomitans) no differences between the tested mouthrinses were found.For biofilm, CHX+CPC showed more inhibition of viable cells compared CHX and CHX+NaF.
EO presented clinical superiority compared to CPC.
Elias-Boneta et al., [41] 1. 0.075% CPC (alcohol-free and fluoride-free) 2. EO (alcohol) The antiplaque and antigingivitis effect in vivo after 6 weeks/ treatment was done twice daily PI and GI Both mouthrinses proved a significant reduction in dental plaque and gingivitis.
Table II -Effect of commercial antimicrobial mouthrinses on periodontitis/gingivitis or bacteria perio-related.