Efficacy of Antimicrobial Agents in Dentifrices: A Systematic Review

The aim of this systematic review was to verify if the presence of different antimicrobial agents in dentifrices is effective in reducing the number of microorganisms for disease prevention. This review followed the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines and was registered with the Open Science Framework (OSF). A search was conducted in the PubMed, Embase, Scopus, and Web of Science databases. Two independent authors reviewed the titles and abstracts according to the inclusion criteria, which comprised in vitro studies published in English that evaluated the efficacy of antimicrobial agents in dentifrices and their antimicrobial activity. A total of 527 articles were found. Of these, 334 were included for reading of the title and abstract, and 69 were selected for reading in full. In the end, 39 articles remained in this review. Triclosan, sodium fluoride, and sodium monofluorophosphate were the most commonly used chemical antimicrobial agents. Among the herbal agents, miswak extract and neem extract were the most commonly used. The presence of antimicrobial agents in dentifrice formulations can promote the reduction of the number of microorganisms involved in oral diseases, but with variations in their effectiveness, depending on the agent used and the microorganism evaluated.


Introduction
The human oral cavity is a complex environment composed of a variety of microorganisms, including commensal bacteria that are part of the healthy oral microbiota and are important for physiology, and pathogenic microorganisms, which are responsible for various infections, the most common and prevalent of which are dental caries and periodontal diseases [1]. When the balance of an individual's oral microbiota is lost, opportunistic pathogens can proliferate, resulting in the development of diseases [2].
Several microorganisms are involved in the progression of oral pathologies. Streptococcus mutans is a Gram-positive bacteria frequently found in the human oral cavity and one of the main microorganisms involved in the etiology of dental caries, along with Lactobacillus spp. [3][4][5]. Candida albicans is the most commonly commensal fungal species from the oral cavity, and under conditions of dysbiosis, can favor the appearance of oral candidiasis, an opportunistic infection, and is also associated with active caries lesions and promotes negative influences on tissue [6,7]. Staphylococcus aureus is another Gram-positive bacterium capable of causing opportunistic infections [8]. On the other hand, periodontal diseases are mainly caused by streptococci and spirochetes [6].
Caries is a multifactorial disease characterized by unbalanced mineralization and demineralization of teeth. S. mutans acts in the fermentation of carbohydrates from food, and this process produces acids that demineralize and degrade the dental element [6]. Biofilm accumulation and cariogenic microbiota are closely related to disease progression [9]. Periodontal diseases include gingivitis and periodontitis, which affect the tissue and structures that protect and support the teeth, respectively. Periodontitis, the most severe form of the disease, can result in loss of the dental element [6]. Biofilm accumulation also plays an important role in this infection [10].
Tooth brushing with dentifrice is one of the most frequently used oral hygiene practices in the world and is an essential measure for the maintenance of oral health [11][12][13]. Through it, it is possible to mechanically remove biofilm and consequently reduce the number of microorganisms [1]. However, it is not always properly performed. Therefore, the incorporation of antimicrobial agents in dentifrices is a prophylactic method of great importance to help control the number of microorganisms present in the oral cavity and thus reduce the chances of occurring infections. They act by slowing the microorganisms' multiplication, preventing bacterial aggregation and rupture of the pathogens' cell walls [14]. Antimicrobial agents such as sodium fluoride and triclosan have as their mechanism of action the inhibition of the activity of different enzymes [15,16]. In addition, herbal dentifrices contain phytochemicals, which are the substances responsible for their antimicrobial and anti-inflammatory effects [17].
Currently, there are several components present in dentifrice formulations with antimicrobial properties, such as triclosan and fluoride agents. However, some chemical antimicrobial agents can cause adverse effects [1,18]. Therefore, natural herbal dentifrices have emerged as a safer alternative to control and reduce the risk of infections and improve oral health. Herbal extracts have phytochemical components, which have antimicrobial and anti-inflammatory properties [17].
Most dentifrices are claimed to have antimicrobial properties, but comparative information on their efficacy is needed. There is a wide variety of dentifrices currently available, with various active substances, especially with the popularization of phytotherapeutic agents. Therefore, the aim of this systematic review was to verify if the presence of different antimicrobial agents in dentifrices is effective in reducing the number of microorganisms involved in oral diseases.

Materials and Methods
This systematic review was structured in accordance with Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines and was registered with the Open Science Framework (OSF). The question of the present review was formulated based on the PICOS: "Is the presence of antimicrobial agents in dentifrices really effective in reducing the number of microorganisms?" In this case, the population was formed by dentifrices without type restriction, the intervention was the presence of antimicrobial agents, the comparison was made with a group without the presence of antimicrobial agents, and the outcome was the evaluation of efficacy through antimicrobial activity, with the study focus being in vitro studies.
The electronic search for articles was conducted in November 2021 in the PubMed, Embase, Scopus, and Web of Science databases. The search was performed using the following terms: (dentifrices OR toothpastes) AND ("anti-infective agents" OR "anti-bacterial agents" OR "antimicrobial agents") AND ("antibacterial activity" OR "antimicrobial activity"). The articles found were exported to Rayyan digital platform (Qatar Computing Research Institute, Doha, Qatar). Table 1 summarizes the database search strategy.
The initial selection was performed by two independent authors. In it, articles were selected based on titles and abstracts, and for studies with insufficient data, the manuscript was obtained and read in full. Studies that did not meet the established eligibility criteria were excluded. The second selection was made by reading the full text. Disagreements in the selection of articles were resolved through discussion between independent authors. Eligibility criteria included in vitro studies published in English that evaluated the efficacy of antimicrobial agents in dentifrices and their antimicrobial activity. The exclusion criteria were applied to studies that did not evaluate dentifrices and studies that did not evaluate antimicrobial activity. Furthermore, in vivo studies, observational studies, studies that used clinical samples, reviews, book chapters, conference abstracts, case reports, surveys, and letters to the editor were also excluded. Data from the included studies were extracted in a table in a Word document (Microsoft Corporation, Redmond, USA) with the following information: (1) authors and year of publication; (2) antimicrobial agents; (3) dentifrices used; (4) microorganisms evaluated; (5) method of antimicrobial activity evaluation; and (6) main conclusions (Table 1). Triclosan containing dentifrice formulations were more effective compared to non-triclosan containing synthetic dentifrices Herbal dentifrices exhibited lower efficacy compared to the others [6] Nanosilver, chitosan and fluoride 3 dentifrices: containing nanosilver (TruCare Nanosilver); containing chitosan (Conybio Plus Chitosan); and containing fluoride (Oral B Pro-Health)

Streptococcus mutans
Modified agar well diffusion method The dentifrice containing nanosilver has the highest antibacterial efficacy against S. mutans, followed by the dentifrice containing fluoride and chitosan [9] Rosemary extract, propolis, mauve, cinnamon, peppermint, triclosan and sodium fluoride The antimicrobial activity of rosemary-based dentifrice (TR) was similar to the commercial dentifrice (TH) For the inhibition of L. rhamnosus, the propolis-based commercial dentifrice was more effective than TR dentifrice [11] Antibiotics 2022, 11, 1413 4 of 17  All formulations showed antimicrobial activity with the average order of highest activity descending being C, SF2, SF1, SFSP1, SFSP2 and G SF1 containing stannous fluoride was less active than C, a conventional dentifrice containing sodium fluoride (NaF) [25] Antibiotics 2022, 11, 1413 6 of 17   The formulated dentifrices exhibited antimicrobial property against all tested microorganisms and showed better and significant antimicrobial effect when compared to commercial dentifrices The dentifrice formulated with S. aromaticum extract alone seems to be much more active among the 3 bioactive materials used for formulation [41] Origanum dubium and Cinnamomum cassia oils, triclosan, sodium fluoride, sodium bicarbonate and plant extracts, such as Calendula officinalis, Aloe barbadensis and Melaleuca alternifolia oil 4 dentifrices: Splat Organic, Splat Biocalcium, Jack N 'Jill and Colgate Total

Streptococcus mutans
Agar diffusion method in disk (pure oil) and well (dentifrice) The antibacterial activity of the dentifrices was higher than positive control Herbal dentifrices showed higher antibacterial activity than their initial forms after the addition of essential oils. C. cassia showed higher antibacterial activity than O. dubium [42] Antibiotics 2022, 11, 1413 9 of 17

Streptococcus mutans
Standard disk diffusion method All dentifrices showed antibacterial activity at both concentrations tested, but better results at full strength compared to the diluted.
The antibacterial activity of the non-fluoridated toothpastes is as good as the fluoridated ones [47]  In vitro multispecies biofilm model Propolis seems to have no effect in respect to reducing CFU in the supragingival biofilm model used, neither in the dentifrice nor in the mouthwash [50] The methodological quality and risk of bias of the included studies were assessed by two authors by the Joanna Briggs Institute quasi-experimental study assessment tool, which was adapted for systematic review of in vitro studies. Each study was classified as low risk, uncertain risk, or high risk. Figure 1 depicts the study selection strategy. A total of 527 articles were identified in the initial search, of which 193 were duplicates. After analyzing the inclusion and exclusion criteria, 69 articles were selected for full-text reading. Of these, 30 were excluded, resulting in the selection of 39 relevant articles.
The most investigated microorganisms were Streptococcus mutans, Candida albicans and Staphylococcus aureus, respectively. In general, most antimicrobial agents were able to reduce the amount of S. mutans, but with great variations in their effectiveness. The largest diameter of the inhibition zones for this microorganism were 5.12 mm, 22 mm, 30 mm and 44.6 mm in the studies of Thounaojam et al. [39], Leite et al. [38], Babu et al. [32] and Evans et al. [22], respectively. The same was observed for S. aureus, where the largest diameter of the inhibition zones were 7 mm, 16.5 mm, 29.67 mm and 49 mm in the studies of Shaheena et al. [28], Leite et al. [38], Anushree et al. [37] and Ali et al. [12], respectively. In five studies [6,29,35,38,48], C. albicans did not show zones of inhibition or showed greater resistance to growth inhibition by plant extracts compared to chemical agents. However, another five studies [2,37,41,43,45] showed better results for some herbal dentifrices compared to chemical antimicrobials. Figures 2 and 3 summarize the results of the quality evaluation of the studies. The risk of bias was evaluated with the use of an adapted quasi-experimental studies appraisal tool by the Joanna Briggs Institute. Of the 39 studies included in this systematic review, most had a low risk of bias. The exception was the criteria "Were outcomes measured in a reliable way?", which showed a high risk of bias. The studies did not report whether the trials were performed by the same technician or the number of raters. pared to chemical antimicrobials. Figures 2 and 3 summarize the results of the quality evaluation of the studies. The risk of bias was evaluated with the use of an adapted quasi-experimental studies appraisal tool by the Joanna Briggs Institute. Of the 39 studies included in this systematic review, most had a low risk of bias. The exception was the criteria "Were outcomes measured in a reliable way?", which showed a high risk of bias. The studies did not report whether the trials were performed by the same technician or the number of raters.  Another criterion that showed a higher risk of bias was "Was appropriate statistical analysis used?" This result is justified by the absence of the statistical methods used [19,20,[23][24][25][26][30][31][32][33]35,40,45]. Finally, some studies [20,22,26,31,32,43,46] had a high risk of bias for the criterion "Was there a control group?" because they had no control group, but rather just compared groups. Because the studies showed heterogeneity in antimicrobial agents, dentifrices used and microorganisms evaluated, statistical analysis or meta-analysis was not possible.

Discussion
The addition of antimicrobial agents to oral care products has been suggested as an important strategy to assist in the control and reduction of microorganisms involved in various diseases, in order to improve oral health. This systematic review investigated the effectiveness of different antimicrobial agents in toothpaste formulations and found that the presence of these components can promote a real reduction in the number of microorganisms involved in oral diseases, but with varying levels of effectiveness.
Fluoride agents were the most frequently used, such as sodium fluoride (NaF) and sodium monofluorophosphate (MFP). Besides acting in the tooth remineralization process and helping to prevent dental caries, fluoride is capable of interfering with bacterial metabolism through the inhibition of enzymes, such as enolase [15]. However, fluoride agents at high levels can cause adverse effects. Excessive exposure to fluoride in children during the formation of permanent teeth can lead to enamel mineralization defects, characterizing dental fluorosis [51]. In a study by Haraszthy et al. [52], the analyzed fluoridecontaining dentifrices showed different antimicrobial effects. In most of the studies included in this review that compared fluoride dentifrices, the diameter of their zones of inhibition also varied [6,12,18,21,22,37,41,43,45,46]. Fluoride is more active and has a greater capacity to interfere with microorganism proliferation under low pH conditions [22]. Since some culture media used have a neutral pH, such as Mueller-Hinton agar, the Another criterion that showed a higher risk of bias was "Was appropriate statistical analysis used?" This result is justified by the absence of the statistical methods used [19,20,[23][24][25][26][30][31][32][33]35,40,45]. Finally, some studies [20,22,26,31,32,43,46] had a high risk of bias for the criterion "Was there a control group?" because they had no control group, but rather just compared groups. Because the studies showed heterogeneity in antimicrobial agents, dentifrices used and microorganisms evaluated, statistical analysis or meta-analysis was not possible.

Discussion
The addition of antimicrobial agents to oral care products has been suggested as an important strategy to assist in the control and reduction of microorganisms involved in various diseases, in order to improve oral health. This systematic review investigated the effectiveness of different antimicrobial agents in toothpaste formulations and found that the presence of these components can promote a real reduction in the number of microorganisms involved in oral diseases, but with varying levels of effectiveness.
Fluoride agents were the most frequently used, such as sodium fluoride (NaF) and sodium monofluorophosphate (MFP). Besides acting in the tooth remineralization process and helping to prevent dental caries, fluoride is capable of interfering with bacterial metabolism through the inhibition of enzymes, such as enolase [15]. However, fluoride agents at high levels can cause adverse effects. Excessive exposure to fluoride in children during the formation of permanent teeth can lead to enamel mineralization defects, characterizing dental fluorosis [51]. In a study by Haraszthy et al. [52], the analyzed fluoride-containing dentifrices showed different antimicrobial effects. In most of the studies included in this review that compared fluoride dentifrices, the diameter of their zones of inhibition also varied [6,12,18,21,22,37,41,43,45,46]. Fluoride is more active and has a greater capacity to interfere with microorganism proliferation under low pH conditions [22]. Since some culture media used have a neutral pH, such as Mueller-Hinton agar, the action of fluoride may have been affected. In in vivo studies, considering the pH conditions of the oral cavity, it is possible to observe a reduction in the plaque index when this antimicrobial agent is used. Binney et al. [53] performed a clinical trial that evaluated the plaque inhibitory properties of five dentifrices, all containing fluoride agents. There was a reduction in the participants' plaque index, which ranged from 2.49 to 2.24 among the dentifrices over a 4-day period. Similarly, in a clinical study by Gupta et al. [54], participants used a fluoride dentifrice and achieved a plaque score reduction ranging from 1.86 to 1.28 over a 4-week period.
Triclosan is a non-ionic phenolic derivative with antimicrobial properties that has also been widely used. In a study by McMurry et al. [16], triclosan showed antimicrobial activity through inhibition of the enzyme enoyl-acyl reductase (ENR) transporter protein, which participates in the synthesis of fatty acids. It also has anti-inflammatory effects, as it acts in the inhibition of the cyclooxygenase/lipoxygenase pathways [55]. Furthermore, triclosan and fluoride are able to damage the bacterial inner membrane [12]. However, great attention has been paid to the possible long-term side effects that this antimicrobial agent can cause [18], including the increase in resistance of microorganisms [22,56]. Some commercial dentifrices have a combination of triclosan and copolymer of maleic acid and polyvinyl methyl ether (PVM/MA), in order to improve its retention in the oral cavity and its solubility in water, resulting in greater substantivity [57,58]. A commercial dentifrice formulation containing triclosan and sodium fluoride has been used in several studies as a positive control and has been shown to be effective in the formation of inhibition zones of various microorganisms [2,11,42,44]. In only three studies in this review [27,43,45] were other dentifrice formulations more effective than those containing triclosan. In the results of Benlatef et al. [43] and Gibraiel et al. [45], herbal dentifrice formulations were more effective than triclosan. Fernández et al. [27], on the other hand, demonstrated that dentifrices containing sodium fluoride and stannous fluoride were more effective. In nine other studies [6,12,18,[20][21][22]24,37,46], dentifrices containing combinations of triclosan and sodium fluoride or sodium monofluorophosphate were more effective than the other compared dentifrices that did not have triclosan in their composition. Similarly to these findings, Haraszthy et al. [52] and Forbes et al. [59] reported that dentifrices containing triclosan as the active agent showed greater antimicrobial activity than the others, such as sodium fluoride. In a study by Haraszthy et al. [52], the minimum inhibitory concentration (MIC) of dentifrice containing only sodium fluoride as an antimicrobial agent was 30 µg/mL for S. mutans, 30 µg/mL for S. aureus, and 75 µg/mL for C. albicans. In contrast, the MIC of the dentifrice containing triclosan and sodium fluoride was 7.5 µg/mL for S. mutans, 15 µg/mL for S. aureus, and 30 µg/mL for C. albicans, showing better results. The minimum inhibitory concentration is the lowest concentration of a substance that inhibits microbial growth. In addition, in a study by Forbes et al. [59], the minimum bactericidal concentration (MBC) against anaerobic bacteria of dentifrice containing only sodium fluoride as antimicrobial agent was 3.3 to 12.5 mg/mL, while the MBC of dentifrice containing triclosan and sodium fluoride was 1.6 to 6.3 mg/mL, the latter being more effective at lower concentrations.
Phytotherapeutic antimicrobial agents can be an alternative to the use of chemical agents in children and adults, in view of the possible adverse effects they can cause. Several studies [2,18,32,37,48] have demonstrated that the antimicrobial activity of herbal dentifrices containing different extracts shows a wide variation. In this review, miswak (Salvadora persica) and neem (Azadirachta indica) extracts were the most present in natural dentifrice formulations. Miswak is obtained from the branches of the Salvadora persica tree and has been used as an oral hygiene device since antiquity [60]. In studies by Mohammed [60], Sivakumar et al. [61] and Adwan et al. [62], dentifrices containing miswak extract showed antimicrobial action against different microorganisms, which is in agreement with the studies evaluated in this review [32,37,44,45,47]. Azadirachta indica, known as neem, is a plant that belongs to the Meliaceae family found in Asia and Africa [63]. From it, a stick is made that is also used as an oral hygiene tool by some Asian and African countries [64]. In a study by Jenner et al. [65], the dentifrice containing neem extract was the most effective among those evaluated. Shafiq et al. [66] reported that dentifrice containing neem extract showed inhibitory activity. In the present review, some included studies [37,44,45,47] also demonstrated antimicrobial activity in dentifrices containing neem extract. Prasanth [6] and Anushree et al. [37] reported that dentifrice containing Azadirachta indica and Salvadora persica extracts formed halos of inhibition, but smaller in comparison to dentifrice containing triclosan. It is important to emphasize that most herbal dentifrices do not contain only one extract, but a combination of them. Thus, the variations in efficacy between herbal dentifrices probably occurs because of the difference between the components present in each brand.
Most of the assays were performed using the agar diffusion test, used to compare the antimicrobial activity of the products. In this method, the physicochemical properties of each antimicrobial agent, such as the diffusion coefficient and solubility, have an influence on its diffusion through the agar matrix and, consequently, on the results obtained [14,18]. However, this is a good option as a screening before performing in vivo studies. The test can be done using paper disks containing the agent or in drilled wells, where the agents are introduced. The disk diffusion method is appropriate for fluid products, but it has been widely used to evaluate the antimicrobial activity of dentifrices, which are semi-solid, but become fluid upon contact with water or saliva [67].
Another point to be considered is the interaction between the different components of dentifrices. The presence of some substances in the formulation of dentifrices can either increase or decrease the substantivity and clinical activity of some component [68]. In a study by Sadeghi and Assar [21], two commercial dentifrices had triclosan and sodium monofluorophosphate as antimicrobial agents, but they differed in other components. Although the antimicrobials used were the same, the antimicrobial activity of the dentifrices was different. Thus, the synergistic interactions between the various ingredients present in the formulations are an important factor not to affect the effectiveness of the product [38]. Sadeghi and Assar [21] observed that the antimicrobial action against the microorganisms evaluated was greater in dentifrices that contained more than one antimicrobial agent in their formulation. Following the same reasoning, in a study by Ali et al. [12], commercial dentifrices that already contained antimicrobial agents showed larger zones of inhibition after the addition of Piper betle extract. Oluwasina et al. [41] reported that dentifrice formulations containing a mixture of three or two extracts showed greater bioactivity. In this study, the dentifrice containing three extracts, Dennettia tripetala, Syzygium aromaticum, and Jatropha curcas latex showed inhibition zones against several microorganisms ranging from 10 to 18.3 mm, while the dentifrice containing two extracts, Syzygium aromaticum and Jatropha curcas latex, showed zones ranging from 9 to 16 mm. In contrast, the dentifrice containing only one extract, Jatropha curcas latex, exhibited inhibition zones ranging from 0 to 10 mm. Also, the dentifrice containing only Dennettia tripetala showed zones of inhibition ranging from 0 to 8 mm, demonstrating the greater efficacy of dentifrices containing three and two extracts.
In addition, five authors [6,32,37,46,47] evaluated dentifrices in different dilutions and reported that the diameter of the inhibition zones decreased with increasing dilution of the dentifrice, which suggests that the efficacy of the antimicrobial agent may be reduced when it is diluted. The evaluation of dentifrice at various concentrations is of interest, since under in vivo conditions, saliva is able to dissolve the product.

Conclusions
Based on the results of this systematic review, some conclusions were established.

1.
The presence of antimicrobial agents in dentifrice formulations can promote the reduction of the number of microorganisms involved in oral diseases, but with variations in their effectiveness, depending on the agent used and the microorganism evaluated.