Introduction
Black extrinsic tooth stain (BETS) is widespread amongst children and affects the aesthetics of teeth (Li et al.
2015). It is distinguished by dark dots or dark lines on the cervical third of enamel in both deciduous and permanent teeth (Koch et al.
2001). BETS is difficult to be wiped off by daily tooth brushing, it can be removed through ultrasonic scaling, polishing and fluoride pumice (Ronay and Attin
2011). Moreover, the cleaning procedure required for its removal can be challenging to dentists, especially pit and fissure BETS (Ronay and Attin
2011). Furthermore, it tends to recur even with good oral hygiene after scaling (Hattab et al.
1999). BETS comprises insoluble ferric sulphide which is formed by the reaction between hydrogen sulphide that is produced by bacteria and the salivary iron (Li et al.
2015).
Previous studies proposed chromogenic bacteria as the causative agents of BETS formation.
Porphyromonas gingivalis,
Prevotella intermedia,
Prevotella melaninogenica and
Prevotella nigrescens are also closely related to BETS (França-Pinto et al.
2012; Slots
1974; Soukos et al.
2005).
Aggregatibacter actinomycetemcomitans (
Aa) is a Gram-negative, facultative anaerobic coccobacillus bacterium of the Pasteurellaceae family (Rahamat‐Langendoen et al.
2011).
Aa is implicated in aggressive periodontitis in a subset of African and Middle Eastern patients (Fine et al.
2018). Saba et al. (
2006) reported the presence of significantly higher prevalence of
Actinomyces and
Aa in BETS. Sakai et al. (
2007) also detected a high prevalence of
Aa in the saliva of children with mixed dentition. However, the association of
Aa in the formation of BETS has not yet been documented. Here, we aim at revealing whether
Aa is related to BETS formation or not.
There is a lack of reports in the dental literature that focus on the treatment and prevention of BETS. Particularly, the unique microbiota of BETS necessitates an antibacterial agent that is capable of inhibiting the growth of the etiological agent. On the other hand, seeking alternative agents allowing for the use of plant-derived essential oils to promote dental health is of particular interest (Pourabbas and Delazar
2010). Hence, the development of a natural antibacterial agent that is safe for the host as well as specific for BETS microbiota is awaited. In a recent study, Arpag et al. (
2020) stated that the antibacterial effect of
Hypericum perforatum essential oil on
Porphyromonas gingivalis and
Aa strains increased synergistically when combined with chlorhexidine. Despite considering chlorhexidine as an effective chemotherapy in controlling plaque flora (Ronanki et al.
2016), taste altering and tooth staining limit its prolonged administration. Lavine et al. (
2018) also reported that virgin coconut oil reduced the viability of
Actinomyces sp. isolated from children aged 4–11 years with BETS.
Cinnamomum zeylanicum belongs to the family Lauraceae; cinnamon essential oil (CEO) is rich in transcinnamaldehyde which has antimicrobial effects against various pathogens. More than 300 volatiles have been found to be the constituents of CEO (Malsawmtluangi et al.
2016). El Atki et al. (
2019) demonstrated a synergistic interaction of CEO and chloramphenicol or ampicillin against
Staphylococcus aureus and against
Escherichia coli. Further, the combination of CEO and streptomycin showed additive effects against
Staphylococcus aureus,
Escherichia coli and
Pseudomonas aeruginosa. However, their study did not evaluate the effect of CEO on oral bacteria. In another study, Panjaitan et al. (
2022) reported that the ethanolic extract of cinnamon with a concentration of 7.5% and 2.5%, respectively, showed effective influence against biofilm formation of
Porphyromonas gingivalis and
Aa. Nevertheless, their study focused on the antimicrobial effect of the ethanolic extract of cinnamon which differs from CEO with respect to the chemical composition, physical properties, solubility and antibacterial activity.
The aims of this work are: (i) to investigate the association of Aa in BETS formation, (ii) to assess the antibacterial efficacy of CEO and its active compounds against planktonic and sessile Aa that has been isolated from BETS and (iii) to evaluate the preventive effect of CEO and its active substances on the formation of BETS using an ex vivo model. To our knowledge, no single report has addressed the in vitro effect of CEO on the formation of BETS and on Aa isolated from BETS.
Discussion
Black extrinsic tooth stain affects the aesthetics of children’s teeth and tends to recur even after removal by conventional procedures (Hattab et al.
1999; Li et al.
2015). In the current study,
Aggregatibacter actinomycetemcomitans was found to be associated with the in vitro formation of black extrinsic tooth stain and three individual compounds of cinnamon essential oil, namely eugenol, cinnamaldehyde and α-methyl cinnamaldehyde showed anti-
Aggregatibacter actinomycetemcomitans activities. The sessile minimum inhibitory concentration value of cinnamon essential oil, eugenol, cinnamaldehyde and α-methyl cinnamaldehyde was also determined and, respectively, showed two-, four-, four- and eightfold increase compared to their planktonic minimum inhibitory concentration. Moreover, at the minimum inhibitory concentrations of cinnamon essential oil, eugenol, cinnamaldehyde and α-methyl cinnamaldehyde, the growth kinetics of both planktonic and sessile
Aggregatibacter actinomycetemcomitans showed a complete inhibition. The time-killing study indicated that the survival rate of planktonic and sessile
Aggregatibacter actinomycetemcomitans cells was reduced by cinnamon essential oil, eugenol and cinnamaldehyde after 15–20 and 25–30 min, respectively. Cinnamon essential oil, eugenol and cinnamaldehyde were also capable of preventing the formation of black extrinsic tooth stain in an ex vivo model.
Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis in a subset of patients from Africa and Middle East (Fine et al.
2018). In few studies
Aggregatibacter actinomycetemcomitans has been also detected in black extrinsic tooth stain in a high prevalence (Saba et al.
2006; Sakai et al.
2007). However, the interrelation between
Aggregatibacter actinomycetemcomitans and the formation of black extrinsic tooth stain has not been yet documented. In the current study,
Aggregatibacter actinomycetemcomitans strain was isolated from black extrinsic tooth stain and the bacterium was identified based on the morphological characteristics, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and 16S rRNA sequencing. Further, the isolate was capable of inducing black extrinsic tooth stain in an ex vivo model of sound fallen deciduous teeth.
Recently, natural agents have gained an excessive importance as therapeutic source against oral pathogens because of the safety concern about synthetic agents and the abuse of antibiotics (Lotfy et al.
2018,
2019). Furthermore, natural agents can be applied for the prevention of pathology due to their antibacterial activities and can also be used as auxiliary therapy to prevent systemic infections caused by transient bacteraemia during dental procedures, sub-clinical dental infection or daily dental brushing (Potron et al.
2010; Rahamat‐Langendoen et al.
2011; Roda et al.
2008; Wang et al.
2010). Therefore, there is a need to search new natural bioactive compound that should demonstrate broad approach of action targeting effectively both free- and biofilm-mode of
Aggregatibacter actinomycetemcomitans cells. In the literature, the antibacterial effect of
Hypericum perforatum (Arpag et al.
2020) essential oil and coconut oil (Baharvand et al.
2021) on
Aggregatibacter actinomycetemcomitans was reported. The antibacterial activity of cinnamon essential oil against
Staphylococcus aureus,
Escherichia coli and
Pseudomonas aeruginosa was also investigated (El Atki et al.
2019). Recently, the effect of the ethanolic extract of cinnamon on biofilm formation of
Porphyromonas gingivalis and
Aggregatibacter actinomycetemcomitans was investigated (Panjaitan et al.
2022). The current work focused on the study of cinnamon essential oil and its individual components against planktonic and sessile
Aggregatibacter actinomycetemcomitans. To our knowledge, no study has reported the in vitro effect of cinnamon essential oil and its individual compounds on
Aggregatibacter actinomycetemcomitans.
The chemical analysis of cinnamon essential oil using gas chromatograph–mass spectrometer has revealed eight compounds. Out of these compounds, only eugenol, cinnamaldehyde and α-methyl cinnamaldehyde demonstrated anti-
Aggregatibacter actinomycetemcomitans activities. Eugenol exhibited the lowest minimum inhibitory concentration and the values of the minimum inhibitory concentrations were in the following order: cinnamon essential oil (421.5 mg/ml) > α-methyl cinnamaldehyde (26.37 mg/ml) > cinnamaldehyde (0.209 mg/ml) > eugenol (0.052 mg/ml). In another study, the ethanolic extract of cinnamon with a concentration of 2.5% was effective against biofilm formation of
Aggregatibacter actinomycetemcomitans. It is likely that the low minimum inhibitory concentration values in this study are due to the difference in chemical composition and physical properties of cinnamon essential oil compared to the ethanolic extract of cinnamon. With respect to the antibacterial activity, eugenol and cinnamaldehyde exert it by permeabilising the cell membrane and interacting with proteins causing leakage of adenosine triphosphate and potassium ions from the cell (Di Pasqua et al.
2007).
Cinnamon essential oil, eugenol and cinnamaldehyde, respectively, exhibited two-, four- and fourfold increase of sessile minimum inhibitory concentration compared to their planktonic minimum inhibitory concentration. This finding clearly showed that cinnamon essential oil, eugenol and cinnamaldehyde are equally effective against both planktonic and sessile modes of
Aggregatibacter actinomycetemcomitans. Moreover, they have an added value over ceftriaxone to which several-fold increase in biofilm resistance was perceived. These results are in agreement with Jafri et al. (
2019) who studied the in vitro efficacy of eugenol on single and mixed biofilms of
Streptococcus mutans (Jafri et al.
2019). However, the results of cinnamaldehyde contradict the findings of Chung et al. (
2018) who found that cinnamaldehyde has no effect on the adhesion and internalisation of
Aggregatibacter actinomycetemcomitans (ATCC 33384) to THP-1 cells (Chung et al.
2018). This divergence is more likely due to the difference in biofilm susceptibility to cinnamaldehyde between the two examined
Aggregatibacter actinomycetemcomitans strains which has been previously reported (De Martino et al.
2009). Further, cinnamaldehyde has multiple targets of activity that can influence strains differently (Nazzaro et al.
2013) as it can inhibit the growth of some Gram-negative bacteria without degeneration of the outer membrane or depletion of intracellular adenosine triphosphate (Nazzaro et al.
2013).
The growth kinetics of both planktonic and sessile
Aggregatibacter actinomycetemcomitans in the presence or absence of cinnamon essential oil, eugenol, cinnamaldehyde and α-methyl cinnamaldehyde was also investigated. A delayed start of the lag phase of planktonic
Aggregatibacter actinomycetemcomitans by 36 and 32 h was observed at half the minimum inhibitory concentrations of cinnamon essential oil and eugenol, respectively. Both planktonic and sessile
Aggregatibacter actinomycetemcomitans cells were completely inhibited at the minimum inhibitory concentrations of cinnamon essential oil, eugenol, cinnamaldehyde and α-methyl cinnamaldehyde. The inhibition of
Aggregatibacter actinomycetemcomitans biofilm formation by cinnamon essential oil, eugenol, cinnamaldehyde and α-methyl cinnamaldehyde at their sub-minimum inhibitory concentrations ranged from 5.3% to 37.4%. These findings are superior to other studies on the effect of coconut oil against
Aggregatibacter actinomycetemcomitans (Baharvand et al.
2021),
Actinomyces sp. (Lavine et al.
2018) and
Prevotella sp. (Gayatri et al.
2017).
Cinnamon essential oil, eugenol and cinnamaldehyde exhibited inhibitory effect against planktonic and sessile
Aggregatibacter actinomycetemcomitans cells which is indicated by the reduction in the survival rate after 15–20 and 25–30 min, respectively. These data suggest that cinnamon essential oil, eugenol and cinnamaldehyde could be used to prevent biofilm formation by
Aggregatibacter actinomycetemcomitans after removal of black extrinsic tooth stain or they could be used as ancillary therapy in preventing bacteraemia of
Aggregatibacter actinomycetemcomitans. Furthermore, cinnamon essential oil, eugenol and cinnamaldehyde are non-toxic and safe for human use at their minimum inhibitory concentration and minimum bactericidal concentration (Singh et al.
2009). Consequently, if they can be incorporated into children’s chewing gums or mouth wash formulations, this will provide a beneficial effect in preventing the adhesion of
Aggregatibacter actinomycetemcomitans to tooth enamel and accordingly preventing black extrinsic tooth stain. In contrast, α-methyl cinnamaldehyde showed a better anti-planktonic than anti-biofilm activity. It seems that the presence of methyl moiety in the structure of α-methyl cinnamaldehyde appears to decrease its activity when compared to cinnamaldehyde. In this case, we propose that it could be a good choice to prevent dissemination of
Aggregatibacter actinomycetemcomitans in the oral cavity or it could be used as an auxiliary therapy in anatomical areas with high risk of bacteraemia. Since various bacteria have been reported to be associated with black extrinsic tooth stain formation (França-Pinto et al.
2012; Slots
1974; Soukos et al.
2005), an ex vivo model was employed using a polymicrobial culture of
Aggregatibacter actinomycetemcomitans,
Prevotella intermedia and
Porphyromonas gingivalis to evaluate the preventive effect of cinnamon essential oil and its active components on the formation of black extrinsic tooth stain. The results revealed that the daily addition of cinnamon essential oil, eugenol and cinnamaldehyde at their minimum inhibitory concentrations for 14 days, totally prevented the formation of black extrinsic tooth stain and, respectively, reduced the survival of sessile bacterial cells to 0.57%, 0.75% and 0.87%. However, the daily incorporation of α-methyl cinnamaldehyde at its minimum inhibitory concentration was not capable of preventing black extrinsic tooth stain formation. Furthermore, α-methyl cinnamaldehyde reduced sessile bacterial cells by 17.63% and black extrinsic tooth stain was visually detectable in the ex vivo model after 10 days of exposure to α-methyl cinnamaldehyde.
A few limitations were encountered in this study. Since the oral cavity is a challenging environment for antimicrobials due to the effects of saliva and the gingival crevicular fluid, further studies will be needed to study the in vivo substantivity of cinnamon essential oil, eugenol, cinnamaldehyde and α-methyl cinnamaldehyde. Furthermore, it is not possible for one study to investigate all the bacterial species associated with black extrinsic tooth stain. Accordingly, future researches should challenge the in vitro effect of cinnamon essential oil, eugenol, cinnamaldehyde and α-methyl cinnamaldehyde on other bacteria from the polymicrobial community of black extrinsic tooth stain.
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