Microbial species associated with dental caries found in saliva and in situ after use of self-ligating and conventional brackets

Abstract Objectives Enamel demineralization is among the main topics of interest in the orthodontic field. Self-ligating brackets have been regarded as advantageous in this aspect. The aim of this study was to evaluate the break homeostasis in the oral environment and the levels of microorganisms associated with dental caries among the different types of brackets. Material and Methods Twenty patients received two self-ligating brackets: In-Ovation®R, SmartClipTM, and one conventional GeminiTM. Saliva was collected before bonding (S0), 30 (S1) and 60 (S2) days after bonding. One sample of each bracket was removed at 30 and 60 days for the in situ analysis. Checkerboard DNA-DNA Hybridization was employed to evaluate the levels of microbial species as-sociated with dental caries. Data were evaluated by nonparametric Friedman and Wilcoxon tests at 5% significance level. Results The salivary levels of L. casei (p=0.033), S. sobrinus (p=0.011), and S. sanguinis (p=0.004) increased in S1. The in situ analyses showed alteration in S. mutans (p=0.047), whose highest levels were observed to the In-Ovation®R. Conclusions The orthodontic appliances break the salivary homeostasis of microorganisms involved in dental caries. The contamination pattern was different between self-ligating and conventional brackets. The In-Ovation®R presented worse performance considering the levels of cariogenic bacterial species.


Introduction
Enamel demineralization and cavities are among the main topics of interest in the orthodontic field. 1,2 Dental caries are caused by the effects of acid products on the carbohydrate metabolism of bacterial species, mainly S. mutans, S. sobrinus, and Lactobacillus ssp. 3,4 These species grow 6 or 12 weeks after orthodontic appliance bonding. 5 Among the microbial species involved in the dental caries, mutans streptococci are the main etiological agent. In dental biofilm, which is correlated with dental caries, Streptococcus sobrinus and Streptococcus mutans are the most frequently isolated microorganisms. 6 Recently, studies found that the coexistence of S. mutans and S. sobrinus is an important risk factor for the multi-colonized patient in the development of dental caries. 7,8 The levels of the microorganism associated with the disruption of oral microbiota homeostasis are essential to determine caries risk and activity.
Simultaneously, the literature emphasizes the importance of S. mitis, S. oralis, S. sanguinis, S. salivarius, and S. gordonii as initial colonizers, since they provide attachment points for other species and could promote or antagonize the existence of S. mutans and Lactobacillus. [9][10][11] In addition, Candida spp. are commonly found colonizing oral cavity with heavy infection by S. mutans and have been related to reduced pH levels. Previous research suggests that the interaction between these species could help develop dental caries. 12,13 Self-ligating brackets were introduced in 1930 to reduce patient discomfort and the time spent chairside . These brackets have progressively become part of the typical orthodontic routine. A systematic review showed that the self-ligating brackets improved oral hygiene because they retain less dental plaque and less bacterial contamination. This is a result of the design of self-ligating brackets and the absence of the elastomeric and metallic ligature. Self-ligating brackets could be divided into two categories: 1) active, which has a spring clip that presses against the archwire in the bracket slot such as In-Ovation ® R (Dentsply -GAC), and 2) passive, in which the clip does not press against the archwire such as the SmartClip TM (3M Unitek). [14][15][16] Previous studies describe that different types of brackets could influence bacterial contamination. [16][17][18][19] However, other authors have not been as concerned in assess the microbial contamination of self-ligating brackets after clinical use. 20,21 To date, according to a recently published systematic review, 22 the authors concluded there is insufficient evidence to support a possible influence of (conventional or self-ligating) brackets on the bacterial colonization, and they are limited to S. mutans. Although the microbial profile seems to be distinct from different brackets, the impact of this condition on the development of dental caries is still not conclusive.
Therefore, the aim of this randomized clinical study was to assess the profile of microbial species colonizing conventional or self-ligating brackets and saliva. Our null hypothesis is that orthodontic appliances do not break the homeostasis in the oral environment, measured by saliva, and that there are no significant differences in the bacterial levels among the different types of brackets on in situ analysis. and number 6 matched the Gemini ™ bracket removed 60 days after bonding (second dental set analyzed). A total of 120 brackets were investigated in this study:
This random assignment also ensured that the In the same way, the statistically significant differences among the three brackets, in situ evaluation, were carried out. Wilcoxon's test was used to determine the differences between the microbial levels at 30 and 60 days after bonding.
Differences were considered significant when p<0.05. The SPSS 21.0.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for data analysis.

Results
A total of fifty participants were recruited. Thirty of them were excluded, and twenty people were enrolled in this study. Figure 2 highlights the flowchart of participants through the trial. showed highest levels before bonding. Significant difference was observed for S. oralis, whose levels decreased 60 days after bonding when compared with S0 (p=0.48) and S1 (p=0.40). An increased level of S. sobrinus (p=0.011) and L. casei (p=0.033) was observed 30 days after bonding when compared with levels before bonding. S. sanguinis, whose levels in S1 were higher than S0 (p=0.004) and S2 (p=0.004) ( Table 1).
The in situ analysis showed the presence of all species in all brackets. Table 2 and Table 3 show the microbial levels, in the different periods of this study.
No significant difference was observed in the contamination levels of Candida ssp. among different brackets by the Friedman test ( Table 3).
The bacterial levels showed a significant difference for the S. mutans 60 days after bonding among the three different brackets, by Friedman test. The highest   Although a statistically significant difference was not found, the highest levels of L. casei and S. sobrinus were also observed in the In-Ovation ® R (Table 2).
When the in situ levels of microbial species were compared 30 or 60 days after bonding, a significant difference occurred in the S. sanguinis, which increased levels for all types of brackets. Table 4 shows the p-value.

Discussion
In this study, we investigated through the    are able to attach and colonize dental surfaces. 27, 28 We must emphasize that L casei is associated with deep carious lesions, thus it has no capacity for adhesion or the ability to be maintained mechanically. 9  saliva. S. sobrinus is a highly cariogenic species. It presents high acidogenicity, synthesizes extracellular polysaccharides from sucrose, promotes attachment points, has capacity to store compounds that could be converted into acid during periods when sugars are not available, and it grows in a low pH environment. 7,8 The in situ analysis of this study showed a different contamination pattern when compared with salivary analysis. While the salivary analysis showed that S. In this article, we focused only on the microbial ecology of different brackets. Dental caries is an endogenous disease, caused by change of mutualistic symbiosis in the microbial ecosystem, associated with local environmental changes, sugar and carbohydrate intake, salivary secretion, and previous dental caries history. Some diseases such as mouth breathing syndrome increased susceptibility to dental caries and other oral infections 33,34 in order to minimize the bias, the sample in this study was not composed of mouth-breathing patients. Considering these aspects, future microbiological studies should focus on all these aspects to better understand the physiological mechanisms that maintain the dynamic stability in dental biofilms and dental caries in orthodontic treatments, as well as the impact on oral health.

Conclusion
Within the limitations of this study, we can conclude that orthodontic appliances disrupted the homeostasis of microorganisms commonly involved in dental caries. The type of bracket may influence the bacterial adhesion, since a significant difference was found for the S. mutans levels among the three brackets over time, with the highest value observed for the self-ligating bracket In-Ovation ® R. A similar pattern of colonization was observed for S. sobrinus and L. casei, whose highest value was detected in the In-Ovation ® R bracket as well.