Salivary concentrations of Streptococcus mutans and Lactobacilli during an orthodontic treatment. An observational study comparing fixed and removable orthodontic appliances

Abstract Aim This study aimed to investigate salivary concentrations of Streptococcus mutans (S. mutans) and some Lactobacilli, and plaque index (PI) in patients wearing fixed versus removable orthodontic appliances. Methods A sample of 90 orthodontic patients (56 males and 34 females) was included in the study: 30 subjects (aged 21.5±1.5 years) were treated with removable clear aligners (CA), while for other 30 cases (aged 23.3±1.6 years) a fixed multibrackets appliance (MB) were utilized, and 30 patients (aged 18.2 ±1.5 years) wearied a removable positioner (RP). Salivary concentrations of S. mutans and Lactobacilli and PI were evaluated prior to start of the orthodontic treatment, after 3 months and 6 months. Results After 6 months, 40% of MB patients (12 subjects over 30) showed a concentration of S. mutans associated to high risk of developing tooth decay (CFU/ml>105), differently from participants wearing removable appliances (odds ratio = 5.05; 95% C.I. = 1.72‐14.78; chi‐square = 9.64; p = 0.0019). The same trens was observed for the concentration of Lactobacilli (odds ratio = 4.33; 95% C.I. = 1.53‐12.3; chi‐square = 8.229; p = 0.004). In addition, over the duration of the study, CA patients maintained PI at 0 level, while MB patients experienced a statistically significant increasing trend of PI over time, and their PI became clinically/statistically relevant after 6 months, respect to CA and RP patients. Conclusions Comparing all the data, while, after 6 months, only about 10% of CA patients and 13.3% of RP patients achieved a microbial colonization which may lead to high risk of caries development, about 40% of MB patients ‐ and 20% after 3 months ‐ showed a high level of vulnerability to developing caries, which require additional strategies for plaque control and microbial colonization to be employed.

3 months -showed a high level of vulnerability to developing caries, which require additional strategies for plaque control and microbial colonization to be employed.

K E Y W O R D S
Clear aligners, Dental Plaque Index, Fixed orthodontic appliances, Lactobacilli, Microbial colony count, Oral Hygiene, Orthodontics, Removable orthodontic appliances, Removable positioner, Saliva, Streptococcus mutans 1 | INTRODUCTION Two literature reviews (Freitas, Marquezan, Nojima, Alviano, & Maia, 2014) (Lucchese, Bondemark, Marcolina, & Manuelli, 2018) show that there is moderate-to-high evidence that orthodontic appliances are able to significantly influence the concentration of oral microbiota, causing an alteration of the quantity of Streptococcus mutans (S. mutans) and Lactobacilli that can basically affect the process of dental caries and tooth enamel demineralization, due to their acid production and tooth adhesive properties. This statement is confirmed both for removable (Mummolo et al., 2014) and fixed (Mummolo et al., 2013) orthodontic appliances.
From a clinical point of view, as a probable consequence of the changes in microbiota, caries incidence increases during orthodontic treatment with multibrackets appliances (MB), as well as the occurrence of white spot lesions, which range, among orthodontic patients, from 2% to 97% (Migliorati et al., 2015;Mummolo, Nota, De Felice, et al., 2018). On the contrary, it was observed that removable orthodontic appliances have less impact on the oral microbiota than fixed ones (Mummolo et al., 2014), but literature still lacks data on clear aligners (CA), a removable appliance that has become highly demanded as an alternative to MB, owing to its desirable characteristics providing aesthetic and comfortability. (Brignardello-Petersen, 2019) The attention on clear aligners has ultimately increased over time, mostly because they are often used to manage pre-prosthetic clinical cases (Dinoi et al., 2015), also affected by temporo-mandibular joint diseases, (Tecco, Festa, Salini, Epifania, & D'Attilio, 2004) or adult patients for which a molar distalization is required, owing a great attention to the periodontal health status (Caruso et al., 2019) But, also in children, this type of removable appliance has been increasingly used, for example, to manage clinical cases with a single tooth anterior crossbite, to prevent further progress of periodontal diseases. (Meuli, Tecco, Nota, Gatto, & Caruso, 2018) (Silvestrini-Biavati et al., 2013 Furthermore, considering previous observations for removable appliances (Lucchese et al., 2018) (Mummolo et al., 2014), there exist a general clinical trend to prefer CA to maintain a more satisfactory level of oral hygiene during the orthodontic treatment. It can be justified that to obtain a better periodontal health the CA appliance can be more desirable than MB appliances.  Due to the lack of empirical data this study aims to provide a solid base for assessment of a preferable trend that rempvable appliance, in particular CA appliance, can provide a more suitable clinical treatment while reducing the concentrations of S. mutans and Lactobacilli in saliva.
In addition, the plaque index (PI) was also evaluated, because as evidence exists for salivary bacterial concentrations being an acceptable approximate representation of dental plaque, they aren't nonetheless an indirect measure of the bacterial threat on the teeth. Thus, the recording of PI parallel with the count of cariogenic species in the saliva could be a validation of the clinical conclusions derived from the data.

| Study population and methodology
This is an observational controlled study, aimed to investigate salivary concentrations of S. mutans and Lactobacilli, and PI in patients wearing fixed versus removable orthodontic appliances. The participants were selected from a population of young adult patients who were going to be treated for their malocclusion in a dental clinic in the geographical region of Abruzzo (Central Italy).
A total sample of 90 patients were included. Demographic data of the sample are described in The orthodontic technique adopted for the treatment of each subject has been already chosen for each partecipant by the expert orthodontists, prior to the beginning of this research project.
The selection of participants was made on the bases of the following inclusion criteria: permanent dentition, adult age, complete dentition, and a malocclusion characterized by Angle class I, with lowmiddle level of crowding (the subjects did not require any orthodontic teeth extraction). The following parameters were taken as exclusion criteria: chronic periodontitis, presence of prosthetic rehabilitations, presence of endodontically treated teeth, history of high grade gingivitis, poor oral hygiene, initial Plaque index (PI) and Bleeding index (BI). The enrollment of the participants was done from January 2016 to April 2018.
All the participants were treated by two expert orthodontists (author F.A. and author S.C.), who exclusively practice the branch of orthodontics for more than 5 years. As the home oral hygiene habits could be considered as a potential confounder, a few days before the beginning of the observational period, a professional oral hygiene procedure was performed on each participants, and accurate oral hygiene instructions were given to each subject to be employed at home. Then, at the day scheduled for the beginning of the orthodontic treatment a salivary sample was taken, and the PI was recorded, from each subject preceding to the bonding procedure. Other salivary samples and recordings of PI were taken after periods of 3 and 6 months. All the salivary samples were taken by the same operator.
The salivary samples were analyzed by CRT ® bacteria (Ivoclar Vivadent Clinical, Schaan, Liechtenstein). The CRT ® bacteria was employed for the bacterial count, as previously published. (Mummolo et al., 2013;Mummolo, Nota, Caruso, et al., 2018) CRT ® bacteria was used to determine the S. mutans and Lactobacilli count in saliva by means of selective culture media. The dentist and skilled personnel professionally conduct the test. Findings of 10 5 CFU or more of Lactobacilli and S.mutans per ml saliva indicate a high caries risk. Leaving the test vial inside an incubator for an additional day or two does not influence the number of CFUs. The preparation of samples and incubation were carried out according to the step-by-step procedure as it was described in its instruction brochure. This test only determines whether or not S. mutans are present in dental saliva.
The CRT ® bacteria can be considered a comprehensive test, whose main benefits are to determine the caries risk status, to create the basis for target treatment and individualized check-up intervals for the long-term maintenance of oral health. This chair-side method is highly specific and sensitive for S. mutans (Sánchez-García et al., 2008) and its only limitation is that at least 48 hours are require for detection of

S. mutans.
At each of the follow-up appointments (at the beginning, after 3 months, and after 6 months), firstly, the PI was recorded. Then, the patient was asked to chew a stimulant paraffin tablet for 30 seconds, then the secreted saliva was collected in a glass tube and used for bacterial count through the CRT ® bacteria. The saliva was placed in culture media (agar). A NaHCO 3 tablet was added to stimulate the growth of bacteria and each culture was placed in an incubator at 35-37 C for 48 hours. S.mutans colonies appeared visible as small blue colonies with a diameter < 1mm on blue agar, while white color Lactobacilli colonies were detected on transparent agar. The presence of a bacterial count higher than 10 5 CFU/ml of saliva indicates a high risk of developing caries (cut-off value for the high risk). Thus, in this study, subjects were dichotomized as S. mutans and Lactobacilli CFU > or < 10 5 CFU/ml, that is considered the cut-off value for the high risk.

| Data analyses
To avoid bias, the data were analyzed by operators who were blind to the fact that each collected data belongs to which group.
The data about the microbiota were examined considering the number of subjects (and percentage) with CFU/ml ≥ or < the cut-off value (i.e. 10 5 CFU/ml). These percentages were compared over time at the beginning of the treatment, after 3 months and after 6 months from the beginning, and differences among the three groups were investigated using the Chi-square test.
PI was handles as a continuous variable, and recorded through 0, 1, 2, and 3 values. Descriptive statistics included mean and standard deviation, and differences among the two groups were tested by the ANOVA statistics, and post-hoc comparisons.
For all the analysis the p value was set at 0.05.

| RESULTS
All the participants completed the study, without any adverse event, and there weren't missing data.    influence the oral ecosystem. (Ferrazzano et al., 2017) This study was subjected to some limitations, for instance only patients with Angle class I malocclusion with low/mild level of crowding were included, and consequently it wasn't possible to analyze the type of malocclusion as a confounding factor, therefore the present results can not be generalized for other type of malocclusions with more severe level of crowding. In addition, smoking patients were not included, while it is known that salivary immunoglobulin and T A B L E 3 Plaque Index (mean and standard deviation) in the three groups at t0, t1 and t2, with intra-group and between groups differences

Clinical Relevance
In general, it was observed that removable appliances have less impact on the oral microbiota than fixed ones, but literature still lacks data on clear aligners (CA), an appliance that has become an increasingly applied treatment of choice because of the great number of patients that desire aesthetic and comfortable alternatives to multibracket appliances (MB).

Principal findings
After 6 months of treatment, MB patients manifested an odds ratio of additional strategies for plaque control and microbial colonization must be controlled just after the first 6 months of treatment. Thus plaque control and salivary microbial colonization must be assessed after three and six months of treatment.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Ethics approval was obtained by the Ethic Committee of the University of L'Aquila, Italy (Document DR206/2013). The consent to the treatment was obtained by the patients before the beginning of the therapy.

CONSENT FOR PUBLICATION
The consent to publish the present data was obtained from the subjects.

AVAILABILITY OF DATA AND MATERIALS
The data that support the findings of this study are available from the archive of the University of L'Aquila, but restrictions apply to the availability of these data, which were used under permission and consent for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the patients and the Ethic Committee of the University of L'Aquila.

COMPETING INTERESTS
The authors declare that they have no competing interests FUNDING This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

AUTHORS' CONTRIBUTIONS
SM: study conceiving, data recording, data analysis, data interpreta-