Salivary levels of Streptococcus mutans and Lactobacilli and other salivary indices in patients wearing clear aligners versus fixed orthodontic appliances: An observational study.

OBJECTIVE: This study aimed to investigate salivary levels of Streptococcus mutans (S. mutans) and Lactobacilli, and other salivary indices in subjects wearing clear aligners (CA) in comparison with multibrackets orthodontic appliances (MB).
MATERIALS AND METHODS: A sample of 80 participants (46 males and 34 females) was included in the study: 40 subjects (aged 20.4±1.7 years) were treated with CA, and 40 (aged 21.3±1.7 years) were treated with MB. Plaque index (PI), salivary flow, buffering power of saliva, and salivary levels of S. mutans and Lactobacilli were evaluated prior to start of orthodontic treatment (t0), after 3 months (t1) and 6 months (t2).
RESULTS: CA patients maintained PI at level 0 over time, while MB participants experienced a statistically significant increasing trend of PI over time. In addition, at t2, 37.5% of MB participants (15 subjects over 40) showed risky salivary levels (CFU/ml>105) of S. mutans (odds ratio = 7.40; 95% C.I. = 1.94-28.25; chi-square = 10.32; p = 0.001) as well as Lactobacilli (odds ratio = 23.40; 95% C.I. = 2.91-188.36; chi-square = 15.31; p = 0.0001).
CONCLUSIONS: Comparing all the data, subjects treated with CA achieved lower salivary microbial colonization after 6 months of treatment compared with MB. Different additional strategies for plaque control and salivary microbial colonization must be triggered considering the type of orthodontic appliance.

Introduction

The bacterial microflora, present in the oral cavity, contributes to the health of the host and prevents infections by potentially pathogenic exogenous microorganisms, thus providing resistance to the colonization of these parasitic species, and by regulating the inflammatory response towards the commensal bacteria that host in the buccal cavity.[1] Two literature reviews [2, 3] showed 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 tooth enamel demineralization [4], due to their acid production and tooth adhesive properties. This statement is confirmed both for removable [5] and fixed [6] orthodontic appliances.

From a clinical point of view, as a consequence of the changes in microbiota, enamel demineralization increases during orthodontic treatment with multibrackets appliances (MB) ranging in orthodontic patients from 2% to 97%. [7]

In general, it was also observed that removable appliances have less impact on the oral microbiota than fixed ones [5], but literature still lacks data on clear aligners (CA), an appliance that has become a highly demanded alternative to MB, due to its desirable characteristics providing aesthetic and comfortability [8]. Furthermore, considering previous observations for removable appliances [3, 5] there is a general clinical trend to prefer CA to maintain a more satisfactory level of oral hygiene during the orthodontic treatment, in respect to MB. A previous study compared the periodontal health status between subjects in orthodontic treatment with CA and MB, observing a better periodontal health in the group treated with CA [9]. On the basis of this background, the purpose of the present study was to investigate salivary levels of S. mutans, Lactobacilli and other salivary indices in patients wearing CA versus MB.

Materials and methods

This is an observational prospective controlled study, aimed to investigate salivary levels of S. mutans, Lactobacilli and other salivary indices in patients wearing CA versus MB. 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 80 subjects were selected, 40 participants were treated with CA (Invisalign, Align Technology, Santa Clara, CA, USA) and the other 40 participants (matched with the previous group for age and gender distribution) were treated with MB (Damon Q2, Ormco, Washington, DC, USA) (Table 1).

Table 1

Demographic information about the whole sample.

	Clear aligners (CA group)	Multibrackets appliance (MB group)
Gender	24 M and 16 F	22 M and 18 F
Numerosity	40	40
Age (mean ±sd)	20.4±1.7 years	21.3±1.7

The orthodontic technique adopted for the treatment of each patient was chosen independently by the expert orthodontists prior to the beginning of this research project. The selection of participants was made on the basis of the following inclusion criteria: permanent dentition, adult age, complete dentition, and a malocclusion characterized by Angle class I, with low-middle level of crowding (the patients did not require any orthodontic teeth extraction). The following parameters were taken as exclusion criteria: presence of oral breathing, presence of active caries, chronic periodontitis, presence of prosthetic rehabilitations, presence of endodontically treated teeth, history of high grade gingivitis, poor oral hygiene, evaluated through initial Plaque index (PI) and Bleeding index (BI). Regarding dental caries as an exclusion criteria, they were assessed by clinical examination with specillum and mirror before the beginning of the study and during the study at each appointment. In particular, the orthodontic treatment was not initiated (brackets or first aligner positionment) if caries were found on the first clinical examination, in this case a conservative treatment of dental caries was performed before beginning the treatment. No caries were then observed in the whole sample during the study period.

A sample power analysis was carried out considering the results of a preliminary pilot study on the first 30 participants (cit.), recording a difference of 29% in the number of participants with a bacterial count for S. mutans higher than 105 CFU/ml between the groups, concluding that a minimum sample size in order to achieve a power of 80% with an alpha of 0.05 should be 39 subjects for each group. All the participants were treated by two expert orthodontists, who have exclusively practiced the branch of orthodontics for more than 5 years. The enrollment of the participants was done from January 2016 to June 2017. The matching criteria were confirmed since no significant difference was detected in gender distribution and mean age between the participants of the two groups.

Data collection and follow-up of the subjects were performed in the following way. A proper informed consent form was signed by each subject during one of the preliminary appointments before beginning the actual treatment. To motivate the participants, a free oral hygiene session (with instructions) was offered. Furthermore, subsequent follow-up visits included in the present protocol were arranged free of charge. This clinical protocol was in accordance with the ethical standards reported in the Declaration of Helsinki of 1975, and the study was ethically approved by the Ethical Committee of the University of L’Aquila (Document DR206/2013).

As the home oral hygiene habits could be potential confounders, a few days before the beginning of the observational period a professional oral hygiene procedure was performed to each of the participants, and accurate home oral hygiene instructions were given to each subject to be employed at home. Then, on the day scheduled for the beginning of the orthodontic treatment (T0), a salivary sample was taken from each subject before the bonding procedure, and other salivary samples were also taken after periods of 3 months (T1) and 6 months (T2). All the salivary samples were taken by the same operator during the morning (9am-12am).

The salivary samples were analyzed by CRT® prevention system (Ivoclar Vivadent Clinical, Schaan, Liechtenstein). the CRT® buffer system was used to evaluate salivary flow and saliva buffering power, and the CRT® bacteria was employed for the bacterial count, as previously published [6, 10]. At each of the follow-up appointments participants were recommended to refrain from eating, drinking alcohol and brushing their teeth for at least one hour prior to the visit, as all these actions could alter the average salivary flow. During every scheduled appointment, first the PI was recorded (clinical recordings were made by the author S.M.). Then, the patient was asked to chew a stimulant paraffin tablet for 30 seconds, then the secreted saliva was collected. These samples were immediately subjected to evaluation of the buffering capacity by using the CRT® buffer, which was expressed, according to the manufacturer’s instructions, in five scores. Subsequently, the participant was invited to chew the paraffin tablet for another 5 minutes, collecting all the produced saliva in a scaled glass tube and then the flow rate was calculated (ml/min). Through this procedure, milliliters of saliva were collected and the salivary flow rate for minutes (ml/min) was calculated. Subsequently, a part of each sample was used for bacterial count through the CRT® bacteria. The saliva was placed in culture media (agar) using pipettes. A NaHCO3 tablet was added to stimulate the growth of bacteria. The culture was then 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 105 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 < 105 CFU/ml, which is considered the cut-off value for the high risk [11, 12].

Data analysis

To avoid bias, the data were analyzed by operators who did not know the origin group of the collected data. The buffering power and the salivary flow were taken as nominal variables. In particular, for the salivary flow, the data were handled in 3 categories, as follows: level 1 = 02. For the buffering power of saliva, the data were categorized in the following way: 1 = low; 2 = medium-low; 3 = medium; 4 = medium-high; 5 = high. These data were compared between the two groups using the chi-square test.

The data about the microbiota were examined considering the number of subjects (and percentage) with CFU/ml ≥ or < the cut-off value (i.e. 105 CFU/ml). These percentages were compared over time at t0, t1 and t2, and differences between the two groups were investigated using the Binomial test.

The Plaque index (PI) was considered as a continuous variable and recorded as 0, 1, 2, and 3 values; for this variable, descriptive statistics showed mean and standard deviation; differences between the two groups were tested by Student t test.

For all the analyses the p value was set at 0.05.

Results

All the participants completed the study, without any adverse event, and there were no missing data. Table 2 shows the distribution of values ​​for the PI, the salivary flow and the buffering power of saliva at t0 in both groups.

Table 2

Data observed before the beginning of the study in the two groups.

	Clear aligners (CA group) (modal value) N = 40	Multibrackets appliance (MB group) (modal value) N = 40	CA Group vs MB Group
PI	0	0	n.s.
Salivary flow	1 (observed in 26 over 40 subjects; 65%)	1 (observed in 21 over 40 subjects; 52.5%)	Chi-square: 13.61; p = 0.01
Buffering power	3	3	n.s.

For the plaque index, the scores are 0.1.2.3.

For salivary flow: low (1 = 02).

For the buffering power of saliva: 1 = low; 2 = medium-low; 3 = medium; 4 = medium-high; 5 = high.

For the salivary flow, a statistically significant difference was found between the two groups at t0, with the CA group showing a low level of salivary flow (0MB group (p = 0.01). Table 3 reports descriptive statistics (mean ± standard deviation) for the PI over time, at t0, t1 and t2, in the two groups. In the CA group, PI remained 0 over the whole follow-up period in all the subjects, while a progressive increase over time was observed in MB group, with a statistically significant increase over time, from t0 to t2, and significant differences with the other group at t2 and t3.

Table 3

Plaque index (mean and standard deviation) in the two groups at t0, t1 and t2, with intra-group and between groups differences.

	t0 Initial	t1 After 3 months	t2 After 6 months	t0 versus t1 comparison	t0 versus t2 comparison	t1 versus t2 comparison
Clear aligners (CA group)	0	0	0	n.s.	n.s.	n.s.
Multibrackets appliance (MB group)	0	0.7 ± 0.55	1.4 ± 0.5	t = -7.85 p = 0.001	t = -5.6 p = 0.001	t = -17.8 p = 0.001
CA group versus MB group comparison	n.s.	t = -7.85 p = 0.001	t = -17.8 p = 0.001

n.s. = not significant

The buffering power of the saliva remained almost constant over the follow-up period in both the two groups (score 5).

Table 4 shows the average values of salivary flow. As seen, the two groups showed a statistically significant difference at t0 (p = 0.01), although the modal value resulted 1 in both the groups. At t1 and t2 no significant difference was observed between the two groups.

Table 4

Salivary flow modal values (number and percentage of subjects with the modal values) at t0, t1 and t2 in the two groups, and significant differences.

	t0 Initial	t1 After 3 months	t2 After 6 months	t0 versus t1 comparison	t0 versus t2 comparison	t1 versus t2 comparison
Clear aligners (CA group)	1 (26 over 40; 65%)	1 (22 over 40; 55%)	1 (22 over 40; 55%)	n.s.	n.s.	n.s.
Multibrackets appliance (MB group)	1 (21 over 40; 52.5%)	1 (24 over 40; 60%)	1 (24 over 40; 60%)	n.s.	n.s.	n.s.
CA group versus MB group Comparison	Chi-square: 13.61; p = 0.01	n.s.	n.s.

n.s. = not significant

Table 5 shows the percentage of subjects with S. mutans and Lactobacilli CFU/ml >105 over time in both groups. In the CA group, the number of subjects with S.mutans CFU/ml >105 slightly increased at t2, without statistical relevance. In the MB group, it increased progressively over time, with a statistically significant difference from t0 to t1 and from t0 to t2. The differences between the two groups were statistically significant at t1 and t2. At t2 only 8% of CA subjects (8 subjects over 40) showed S.mutans CFU/ml >105, whereas 37.5% of MB subjects (15 subjects over 40) showed it. Thus, MB subjects manifested an odds ratio of 7.40 (95% C.I. = 1.94–28.25; chi-square = 10.323; p = 0.001) in respect to CA subjects, which suggests that MB subjects after 6 months of treatment are at a higher risk of developing caries, due to the salivary concentrations of S. mutans.

Table 5

Number (and percentage) of subjects with S. mutans and Lactobacilli CFU >105, at t0, t1 and t2, with intra-group and between-groups differences.

S.Mutans CFU > 105
	t0 Initial	t1 After 3 months	t2 After 6 months	t0 versus t1 comparison	t0 versus t2 comparison	t1 versus t2 comparison
Clear aligners (CA group)	0 over 40 (0%)	0 over 40 (0%)	3 over 40 (8%)	n.s.	n.s.	n.s.
Multibrackets appliance (MB group)	0 over 40 (0%)	8 over 40 (20%)	15 over 40 (37.5%)	Chi-square = 8.88 p = 0.002	Chi-square = 18.462 P = 0.001	n.s.
CA group versus MB group Comparison	n.s.	Chi-square = 8.88 p = 0.002	Chi-square = 10.32 p = 0.001
Lactobacilli CFU > 105
	t0 Initial	t1 After 3 months	t2 After 6 months	t0 versus t1 comparison	t0 versus t2 comparison	t1 versus t2 comparison
Clear aligners (CA group)	0 over 40 (0%)	0 over 40 (0%)	1 over 40 (2.5%)	n.s.	n.s.	n.s.
Multibrackets appliance (MB group)	0 over 40 (0%)	8 over 40 (20%)	15 over 40 (37.5%)	Chi-square = 8.88 p = 0.002	Chi-square = 18.462 p = 0.001	n.s.
CA group versus MB group Comparison	n.s.	Chi-square = 8.88 p = 0.002	Chi-square = 15.313 p = 0.0001

n.s. = not significant

For the Lactobacilli CFU/ml >105 the differences between the two groups were also statistically significant at t1 and t2. Thus, the MB group manifested a 23.40 odds ratio(95% C.I. = 2.91–188.36; chi-square = 15.313; p = 0.0001) of showing risky values of Lactobacilli colonization after 6 months of treatment compared to the CA group.

Discussion

This study aimed to investigate PI, the salivary levels of S. mutans and Lactobacilli, and other salivary indices in subjects wearing CA versus MB. In fact, scientific evidence showed that the early stage of caries is highly affected by S. mutans and by visible dental plaque on maxillary incisors whereas cavities are strongly related to lactobacilli [13].

From the present data, it results that PI did not show statistically significant changes over time in the CA group, while the MB group experienced a statistically significant increasing trend, with a clinically relevant PI at t2 (mean PI>1) as reported in Table 3, with statistically significant differences compared to the other group at t1 and t2. This result suggests that CA allow the maintenance of a better oral hygiene level, compared to MB. This observation agrees with previous data from literature, indicating that teenagers treated with CA show a significantly lower plaque index than those treated with MB, even at a follow up of 12 months [14]. In addition, a systematic review pointed out, with a level of moderate evidence, that periodontal health indexes are significantly improved with CA compared to MB [15].

Regarding the buffering power of saliva, described in Table 4, the present survey data show that it remained stable over time in both the two groups, as expected, because this is a physical property of saliva. It prevents the colonization of pathogenic microorganisms in the mouth and neutralizes acids produced by acidogenic bacteria, and enamel demineralization [10, 16]. The present data confirm previous reports in literature, which used the same methods to evaluate the buffering capacity of saliva in the course of removable orthodontic treatment [5] and with passive self-ligating brackets [6] and reported no change for this parameter over the follow up period. Thus, it could be stated that CA are unable to influence the buffering power of saliva.

Similarly, the salivary flow (Table 4) showed no difference between the two groups at t1 and t2. The great part of participants showed a normal value of salivary flow (<1.5 ml). This observation suggests that the type of orthodontic appliance is unable to influence the salivary flow. At the authors’ knowledge no previous study was performed about the buffering power of saliva and salivary flow in subjects treated with CA.

For the microbial colonization, CRT® bacteria was used to determine the S. mutans and Lactobacilli count in saliva by means of selective culture media. 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 in quantity that determines an increase of the caries risk.

The CRT package 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 and it is only limited by the 48 hours required for detection of S.mutans [17].

A different trend of bacterial colonization in the two groups was observed (Table 5). All the CA participants showed S. mutans and Lactobacilli colonies under the risky values (i.e. 105 CFU/ml) at t0 and t1. While at t2, only 8% of the group (3 participants over 40) showed a risky value. While the MB group showed a progressive increase over time from t0 to t1 (p<0.01) and at t2, when the 37.5% of participants (15 participants over 40) showed risky values. As seen, the count of Lactobacilli colonies showed a trend similar to S. mutans (Table 5) in the two groups. These trends suggest that CA do not increase the bacterial growth, and consequently the caries risk, in about 90% percent of the treated subjects, even after six months of treatment. These results are in agreement with a recent study by Wang et al. (Wang et al. 2019) that performed a microbiological analysis of salivary bacteria in subjects in orthodontic treatment with CA compared with MB and untreated control group, observing a lower abundance of Firmicutes (the phylum of both S. Mutans and Lactobacilli) in the CA group compared with the MB group and the absence of differences with the control group. On the contrary, a recent prospective study, performed by quantitative polymerase chain reaction, found no differences in the identification of S. mutans and Lactobacillus acidophilus in the saliva of participants wearing thermoplastic retainers compared with subjects in orthodontic treatment with fixed appliances. But in that study no quantitative analysis was possible, as almost no Lactobacillus acidophilus was identified in the collected samples [18].

From a clinical point of view, comparing all the data, it can be stated that only about 8% of CA participants achieve risky values of microbiota colonies after 6 months of treatment, with a stable plaque control, differently from MB participants. The maintenance of a better macroscopically (plaque index) and microscopically (S. mutans and Lactobacilli CFU) oral hygiene levels in CA participants, compared with MB participants, should be related to the absence of retentive surfaces on the patient’s teeth and the consequent facilitation of oral hygiene even comparable with an untreated subject [6]. While in about 40% of MB participants there is an increased risk of dental caries and demineralizations and so additional strategies for plaque control must be applied after the first six months of treatment (and for 20% just after 3 months), the use of CA seems to significantly limit this risk to less than 10% of the subjects. Additional strategies in subjects with risk of developing demineralization could be the use of disinfecting and mineralizing substances, to be used at all times when the patient has no social interactions, preserving the potential aesthetic of therapy [16]. An additional aid can be provided by the use of food, like some yoghurt that can positively influence the oral ecosystem [19].

Literature has not clarified yet whether there is a direct proportion between the count of bacterial colonies in the saliva and the appearance/increase of decalcifications on the teeth during orthodontic treatment. This correlation is actually derived from a clinical observation, according to which decalcifications near to orthodontic brackets can increase during orthodontic treatment [20], and from the laboratory observation that the number of bacterial colonies in the saliva is linked to a higher risk of enamel decalcification [11]. From one of the classical studies in literature dated 1989, S. mutans has been detected in the dental plaque near an orthodontic band both in areas interested by enamel dissolution and in other areas of the teeth not interested by enamel dissolution, without any relationship between the presence of S. mutans and enamel dissolution [21]. Similarly, while it must be observed that the literature is poor about the topic, it seems that there is no direct relationship between salivary and dental plaque microbiome [22], both plaque amount and saliva microbiomes, analyzed in the present study, are associated with the development of dental caries [11, 23].

A considerable limitation of the present study, related with the absence of a proper randomization, is the presence of a selection bias related with the possible higher socioeconomic status of subjects treated with CA rather than MB considering that there was a reported association between oral hygiene level and socioeconomic status [24].

While exclusion criteria were carefully applied to exclude potential confounders [25], this study has some limitations because only subjects 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 confounding factor and the present results are limited to young adult subjects with Angle class I malocclusion and low/mild level of crowding and further studies are needed to generalize them to other types of malocclusion. Similarly, no certain data were included about the compliance of the participants regarding home oral hygiene procedure, although proper instructions were given to all the participants. Furthermore, other limitations of the present study were the use of the CRT® prevention system (Ivoclar Vivadent Clinical, Schaan, Liechtenstein) as an in-office test rather than other laboratory tests with the related limitations, among them the use of paraffin wax to stimulate salivary flow, that may have slightly modified the results by detaching bacteria from surfaces.

Conclusions

Comparing all the data, orthodontic treatment with CA appliances allow the maintenance of a better oral hygiene level, compared to MB. Only 8% of CA participants against approximately 40% of MB participants showed high concentrations of S. Mutans after 6 months of treatment requiring additional strategies for plaque control considering the type of orthodontic appliance.

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26 Sep 2019

PONE-D-19-21131

Salivary levels of Streptococcus mutans and Lactobacilli and other salivary indices in patients wearing clear aligners versus fixed orthodontic appliances: an observational study

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Reviewer #1: Thank you for your submission

A few issues - firstly, the English is not easy to read, so i suggest you recruit a native English speaker knowledgeable in cariology or microbiology

the term 'patient' should be avoided and replaced with 'participant'

Streptococcus mutans in italics or S. mutans (space between S. and mutans) should be used - you have a variety of iterations of these

On page 4, you mention 'caries' incidence increases as well as white spot lesions - white spot lesions are carious lesions - please correct terminology - maybe refer to Innes et al JDR 2016 caries terminology paper

Did you attempt to categorise the amount of crowding apart from 'low-middle'? Crowding features such as rotations and imbrications can have an effect on oral hygiene effectiveness

Did you collect dietary data, as the type of appliance could influence dietary habits?

You do not discuss the limitations of the simplistic microbiological testing methodology you have used - also, is salivary microbiome representative of the plaque microbiome? this should be discussed

Are Lactobacilli involved in the early development of carious lesions?

In the cut off values for bacterial CFUs, you cite 4 papers, none of which investigated bacterial counts with carious lesion development - do you think these are appropriate references?

Is there any evidence that salivary buffering capacity tested as you have done is correlated with caries risk?

Did you examine the participants for carious lesions? if not - why not?

If you did, why isn't it reported?

Of the four references you use to justify the cut-oof values for CFUs, only one correlated Lactobacilli counts to carious lesions, and the majority of children had cavitated lesions - which i assume is not the case in your study- these are not sound references to use with early carious lesions

Reviewer #2: Is there any correlation between number of bacteria and decalcification lesions? if so, please discuss

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Reviewer #1: No

Reviewer #2: Yes: Phimon Atsawasuwan

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Submitted filename: Peer review PLOS ONE 19-21131.docx

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12 Nov 2019

Reviewer #1: Thank you for your submission

A few issues - firstly, the English is not easy to read, so i suggest you recruit a native English speaker knowledgeable in cariology or microbiology

R: The manuscript was fully proofreaded by a native English speaker dentist

The term 'patient' should be avoided and replaced with 'participant'

R: thanks for the suggestion. It was changed all over the manuscript

Streptococcus mutans in italics or S. mutans (space between S. and mutans) should be used - you have a variety of iterations of these

R: thanks for the suggestion. It was changed all over the manuscript

On page 4, you mention 'caries' incidence increases as well as white spot lesions - white spot lesions are carious lesions - please correct terminology - maybe refer to Innes et al JDR 2016 caries terminology paper

R: thanks to the reviewer for this suggestion. We considered the terminology referring to Innes et al (2016) Innes NP, Frencken JE, Schwendicke F. Don't Know, Can't Do, Won't Change: Barriers to Moving Knowledge to Action in Managing the Carious Lesion. J Dent Res. 2016 May;95(5):485-6. doi: 10.1177/0022034516638512. PMID: 27099269)

Did you attempt to categorise the amount of crowding apart from 'low-middle'? Crowding features such as rotations and imbrications can have an effect on oral hygiene effectiveness

R: thanks to the reviewer for this suggestion. Unfortunately, in this study, we included only patients with "low-middle" degree of crowding, in order to maintain the homogeneity between the two groups. Surely this inclusion criterion represents a limit for the present study. The reviewer with this observation gives us an indication to future studies that also include patients with “moderate/ severe” crowding. This was also already reported among the limits of the study at the end of the discussion section.

Did you collect dietary data, as the type of appliance could influence dietary habits?

R: unfortunately we have not investigated patients' eating habits. The reviewer with this observation gives us an indication to future studies.

You do not discuss the limitations of the simplistic microbiological testing methodology you have used

R: thanks to the reviewer for the suggestion. The following sentence was added in the Discussion: “CRT® bacteria was used to determine the S. mutans and Lactobacilli count in saliva by means of selective culture media. 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 saliva in quantity that determines an increase of the caries risk.

The CRT package 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 and its only limitation is that at least 48 hours are required for detection of S. mutans.

Sánchez-García S, Gutiérrez-Venegas G, Juárez-Cedillo T, Reyes-Morales H, Solórzano-Santos F, García-Peña C. A simplified caries risk test in stimulated saliva from elderly patients. Gerodontology. 2008;25:26–33. doi:10.1111/j.1741-2358.2007.00184.x.

Also, is salivary microbiome representative of the plaque microbiome? this should be discussed

R: The literature is poor on this topic and an old study showed that there isn’t a direct correlation between the plaque amount and the saliva microbiome (Jalil RA. Correlating Streptococcus mutans counts in saliva with plaque amount, gingival inflammation and caries experience in school children. Singapore Dent J. 1995 Jul;20(1):16-20.).

Anyway both plaque and saliva microbiomes are associated with the development of dental caries. In the present study the evaluation of the PI, and the saliva microbioma, gives to the literature a new step in this field.

This point was added to the discussion following the suggestion of the reviewer.

Are Lactobacilli involved in the early development of carious lesions?

R: The evidence remains weak or absent about the association between initial dental caries and Lactobacilli. (Neves BG, Stipp RN, Bezerra DDS, Guedes SFF, Rodrigues LKA.Quantitative analysis of biofilm bacteria according to different stages of early childhood caries.Arch Oral Biol. 2018 Dec;96:155-161. doi: 10.1016/j.archoralbio.2018.09.007.

Periodontol 2000. 2016 Feb;70(1):128-41. doi: 10.1111/prd.12100. Salivary biomarkers for dental caries. Gao X, Jiang S, Koh D, Hsu CY)

On the other hand it seems that the early stage of caries is highly affected by mutans streptococci and visible dental plaque on maxillary incisors whereas cavities are strongly related to lactobacilli that are then involved in the lesions development (Parisotto TM, Steiner-Oliveira C, Duque C, Peres RC, Rodrigues LK, Nobre-dos-Santos M. Relationship among microbiological composition and presence of dental plaque, sugar exposure, social factors and different stages of early childhood caries.Arch Oral Biol. 2010 May;55(5):365-73.)

This was also added to the discussion in order to make more clear for the reader the involvement of each considered parameter.

In the cut off values for bacterial CFUs, you cite 4 papers, none of which investigated bacterial counts with carious lesion development - do you think these are appropriate references?

R: 3 out of 4 cited papers are previously published studies that uses the same microbiological technique and cutoff values for caries risk evaluation. Anyway, according to what suggested by the reviewer we removed these 3 citations and added another citation that observed the CFU cutoff value applied in the present study.

(Gábris K, Nagy G, Madléna M, Dénes Z, Márton S, Keszthelyi G, Bánóczy J.Associations between microbiological and salivary caries activity tests and caries experience in Hungarian adolescents. Caries Res. 1999 May-Jun;33(3):191-5.)

Is there any evidence that salivary buffering capacity tested as you have done is correlated with caries risk?

R: Salivary buffering capacity is correlated with caries risk as reported by different articles as Fernando S, Tadakamadla SK, Bakr M, Scuffham PA, Johnson NW. Indicators of Risk for Dental Caries in Children: A Holistic Approach. JDR Clin Trans Res. 2019 Oct;4(4):333-341. that used a similar caries risk analysis kit and was reported in the manuscript methods section.

The reference was also added to the manuscript.

Did you examine the participants for carious lesions? if not - why not?

If you did, why isn't it reported?

R: All subjects were examined for carious lesions and were free from caries before being included in the study and checked during the therapy.

We thank the reviewer for reminding us to include this as an inclusion criteria in the manuscript, it wasn’t previously reported because for every orthodontist this is a criteria for starting an orthodontic treatment and is considered a routine check.

Of the four references you use to justify the cut-off values for CFUs, only one correlated Lactobacilli counts to carious lesions, and the majority of children had cavitated lesions - which i assume is not the case in your study- these are not sound references to use with early carious lesions

R: As previosly stated, 3 out of 4 cited papers are previously published studies that uses the same microbiological technique and cutoff values for caries risk evaluation. Anyway, according to what suggested by the reviewer we removed these 3 citations and added another citation that observed the CFU cutoff value applied in the present study. (Gábris K, Nagy G, Madléna M, Dénes Z, Márton S, Keszthelyi G, Bánóczy J.Associations between microbiological and salivary caries activity tests and caries experience in Hungarian adolescents. Caries Res. 1999 May-Jun;33(3):191-5.)

Reviewer #2:

This manuscript described to investigate salivary levels of Streptococcus mutans (S.mutans) and Lactobacilli, and other salivary indices in patients wearing clear aligners (CA) versus multibrackets orthodontic appliances (MB).The authors determined Plaque index (PI), salivary flow, buffering power of saliva, and

salivary levels of S.Mutans and Lactobacilli were evaluated before the beginning of the orthodontic treatment (t0), and after 3 months (t1) and 6 months (t2) from the beginning. The authors recruited 80 subjects with equal numbers of Mb and CA treatments.

Overall this study is an interesting area of investigation; however, there were some limitations and questions that the author should clarify for the comprehensive understanding of the manuscript.

1. Please provide the time of saliva collection since it affects the composition and flow of saliva.

R: Saliva was collected during morning (9-12am). This data was added in the Material and Methods section.

2. Please discuss the association between number of bacteria and incidence of decalcification in the discussion.

R: Literature has not clarified yet, whether there is a direct proportion between the count of bacterial colonies in the saliva and the appearance/increase of decalcifications on the teeth during an orthodontic treatment. This correlation is actually derived from a clinical observation according to which decalcifications near to orthodontic brackets can increase during an orthodontic treatment (Morrier JJ. White spot lesions and orthodontic treatment. Prevention and treatment. Orthod Fr. 2014 Sep;85(3):235-44. doi: 10.1051/orthodfr/2014016. Epub 2014 Aug 28), and from the laboratory observation that the number of bacterial colonies correlates in the saliva is linked to a higher risk to develop enamel decalcification (Messer LB. Assessing caries risk in children. Aust Dent J. 2000;45:10–6.). While from one of the classical study in literature dated 1989, S.mutans has been detected in the dental plaque near an orthodontic band both in areas interested by enamel dissolution, than in other areas of the teeth not interested by enamel dissolution, without any relationship between the presence of S.mutans and enamel dissolution. (Boyar RM1, Thylstrup A, Holmen L, Bowden GH. The microflora associated with the development of initial enamel decalcification below orthodontic bands in vivo in children living in a fluoridated-water area.J Dent Res. 1989 Dec;68(12):1734-8.)

3. Normally the orthodontic treatment will take about 12 months, would the author follow up after 6 months. If so, would the author observe any difference or trend of bacteria level?

R: Patients were followed for orthodontic treatment beyond the period of this study. However, no salivary sample measurements were performed. Unfortunately, therefore, no data are available on the saliva microbiota beyond the period included in the follow-up of this study. Future research protocols could include this monitoring beyond the time of 6 months.

4. Would the author explain the randomization process of subjects?

R: Unfortunately no randomization was performed. The type of orthodontic treatment had been chosen by the clinician in agreement with the patient, but the subjects were included in the samples matching age and gender distributions as already reported in the materials and methods section.

Thank you for the opportunity to review this manuscript.

Submitted filename: R1.docx

Click here for additional data file.

12 Dec 2019

PONE-D-19-21131R1

Salivary levels of Streptococcus mutans and Lactobacilli and other salivary indices in patients wearing clear aligners versus fixed orthodontic appliances: an observational study

PLOS ONE

Dear Dr. Nota,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Three main issues need to be addressed:

1) Please consider more recent publications (Reviewer 2),

2) Address concerns on study limitations and explanation on the limitations,

3) Also since there are only 2 bacterial species mentioned, please explain how the results would be applicable to the overall microbiome (Reviewer 2).

4) The writing and organization of the manuscript need to be improved.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Thank you for your responses to our comments and questions. There are still outstanding issues with the paper

The written English is still poor

The examination for caries was raised - and you have included a statement that active caries was an exclusion criteria - how was this determined?

you still have not included caries data in the paper - if you are relating bacterial counts to carious lesion development, you should present the caries data and correlate it to the bacterial counts

Your comment regarding the validity of the CRT system may be challenged by microbiologists

Regarding the Table s- i believe that several could be combined (such as Tables 6 and 7), or entered into the text (such as Table 4)

Your conclusions state that the CA are at lower risk of caries development - you cannot conclude this as you have not provided caries data - all you can conclude is about what you have measured - two bacterial counts and saliva characteristics

Reviewer #2: A recent article published in American Journal of Orthodontics and Dentofacial Orthopedics demonstrated the oral microbiome comparison between fixed, clear aligner and control groups and showed a different result from this current study. Please refer to the AJODO study and discuss about the different result so the manuscript will provide more information.

The selection bias could affect the result and conclusion. There wa a reported relationship between oral hygiene status and socioeconomic status and attitude toward types of appliances. Please state the limitation of your subject recruitment in the discussion since there is no random sampling in the process.

The conclusion should be rewritten to be specific to the species studied in the manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

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16 Jan 2020

Editor

Three main issues need to be addressed:

1) Please consider more recent publications (Reviewer 2),

We were happy to add the recent publication that was indicated by the reviewer 2 and was previously not included because it was published in November 2019 after the submission of this manuscript.

2) Address concerns on study limitations and explanation on the limitations,

Following the reviewers suggestions other study limitations principally involved with the absence of randomization and the use of the CRT system. Another limitation was the fact that only two bacterial species are mentioned

3) Also since there are only 2 bacterial species mentioned, please explain how the results would be applicable to the overall microbiome (Reviewer 2).

The conclusions were limited to the 2 considered bacterial species that are involved in caries development, while information about the overall microbiome are not obtainable from the results of the present study.

4) The writing and organization of the manuscript need to be improved.

In the present new version of the manuscript, we tried to better organize the entire manuscript

Thanks to the Editor for this suggestion. In this revised version of the manuscript we tried to improve the writing and the organization of the manuscript, mostly for the Discussion section, where the order of the paragraphs was changed. beginning the discussion with the data about the PI, and then about the other properties of saliva (the salivary flow and the buffering capacity) and finally discussing the data about the microbial colonization. Then limitations and conclusions were stated.

Reviewer #1: Thank you for your responses to our comments and questions. There are still outstanding issues with the paper

The written English is still poor

R: English was carefully revised.

The examination for caries was raised - and you have included a statement that active caries was an exclusion criteria - how was this determined?

R: “Regarding dental caries as an exclusion criteria, they were assessed by clinical examination with specillum and mirror before the beginning of the study, and during the study period at each appointment. In particular, the orthodontic treatment was not started (brackets or first aligner positionment) if caries were found at the first clinical examination, in that case, a conservative treatment of dental caries was performed before beginning the treatment. Consequently, no active caries were observed in the whole sample during the study period.”. This sentence regarding the caries management was added in the materials and methods section.

You still have not included caries data in the paper - if you are relating bacterial counts to carious lesion development, you should present the caries data and correlate it to the bacterial counts

R: As reported in the previous answer and now reported in the study, no caries were observed during the study period, for this reason no further caries data were reported. The relationship between carious lesion development and bacterial count come from the previous literature, in the present study, a 6 months follow-up in subjects with medium to very good oral hygiene (as related to exclusion criteria) did not show the development of carious lesions that anyway wasn’t one of the objectives of the study.

Your comment regarding the validity of the CRT system may be challenged by microbiologists

R: Thanks to the reviewer for this comment. The use of the CRT system was explicitely reported as a limitation of the study according to the suggestion of the reviewer. In particular, it was used because it is a method that every dentist can adopt in his/her daily clinical routine, to monitor his/her patients during orthodontic treatment in a very simple way, differently from microbiological tests that are more difficult to be used in the daily routine

Regarding the Table s- i believe that several could be combined (such as Tables 6 and 7), or entered into the text (such as Table 4)

R: Thanks to the reviewer for this suggestion. In the present version of the paper, table 4 was deleted, as its data were entered into the text, while tables 6 and 7 were combined

Your conclusions state that the CA are at lower risk of caries development - you cannot conclude this as you have not provided caries data - all you can conclude is about what you have measured - two bacterial counts and saliva characteristics

R: The conclusions were restructured deleting the sentence about dental caries and reporting only what observed about oral hygiene and bacterial count.

Reviewer #2:

A recent article published in American Journal of Orthodontics and Dentofacial Orthopedics demonstrated the oral microbiome comparison between fixed, clear aligner and control groups and showed a different result from this current study. Please refer to the AJODO study and discuss about the different result so the manuscript will provide more information.

R: We really thank the reviewer for showing us the publication of a very recent paper (November 2019) about oral microbiome in clear aligners treatment (Alterations of the Oral Microbiome in Patients Treated with the Invisalign System or with Fixed Appliances.” American journal of orthodontics and dentofacial orthopedics. 156.5 (2019): 633–640.). After a careful evaluation of the article that analyzed a much wider microbiome spectrum, they state that “changes in the microbiome associated with Invisalign treatment showed different effects on oral health, with some beneficial and some harmful” and in particular they observed some negative results about periodontal harmful bacteria. Regarding caries involved bacteria, perfectly in agreement with this study, they observed that “For the Invisalign group, the abundance of Firmicutes was less than the fixed appliance group (I vs F) with no difference than the control group (I vs C)” and Firmicutes represents the Phylum of both S. Mutans and Lactobacilli analyzed in this study, related with caries risk rather than periodontal aspects. Furthermore, the sample size of that study is small (7 subjects with aligners and 14 with fixed appliances) and it is probably underpowered, in fact, no sample power analysis was reported, thus, the trustworthiness of that study (especially when they state an absence of significant differences between the microbiota of the two groups) is limited, this could be the reason of its contrast with the most complete systematic review on this topic that showed a better periodontal health in subjects in orthodontic treatment with clear aligners.

For this reason, we discussed the results of that recent study regarding caries related bacteria (i.e. Firmicutes - S. Mutans and Lactobacilli) that are the outcome of our study in in the proper section.

The selection bias could affect the result and conclusion. There was a reported relationship between oral hygiene status and socioeconomic status and attitude toward types of appliances. Please state the limitation of your subject recruitment in the discussion since there is no random sampling in the process.

R: Thank you for the interesting observation, it was added to the limitations of the study.

The conclusion should be rewritten to be specific to the species studied in the manuscript.

R: The conclusions were rewritten in order to be specific to the outcomes of the present study.

Submitted filename: R2.docx

Click here for additional data file.

24 Jan 2020

Salivary levels of Streptococcus mutans and Lactobacilli and other salivary indices in patients wearing clear aligners versus fixed orthodontic appliances: an observational study

PONE-D-19-21131R2

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Academic Editor

PLOS ONE

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Thank you for your responses to the reviewers. All comments have been addressed.

Reviewers' comments:

13 Apr 2020

PONE-D-19-21131R2

Salivary levels of Streptococcus mutans and Lactobacilli and other salivary indices in patients wearing clear aligners versus fixed orthodontic appliances: an observational study.

Dear Dr. Nota:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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