Effect of 8% arginine toothpaste on Streptococcus mutans in patients undergoing fixed orthodontic treatment: randomized controlled trial

ABSTRACT Objective: To assess the effect of toothpaste containing 8% arginine on Streptococcus mutans (S. mutans) in dental plaque around orthodontic brackets, and to draw a comparison with a regular fluoride toothpaste. Trial design: A single-center, parallel-arm, triple-blind, randomized controlled trial was conducted. Methods: The clinical trial was conducted at the Orthodontic Clinic, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran. Seventy-two patients (age range: 15-30 years) who required fixed orthodontic treatment were recruited and randomly assigned to arginine and fluoride groups. Randomization was performed using RANDOM.ORG online software, and the participants were divided into two parallel groups, with a 1:1 allocation ratio. Patients were requested to brush their teeth twice daily for 30 days with an experimental toothpaste. Plaque sampling was performed at two intervals, namely at the beginning of the study (T0) and 30 days later (T1). Real-time PCR was used to assess plaque samples in terms of the number of S. mutans surrounding stainless steel brackets in orthodontic patients. A triple-blind design was employed. Results: The baseline characteristics (age, sex, and the relative number of S. mutans) between the groups were similar (p>0.05). Only the arginine group showed a significant decrease in the relative number of bacteria between T0 and T1 (p=0.02). Conclusion: Arginine is an important prebiotic agent in maintaining healthy oral biofilms, and prevent dental caries during fixed orthodontic treatments. Trial registration: The trial was registered at the Iranian Registry of Clinical Trials (IRCT20181121041713N2), https://en.irct.ir/user/trial/42409/view.


INTRODUCTION
Despite improvements in the design of orthodontic appliances, concerns exist about the treatment protocol and patient care during orthodontic treatment and the risk of enamel demineralization and white spot lesions. In terms of enamel demineralization during orthodontic treatment, incidences of up to 50% of patients have been reported. 1,2 A variety of techniques have been proposed to prevent or reduce the occurrence of enamel demineralization during fixed orthodontic treatment, such as educating patients about dental plaque management, recommending the use of fluoride mouth rinse and dentifrice, prescribing probiotics and antibiotics, using fluoride-releasing restorative materials, and the use of nanoparticles, due to antibacterial properties. 1,[3][4][5][6] In recent years, biological methods such as antibiotics, antimicrobial therapy (with chlorhexidine, povidone-iodine, fluoride, and penicillin), and the use of live microbial dietary supplements or toothpastes have gained importance. 1,7,8 Oral biofilms are highly organized communities of a physiologically and genetically diverse group of microorganisms, many of which are either commensal (i.e., overtly beneficial) or opportunistically pathogenic. 9 It is well-known that the consumption of fermentable carbohydrates initiates caries formation by acidogenic bacteria living in the biofilm on tooth surface. A healthy biofilm can become cariogenic through a Razeghian-Jahromi I, Babanouri N, Ebrahimi Z, Najafi HZ, Sarbaz M, Montazeri-Najafabady N Effect of 8% arginine toothpaste on Streptococcus mutans in patients undergoing fixed orthodontic treatment: randomized controlled trial 5 protracted presence of acid in the biofilm, which in turn promotes the growth of acid-tolerant bacteria (e.g., Streptococcus mutans and lactobacilli), leading to dental caries. 10,11 In contrast, the presence of bacteria that are less acid-tolerant (e.g., Streptococcus sanguinis and Streptococcus gordonii) is associated with good oral health. 12,13 Oral commensal bacteria stimulate an oral biofilm environment in various ways, to inhibit the presence of pathogenic species. Compared to S. mutans, commensal streptococci are less acid-tolerant and can catabolize urea using urease enzymes and/or arginine via the arginine deiminase system (ADS). As a result, ammonia, which alkalinizes the cytoplasm of the commensal, is released and thereby raises the pH in biofilm communities. Furthermore, hydrolysis of urea by ureases and arginine via ADS provides bioenergetic advantage to the commensal, which in turn improves the stability and health of biofilms. 9 Prebiotics are compounds that have a positive effect on human health by selectively stimulating the growth or activity of beneficial microbes. 14 Arginine is a prebiotic-based organic compound that, in combination with fluoride and calcium compounds, provides significant anti-caries benefits compared to matched formulations containing fluoride alone. 15 Various studies have reported the beneficial effects of toothpaste containing arginine and fluoride on the de/remineralization Razeghian-Jahromi I, Babanouri N, Ebrahimi Z, Najafi HZ, Sarbaz M, Montazeri-Najafabady N Effect of 8% arginine toothpaste on Streptococcus mutans in patients undergoing fixed orthodontic treatment: randomized controlled trial 6 balance. Compared to toothpaste with fluoride alone, this combination has superior efficacy in caries prevention. 16,17 A previous in vitro study showed that the ecology of the biofilm bacteria grown under cariogenic conditions changes when exposed to L-arginine. In the presence of 1.5% exogenous L-arginine, the growth of Streptococcus gordonii (commensal) is more enhanced than of cariogenic S. mutans. 9 Another study reported that toothpaste containing 1.5% arginine combined with 1,450 ppm fluoride provided greater levels of protection against the formation and progression of lesions, and promoted remineralization better than a dentifrice with 1,450 ppm fluoride alone. 10 It has been shown that the level of acidogenic bacteria present in plaque, particularly S. mutans, is higher in orthodontic patients. 18,19 However, to date, the possible effects of arginine toothpaste on dental biofilm in patients undergoing orthodontic treatment have not been investigated. Hence, the present randomized controlled trial was performed to evaluate possible additional benefits of arginine-containing toothpaste, compared with regular fluoride toothpaste.

SPECIFIC OBJECTIVES OR HYPOTHESES
The primary purpose of the present study was to assess the effect of short-term usage of toothpaste containing 8.0% arginine on the level of S. mutans in plaque surrounding stainless steel brackets in orthodontic patients, and to draw a comparison with a toothpaste containing fluoride alone. The null hypothesis was that the 8.0% arginine toothpaste would not have any impact on the number of S. mutans, compared to the fluoride-containing toothpaste.

TRIAL DESIGN
The present study was a single-center, triple-blind (patients, examiner, assessor) randomized controlled trial, with a 1:1 allocation ratio.

CEALMENT, IMPLEMENTATION)
The block randomization method was used with a block size of 6 (RANDOM.ORG online software) to allocate patients to a control and an intervention group with a 1:1 ratio. Subsequently, the random sequences to either fluoride or arginine group were concealed in opaque, sealed envelopes and shuffled before the intervention, to increase the unpredictability of the random allocation sequence and avoid selection bias.
Each patient was asked to pick an envelope for allocation to either the arginine or fluoride toothpaste group.  Table 2.  Razeghian-Jahromi I, Babanouri N, Ebrahimi Z, Najafi HZ, Sarbaz M, Montazeri-Najafabady N Effect of 8% arginine toothpaste on Streptococcus mutans in patients undergoing fixed orthodontic treatment: randomized controlled trial

INTERIM ANALYSIS AND STOPPING GUIDELINES
Not applicable.

STATISTICAL ANALYSIS
The data were analyzed using the Statistical Package for Social

PARTICIPANTS FLOW
During the trial, eight patients in the arginine group and ten patients in the fluoride group were excluded from the study due to irregular attendance, inadequate DNA concentration, or unsuccessful DNA amplification (Fig 1).

BASELINE DATA
Data regarding age, sex, and duration of alignment phase of the participants are listed in Table 3. The results showed no significant difference between the study groups in terms of age and sex and duration of alignment phase. Since the variables had non-normal distribution, non-parametric tests were used in the statistical analysis. There was no significant difference between the two study groups in terms of the S. mutans number at T 0 (p = 0.8456) (Fig 2). However, the results showed that the bacterial number significantly decreased in the arginine group at T 1 after using the toothpaste (p = 0.0229), while there was no significant difference between the two intervals (p = 0.1899) in the fluoride group (Table 4).

ADVERSE EFFECTS
No harm or adverse effects were observed during the trial.

MAIN FINDINGS IN THE CONTEXT OF THE EXISTING EVIDENCE
The present clinical trial investigated the effects of toothpaste containing 8.0% arginine on S. mutans in dental biofilm around the fixed orthodontic brackets, and compared the results with the efficacy of regular fluoride toothpaste.
It is well known that during fixed orthodontic treatment the As previously mentioned, the complexity of fixed orthodontic attachments and reduction in self-cleaning capacity of tooth surface during the orthodontic treatment generate more bacterial colonization and increase development of white spot lesions. Fluoride has been used as a preventive method, but it has not been enough to avoid its occurrence. 6 So, it seems beneficial to incorporate it with the other antibacterial substances.
Razeghian-Jahromi I, Babanouri N, Ebrahimi Z, Najafi HZ, Sarbaz M, Montazeri-Najafabady N Effect of 8% arginine toothpaste on Streptococcus mutans in patients undergoing fixed orthodontic treatment: randomized controlled trial 20 This study in line with the previous studies demonstrating the adverse effects of arginine on the growth of S. mutans in patients with fixed orthodontic treatment that can have a potent influence on oral biofilm ecology and reduction in withe spot lesions. However, its exact efficacy in reduction of enamel remineralization, optimal delivery method (varnish, toothpaste, or mouthwash) and also optimal daily dose have been established neither in this study nor in the previous studies. In addition, long-term use of arginine may pose a risk of increased plaque alkalization and overgrowth of oral anaerobes such as Porphyromonas gingivalis, a keystone pathogen associated with periodontitis. 8 So, it remains unclear whether or not this really is beneficial for the patients.
In the present study, biofilm samples were collected from the maxillary lateral incisors since these teeth are most susceptible to enamel demineralization due to decreased salivary clearance and less space between the bracket and gingiva. 1,29 Considering limited salivary clearance in the anterior region, The real-time PCR method was used to evaluate the number of S. mutans in the biofilm samples. This was an innovative step since the method permitted the determination of even a minimal quantity of bacteria. In detecting S. mutans, PCR assay is more specific, compared to the conventional culture methods. 32

GENERALIZABILITY
The results showed that 8% arginine toothpaste decreased the number of S. mutans in the dental biofilm of patients undergoing fixed orthodontic treatment. However, the short duration of the trial, single-center study, and the inclusion of patients with good oral hygiene limited the generalizability of the present findings.