Real‐time polymerase chain reaction quantification of the salivary levels of cariogenic bacteria in patients with orthodontic fixed appliances

Abstract Aim The aim was to investigate the salivary detection frequencies and quantities of caries‐associated bacteria from patients with orthodontic brackets. Methods Patients wearing orthodontic brackets (n = 40, mean age = 26 years) and healthy controls without brackets (n = 40, mean age = 17 years) were enrolled in the study. Saliva samples from each patient was collected. After DNA purification, target species comprising streptococci and a Lactobacillus species were detected and quantified from the samples using polymerase chain reaction (PCR) and real‐time quantitative PCR. Results Detection frequencies did not differ between the orthodontic patients and the control subjects for any target species except for Streptococcus sobrinus, which showed significantly lower detection rates in orthodontic patients (p < .05). Lactobacillus casei and Streptococcus gordonii were found at the highest detection frequencies with both species being detected in 38 (95%) of the saliva samples of orthodontic patients. Similarly, L. casei and Streptococcus salivarius were the species with highest detection frequencies (35, 87.5%) in the control subjects. Real‐time PCR revealed that Streptococcus mutans and S. salivarius quantities were significantly higher in orthodontic patients than in the control subjects (p < .05). Conclusions Application of orthodontic brackets for 12 months leads to increased salivary levels of cariogenic bacteria and may serve as a potential risk factor for caries initiation.

There are also a few reports showing that fixed appliances might impede effective oral hygiene and cause an increased risk for caries initiation (Bretas, Macari, Elias, Ito, & Matsumoto, 2005;Turkkahraman et al., 2005). Furthermore, based on the difficulty of maintaining oral hygiene, the oral microbiota may also be influenced by orthodontic appliances (Naranjo, Trivino, Jaramillo, Betancourth, & Botero, 2006;Thornberg et al., 2009). These variables would possibly lead to microbial dysbiosis leading to a shift in increased colonization by pathogenic bacteria possessing potent virulence factors that contribute to gingival inflammation, periodontal support destruction (Naranjo et al., 2006;Thornberg et al., 2009), and changes in enamel surface (Bretas et al., 2005;Sukontapatipark, el-Agroudi, Selliseth, Thunold, & Selvig, 2001;Turkkahraman et al., 2005). Further, there are a number of studies on orthodontic treatment and the risk for adverse effects, such as the development of white spot lesions (Khoroushi & Kachuie, 2017;Srivastava, Tikku, Khanna, & Sachan, 2013). Hence, the orthodontic patients are considered to be "at-risk" patients.
There is considerable evidence in the literature, demonstrating that the presence of fixed orthodontic appliances in the oral cavity of dental patients could influence changes in oral microbial profiles (Freitas et al., 2014). Following the application of orthodontic appliances, the structure, metabolism, and composition of dental plaque would change, leading to a general increase in the levels of microbial population, particularly Streptococcus and Lactobacillus (Sukontapatipark et al., 2001).
Bacterial cells are continuously detached from dental plaque biofilms. Saliva constantly washes mucosal and dental surfaces and collects detached bacteria. Saliva with live bacteria (von Troil-Linden, Torkko, Alaluusua, Jousimies-Somer, & Asikainen, 1995) is the likely vehicle for intraoral but also person-to-person transmission of periodontitis-associated bacteria, with their levels being a determinant for successful transmission (Asikainen, Alaluusua, & Saxen, 1991).
Free planktonic bacteria cannot multiply in saliva since they get swallowed constantly, but after each swallowing incident part of whole saliva remains on oral surfaces (Lee, Crouse, & Kline, 2010), in this case, orthodontic appliances, which may result in reattachment of bacteria. Despite the development of new orthodontic appliances, new bonding techniques and material, it has not yet been possible to reduce dental plaque retention (Ristic et al., 2007). This study may provide important new information not only on the detection frequencies but also the absolute quantities of major cariogenic bacteria that colonize around Damon Q self-ligating brackets. Eventually, this may help orthodontists reinforce proper oral hygiene measures for the orthodontic patients. Thus, the aim of this study was to assess the detection frequencies and quantities of species belonging to the genera Streptococcus and Lactobacillus, which are known to play a major role in dental caries, from the saliva samples of patients wearing orthodontic appliances.

| Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study's experimental design and protocol were approved by the Ethical Committee of the Health Sciences Center of Kuwait University.
The ethical approval was obtained on July 4th, 2017. A written informed consent was obtained from all the patients.

| Study subjects
Forty patients (8 males, 32 females), with a mean age of 26 years (SD: 8.05), receiving orthodontic treatment with fixed orthodontic brackets were recruited to the study. The control group without orthodontic brackets consisted of 40 subjects (11 males, 29 females) with a mean age of 17 years (SD: 9.53). None of the approached patients declined to participate. The sample size was estimated based on previous literature and by using the online tool "ClinCalc" (https://clincalc.com/ stats/samplesize.aspx). For a sample size that would allow us to detect the differences between the groups, a test power of 90% and α = 5% were considered.
The inclusion criteria involved patients with intact maxillary and mandibular teeth with fixed orthodontic appliances for at least 12 months. The fixed orthodontic appliances included Damon Q. 022 slot self-ligating stainless steel brackets, which were placed on the labial surfaces of the maxillary and the mandibular permanent teeth up to the first molars, which received bondable tubes.
The steps for bracket bonding were the same for all patients receiving orthodontic treatment with fixed appliances. Prior to bonding, the teeth were cleaned using pumice paste, and then, the teeth were carefully washed and dried. Proper isolation was obtained with the use of cheek retractors, cotton rolls, and saliva ejectors.
First, the enamel of the teeth receiving the brackets was etched with 35% phosphoric acid (Ultra-Etch) for 30 s. Then, the enamel surfaces were adequately rinsed with water for 10 s and thoroughly dried until the enamel appeared chalky white. After that a bonding agent was applied on the enamel surfaces and was gently dried (3 M Unitek), and the enamel surfaces were light cured for 10 s. Last, each bracket was bonded using Transbond Plus Light Cure Orthodontic Adhesive (3 M Unitek), and every bracket was light cured for 20 s.
Patients with gingival inflammation, missing teeth, active carious lesions, and prosthetic crowns were excluded from the study. Moreover, patients with systemic diseases, patients receiving systemic medication, and pregnant women were excluded from the study. All patients were given the same oral hygiene instructions at the start of the study.

| Clinical parameters and sample collection
Plaque index scores from the orthodontic patients and the controls were recorded on a scale of 0-3 (Silness & Loe, 1964).
For collecting the saliva sample, each patient was given a paraffin wax pellet (Ivoclar Vivadent AG, Liechtenstein, Germany) to chew on while providing the saliva. While chewing on the wax pellet, the patient was asked to gradually expectorate the saliva in a sterile 50 ml OMG 3184 were cultured on brucella blood agar media plates containing 5% sheep blood and incubated at 37 C in 5% CO 2 in air for 2 days.

| Laboratory processing of samples
Samples collected were processed immediately or maximum within 24 hr of sampling. Saliva samples were centrifuged at 5000×g for 10 min, supernatants were discarded and the pellets were resuspended in 1 ml of phosphate buffered saline. The saliva samples were centrifuged at 18000×g for 5 min to recover pellets. Supernatants were discarded and the pellets were stored at −80 C until further testing.

| DNA purification
Total DNA from the saliva samples and from the reference bacterial strains were purified using DNeasy Blood and Tissue Kit according to manufacturer's instructions (Qiagen). In brief, bacterial cells in the sample were lysed by incubating at 56 C for 1 hr in 180 μl of animal tissue lysis buffer and 20 μl of Proteinase-K. DNA in the lysate was further precipitated in lysis buffer (200 μl) and absolute etha- The lysed sample was loaded on the membrane column provided with the kit and washed with wash buffers to remove contaminants and enzyme inhibitors, followed by centrifugation at 18000×g. Finally, the DNA bound to the membrane was eluted in 200 μl nuclease free water on centrifugation at 6000×g for 1 min. DNA concentration was measured by UV spectrometry method on NanoDrop 1000 (Thermo Scientific).

| Validation of species-specificity of 16S rDNA primers
The specificities of the primers for polymerase chain reaction (PCR) and quantitative PCR (qPCR) chosen from the literature were confirmed by in silico analysis (

| Polymerase chain reaction
Target bacterial genes in the purified DNA from saliva samples were amplified by PCR using 16 s rDNA species specific primers chosen from literature (Table 1) and their specificity revalidated using in silico analysis tools (Karched, Bhardwaj, Inbamani, & Asikainen, 2015). Reaction was carried out on ABI Fast RT-PCR machine using the following thermal cycling profile: After a 10-min initial denaturation at

| RESULTS
The plaque index scores from the patient group ranged between 2 and 3, while the scores for healthy subjects were 0-1. PCR amplification of 16S rRNA genes from the reference strains of the target species showed bands of expected sizes deduced from the respective DNA sequences (Figure 1 and Table 1). All reference strains produced single bands confirming the absence of nonspecific amplification. casei and S. gordonii were found at the highest detection frequencies with both species being detected in 38 (95%) of the saliva samples.
Bacterial detection frequencies from saliva samples differed significantly (p < .05) between the target species with the exception that S. salivarius detection rates did not differ from those of S. mutans, S. gordonii, and L. casei (p > .05). Similarly, detection rates between L. casei and S. gordonii did not show significant difference.
Since no significant differences in the detection frequencies between the orthodontic patients and the controls were found, we next sought to determine quantities of the target species in the study group and the control group. For this purpose, we chose S. mutans and S. salivarius, two of the most important species in dental caries. As quantified by the real-time PCR method (Figure 2), both S. mutans and S. salivarius were in significantly higher quantities in orthodontic patients than in control subjects (p < .05). The median cells per ml for S. mutans were 1.8 × 10 3 (ortho patients) and 3.3 × 10 2 (controls).

| DISCUSSION
While detection frequencies of the target streptococci and Lactobacillus species generally did not differ significantly between the controls and the orthodontic patients, real-time qPCR revealed that quantities of S. mutans and S. salivarius were significantly higher in orthodontic patients than in control subjects. In oral infections, which are polymicrobial in nature, it is alterations in quantities of specific bacterial species, not mere presence or absence that indicates dysbiosis and sets up initiation of infection process.
Primer specificity is critical in getting accurate information of the quantities of target species from clinical samples. Although we chose the primers from literature based on their reported specificity for target species, we confirmed the specificity in silico using bioinformatics tools and databases. Each primer pair was found to be species-specific except for L. casei, in which, the forward primer had a binding site in Lactobacillus paracasei as well. Therefore, while detection/quantification of L. casei using the present primer pair in this study must be considered cautiously, new primer pair with absolute specificity for L. casei need to be optimized in future studies.
Several studies examined the association between orthodontic appliances and oral infections such as caries. While a number of longitudinal studies have reported a significant increase in the counts of major oral bacteria associated with caries or periodontitis (Maret et al., 2014;Pan et al., 2017), the aim of this study was to investigate the occurrence frequencies and quantities of select cariogenic bacterial species in saliva from patients wearing orthodontic fixed brackets in Kuwait.
In the current study, we found that both S. mutans and S. salivarius were in significantly higher quantities in orthodontic patients than in control subjects. It is plausible that plaque accumulated around orthodontic appliance is the source of elevated levels of these bacterial species in the saliva. In fact, it has been long known that free planktonic cells are continuously detached from mature biofilms (Costerton et al., 2003). Increased occurrence and quantities of cariogenic streptococcal species following the application of orthodontic appliances has been well-documented. As discussed elsewhere (Ristic et al., 2007), perhaps it is imperative that this is due to plaque retention caused by orthodontic appliances. It is important to clarify that plaque retention in patients wearing orthodontic appliances is not because of poor oral hygiene practice, but rather due to inappropriate or incomplete oral hygiene procedures. Higher salivary levels of cariogenic streptococci warrants better oral hygiene approaches, for example, more efficient mouth wash liquids to diminish salivary bacterial loads.
Another study assessed the microbial changes in the saliva of children with fixed orthodontic appliances compared with a control group of children without orthodontic treatment. Although this study was conducted on children, it was demonstrated that wearing a fixed orthodontic appliance for 6 months was associated with high levels of S. mutans and Lactobacillus species (Maret et al., 2014). This finding was in agreement with our study, which showed that there is an increase in S. mutans and S. salivarius after 12 months of treatment with fixed orthodontic appliances. There is evidence in the literature that the presence of fixed appliances influences the quantity and quality of oral microbiota (Lucchese, Bondemark, Marcolina, & Manuelli, 2018). Moreover, orthodontic treatment alters the oral environmental factors, leading to an increase in stimulated flow rate, buffer capacity, and salivary pH, which can affect the anti caries activity of saliva (Chang et al., 1999). Therefore, it seems necessary to evaluate the microbial parameters and stress oral hygiene preventive measures until the termination of orthodontic treatment.
An important limitation of this study was that we investigated only a few bacterial species of importance in caries development and progression. With the increasing utility of NextGen sequencing technologies that reveal total microbiota of the samples in question, several novel bacterial species whose role in caries was hitherto unknown have been uncovered during the recent years. Sequencing techniques like 16S rRNA gene metagenomics would have provided a comprehensive delineation of the microbiota in the dental plaque retained around the orthodontic braces.

| CONCLUSIONS
Significantly higher levels of cariogenic streptococci in the saliva of our orthodontic patients, suggests that patients should be prescribed to regularly use mouth washes that are clinically proven to be efficient not only in reducing plaque accumulation but also in controlling salivary levels of cariogenic bacteria.