INTRODUCTION

Microbes are present in both healthy and diseased environments. Synergistic, mutualistic, and antagonistic interactions among the microorganisms contribute to the development of a polymicrobial biofilm.^1,2 Initial biofilm formation plays an important role in understanding the growth and proliferation of microorganisms.^3–6

The literature describes that 60% of all orthodontic patients experience some alteration in biofilm accumulation after the bonding of orthodontic appliances. Authors emphasize a need for preventive measures to prevent biofilm-related complications during orthodontic treatment. Knowledge of the microbial dynamics in the early stages of orthodontic treatment can assist in the adoption of effective measures to prevent these changes.⁷

Studies investigating microbial contamination during orthodontic treatment have evaluated the species that are related to dental caries^8–11 or periodontal disease.^11,12 Few studies have evaluated the microbial ecology in the oral cavity after the bonding of orthodontic appliances.^13,14

According to Socransky and Haffajee,¹⁵ there are specific associations among bacterial species in the supragingival biofilm. Based on this relationship, they are grouped into different complexes. Purple and green complex species are present in early biofilm formation because of the distinct patterns of coaggregation. Yellow complex returns very quickly after cleaning surfaces of the teeth; this is an intermediate complex that improves the attachment points of adhesion. The orange and red complexes are directly involved with periodontal disease and are frequently associated with the soft tissue inflammation observed during orthodontic treatment.^16,17

The aim of this clinical study was to assess periodontal parameters and to examine microbial communities in saliva in the early stages of orthodontic treatment in patients who received oral hygiene instructions, monitoring, and motivation throughout the study.

MATERIALS AND METHODS

The Institutional Research Ethics Committee granted approval for the research project (Process 0062.0.138.000-10). Fifteen patients of both sexes (one male and 14 females) aged 11 to 41 years (mean age = 17.53 ± 8.0 years) with permanent dentition were screened.

The following inclusion criteria were considered in this study: no previous orthodontic treatment; no use of antibiotics, antimicrobial mouthwashes, or any systemic medication within 3 months prior to the study; no periodontal treatment within the previous 3 months; no smoking; no clinical signs of gingivitis; and no systemic disorder that could alter the periodontal conditions prior to bracket bonding.

The plaque index (PI) and gingival index (GI) were measured at three sites per tooth (mesiobuccal = MB, buccal = B, and distobuccal = DB),¹⁸ and gingival bleeding index (GBI)¹⁹ using a PCPUNC-BR15 probe (HuFriedy of Brazil, Rio de Janeiro, RJ, Brazil) was determined to the nearest millimeter. These indices were measured before bonding (T0) and 30 (T1), 60 (T2), and 90 (T3) days after bonding in the upper and lower arches.

Standardized hygiene instructions (modified Bass brushing technique) were given to all patients by the same investigator. The subjects were asked to brush three times daily, after meals, and were instructed not to use any hygiene products other than toothpaste and dental floss. Recall visits were scheduled at 30 days, at which time the instructions were reinforced.

The patients received edgewise metallic orthodontic brackets (0.022 × 0.028-inch slot) (Dental Morelli, Sorocaba, SP, Brazil) in the upper and lower arches. The brackets were bonded with orthodontic light-cured adhesive (Transbond XT, 3M Unitek, Monrovia, Calif).

Checkerboard DNA-DNA Hybridization

The levels of five Candida species and 38 bacterial species were analyzed (Table 1). After thawing, the samples were boiled for 5 minutes. After cooling, 800 μL of 5 M ammonium acetate was added, and the contents were applied to the extended slot in the MiniSlot apparatus (Immunetics Inc, Boston, Mass) and then concentrated onto a 15 × 15-cm nylon membrane (Yond Nþ, Amershan Biosciences, Buckinghamshire, UK), followed by baking for 2 hours at 80°C. Control samples defined amounts of genomic DNA corresponding to either 10⁵ or 10⁶.

Table 1 Microbial Count (μg × 10⁵) in the Saliva, Before Bonding Brackets and 30, 60, and 90 Days After Bonding Brackets^a

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The membranes were prehybridized (buffer hybridization; NaCl 0.5 M; blocking reagent 0.4% [w/v]). After prehybridization, the membranes were placed in a Miniblotter 45 (Immunetics). Defined amounts of fluorescein-labeled whole genomic probes were diluted in 150 mL of hybridization solution, applied in individual lanes of the Miniblotter, and the whole apparatus was placed in a sealed plastic bag containing sheets of wetted paper towel. Hybridization was performed overnight at 60°C with gentle agitation. The following day, the membranes were washed twice in a solution of 2 M urea, 0.1% sodium dodecyl sulfate, 50 mM NaH₂PO₄ (pH 7.0), 150 mM NaCl, 1 mM MgCl₂, and 0.2 blocking reagent at 65°C for 30 minutes and were also washed twice in a solution of 1 M Tris base, 2 M NaCl, and 1 M MgCl₂ for 15 minutes at room temperature.

The hybrids were detected by chemiluminescence using the Gene Images CDP-Star detection module (GE Healthcare, Buckingham, UK). Chemiluminescent signals were detected by exposing the membrane to ECL Hyperfilm MP (GE Healthcare) for 10 minutes. The image obtained on the hyperfilm was digitized and analyzed by the TotalLab™ Quant v13 software (TotalLab Ltd, Newcastle, UK).

RESULTS

No statistically significant difference was observed in PI, GI, and GBI scores throughout the study. Figure 1 shows the median, first, and third quartiles of these indexes for all evaluated periods.

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All microbial complexes showed significant changes over the study period (Friedman test P = .00001), with a decrease in the levels of the microorganisms throughout the evaluated observational period (Figure 2). Purple and green complexes decreased levels over the observational period. A significant decrease of the purple started from T2, and the lowest level of the purple complex was also identified at T2. Green complex showed its lowest level at T3. The yellow and orange complex levels decreased significantly only at T3. The red complex and Candida spp decreased significantly at T2, and the lowest value was observed at T3 (Table 2).

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Table 2 Post Hoc Dunn's Test^a

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Regarding individual analysis of the microorganisms, only eight species' levels did not change throughout the study (Table 1). There was no positive test observed for anaerobic species such as Prevotella melaninogenica, Parvimonas micra, and Capnocytophaga gingivalis. Some opportunistic species (Bacteroides fragilis, Escherichia coli) were not observed at T0. After bonding, significant changes were observed for P melaninogenica (T2-T0, T2-T1, and T2-T3; P = .00001), P micra (T2-T0 and T2-T1; P = .034), and B fragilis (T2-T0 and T2-T1; P = .007: T2-T3; P = .048), which had their highest levels identified at T2 and which decreased significantly at T3. Out of 15 species related to deep pockets and the status of advanced periodontitis, eight showed significant decreases in levels at T2 and T3: A.a.b, Prevotella nigrescens, Pseudomonas putida, Fusobacterium periodonticum, Pseudomonas aeruginosa, Peptostreptococcus anaerobius, Treponema dentícola, and Tanerella forsythia (Table 3). Only P gingivalis increased at T2, and its highest level occurred at T3 (T2-T0 [P = .048]; T2-T1 [P = .020]; T3-T0 [P = .001]; T3-T1 [P = .00001]).

Table 3 Periodontal Pathogens in Which Levels Decreased^a

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Among nine species of the Streptococcus group analyzed, six species showed significantly decreased levels throughout the study: S sobrinus, S mutans, S sanguinis, S salivarius, S parasanguinis, and S oralis (Table 4). Lactobacilos casei increased significantly at T2 (T2-T0; P = .002: T2-T1; P = .00001). The levels decreased significantly at T3 (T3-T2; P = .011).

Table 4 Median Values (μg × 10⁵)—Streptococcus Group Post Hoc Dunn's Test^a

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Candida species C albicans, C dubliniensis, C glabrata, and C krusei showed a significant alteration in their levels (P = .00001). However, the behavior of C albicans was different from that of the others: its levels decreased significantly only at T3, while the others showed a decrease starting from T2 (Table 1).

DISCUSSION

Previous literature^1,2,21–23 emphasized the importance of knowing the relationship between microbial species and the complex oral cavity environment for the early diagnosis of dental caries and periodontal disease. When the balance of the oral environment was broken, a dynamic fluctuation of microbial levels was identified.^24–26 Thus, the increase in levels of individual species should be viewed with caution in the orthodontic appliance environment.²⁷

In this study, immediately after installation of the orthodontic appliance, there was an increase in the levels of purple and yellow complexes and Candida spp (T1). At T2, levels were mildly reduced, and at T3, the lowest levels were identified, indicating that homeostasis was recovered.

Some periodontal pathogenic species, such as P melaninogenica and P intermedia, were not identified at the baseline but showed an increase in levels at T2. Some anaerobic species have been shown^26–29 to be unable to live for long periods in aerobic sites. This is in agreement with the current data, which showed a decrease in the levels of several pathogenic species (A.a.b, P nigrescens, P putida, F periodonticum, P aeruginosa, P anaerobius, T dentícola, and T forsythia) over the study period. P gingivalis increased levels at T2 and T3. The levels may have altered according to the presence of oxygen and nutrients. Some species are more sensitive to changes in these conditions, which could explain the different behaviors of these periodontal pathologic species.

In the Streptococcus group, a significant decrease occurred to most species at T3. This agrees with the findings of previous literature, which described that these species were present in the initial stage of biofilm development. They were the primary colonizers that then co-aggregated with other bacteria, thus leading to the development of a mature biofilm.^23,30,31

S mutans and S sobrinus exhibited higher levels before bonding. S mutans levels decreased significantly at T2 and showed a mild increase at T3, while S sobrinus levels decreased significantly only at T3. This species has high virulence because of high adhesion capability, acidogenicity, and acid-uric properties. High levels of both species in a patient indicated more susceptibility to caries incidence than for patients who only had the presence of one species.^31,32 Therefore, in this study, this high risk for the incidence of caries was observed before bonding. However, the oral hygiene program implemented in the study was successful in reducing the levels, and control was obtained.

S sanguinis is considered a beneficial bacterium with regard to dental caries because it is an antagonist to S mutans. Epidemiological studies^31,33 of dental caries demonstrated that early colonization and high levels of S sanguinis in a patient's oral cavity correlated with significantly delayed colonization by S mutans. The current results were in agreement with those studies, since the highest levels of S sanguinis were observed at T0, decreased significantly at T2, and reached their lowest levels at T3, when the S mutans levels had a mild increase. This result highlighted the antagonism between species and emphasized the predisposition of orthodontic patients to dental caries even after instruction, motivation, and supervision of oral hygiene throughout the study period.

The L casei cariogenic species correlated with deep cavities and increased significantly at T2, but decreased significantly at T3, indicating a return to baseline levels. This species is correlated with carious dentin,³² and the sample did not show cavity activity.

Candida spp are frequently correlated with a decrease of pH, increase of orthodontic appliance deterioration due to the release of metallic ions, and secondary infections.^34,35 In this study, the general levels of Candida spp decreased after T2. However, C albicans, the most frequently identified fungus, which is responsible for 75% of opportunist systemic infections and has an indirect role in gingivitis, periodontitis, and dental caries,³⁶ showed decreased levels only at T3. In turn, L casei, P gingivalis, and P intermedia, species that are favored with the presence of C albicans, showed an increase starting from T2. These data may suggest that C albicans proliferation could trigger imbalance in the oral environment.

The sample enrolled was composed of healthy patients, with GI, PI, and GBI indices that indicated health, and the patients' oral hygiene was supervised and monitored monthly. Despite the absence of significant changes in the clinical indices used in the present study, there was a difference in the microbiological parameters between the initial timepoint T0, T1, and T2, showing the important role of adequate mechanical hygiene in these patients. In this study, the Hawthorne Effect might have been expected to play some role in motivating patients to perform better oral hygiene. At the first observational period (30 days), the Hawthorne Effect should have had its greatest influence, because this was a novel situation, but at that timepoint, an increase in the measured parameters was observed. Patient motivation and the reinforcement of oral hygiene instruction resulted in oral hygiene improvements, resulting in subsequent decreases in the parameters evaluated. The limitations of this study, including the small number of enrolled subjects and the large variability in the ages of the patients, must be considered. Future research considering the effects of age, longer observation periods, and additional monitoring may add important new information to the current findings.

CONCLUSION

• A dynamic change in microbial levels was found. The decrease in the levels of complexes present was only possible because of the mechanical method of oral hygiene implemented in this sample.