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Dysbiotic changes of periodontal pathogens in patients wearing conventional and self-ligating orthodontic appliances

Dysbiotic changes of periodontal pathogens in patients wearing conventional and self-ligating... IntroductionMalocclusion is a common dental condition that may be corrected by orthodontic treatment involving fixed or removable appliances.1 Preserving the integrity of the periodontal tissues is a priority during treatment, and has led to the definition of specific hygiene protocols for orthodontic patients.2 The principle of orthodontic treatment is based on corrective tooth movement, which is an occlusal reconstruction process involving alveolar bone remodelling and changes in bone morphology whose objectives are to correct function and establish dental and facial aesthetics.3,4Fixed appliances may offer conventional-ligation (CLA) or self-ligation (SLA) mechanisms. The main consideration of CLA is to achieve better positional crown control in addition to improving root position following the closure of spaces. To retain an arch wire within a bracket slot, either stainless steel ligatures which can vary in size (0.009 to 0.014 inches) or elastic modules (circular shaped elastomers that can be deformed to engage bracket tiewings and maintain the arch wire), may be used.5 As an alternative to CLA, two types of SLA have been developed: an active closing clip that exerts pressure on the arch wire, which in turn improves rotation control, and a passive bracket with a closing mechanism that transforms an open slot into a tube.6The biggest challenge of orthodontic therapy is to complete treatment with the least impact on the oral tissues (Figure 1). Periodontal disease is a chronic inflammatory process, initiated by a dysbiotic bacterial biofilm7 that leads to the destruction of investing tissue. Even though periodontal disease has been linked to a defined microbial biofilm composition, the effect of polymicrobial disruption on host homeostasis to periodontitis progression remains poorly understood.8 Recently, metagenomic studies have estimated that the number of bacteria found in the periodontal tissues is up to 600 species.9Figure 1.Orthodontic appliances impact oral health since patients must change their eating habits. Braces create oral hygiene difficulty and produce changes in salivary pH and disruption of the oral biofilm leading to inflammatory changes, plus damage to the periodontal tissues.Fixed orthodontic appliance materials affect the adhesion of bacteria and the accumulation of biofilm as a result of the roughness of the devices, the free energy of the surfaces, and other physicochemical properties of the biomaterials which influence the retentive capacity of the biofilm.10 In addition to enamel decalcification that leads to incipient caries, periodontal damage can also occur11 with associated local and systemic effects identified as root resorption, psychological disorders, gastrointestinal complications, allergic reactions, infective endocarditis, and chronic fatigue syndrome.12Knowing which microbial species colonises orthodontic appliances is important to plan strategies and to implement specific preventive control measures during treatment.13 Few studies have investigated periodontal pathogens when different types of fixed appliances have been placed. Previous research has determined that the microorganisms of Socransky’s ‘red’ complex are considered more pathogenic compared to the ‘orange’ complex regarding the aetiology of periodontal disease.14 It is therefore, important to assess the changes in the frequency and load of Socransky’s red and orange complexes that colonise orthodontic appliances and to know the periodontal risk.15 The present study aimed to explore dysbiotic changes in periodontal pathogenic bacteria associated with different types of fixed orthodontic appliances in a Northwestern Mexican population.Materials and methodsExperimental designBiofilm samples were collected from systemically healthy patients of both genders, who provided signed written informed consent. The study was approved by the ethics committee of the Autonomous University of Sinaloa and complied with the principles of the Declaration of Helsinki. The type of sampling was non-probabilistic. DNA concentrations up to 100 ng or greater were selected using spectrophotometry (nanodrop) and corroborated with electrophoresis tests. In total, 92 biofilm samples from patients were collected which subsequently formed three groups: patients without orthodontic appliances (N:31, group C) patients with conventional-ligating appliances (N:30, group CLA), and patients with self-ligating appliances (N:31, group SLA). The exclusion criteria were patients with an active diagnosis of COVID-19, who had received antibiotic treatment within the previous two weeks, those diagnosed with convulsive diseases, those with injuries or oral pathologies that were not the product of orthodontic treatment (trauma, lacerations, surgeries), those with appliances other than conventional or self-ligating, pregnant patients, or those who, during the previous 2 hours, had carried out oral hygiene, ingested food or administered mouthwash. The clinical characteristics of the patients selected for biofilm sampling were those undergoing orthodontic treatment without extractions, those with mild or moderate crowding whose periodontal status was clinically healthy at the beginning of the treatment and at the time the sample was taken, patients without bleeding at the time of sample collection and who did not require any type of surgical procedure, and patients with a class I molar, class II and III molar relationship of no more than 4 mm.Biofilm sample collectionThe patients refrained from eating, drinking, brushing their teeth, and chewing gum for at least 2 hr before the sampling procedure. Sample collection was performed between 8:30 am and 10:30 am before any oral examination or manipulation, thus avoiding disrupting the oral microbiota.16 Biofilm samples were taken from the areas adjacent to the fixed orthodontic appliances on the lower incisors using a CK6 instrument. The biofilm samples were placed in microtubes containing 500 µL of phosphate-buffered saline (PBS) 1X. The samples were kept at 4°C during transportation and stored at -80°C until DNA extraction.DNA extractionGenomic DNA was obtained from the biofilm samples using the Puregene Gentra Systems kit (DNA Isolation Kit, Minneapolis, MN, USA), according to the manufacturer’s instructions and following their protocol.Quantitative PCRThe presence and load of periodontal pathogenic bacteria were evaluated by quantitative polymerase chain reaction (qPCR). Sequences of the primers and the conditions applied for real-time PCR are shown in Table I. All species-specific primers targeted the variable regions of the 16S ribosomal RNA gene of the three strains. The qPCR conditions are listed in Table II. All assays were performed using SoFast EvaGreen Supermix (Bio-Rad) in a real-time PCR system (QuantStudio 12K Flex Real-Time PCR System, Thermo Fisher Scientific, Waltham, MA, USA) and the load was determined using the Ct threshold method, assigning 100% of the bacterial load to the C group.14Table I.Primers and qPCR conditions to measure bacterial load.Specific initiatorsAmplified products (pb)BacteriaFTd 5′-TAATACCGAATGTGCTCATTTACAT-3′316Treponema denticolaRTd 5′-TCAAAGAAGCATTCCCTCTTCTTCTTA-3;    FFn 5′-AGAGTTTGATCCTGGCTCAG-3′408Fusobacterium nucleatumRFn 5′-GTCATCGTGCACACAGAATTGCTG-3    FPi 5′-TTTGTTGGGGAGTAAAGCGGG-3′576Prevotella intermediaRPi 5′-TCAACATCTCTGTATCCTGCGT-3′Table II.qPCR conditions to measure bacterial load.Treponema denticolaFusobacterium nucleatumPrevotella intermediaCyclesReading melt curveDesn. initial96°C-5 m96°C-5 m96°C-5 m3080°CDesn. nat95°C-30 s95°C-30 s95°C-30 sAlign55°C-45 s56°C-45 s56°C-45 sExt72°C-45 s72°C-45 s72°C-45 sExt final72°C-10 m72°C-10 m72°C-10 mConservation4°C4°C4°CStatistical analysisFor a comparison of frequencies between groups, a chi-squared analysis was applied; for bacterial load analysis, a one-way ANOVA was followed by a Dunnet test to compare the conventional and self-ligating groups with the control group. P-values <0.05 were considered significant. All statistical analyses were performed using the software Graphpad Prism 8 (GraphPad Software, San Diego, CA, USA).ResultsDysbiotic changes were seen in patients wearing orthodontic appliances: F. nucleatum frequency was significantly higher in CLA and with a greater load in SLAThe presence of periodontal pathogens was observed in all groups, but F. nucleatum was identified as the bacteria of highest frequency in all groups (C 96.7%, CLA 100%, and SLA 67.7%, respectively). It was noted that all samples of the CLA group were positive for F. nucleatum (Figure 2). T. denticola was the bacteria of lowest frequency, however, it was high in the CLA group (76.6%). P. intermedia was present in 77.4%, 83.3% and 83.8% of the samples of the C, CLA and SLA groups, respectively.Figure 2.A, Presence of bacteria in C group. B, The bacterial presence of F. nucleatum is significantly higher in the CLA group displaying 100% of positivity when compared to SLA. C, Bacterial presence is lower in the SLA group.The percentages of bacterial load obtained using the Ct threshold method, are summarised in Figure 3. Although the differences in T. denticola were not significant, it was possible to observe a load difference between the groups, in which the SLA group showed 12.02% greater load than C and 14.68% greater load than the CLA group. P. intermedia in the CLA group showed a 7.27% greater load than the C group and 6.21% above the SLA group. F. nucleatum load showed significant differences between the groups. SLA had a higher load, with 31.60% above the C group and 14.99% above the CLA group. These results show that F. nucleatum frequency was high in the CLA group, but its load was higher in the SLA group.Figure 3.Percentages of bacterial load in the groups related to Ct, for T. dentícola (A, B), P. intermedia (C, D) and F. nucleatum (E,F). F. nucleatum showed statistical significance for CLA and SLA groups when compared to C group. Stars indicate statistical significance.Female participants showed higher frequencies of periodontal pathogens while male subjects were more affected by F. nucleatumT. denticola was found more frequently in females than males for all groups tested: C (95% vs 72%; p=0.0001), CLA (86% vs 66%; p=0.0008), and SLA (94% vs 76%; p=0.0002), respectively (Figure 4). In addition, the female higher percentage was found in the C group. For P. intermedia (Figure 5), the results for female vs male were C (65% vs 100%; p=0.0001), CLA (100% vs 73%; p=0.0001), and SLA (84% vs 84%; p=0.8449), respectively. Group C showed a 100% frequency in male subjects, while group CLA had a 100% frequency in females and SLA showed the same frequency between the genders. The F. nucleatum results for female vs male were C (95% vs 100%; p=0.0235), CLA (100% vs 100%; p=0.9999), and SLA (77% vs 94%; p=0.1510), respectively (Figure 6). All groups wearing appliances showed almost 100% positivity for F. nucleatum in the male group (Figure 4).Figure 4.T. denticola was found with higher positive percentages in the female gender when compared to the male gender (female vs male) for all groups. Nevertheless, the higher percentage was found in the C group.Figure 5.In P. intermedia, C group showed 100% of positivity in males. However, the CLA group had 100% of positivity for the female gender, meanwhile, SLA showed no significant differences between genders.Figure 6.In the case of F. nucleatum, all groups showed almost 100% of positivity in the male group. *Statistical significance.Increased treatment time correlated with a higher frequency of periodontal pathogensThe CLA and SLA groups were subdivided into those with <12 months and >12 months of treatment. The frequency of T. denticola (Figure 7) trended high in CLA (17 positive samples (74%)) and SLA (14 positives (78%)) groups after 12 months of treatment (the difference between the groups was not significant). The frequency of P. intermedia was significantly high in both the CLA (18 positives (78%)) and SLA (16 positives (89%)) groups after >12 months of treatment (Figure 8). Of note, F. nucleatum was found in 100% of CLA samples independently of treatment time and was more frequent in the SLA group after 12 months of treatment (Figure 9).Figure 7.Differences between groups according to time of treatment. T. denticola has greater presence in CLA and SLA after 12 months of treatment.Figure 8.P. intermedia is higher after 12 months in CLA and SLA.Figure 9.F. nucleatum is present in 100% of CLA samples before and after 12 months of treatment and is higher in the SLA group after 12 months.DiscussionThe present results confirm that dysbiotic changes are seen in patients wearing CLA and SLA. A dysbiotic microbiome is present when the diversity and proportions of normal species are disturbed.17 In this regard, dysbiosis generated by fixed orthodontic appliances is responsible for enamel demineralisation but also periodontal disease.18 The current results revealed that all tested bacteria showed a higher load in SLA when compared with the C group. P. intermedia and F. nucleatum in those wearing CLA, showed a higher load when compared to the C group. These results support the concept that dysbiosis leading to periodontal disease can be caused by the rise of a dominant bacterial species, instead of the appearance of a new species.8 All changes in oral bacterial composition accompanying dysbiosis and periodontal damage, have been related to other diseases including colo-rectal disease, cardiovascular diseases, Alzheimer’s disease, rheumatoid arthritis, meningitis, lung, brain, liver or splenic abscesses, appendicitis, pneumonia, and diabetes.9,19,20It is known that after the placement of fixed appliances, an initial biofilm is formed on material surfaces, often resulting in a worsening of the periodontal status. After an initial period of change, the host-microorganism balance can be restored.21 However, the appliance material surface may be altered over time because of contact with food and drink, abrasive oral hygiene measures, or corrosive processes.22 Since the attachment of appliances to the teeth makes it difficult to mechanically remove debris, the environment favours the formation of a biofilm.10,23 Clinically, the bacterial side effects of the appliances become apparent as plaque-associated gingivitis, an increase in pocket probe depth, and bleeding on probing.24 A recent study has explored the presence of bacteria in saliva and while salivary bacterial levels are helpful to indicate risk factors for the development of disease, in situ levels should also help to differentiate contamination patterns associated with the different appliance designs.25The present study focused on metal braces for both CLA and SLA, to minimise the intrinsic variability associated with other materials such as ceramics. Previous studies have reported a greater accumulation of biofilm in CLA metal braces compared to aesthetic material braces.10 A likely reason for a greater accumulation of biofilm on metal braces compared to aesthetic braces might relate to the nature of the ceramic surface, although aesthetic braces are usually larger and therefore have a greater surface area for biofilm adherence. However, ceramic surfaces are smoother, which makes it difficult for the biofilm to adhere and therefore facilitates mechanical removal.26More recently, Gujar et al. investigated the infection levels of Socransky’s orange and red bacterial complexes in patients wearing orthodontic appliances which included those with CLA. The findings showed that T. denticola had a higher frequency than other bacteria types in CLA patients.14 In agreement, the present study also found a higher frequency of T. denticola (C 67.7%; CLA 76.6%; SLA 67.7%) in the CLA group. T. denticola has been associated with the red complex (along with Bacteroides forsythum and Porphyromonas gingivalis),14 and implicated in severe forms of periodontal disease.27–31 The present results agreed with those of Pejda et al. who showed more positivity of red bacterial complexes on metal CLA appliances compared to metal SLA braces.24 Although the load of T. denticola in the present study was not the highest, its presence was an important finding considering its known virulence which sets it apart from other bacteria.32 In this regard, T. denticola, above threshold values of around 10–15% of the microbial biomass removed from the pocket, was a predictor of imminent attachment loss in a longitudinal prospective clinical trial.33 It may be speculated that the current finding of T. denticola also assumes the presence of P. gingivalis, as a recognised pathobiont and synergistic partner that normally co-localise in vivo.34–36 In addition, the bacteria can produce biofilms with increased biomass when grown together in vitro and have increased pathogenicity during co-infection in animal models of disease.33 More importantly, the levels of this dysbiotic signature of the biofilm could be used as a biomarker/predictor of site disease activity and imminent progression.37While P. gingivalis was not specifically identified, F. nucleatum and P. intermedia, two members of the orange complex (along with Parvimonas micra, Campylobacter rectus, and Eubacterium nodatum), were studied.38 The results indicated a significant presence of F. nucleatum in CLA as previously reported,39 and also a higher load in SLA. The findings agreed with those of Bergamo et al. in which the species related to the red and orange complexes, including F. nucleatum, showed the highest in situ levels on self-ligating appliances 30 and 60 days after placement.25 Clinically, this is important since, in conjunction with Actinobacillus actinomycetemcomitans (serotypes a and b), F. nucleatum, P. gingivalis, P. intermedia, T. forsythia, and T. denticola have been associated with the progress and severity of periodontal disease.40 In this regard, it has been reported that SLA braces cause more changes in microbial, plaque, and bleeding indices than conventional braces over time.41While females displayed a greater positivity for periodontal pathogens than males, except for F. nucleatum, Zorina et al. previously showed that females and males had equal periodontal pathogen colonisation; however, females had a higher risk of developing chronic periodontitis. The present results indicated that P. intermedia was present in 100% of the female samples in the CLA group. Nevertheless in males, the complex of T. denticola and P. intermedia has been correlated with the onset of chronic periodontitis.42 The present results indicate that, in the case of F. nucleatum, the presence of fixed metal appliances does not significantly change the percentage of positivity between the groups. However, it must be considered that an alteration in the oral microbiota can promote inflammatory reactions in the periodontal tissues and the different types of appliances interfere with bacterial adherence.The present study is limited by the lack of follow-up of patients after sample collection and by the clinical variables present over time to corroborate that dysbiotic changes lead to periodontal disease. Further studies need to be conducted to support this hypothesis and must consider different types of appliances, their design, orthodontics treatment, environmental, and cultural factors, related to diet, smoking, and oral hygiene.ConclusionsThe periodontal pathogens identified as F. nucleatum, T. denticola, and P. intermedia are found in healthy periodontal tissues; however, the present study showed that dysbiotic changes may be seen in patients wearing orthodontic appliances. F. nucleatum frequency was significantly higher in CLA and with a greater load in SLA which could affect the periodontal tissues. In addition, female participants showed higher frequencies of periodontal pathogens while male subjects were more affected by F. nucleatum. As expected, treatment lasting longer than 12 months correlated with a higher frequency of all periodontal pathogens. The results support the concept that dysbiosis, which leads to periodontal disease, can be caused by the rise of a dominant bacterial species, instead of the appearance of a new species. The evidence emerging from the present study should guide clinicians in modulating and enhancing patient motivation to prevent dysbiosis, thus reducing the risk of periodontal disease. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Australasian Orthodontic Journal de Gruyter

Dysbiotic changes of periodontal pathogens in patients wearing conventional and self-ligating orthodontic appliances

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References (31)

Publisher
de Gruyter
Copyright
© 2023 Juan Cota-Quintero et al., published by Sciendo
eISSN
2207-7480
DOI
10.2478/aoj-2023-0006
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Abstract

IntroductionMalocclusion is a common dental condition that may be corrected by orthodontic treatment involving fixed or removable appliances.1 Preserving the integrity of the periodontal tissues is a priority during treatment, and has led to the definition of specific hygiene protocols for orthodontic patients.2 The principle of orthodontic treatment is based on corrective tooth movement, which is an occlusal reconstruction process involving alveolar bone remodelling and changes in bone morphology whose objectives are to correct function and establish dental and facial aesthetics.3,4Fixed appliances may offer conventional-ligation (CLA) or self-ligation (SLA) mechanisms. The main consideration of CLA is to achieve better positional crown control in addition to improving root position following the closure of spaces. To retain an arch wire within a bracket slot, either stainless steel ligatures which can vary in size (0.009 to 0.014 inches) or elastic modules (circular shaped elastomers that can be deformed to engage bracket tiewings and maintain the arch wire), may be used.5 As an alternative to CLA, two types of SLA have been developed: an active closing clip that exerts pressure on the arch wire, which in turn improves rotation control, and a passive bracket with a closing mechanism that transforms an open slot into a tube.6The biggest challenge of orthodontic therapy is to complete treatment with the least impact on the oral tissues (Figure 1). Periodontal disease is a chronic inflammatory process, initiated by a dysbiotic bacterial biofilm7 that leads to the destruction of investing tissue. Even though periodontal disease has been linked to a defined microbial biofilm composition, the effect of polymicrobial disruption on host homeostasis to periodontitis progression remains poorly understood.8 Recently, metagenomic studies have estimated that the number of bacteria found in the periodontal tissues is up to 600 species.9Figure 1.Orthodontic appliances impact oral health since patients must change their eating habits. Braces create oral hygiene difficulty and produce changes in salivary pH and disruption of the oral biofilm leading to inflammatory changes, plus damage to the periodontal tissues.Fixed orthodontic appliance materials affect the adhesion of bacteria and the accumulation of biofilm as a result of the roughness of the devices, the free energy of the surfaces, and other physicochemical properties of the biomaterials which influence the retentive capacity of the biofilm.10 In addition to enamel decalcification that leads to incipient caries, periodontal damage can also occur11 with associated local and systemic effects identified as root resorption, psychological disorders, gastrointestinal complications, allergic reactions, infective endocarditis, and chronic fatigue syndrome.12Knowing which microbial species colonises orthodontic appliances is important to plan strategies and to implement specific preventive control measures during treatment.13 Few studies have investigated periodontal pathogens when different types of fixed appliances have been placed. Previous research has determined that the microorganisms of Socransky’s ‘red’ complex are considered more pathogenic compared to the ‘orange’ complex regarding the aetiology of periodontal disease.14 It is therefore, important to assess the changes in the frequency and load of Socransky’s red and orange complexes that colonise orthodontic appliances and to know the periodontal risk.15 The present study aimed to explore dysbiotic changes in periodontal pathogenic bacteria associated with different types of fixed orthodontic appliances in a Northwestern Mexican population.Materials and methodsExperimental designBiofilm samples were collected from systemically healthy patients of both genders, who provided signed written informed consent. The study was approved by the ethics committee of the Autonomous University of Sinaloa and complied with the principles of the Declaration of Helsinki. The type of sampling was non-probabilistic. DNA concentrations up to 100 ng or greater were selected using spectrophotometry (nanodrop) and corroborated with electrophoresis tests. In total, 92 biofilm samples from patients were collected which subsequently formed three groups: patients without orthodontic appliances (N:31, group C) patients with conventional-ligating appliances (N:30, group CLA), and patients with self-ligating appliances (N:31, group SLA). The exclusion criteria were patients with an active diagnosis of COVID-19, who had received antibiotic treatment within the previous two weeks, those diagnosed with convulsive diseases, those with injuries or oral pathologies that were not the product of orthodontic treatment (trauma, lacerations, surgeries), those with appliances other than conventional or self-ligating, pregnant patients, or those who, during the previous 2 hours, had carried out oral hygiene, ingested food or administered mouthwash. The clinical characteristics of the patients selected for biofilm sampling were those undergoing orthodontic treatment without extractions, those with mild or moderate crowding whose periodontal status was clinically healthy at the beginning of the treatment and at the time the sample was taken, patients without bleeding at the time of sample collection and who did not require any type of surgical procedure, and patients with a class I molar, class II and III molar relationship of no more than 4 mm.Biofilm sample collectionThe patients refrained from eating, drinking, brushing their teeth, and chewing gum for at least 2 hr before the sampling procedure. Sample collection was performed between 8:30 am and 10:30 am before any oral examination or manipulation, thus avoiding disrupting the oral microbiota.16 Biofilm samples were taken from the areas adjacent to the fixed orthodontic appliances on the lower incisors using a CK6 instrument. The biofilm samples were placed in microtubes containing 500 µL of phosphate-buffered saline (PBS) 1X. The samples were kept at 4°C during transportation and stored at -80°C until DNA extraction.DNA extractionGenomic DNA was obtained from the biofilm samples using the Puregene Gentra Systems kit (DNA Isolation Kit, Minneapolis, MN, USA), according to the manufacturer’s instructions and following their protocol.Quantitative PCRThe presence and load of periodontal pathogenic bacteria were evaluated by quantitative polymerase chain reaction (qPCR). Sequences of the primers and the conditions applied for real-time PCR are shown in Table I. All species-specific primers targeted the variable regions of the 16S ribosomal RNA gene of the three strains. The qPCR conditions are listed in Table II. All assays were performed using SoFast EvaGreen Supermix (Bio-Rad) in a real-time PCR system (QuantStudio 12K Flex Real-Time PCR System, Thermo Fisher Scientific, Waltham, MA, USA) and the load was determined using the Ct threshold method, assigning 100% of the bacterial load to the C group.14Table I.Primers and qPCR conditions to measure bacterial load.Specific initiatorsAmplified products (pb)BacteriaFTd 5′-TAATACCGAATGTGCTCATTTACAT-3′316Treponema denticolaRTd 5′-TCAAAGAAGCATTCCCTCTTCTTCTTA-3;    FFn 5′-AGAGTTTGATCCTGGCTCAG-3′408Fusobacterium nucleatumRFn 5′-GTCATCGTGCACACAGAATTGCTG-3    FPi 5′-TTTGTTGGGGAGTAAAGCGGG-3′576Prevotella intermediaRPi 5′-TCAACATCTCTGTATCCTGCGT-3′Table II.qPCR conditions to measure bacterial load.Treponema denticolaFusobacterium nucleatumPrevotella intermediaCyclesReading melt curveDesn. initial96°C-5 m96°C-5 m96°C-5 m3080°CDesn. nat95°C-30 s95°C-30 s95°C-30 sAlign55°C-45 s56°C-45 s56°C-45 sExt72°C-45 s72°C-45 s72°C-45 sExt final72°C-10 m72°C-10 m72°C-10 mConservation4°C4°C4°CStatistical analysisFor a comparison of frequencies between groups, a chi-squared analysis was applied; for bacterial load analysis, a one-way ANOVA was followed by a Dunnet test to compare the conventional and self-ligating groups with the control group. P-values <0.05 were considered significant. All statistical analyses were performed using the software Graphpad Prism 8 (GraphPad Software, San Diego, CA, USA).ResultsDysbiotic changes were seen in patients wearing orthodontic appliances: F. nucleatum frequency was significantly higher in CLA and with a greater load in SLAThe presence of periodontal pathogens was observed in all groups, but F. nucleatum was identified as the bacteria of highest frequency in all groups (C 96.7%, CLA 100%, and SLA 67.7%, respectively). It was noted that all samples of the CLA group were positive for F. nucleatum (Figure 2). T. denticola was the bacteria of lowest frequency, however, it was high in the CLA group (76.6%). P. intermedia was present in 77.4%, 83.3% and 83.8% of the samples of the C, CLA and SLA groups, respectively.Figure 2.A, Presence of bacteria in C group. B, The bacterial presence of F. nucleatum is significantly higher in the CLA group displaying 100% of positivity when compared to SLA. C, Bacterial presence is lower in the SLA group.The percentages of bacterial load obtained using the Ct threshold method, are summarised in Figure 3. Although the differences in T. denticola were not significant, it was possible to observe a load difference between the groups, in which the SLA group showed 12.02% greater load than C and 14.68% greater load than the CLA group. P. intermedia in the CLA group showed a 7.27% greater load than the C group and 6.21% above the SLA group. F. nucleatum load showed significant differences between the groups. SLA had a higher load, with 31.60% above the C group and 14.99% above the CLA group. These results show that F. nucleatum frequency was high in the CLA group, but its load was higher in the SLA group.Figure 3.Percentages of bacterial load in the groups related to Ct, for T. dentícola (A, B), P. intermedia (C, D) and F. nucleatum (E,F). F. nucleatum showed statistical significance for CLA and SLA groups when compared to C group. Stars indicate statistical significance.Female participants showed higher frequencies of periodontal pathogens while male subjects were more affected by F. nucleatumT. denticola was found more frequently in females than males for all groups tested: C (95% vs 72%; p=0.0001), CLA (86% vs 66%; p=0.0008), and SLA (94% vs 76%; p=0.0002), respectively (Figure 4). In addition, the female higher percentage was found in the C group. For P. intermedia (Figure 5), the results for female vs male were C (65% vs 100%; p=0.0001), CLA (100% vs 73%; p=0.0001), and SLA (84% vs 84%; p=0.8449), respectively. Group C showed a 100% frequency in male subjects, while group CLA had a 100% frequency in females and SLA showed the same frequency between the genders. The F. nucleatum results for female vs male were C (95% vs 100%; p=0.0235), CLA (100% vs 100%; p=0.9999), and SLA (77% vs 94%; p=0.1510), respectively (Figure 6). All groups wearing appliances showed almost 100% positivity for F. nucleatum in the male group (Figure 4).Figure 4.T. denticola was found with higher positive percentages in the female gender when compared to the male gender (female vs male) for all groups. Nevertheless, the higher percentage was found in the C group.Figure 5.In P. intermedia, C group showed 100% of positivity in males. However, the CLA group had 100% of positivity for the female gender, meanwhile, SLA showed no significant differences between genders.Figure 6.In the case of F. nucleatum, all groups showed almost 100% of positivity in the male group. *Statistical significance.Increased treatment time correlated with a higher frequency of periodontal pathogensThe CLA and SLA groups were subdivided into those with <12 months and >12 months of treatment. The frequency of T. denticola (Figure 7) trended high in CLA (17 positive samples (74%)) and SLA (14 positives (78%)) groups after 12 months of treatment (the difference between the groups was not significant). The frequency of P. intermedia was significantly high in both the CLA (18 positives (78%)) and SLA (16 positives (89%)) groups after >12 months of treatment (Figure 8). Of note, F. nucleatum was found in 100% of CLA samples independently of treatment time and was more frequent in the SLA group after 12 months of treatment (Figure 9).Figure 7.Differences between groups according to time of treatment. T. denticola has greater presence in CLA and SLA after 12 months of treatment.Figure 8.P. intermedia is higher after 12 months in CLA and SLA.Figure 9.F. nucleatum is present in 100% of CLA samples before and after 12 months of treatment and is higher in the SLA group after 12 months.DiscussionThe present results confirm that dysbiotic changes are seen in patients wearing CLA and SLA. A dysbiotic microbiome is present when the diversity and proportions of normal species are disturbed.17 In this regard, dysbiosis generated by fixed orthodontic appliances is responsible for enamel demineralisation but also periodontal disease.18 The current results revealed that all tested bacteria showed a higher load in SLA when compared with the C group. P. intermedia and F. nucleatum in those wearing CLA, showed a higher load when compared to the C group. These results support the concept that dysbiosis leading to periodontal disease can be caused by the rise of a dominant bacterial species, instead of the appearance of a new species.8 All changes in oral bacterial composition accompanying dysbiosis and periodontal damage, have been related to other diseases including colo-rectal disease, cardiovascular diseases, Alzheimer’s disease, rheumatoid arthritis, meningitis, lung, brain, liver or splenic abscesses, appendicitis, pneumonia, and diabetes.9,19,20It is known that after the placement of fixed appliances, an initial biofilm is formed on material surfaces, often resulting in a worsening of the periodontal status. After an initial period of change, the host-microorganism balance can be restored.21 However, the appliance material surface may be altered over time because of contact with food and drink, abrasive oral hygiene measures, or corrosive processes.22 Since the attachment of appliances to the teeth makes it difficult to mechanically remove debris, the environment favours the formation of a biofilm.10,23 Clinically, the bacterial side effects of the appliances become apparent as plaque-associated gingivitis, an increase in pocket probe depth, and bleeding on probing.24 A recent study has explored the presence of bacteria in saliva and while salivary bacterial levels are helpful to indicate risk factors for the development of disease, in situ levels should also help to differentiate contamination patterns associated with the different appliance designs.25The present study focused on metal braces for both CLA and SLA, to minimise the intrinsic variability associated with other materials such as ceramics. Previous studies have reported a greater accumulation of biofilm in CLA metal braces compared to aesthetic material braces.10 A likely reason for a greater accumulation of biofilm on metal braces compared to aesthetic braces might relate to the nature of the ceramic surface, although aesthetic braces are usually larger and therefore have a greater surface area for biofilm adherence. However, ceramic surfaces are smoother, which makes it difficult for the biofilm to adhere and therefore facilitates mechanical removal.26More recently, Gujar et al. investigated the infection levels of Socransky’s orange and red bacterial complexes in patients wearing orthodontic appliances which included those with CLA. The findings showed that T. denticola had a higher frequency than other bacteria types in CLA patients.14 In agreement, the present study also found a higher frequency of T. denticola (C 67.7%; CLA 76.6%; SLA 67.7%) in the CLA group. T. denticola has been associated with the red complex (along with Bacteroides forsythum and Porphyromonas gingivalis),14 and implicated in severe forms of periodontal disease.27–31 The present results agreed with those of Pejda et al. who showed more positivity of red bacterial complexes on metal CLA appliances compared to metal SLA braces.24 Although the load of T. denticola in the present study was not the highest, its presence was an important finding considering its known virulence which sets it apart from other bacteria.32 In this regard, T. denticola, above threshold values of around 10–15% of the microbial biomass removed from the pocket, was a predictor of imminent attachment loss in a longitudinal prospective clinical trial.33 It may be speculated that the current finding of T. denticola also assumes the presence of P. gingivalis, as a recognised pathobiont and synergistic partner that normally co-localise in vivo.34–36 In addition, the bacteria can produce biofilms with increased biomass when grown together in vitro and have increased pathogenicity during co-infection in animal models of disease.33 More importantly, the levels of this dysbiotic signature of the biofilm could be used as a biomarker/predictor of site disease activity and imminent progression.37While P. gingivalis was not specifically identified, F. nucleatum and P. intermedia, two members of the orange complex (along with Parvimonas micra, Campylobacter rectus, and Eubacterium nodatum), were studied.38 The results indicated a significant presence of F. nucleatum in CLA as previously reported,39 and also a higher load in SLA. The findings agreed with those of Bergamo et al. in which the species related to the red and orange complexes, including F. nucleatum, showed the highest in situ levels on self-ligating appliances 30 and 60 days after placement.25 Clinically, this is important since, in conjunction with Actinobacillus actinomycetemcomitans (serotypes a and b), F. nucleatum, P. gingivalis, P. intermedia, T. forsythia, and T. denticola have been associated with the progress and severity of periodontal disease.40 In this regard, it has been reported that SLA braces cause more changes in microbial, plaque, and bleeding indices than conventional braces over time.41While females displayed a greater positivity for periodontal pathogens than males, except for F. nucleatum, Zorina et al. previously showed that females and males had equal periodontal pathogen colonisation; however, females had a higher risk of developing chronic periodontitis. The present results indicated that P. intermedia was present in 100% of the female samples in the CLA group. Nevertheless in males, the complex of T. denticola and P. intermedia has been correlated with the onset of chronic periodontitis.42 The present results indicate that, in the case of F. nucleatum, the presence of fixed metal appliances does not significantly change the percentage of positivity between the groups. However, it must be considered that an alteration in the oral microbiota can promote inflammatory reactions in the periodontal tissues and the different types of appliances interfere with bacterial adherence.The present study is limited by the lack of follow-up of patients after sample collection and by the clinical variables present over time to corroborate that dysbiotic changes lead to periodontal disease. Further studies need to be conducted to support this hypothesis and must consider different types of appliances, their design, orthodontics treatment, environmental, and cultural factors, related to diet, smoking, and oral hygiene.ConclusionsThe periodontal pathogens identified as F. nucleatum, T. denticola, and P. intermedia are found in healthy periodontal tissues; however, the present study showed that dysbiotic changes may be seen in patients wearing orthodontic appliances. F. nucleatum frequency was significantly higher in CLA and with a greater load in SLA which could affect the periodontal tissues. In addition, female participants showed higher frequencies of periodontal pathogens while male subjects were more affected by F. nucleatum. As expected, treatment lasting longer than 12 months correlated with a higher frequency of all periodontal pathogens. The results support the concept that dysbiosis, which leads to periodontal disease, can be caused by the rise of a dominant bacterial species, instead of the appearance of a new species. The evidence emerging from the present study should guide clinicians in modulating and enhancing patient motivation to prevent dysbiosis, thus reducing the risk of periodontal disease.

Journal

Australasian Orthodontic Journal – de Gruyter

Published: Jan 1, 2023

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