Microbial profile in different orthodontic appliances by checkerboard DNA-DNA hybridization: An in-vivo study

Introduction

The design of the orthodontic bracket or appliance is 1 of the most important factors for creating retentive areas for biofilm formation. In orthodontics, this would be the first study to compare the microbial level changes in 3 different types of orthodontic appliances using checkerboard DNA-DNA hybridization technique. The purpose of this study was to evaluate and compare the extent of appearance of orange and red microbial complexes in patients undergoing orthodontic treatment using aligners, conventional metallic fixed labial appliances, and lingual fixed appliances.

Methods

A total of 60 patients, of which 20 patients were undergoing treatment with aligners, 20 patients with labial fixed appliances, and 20 patients with lingual fixed appliances, were included in our study. After 30 days, debonded brackets and rinsed aligners were stored and processed for analysis with checkerboard DNA-DNA hybridization.

Results

Most bacterial species showed moderate counts, with the exception of Treponema denticola, which showed a higher count in all 3 types of appliances. Fusobacterium nucleatum , Porphyromonas gingivalis , and T denticola were present in a higher percentage in the lingual appliance. Fusobacterium periodontium and Prevotella intermedia were present in a higher percentage in the labial fixed appliance. Campylobacter rectus , Tannerella forsythia , and Prevotella melaninogenica counts were moderate in all 3 appliances, with the first 2 microbes showing slightly higher counts in aligners. The association between all the microorganisms were statistically insignificant, with the exception of F nucleatum , which showed a strong statistically significant association in all 3 types of appliances.

Conclusions

The microbial contamination in metallic brackets was higher than that of aligners, when used for a month. Lingual fixed appliances showed more microbial contamination than labial fixed appliances followed by aligners.

Highlights

  • Microbial colonies inhabiting orthodontics appliances were profiled with checkerboard DNA-DNA hybridization.

  • Metallic brackets had more microbial colonization than aligners.

  • Colonization patterns were different for labial and lingual fixed appliances and aligners.

Specific alterations take place in the oral environment because of fixed orthodontic treatment. There is an increased accumulation of plaque, an increase in the count of microorganisms, and a decrease in the level of pH, which leads to an increased risk of caries and periodontal diseases. Apart from white spot lesions and caries, there can be harmful effects on the periodontium owing to the decalcification of enamel. The microbial adhesion and accumulation of plaque depend on many variables such as surface characteristics, but bracket design plays the most important role among them. Supragingival plaque gets accumulated because of reduced efficiency of self-cleaning that results on account of complex brackets designs. The formation of the biofilm could be influenced by the placement of the orthodontic brackets. Many studies have shown a shift in the microbial population because of fixed orthodontic appliances, but very limited information is available on bracket material being prone to bacterial species adhesion and accumulation of plaque. The periodontal health and the relationship between periodontal status during and after orthodontic treatment is a big challenge.

The microbial plaque retention also depends on the method of ligation. Elastic or stainless steel ligatures, which are used for holding the wire onto the brackets, contribute to the retention of supragingival plaque. Because of increasing esthetic demand, lingual orthodontic appliances and clear aligners are in high demand today. The accumulation of plaque varies in both these appliances.

Various microorganisms constituting the 6 complexes (blue, purple, green, yellow, orange, and red) are involved in causing gingival and periodontal diseases. Out of these complexes, the red and orange complexes are subgingival pathogens. The microorganisms in the red complex are considered most pathogenic compared with the orange complex in causing periodontal diseases; hence, it is important to evaluate the changes in the levels of orange and red complexes because of different orthodontic appliances and know its risk in causing periodontal diseases. The microorganisms constituting the orange complex are Campylobacter rectus , Fusobacterium nucleatum, Fusobacterium periodontium , Prevotella intermedia , and Prevotella melaninogenica , whereas those constituting the red complex are Porphyromonas gingivalis, Tannerella forsythi a, and Treponema denticola .

The microbial flora has been assessed with many different methods, such as microbiologic culture techniques and polymerase chain reaction (PCR). The advancement in the techniques of molecular biology made the identification of microbial species very precise with the help of DNA probes. Because it makes use of many DNA probes at the same time and allows for the determination of many bacterial species simultaneously in single or multiple samples, which can be stored for a long time, it is considered to be much faster than PCR. In orthodontics, this would the first study comparing the microbial level changes in 3 different types of orthodontic appliances using checkerboard DNA-DNA hybridization (CDDH) technique.

Therefore, the present study was undertaken to determine and compare the changes in levels of orange and red microbial complexes in patients undergoing orthodontic treatment using aligners, conventional metallic fixed labial appliances, and lingual fixed appliances.

Material and methods

The patients undergoing orthodontic treatment with aligners, metallic fixed labial appliances, and lingual fixed appliances in the Department of Orthodontics and Dentofacial Orthopedics in our institution were selected for the study. Considering the effect size to be measured at 0.40, the power of the study at 80%, and an alpha level of 0.05 and beta value as 0.20, the total sample size was calculated to be 60 using G*Power analysis software (version 3.0.1, Heinrich-Heine-Universität Düsseldorf, Germany). The sample consisted of 60 patients with ages ranging between 11 and 29 years in all 3 groups; 20 patients were undergoing treatment with aligners, 20 patients were undergoing treatment with fixed labial appliances, and 20 patients were undergoing treatment with the lingual fixed appliances.

The patients with good general health requiring orthodontic treatment, with lack of antibiotic therapy within the past 6 months, with no use of anti-inflammatory drugs in the month preceding the study, having periodontal health with a gingival index (GI) score <1, no pockets with generalized probing depths ≤3 mm, and no radiographic evidence of periodontal bone loss were included in our study. The patients with smoking habits, signs of gingivitis or periodontitis, and a diagnosis of systemic disease were excluded from our study.

Following the institutional ethics committee approval, eligible participants were selected from the patients of both sexes with complete dentitions. A formal detailed consent was obtained from all the patients. The selected patients were informed about keeping good oral hygiene. Motivation was maintained during the entire study. At each presentation of the patient, the GI was measured. GI < 1 was considered an expression of good oral hygiene.

Twenty patients were bonded with conventional MBT metallic labial orthodontic brackets (0.022 × 0.028-inch slot; 3M Unitek, Monrovia, Calif) using orthodontic light cured adhesive (Transbond XT; 3M Unitek) in both maxillary and mandibular arches. Maxillary and mandibular archwires (0.016-inch nickel titanium) were placed for initial alignment and leveling. Twenty patients were bonded with lingual metallic brackets (Ormco Corporation, Glendora, Calif) using orthodontic light cured adhesive (Transbond XT) in both maxillary and mandibular arches. Maxillary and mandibular archwires (0.012-inch nickel titanium) were placed for initial alignment and leveling. All the patients receiving metallic labial and lingual brackets were ligated using elastomeric ligatures (3M Unitek). Twenty patients were given both maxillary and mandibular Invisalign aligners (Align Technology, San Jose, Calif).

After 30 days, randomly 1 bracket each from the maxillary arch was debonded (without removing the elastic ligatures on them) from all 20 patients undergoing treatment with labial and lingual bracket appliances. The debonded brackets were placed into coded sterile microtubes containing 150 μL of Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 7.6) and mixed in a Vortex-Genie 2 G560 (Scientific Industries, Inc., Bohemia, NY). The brackets were removed using sterilized pliers followed by the addition of 100 μL of 0.5 M NaOH and stored at −20°C until CDDH was performed, according to Bergamo et al. The maxillary aligner was removed after 30 days and rinsed in 150 μL of Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 7.6) and mixed in a Vortex in a Petri dish, and the rinsed solution was stored in a container at −20°C until CDDH was performed. The samples collected from patients using Invisalign aligners were collected in bottles coded as B1-B20. The samples collected from the patients using labial fixed appliances (debonded metallic labial brackets) were collected in vials (microtubes) coded as V1-V20. The samples collected from patients using lingual fixed appliances (debonded metallic lingual brackets) were collected in vials (microtubes) coded as VL1-VL20. After that stage, all the debonded teeth received new brackets, and the next set of aligners was given to the patient.

The patients were coded to avoid their direct identification. The data analyst also was blinded from the identification.

Using the CDDH method, the levels of orange complex and red complex was determined. The microorganisms constituting the orange complex were C rectus , F nucleatum , F periodontium , P intermedia , and P melaninogenica , whereas those constituting the red complex were P. gingivalis , T forsythia , and T denticola .

For DNA-DNA hybridization, genomic DNA was isolated from the saliva samples. The quality of genomic DNA was checked by loading it on a 1% agarose gel ( Fig 1 ). The ∼1.3 kb/1.5 kb, 16S-rDNA fragment was amplified using high-fidelity PCR polymerase (Catalog number PCR08; Chromous Biotech, Bangalore, India). Biotinylated probes specific to each microbe were designed and synthesized. The amplified products were purified, and DNA samples were spotted on a nylon membrane. Detection of the given 8 microorganisms over 60 samples by southern blotting technique was done.

Fig 1
A, Genomic DNA loaded on 1% agarose gel for samples B1-B20; B, Genomic DNA loaded on 1% agarose gel for samples V1-V20; C, Genomic DNA loaded on 1% agarose gel for samples VL1-VL20.

As standard controls, defined amounts of DNA (PCR amplicons) of given samples were prepared, denatured by heating at 95°C for 10 minutes and snap-chilled on the ice, precipitated, and spotted onto parallel lanes.

  • Nylon membranes were prepared with dimensions 10 × 10 cm and were divided into a grid of 100 squares (10 × 10 squares of dimension 1 × 1 cm).

  • DNA amplicons from 60 samples along with 2 control amplicon DNA samples were spotted; total spots per membrane were 62.

  • DNA samples were spotted in parallel lanes on nylon membrane as shown in Figure 2 .

    Fig 2
    Parallel lanes on nylon membrane for spotting of DNA samples.
  • DNA was fixed onto the membrane by baking the membrane for 2 hours at 80°C.

  • The membrane was then placed on an ultraviolet transilluminator (spotted side down) for 1-5 minutes because this favors the cross-linking of the DNA to the membrane.

  • The membranes were then prehybridized at 60°C for 2 hours in prehybridization buffer containing 1 M NaCl and 4% blocking reagent.

  • The membranes were hybridized with specific probes.

  • Hybridizations were performed at 60°C for 16 hours, under gentle agitation.

  • The membranes were subsequently washed twice at 65°C for 30 minutes in primary wash buffer and twice in secondary wash buffer, at room temperature for 15 minutes.

  • After washing, the hybrids were detected using streptavidin–horseradish peroxidase conjugate.

The results obtained with CDDH technique were analyzed descriptively by using the Statistical Analysis System software (SPSS statistics for windows, version 22.0, IBM Corp. Armonk, NY) to evaluate the level of contamination of the brackets by each microorganism.

Results

The developed membranes were screened for the difference in intensity of the spots developed, and results were tabulated. The images for the developed membranes are shown in Figure 3 .

Fig 3
Membrane specific to ( A) C rectus , (B) F nucleatum , (C) F periodontium , (D) P intermedia , (E) P melaninogenica , (F) P gingivalis , (G) T forsythia , and (H) T denticola .

Figure 3 , A shows the membrane for probe 1, which was specific to C rectus . Samples V7 and VL15 showed very prominent spots, indicating a high level of infection with C rectus , whereas samples B2, V4, V5, and VL12 showed no development of the spots, indicating the absence of infection with C rectus . However, the remaining samples showed very faint spot development, indicating they might be moderately infected by C rectus .

Figure 3 , B shows the membrane for probe 2, which was specific to F nucleatum . Samples B1, B2, B3, B4, B5, B8, B9, B14, B15, B18, B20, V5, V6, V7, V10, V11, V14, and V20 showed very prominent spots, indicating a high level of infection with F nucleatum , whereas sample V17, VL2, VL8, VL12, VL14, and VL20 showed no development of the spots, indicating the absence of infection with F nucleatum . However, the remaining samples showed very faint spot development, indicating they might be moderately infected by F nucleatum .

Figure 3 , C shows the membrane for probe 3, which was specific to F periodontium . Samples B10, B15, B16, V4, V9, V10, V11, and V19 showed very prominent spots, indicating high level of infection with F periodontium , whereas samples B4, B5, B6, B8, B9, B13, B14, VL8, and VL9 showed no development of the spots, indicating the absence of infection with F periodontium . However, the remaining samples showed very faint spot development, indicating they might be moderately infected by F periodontium .

Figure 3 , D shows the membrane for probe 4, which was specific to P intermedia . Samples B6, B19, B20, V17, and VL8 showed very prominent spots, indicating a high level of infection with P intermedia , whereas samples B2, B16, B17, VL5, and VL18 showed no development of the spots, indicating the absence of infection with P intermedia . However, the remaining samples showed very faint spot development, indicating they might be moderately infected by P intermedia .

Figure 3 , E shows the membrane for probe 5, which was specific to P melaninogenica . Samples B10, V17, VL1, VL7, VL9, VL10, VL11, and VL20 showed very prominent spots, indicating high level of infection with P melaninogenica , whereas samples B2, B3, B4, B11, B16, V2, V3, V10, V11, V13, VL15, and VL16 showed no development of the spots, indicating the absence of infection with P melaninogenica . However, the remaining samples showed very faint spot development, indicating they might be moderately infected by P melaninogenica .

Figure 3 , F shows the membrane for probe 6, which was specific to P gingivalis . Samples B1, B2, B5, B7, B9, B10, B17, B18, V7, V11, V18, V20, VL6, VL7 and VL10 showed very prominent spots, indicating a high level of infection with P gingivalis , whereas samples V1, V9, and V14 showed no development of the spots, indicating the absence of infection with P gingivalis . However, the remaining samples showed very faint spot development, indicating they might be moderately infected by P gingivalis .

Figure 3 , G shows the membrane for probe 7, which was specific to T forsythia . Samples B13, V18, V19, V20, VL15, VL16, VL17, VL18, and VL20 showed very prominent spots, indicating a high level of infection with T forsythia , whereas sample B18 showed no development of the spots, indicating the absence of infection with T forsythia . However, the remaining samples showed very faint spot development, indicating they might be moderately infected by T forsythia .

Figure 3 , H shows the membrane for probe 8, which was specific to T denticola . Samples B2, B4, B12, and V12 showed no development of the spots, indicating the absence of infection with T denticola . The remaining samples showed very prominent spots, indicating a high level of infection with T denticola .

Association of the appearance of individual bacteria with the different study groups was analyzed using a chi-square test. All the study groups (ie, groups B, V, and VL) had moderate C rectus count, but the association was found to be statistically insignificant ( P >0.05; Table I ). F nucleatum count was moderate in all the 3 study groups with a comparatively higher percentage in group VL. Its expression varied over the other groups, and it was statistically significant ( P <0.05; Table I ). Comparison of the appearance of F periodontium over the other groups was found to be statistically significant ( P <0.05; Table I ). It was also observed that its expression was more skewed (moderate in number) in all 3 study groups with a slightly high percentage in group V, followed by group VL and B. P intermedia count was moderate in all 3 study groups with a comparatively higher percentage in group V, followed by group VL and group B, but the association was found to be statistically insignificant ( P >0.05; Table I ). P melaninogenica count was moderate in all 3 study groups, but the association was found to be statistically insignificant ( P >0.05; Table I ). P gingivalis count was moderate in number in all 3 groups with a slightly high percentage in group VL followed by group V and group B, but the association was found to be statistically insignificant ( P >0.05; Table I ). T forsythia count was moderate in all 3 groups with a comparatively higher percentage in group B, followed by group V and group VL, but the association was found to be statistically insignificant ( P >0.05; Table I ). T denticola count was high in all 3 groups with a comparatively higher percentage in group VL, followed by group V and group B, but the association was found to be statistically insignificant ( P >0.05; Table I ).

Table I
Comparison of microorganisms in all 3 groups
Group B (n = 20) Group V (n = 20) Group VL (n = 20) Total (n = 60) P value
Orange complex
Campylobacter rectus
High in number 0 (0) 1 (5) 1 (5) 2 (3.3)
Moderate in number 19 (95) 17 (85) 18 (90) 54 (90) 0.890
Negative/absent 1 (5) 2 (10) 1 (5) 4 (6.7)
Fusobacterium nucleatum
High in number 11 (55) 7 (35) 0 (0) 18 (30)
Moderate in number 9 (45) 12 (60) 16 (80) 37 (61.7) <0.001
Negative/absent 0 (0) 1 (5) 4 (20) 5 (8.3)
Fusobacterium periodontium
High in number 3 (15) 1 (5) 0 (0) 4 (6.7)
Moderate in number 10 (50) 19 (95) 18 (90) 47 (78.3) 0.001
Negative/absent 7 (35) 0 (0) 2 (10) 9 (15)
Prevotella intermedia
High in number 3 (15) 1 (5) 1 (5) 5 (8.3)
Moderate in number 14 (70) 19 (95) 17 (85) 50 (83.3) 0.306
Negative/absent 3 (15) 0 (0) 2 (10) 5 (8.3)
Prevotella melaninogenica
High in number 1 (5) 1 (5) 6 (30) 8 (13.3)
Moderate in number 14 (70) 14 (70) 12 (60) 40 (66.7) 0.140
Negative/absent 5 (25) 5 (25) 2 (10) 12 (20)
Red complex
Porphyromonas gingivalis
High in number 8 (40) 4 (20) 2 (10) 14 (23.3)
Moderate in number 12 (60) 14 (70) 18 (90) 44 (73.3) 0.058
Negative/absent 0 (0) 2 (10) 0 (0) 2 (3.3)
Tannerella forsythia
High in number 1 (5) 3 (15) 5 (25) 9 (15)
Moderate in number 19 (95) 17 (85) 15 (75) 51 (85) 0.265
Negative/absent 0 (0) 0 (0) 0 (0) 0 (0)
Treponema denticola
High in number 17 (85) 19 (95) 20 (100) 56 (93.3)
Moderate in number 0 (0) 0 (0) 0 (0) 0 (0) 0.310
Negative/absent 3 (15) 1 (5) 0 (0) 4 (6.7)
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Jan 7, 2020 | Posted by in Orthodontics | Comments Off on Microbial profile in different orthodontic appliances by checkerboard DNA-DNA hybridization: An in-vivo study
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