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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 53  |  Issue : 1  |  Page : 38-42

Analysis of plaque microflora in patients pretreatment and during treatment with fixed orthodontic appliances: An In vivo Study


1 PG Student, M. A. Rangoonwala College of Dental Sciences and Research Centre, Pune, Maharashtra, India
2 Prof., M. A. Rangoonwala College of Dental Sciences and Research Centre, Pune, Maharashtra, India
3 Prof. and HOD, M. A. Rangoonwala College of Dental Sciences and Research Centre, Pune, Maharashtra, India

Date of Submission02-Jul-2018
Date of Acceptance14-Nov-2018
Date of Web Publication04-Feb-2019

Correspondence Address:
Dr. Shymal Vairagi
M. A. Rangoonwala College of Dental Sciences and Research Centre, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jios.jios_104_18

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  Abstract 


Aim: The study analyzed plaque microflora to compare the growth of periodontopathogens-Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella nigrescens and Campylobacter rectus in patients undergoing fixed orthodontic treatment. Materials and Methods: Samples of plaque microflora from gingival crevices from incisors, and molars were collected by sterile periodontal probe at four different time periods-first was taken before placement of fixed appliances (T0) considered to be control group, then at 1 week (T1), 1 month (T2), and 5 months of starting fixed treatment (T3).The samples were transported to the laboratory and processed within 24 hr using the polymerase chain reaction technique. Results: The statistical significance of intragroup comparisons was done using test RMNOVA. The colony-forming units at different follow-up intervals showed a significant increase in all tested periodontopathogens except A. actinomycetemcomitans which showed no significant increase. Conclusion: Fixed orthodontic treatment can interfere in normal oral hygiene which can initiate plaque accumulation further leading to inflammation due to increase in periodontopathogens. Therefore, oral hygiene should be constantly monitored for plaque control.

Keywords: Fixed orthodontic appliances, microflora, plaque


How to cite this article:
Ahmed A, Hegde A, Gautam R, Kalia A, Nene S, Vairagi S. Analysis of plaque microflora in patients pretreatment and during treatment with fixed orthodontic appliances: An In vivo Study. J Indian Orthod Soc 2019;53:38-42

How to cite this URL:
Ahmed A, Hegde A, Gautam R, Kalia A, Nene S, Vairagi S. Analysis of plaque microflora in patients pretreatment and during treatment with fixed orthodontic appliances: An In vivo Study. J Indian Orthod Soc [serial online] 2019 [cited 2019 Jun 16];53:38-42. Available from: http://www.jios.in/text.asp?2019/53/1/38/251540




  Introduction Top


Fixed orthodontic appliances make conventional oral hygiene procedures more difficult, and it leads to more plaque formation on the tooth surface. The plaque microflora comprises more than 600 bacteria and numerous environmental variations alter their composition.[1] Most growth sites for these bacteria are on the gingival margins and edges of orthodontic bands.[2] Accrued food debris, particularly fermentable carbohydrates, causes growth of the cariogenic bacteria such as Streptococcus mutans and Lactobacillus[3] and bacteria such as Eubacterium, Fusobacterium and Treponema which cause periodontitis.[4]

It has been found that brackets and bands can cause difficulty in maintaining good oral hygiene causing plaque formation and further leading to the development of white-spot lesions and caries. It can also favor the growth of periodontopathogens in subgingival pockets and the production of pro-inflammatory cytokines.[5],[6] Bacteria, such Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum and Treponema denticola which cause periodontitis are commonly seen in the subgingival plaque.[7] Removable appliances in comparison allow patients to maintain good oral hygiene, thereby minimizing the risk of developing dental or periodontal problems.[8] The rate at which the bacteria colonize is dependent on the host environment and bacterial factors such as proteins in the gingival crevicular fluid and the fimbriae.[9]

Previous studies had limitations such as, limited number of species were tested and very few studies used accurate detection methods such as polymerase chain reaction (PCR).[10] PCR technique was found to be more reliable than culture methods as it is highly sensitive and consistent means of quantifying bacterial DNA.[11]

This study aimed at determining the changes in the bacterial concentrations and composition of the plaque microflora, especially the periodontopathogens i.e., A. actinomycetemcomitans, P. gingivalis, Tannerella forsythia, Prevotella nigrescens, and Campylobacter rectus before and after initiation of fixed orthodontic treatment.


  Materials and Methods Top


In this study, 20 random Indian patients were selected who visited the Department of Orthodontics for fixed orthodontic treatment. The sample size calculation was based on the effect sizes from the previously published study and the sample size of 20 had 82.5% power. Ethical approval number was MCES/EC/MARDC/ORTHO/124/2014.

Inclusion criteria

  1. Age between 12 and 35 years, patients with full complement of intact permanent dentition
  2. Patients requiring fixed orthodontic therapy, with favorable oral hygiene that is having oral hygiene index score from good to fair (0.0–3.0)
  3. Patients in whom orthodontic treatment was to be initiated with leveling and aligning phase without changing the wire for 5 months after start of treatment.


Exclusion criteria

  1. Patients with gingival pockets of more than 3 mm or alveolar bone loss
  2. Patients with deleterious habits such as pan chewing, gutka chewing, and/or smoking
  3. Patients who were under antibiotic therapy within 3 weeks before sample collection, immunosuppressants, nonsteroidal anti-inflammatory drugs, or radiation therapy, patients with pre-existing gum diseases.


Method of data collection

The samples of subgingival microbial plaque were collected from two sites:

  1. Gingival crevice of the incisors
  2. Mesiobuccal gingival crevice of molars.


Samples were collected from four groups: control group, which was taken before bonding fixed appliance (T0), 1 week (T1), 1 month (T2), and 5 months of placement of fixed appliance (T3) during leveling and aligning phase of orthodontic treatment without changing wire.

Method

Sterile periodontal probe [Figure 1] was gently inserted in gingival crevice of the incisors and mesio-buccal gingival crevice of the molars [Figure 2] to collect the plaque sample making sure not to scrape it. The samples were transported in transport medium and processed in the laboratory within 24 h.
Figure 1: Collection of subgingival microbial samples in incisor region

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Figure 2: Collection of subgingival microbial samples in molar region

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Step 1

Sample collection: samples were collected 1 week before, and after 1 week, 1 month and 5 months of placement of fixed appliance.

Plaque specimen was collected in vial tubes, and coding was done serially based on the group and transported to the department of molecular biology and immunology for PCR analysis. The coding was not revealed to the laboratory staff to minimize experimental bias.

The same procedure was repeated at the completion of 1 week, 1 month, and 5 months to collect plaque samples.

Step 2: Laboratory procedure

DNA extraction, PCR analysis-based 16s RNA was done to evaluate the colonies of A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. nigrescens, and C. rectus as colony forming units (CFU) per milliliter (CFU/ml) in the plaque specimens. Procedure-DNA extraction was done according to the process suggested by Mättö et al.[12],[13]

Statistical analysis

The statistical significance of intragroup comparisons [t0 (baseline) v/s T1, T2 and T3 (post-treatment follow-up comparisons)] was done using repeated measures analysis of variance (RMANOVA). The underlying normality assumption was tested before subjecting the parameters to RMANOVA. The data was entered in MS excel before statistical analysis was done in SPSS (SPSS 11.5, Inc., Chicago, Illinois, USA). The statistical values were presented as mean ± standard error of mean and P < 0.05 are considered to be statistically significant.


  Results Top


A. actinomycetemcomitans organism CFUs at T1, T2, and T3 follow-ups did not differ significantly compared to the average baseline (T0) (P > 0.05 for all). P. gingivalis organism CFUs at T1 follow-up did not differ significantly compared to the baseline (T0) (P > 0.05) but at T2 (0.005) and T3 (0.008) follow-ups differed significantly compared to average baseline (T0) (P < 0.01 for both). T. forsythia organism CFUs at T1 follow-up did not differ significantly compared to the average baseline (T0) CFU (P > 0.05) but CFUs at T2 (0.010) and T3 (0.006) follow-ups differed significantly compared to the average baseline (T0) CFU (P < 0.01 for both) P. nigrescence CFUs at T1 (0.001), T2 (0.039) and T3 (0.001) follow-ups differed significantly compared to the average baseline (T0).C. rectus CFUs at T1 (0.001), T2 (0.001) and T3 (0.001) follow-ups differed significantly compared to the average baseline (T0) (P < 0.001 for all) summarized in [Table 1].
Table 1: The pair-wise statistical comparison of the average colony forming units

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CFU of P. gingivalis and T. forsythia organism showed significant percentage change from baseline T0 to T2 and T3 follow up and in P. nigrescence and C. rectus organism significant percentage change was seen at T1, T2 and T3 follow-ups from baseline T0 summarized in [Table 2].
Table 2: The pair-wise distribution of average colony forming units

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The pair-wise distribution of the average CFU of all organisms showed no significant increase in A. actinomycetemcomitans, but other periodontopathogens such as P. gingivalis, T. forsythia, P. nigrescens, and C. rectus showed a significant increase summarized in [Graph 1].




  Discussion Top


This study analyzed the subgingival plaque microflora from the gingival crevice of the incisors and mesio-buccal gingival crevice of molars at four different time periods, i.e., before bonding of fixed appliances (T0), and 1 week (T1), 1 month (T2), and 5 months after placement of fixed appliances (T3). The gingival crevice of incisors and molars in patients with fixed appliances were probed for sample collection as there is increased prevalence of periodontopathogens in these areas.[4] The specific periodontopathogens were considered in this study, as it has been found to be prevalent in patients with fixed treatment.[7] As PCR method is highly sensitive compared to conventional techniques in detecting anaerobic bacteria it was employed in this study.[12],[13]

The CFU of A. actinomycetemcomitans did not increase significantly in study similar to Demling et al.[14] Some authors report that Orthodontic patients have a very high frequency of A. actinomycetemcomitans.[10] The difference could be due to alterations in the gingival health of patients, ability, and determination to maintain oral hygiene and susceptibility to A. actinomycetemcomitans.

The results showed that P. gingivalis organism CFUs at T1 follow-up did not differ significantly compared to the baseline (T0), but CFUs at T2 and T3 showed a significant increase. Increased incidence of P. gingivalis could be attributed to the fixed appliance or patients age.

In T. forsythia, it was found that there was no sudden change in frequency after the placement of orthodontic appliances but over period of time, it increased significantly at T2 and T3 follow-up, showing it required a longer time to colonize after the placement of orthodontic appliances whereas it appeared C. rectus and P. nigrescens colonized immediately. It was reported T. forsythia is higher in subjects with gingivitis.[9],[13]

In C. rectus and P. nigrescens, it was found that average baseline count differed significantly across the samples (P > 0.05 for all). Which may indicate they are too sensitive to environmental changes than by duration of orthodontic treatment. While other studies had also shown its high prevalence in subjects with gingivitis or periodontitis,[15] there are studies which showed after fixed orthodontic appliance removal periodontol pathogens were significantly reduced within 3 months.[4]

Therefore, to prevent periodontitis, patients must be educated for good oral hygiene maintenance and maximum plaque control after initiation of fixed orthodontic treatment. As the study has shown after placement of orthodontic appliances, there is increase in the prevalence of periodontopathogens which carries a risk of periodontal deterioration. It has been reported that anerobic periodontopathic bacteria showed a negative association with gram-positive species hence due to the absence of antagonist species it may have led to increase in periodontal risk.[7]

The present study showed qualitative comparison of CFU of various organisms, and it concluded that CFU of P. gingivalis and T. forsythia was maximum as compared to P. nigrescence and C. rectus. In comparison, the CFU of A. actinomycetemcomitans was minimum.

Therefore, to control plaque and maintain periodontal condition in fixed treatment, it is necessary to monitor oral hygiene regularly as it may result in accumulation of plaque leading to inflammation and in some cases causing bleeding. Various instruments such as interdental toothbrushes and floss are also available to improve plaque control.[16] Still, further studies are required to effectively control periodontopathic bacteria during fixed orthodontic treatment.


  Conclusion Top


The study concludes that during fixed orthodontic treatment; there was no significant increase in the occurrence of A. actinomycetemcomitans, but other periodontal pathogens such as P. gingivalis, T. forsythia, P. nigrescens, and C. rectus in gingival crevices showed a significant increase after 1 and 5 months of orthodontic treatment thereby heightening the risk of periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001;183:3770-83.  Back to cited text no. 1
    
2.
Andlaw RJ. The relationship between acid production and enamel decalcification in salivary fermentations of carbohydrate foodstuffs. J Dent Res 1960;39:1200-9.  Back to cited text no. 2
    
3.
Bloom RH, Brown LR Jr., A study of the effects of orthodontic appliances on the oral microbial flora. Oral Surg Oral Med Oral Pathol 1964;17:658-67.  Back to cited text no. 3
    
4.
Kim SH, Choi DS, Jang I, Cha BK, Jost-Brinkmann PG, Song JS, et al. Microbiologic changes in subgingival plaque before and during the early period of orthodontic treatment. Angle Orthod 2012;82:254-60.  Back to cited text no. 4
    
5.
Bollen AM, Cunha-Cruz J, Bakko DW, Huang GJ, Hujoel PP. The effects of orthodontic therapy on periodontal health: A systematic review of controlled evidence. J Am Dent Assoc 2008;139:413-22.  Back to cited text no. 5
    
6.
Ren Y, Vissink A. Cytokines in crevicular fluid and orthodontic tooth movement. Eur J Oral Sci 2008;116:89-97.  Back to cited text no. 6
    
7.
Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: Current concepts. J Periodontol 1992;63:322-31.  Back to cited text no. 7
    
8.
Miethke RR, Brauner K. A comparison of the periodontal health of patients during treatment with the invisalign system and with fixed lingual appliances. J Orofac Orthop 2007;68:223-31.  Back to cited text no. 8
    
9.
Socransky SS, Haffajee AD. Dental biofilms: Difficult therapeutic targets. Periodontol 2000 2002;28:12-55.  Back to cited text no. 9
    
10.
Paolantonio M, Festa F, di Placido G, D'Attilio M, Catamo G, Piccolomini R, et al. Site-specific subgingival colonization by Actinobacillus actinomycetemcomitans in orthodontic patients. Am J Orthod Dentofacial Orthop 1999;115:423-8.  Back to cited text no. 10
    
11.
Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986-94.  Back to cited text no. 11
    
12.
Mättö J, Saarela M, Alaluusua S, Oja V, Jousimies-Somer H, Asikainen S, et al. Detection of Porphyromonas gingivalis from saliva by PCR by using a simple sample-processing method. J Clin Microbiol 1998;36:157-60.  Back to cited text no. 12
    
13.
Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol 1996;11:266-73.  Back to cited text no. 13
    
14.
Demling A, Demling C, Schwestka-Polly R, Stiesch M, Heuer W. Short-term influence of lingual orthodontic therapy on microbial parameters and periodontal status. A preliminary study. Angle Orthod 2010;80:480-4.  Back to cited text no. 14
    
15.
Darveau RP, Tanner A, Page RC. The microbial challenge in periodontitis. Periodontol 2000 1997;14:12-32.  Back to cited text no. 15
    
16.
Kossack C, Jost-Brinkmann PG. Plaque and gingivitis reduction in patients undergoing orthodontic treatment with fixed appliances-comparison of toothbrushes and interdental cleaning aids. A 6-month clinical single-blind trial. J Orofac Orthop 2005;66:20-38.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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