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 Table of Contents  
Year : 2017  |  Volume : 51  |  Issue : 3  |  Page : 147-151

Quantitative changes in anaerobic subgingival microbiota in patients, before and during fixed orthodontic treatment

1 Senior Lecturer, Maharaja Gangasingh Dental College, Sri Ganganagar, Rajasthan, India
2 Prof. and HOD, Department of Orthodontics and Dentofacial Orthopedics, Saraswati Dental College, Lucknow, Uttar Pradesh, India
3 Reader, Department of Orthodontics and Dentofacial Orthopedics, Saraswati Dental College, Lucknow, Uttar Pradesh, India

Date of Submission07-Oct-2016
Date of Acceptance04-Apr-2017
Date of Web Publication17-Jul-2017

Correspondence Address:
Amit Sidana
Department of Orthodontics and Dentofacial Orthopedics, Saraswati Dental College, Tiwari Ganj, Lucknow  -  226  016, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jios.jios_197_16

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Aim: The aim of this study was to compare the growth of Actinobacillus actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, and Veillonella before and during fixed orthodontic treatment. Materials and Methods: The study sample consisted of 40 patients (20 males, 20 females). The patients were divided into two groups, Group 1 (pretreatment group) before any fixed orthodontic treatment and Group 2 (active treatment group) 6 months into fixed treatment. A. actinomycetemcomitans was cultured on tryptic soy bacitracin vancomycin agar, and for the rest and total aerobic count, Columbia agar was used. The culture plates were incubated anaerobically for 72 h. Bacteria were identified by their physical and microscopic appearances. Further, specific bacteria were identified by VITEK 2 Compact Automated Identification System (Biomerieux). Using the magnifying glass, the total number of bacteria was determined. Result: For all the microbes, P. gingivalis, P. intermedia, Veillonella (excepting A. actinomycetemcomitans), a significant increase in count was observed. Mean change was highest for P. intermedia (0.64 ± 0.74 × 104 CFU/ml) and minimum for P. gingivalis (0.12 ± 0.35 × 104 CFU/ml). For A. actinomycetemcomitans, at both before and active treatment phases, the count was 0 ± 0 × 104 CFU/ml. For different microbes, change in microbial count ranged from 18.8% (P. gingivalis) to 52% (P. intermedia). For Veillonella, the change was 51.4%. Conclusion: Orthodontic appliance serves as different loci for bacterial growth. In this in vivo study, significant differences were noted between the bacterial count in pretreatment group and active treatment group. Adequate oral prophylaxis instructions should be given to patients before starting fixed orthodontic treatment so that oral hygiene can be maintained.

Keywords: Actinobacillus actinomycetemcomitans, fixed orthodontics, subgingival fluid

How to cite this article:
Sidana A, Tandon R, Srivastava SC. Quantitative changes in anaerobic subgingival microbiota in patients, before and during fixed orthodontic treatment. J Indian Orthod Soc 2017;51:147-51

How to cite this URL:
Sidana A, Tandon R, Srivastava SC. Quantitative changes in anaerobic subgingival microbiota in patients, before and during fixed orthodontic treatment. J Indian Orthod Soc [serial online] 2017 [cited 2019 May 25];51:147-51. Available from: http://www.jios.in/text.asp?2017/51/3/147/210905

  Introduction Top

Orthodontic treatment is associated with some amount of deterioration in periodontal health due to plaque accumulation. This compromised periodontal health status occurs due to increase in pathologic bacteria.[1],[2],[3] Orthodontic treatment makes the process of maintaining oral hygiene more difficult and more time consuming. The bonded appliances have many undercuts which act as sites for food lodgments and plaque colonization. The overall ecology of the oral cavity is modified greatly by increase in these pathologic bacteria.[4],[5] Poor oral hygiene has resulted in enamel decalcification and gingival inflammation. Gingivitis and periodontitis are associated with pathologic microflora. Actinobacillus is an age-related microorganism which is present in the oral cavity.[6],[7],[8],[9] It is highly pathologic and is responsible for causing localized juvenile periodontitis.[10],[11]Tannerella forsythia, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Prevotella intermedia/nigrescens have also been found in patients with inflammation and periodontal pockets as well as in healthy individuals.[12] Literature has shown that there are very limited studies which determine the rise of Actinobacillus in patients wearing fixed orthodontic appliance.[8],[13] Similarly, there are many other anaerobic pathologic bacteria which also increase during fixed orthodontic treatment which have not been investigated in literature. Studies on other pathogens such as Prevotella and P. gingivalis have been scantly reported.[13],[14],[15] To fill this lacuna, the purpose of this study was to quantitatively evaluate the increase in these specific pathogens during orthodontic treatment.

  Materials and Methods Top

Written consent forms were taken from each of the patients after being informed about the nature of the study in detail. The study was approved by the Local Ethical Committee of Saraswati Dental College and Hospital, Lucknow. The sample consisted of 40 patients (20 males and 20 females) who reported to Department of Orthodontics and Dentofacial Orthopaedics, Saraswati Dental College, Lucknow, for treatment.

  • Inclusion criteria:

    • DMFT index should be zero
    • Good oral hygiene
    • No deleterious habits such as smoking and tobacco chewing
    • No oral and systematic diseases or syndromes.

  • Exclusion criteria:

    • Gingivitis, periodontitis, or even mild inflammation
    • Antibiotic intake in the last 6 months
    • Pregnancy, systemic illness, and fluoride use.

The individuals were divided into two groups: Group A (pretreatment group) before any fixed orthodontic treatment and Group B (active treatment group) 6 months of active fixed orthodontic treatment. The samples were numbered 1–40 with respective to Groups A and B. Subgingival fluid samples were taken from the proximal site of the banded molar before and after band placement. Sterile paper point was inserted to the bottom of the periodontal sulcus and kept in place for 20 s and then put in sterile saline. Tryptic soy serum bacitracin vancomycin agar is an enriched selective medium for the isolation and presumptive identification of A. actinomycetemcomitans. TSBV medium contains bacitracin and vancomycin at a concentration that inhibits most Gram-positive and most Gram-negative anaerobes, except for A. actinomycetemcomitans among others.[16] TSBV agar was prepared by adding 3.25 g of tryptic soy agar in 100 ml distilled water. The culture medium was sterilized by autoclaving for 15 min at 121°C under 15 lbs pressure. This hot media was poured in sterilized petri plates under sterilized laminar air flow. After 15 min, the liquid was cooled to room temperature, and 1.2 ml of stock solution of bacitracin and 1 ml of 50 mg/mL stock solution of vancomycin were added and mixed by gently shaking the container. Preformed agar plate of Columbia blood agar was used for total anaerobic culture. The subgingival fluid sample was vortexed for 15–20 s. Adequate amount of subgingival fluid sample was evenly spread onto both culture mediums. Both culture plates were incubated anaerobically in anaerobic jars using gas packs for 72 h. The appearance of small, translucent, and slightly convex circular colonies with a star-like inner structure was seen in case of A. actinomycetemcomitans in the TSBV medium [Figure 1]. Using the magnifying glass, the total number of the colonies of Actinobacillus was calculated. After 3 days, culture plates were examined for evaluating the total anaerobic count [Figure 2] and specific count of three anaerobic bacteria (P. gingivalis, P. intermedia, and Veillonella).
Figure 1: Actinobacillus actinomycetemcomitans culture

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Figure 2: Anaerobic culture

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Specific bacteria were identified by their physical and microscopic appearance. Further, bacteria were identified by VITEK 2 Compact Automated Identification System (Biomerieux) [Figure 3]. The VITEK 2 is an automated microbiology system utilizing growth-based technology. Individual substrates had been measured based on various metabolic activities such as acidification, alkalinization, enzyme hydrolysis, and growth in the presence of inhibitory substances. There are reagent cards available for the identification of different organism classes as follows: GN - Gram-negative fermenting and nonfermenting bacilli, GP - Gram-positive cocci and nonspore-forming bacilli, ANC - Anaerobic card [Figure 3], YST - yeasts and yeast-like organisms, and BCL - Gram-positive spore-forming bacilli. Method of VITEK 2 system was as follows, from anaerobic cultured plate colony was abstracted and subcultured again on anaerobic blood agar. After 3 days, pure culture of specific bacteria was procured. 1–3 colonies were abstracted, and a uniform suspension was prepared in ID tube. Suitable card was placed in respective tubes in barcoded cassettes. At the end, the cassette was loaded in VITEK 2 Compact system which incubates and interprets the results automatically.
Figure 3: VITEK 2 ANC card

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Statistical analysis

The statistical analysis was done using Statistical Package for Social Sciences version 15.0 statistical analysis software (SPSS Inc., IBM, Armonk, New York, US).  The values were represented in number (%) and mean ± standard deviation (SD). All variances are reported as SDs difference among means were analyzed by the paired t-test.

  Results Top

Pretreatment baseline microbial count for P. intermedia was 1.25 ± 1.04 × 104 CFU/ml, for Veillonella was 0.70 ± 0.61 × 104 CFU/ml, and for P. gingivalis was 0.64 ± 0.64 × 104 CFU/ml. A.actinomycetemcomitans was the least common microbe with a mean count of 0.00 ± 0.00 × 104 CFU/ml [Table 1]. Mean anaerobic count was 2.69 ± 1.27 × 105 CFU/ml. In active treatment, microbial count for P. intermedia was 1.89 ± 1.10 × 104 CFU/ml, for Veillonella was 1.06 ± 0.63 × 104 CFU/ml, and for P. gingivalis was 0.76 ± 0.72 × 104 CFU/ml. A. actinomycetemcomitans was the least common microbe with a mean count of 0.00 ± 0.00 × 104 CFU/ml [Table 2]. Total anaerobe count was 2.93 ± 1.33 × 105 CFU/ml. For different microbes, change in microbial count is explained in [Table 3] and [Graph 1 [Additional file 1]]. For anaerobes, the change was higher both for mean count as well as proportional change [Table 4] and [Graph 2 [Additional file 2]].
Table 1: Baseline microbial count in salivary specimen of patients enrolled in the study (n=40)

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Table 2: Microbial count in salivary specimen of patients enrolled in the study during active treatment (n=40)

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Table 3: Comparison of change in count of different microbes from baseline to active phase of treatment

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Table 4: Comparison of change in total microbial count of anaerobes

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  Discussion Top

During active treatment, the important microbial count for the different bacteria had increased except for A. comitans. Same results were seen in a previous study done by Haffajee and Socransky.[12] They reported that important periodontal microorganisms such as P. gingivalis, P. intermedia, and Fusobacterium species were higher after bracket placement. A. actinomycetemcomitans was detected in 7.5% of the total sample in the active treatment group. One of the major reasons for low level of A. actinomycetemcomitans was the presence of increased level of P. gingivalis. A. actinomycetemcomitans and P. gingivalis produce mutual antagonistic products. This result was also seen in the study done by Naranjo et al.[17] They found statistically insignificant findings regarding A. actinomycetemcomitans. Paolantonio et al.[15] did a cross-sectional study on 34 individuals; in which, 20 individuals (test group) underwent orthodontic treatment with fixed appliances for at least 6 months. 85% of test individuals and 47.5% of test sites were positive for A. actinomycetemcomitans in comparison to 28.5% of control individuals and 3.5% of control sites. Paolantonio et al.[18] did longitudinal study and obtained the same result as in cross-sectional study. A. actinomycetemcomitans as a possible pathogen in localized juvenile periodontitis and P. gingivalis were suggested to be important in adult periodontitis. Asikainen et al.[19] isolated the A. actinomycetemcomitans from 2% of sites from periodontally healthy teenagers.

In this study, we compared the subgingival microbiota composition in patients who were scheduled for orthodontic treatment before and after placement of brackets. After 6 months, the change in proportion of anaerobic bacteria is statistically significant. These changes could be observed within 3 months after placement of appliances, which is in accordance with other reports discuss the effect of orthodontic treatment on the periodontium.[20],[21] The diversity in subgingival microbiota described using a microbial cultural technique in patients having orthodontic treatment. Other method to this study particularly found important microorganism that is related to inflammation and periodontal diseases grouped into complex according to their colonization capacities and virulence.

In patients undergoing orthodontic treatment, their periodontal condition should be supervised carefully. Removable and fixed orthodontic appliances block correct oral hygiene resulting in more plaque accumulation, inflammation, and bleeding. To inhibit the environment which is favorable for bacterial growth, appropriate oral hygiene method and instruments should be used. Powered toothbrush, mouthwashes, and floss have been shown to improve control in the orthodontic patient.[22] Choi et al.[23] did a study to evaluate changes that occur in the subgingival microbiota after removal of fixed orthodontic appliances. They concluded that periodontopathogens during orthodontic treatment were significantly reduced within 3 months of appliance removal. A reduction of all bacterial counts was detected after the 3-month follow-up in all groups. Lower counts of P. gingivalis were achieved from 1 week after treatments. The 2% chlorhexidine concentration seemed to contribute to lower A. actinomycetemcomitans levels and increase Streptococcus mutans levels.[24]

  Conclusion Top

  • Maximum increase in number was seen for P. intermedia and least for P. gingivalis
  • For different microbes, change in microbial count ranged from 18.8% (P. gingivalis) to 52% (P. intermedia). For Veillonella, the change was 51.4%
  • For anaerobes, the change was higher both for mean count as well as proportional change before and during treatment.

It can be concluded that orthodontic band placement can influence the aggregation and content of subgingival microbes, resulting in inflammation, bleeding, and periodontal damage. Hence, periodontal disease occurring during active orthodontic treatment should be prevented by giving patient special oral hygiene care.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Zachrisson S, Zachrisson BU. Gingival condition associated with orthodontic treatment. Angle Orthod 1972;42:26-34.  Back to cited text no. 1
Zachrisson BU, Alnaes L. Periodontal condition in orthodontically treated and untreated individuals. I. Loss of attachment, gingival pocket depth and clinical crown height. Angle Orthod 1973;43:402-11.  Back to cited text no. 2
Zachrisson BU. Cause and prevention of injuries to teeth and supporting structures during orthodontic treatment. Am J Orthod 1976;69:285-300.  Back to cited text no. 3
Diamanti-Kipioti A, Gusberti FA, Lang NP. Clinical and microbiological effects of fixed orthodontic appliances. J Clin Periodontol 1987;14:326-33.  Back to cited text no. 4
Huser MC, Baehni PC, Lang R. Effects of orthodontic bands on microbiologic and clinical parameters. Am J Orthod Dentofacial Orthop 1990;97:213-8.  Back to cited text no. 5
Slots J. Microflora in the healthy gingival sulcus in man. Scand J Dent Res 1977;85:247-54.  Back to cited text no. 6
Slots J. The predominant cultivable microflora of advanced periodontitis. Scand J Dent Res 1977;79:105-12.  Back to cited text no. 7
Slots J, Feik D, Rams TE. Actinobacillus actinomycetemcomitans and bacteroides intermedius in human periodontitis: Age relationship and mutual association. J Clin Periodontol 1990;17:659-62.  Back to cited text no. 8
Savitt ED, Kent RL. Distribution of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by subject age. J Periodontol 1991;62:490-4.  Back to cited text no. 9
Mandell RL, Socransky SS. A selective medium for Actinobacillus actinomycetemcomitans and the incidence of the organism in juvenile periodontitis. J Periodontol 1981;52:593-8.  Back to cited text no. 10
Zambon JJ, Christersson LA, Slots J. Actinobacillus actinomycetemcomitans in human periodontal disease. Prevalence in patient groups and distribution of biotypes and serotypes within families. J Periodontol 1983;54:707-11.  Back to cited text no. 11
Haffajee AD, Socransky SS. Microbial etiological agents of destructive periodontal diseases. Periodontol 2000 1994;5:78-111.  Back to cited text no. 12
Paolantonio M, Pedrazzoli V, di Murro C, di Placido G, Picciani C, Catamo G, et al. Clinical significance of Actinobacillus actinomycetemcomitans in young individuals during orthodontic treatment. A 3-year longitudinal study. J Clin Periodontol 1997;24(9 Pt 1):610-7.  Back to cited text no. 13
Wolff LF, Liljemark WF, Bloomquist CG, Pihlstrom BL, Schaffer EM, Bandt CL. The distribution of Actinobacillus actinomycetemcomitans in human plaque. J Periodontal Res 1985;20:237-50.  Back to cited text no. 14
Paolantonio M, di Girolamo G, Pedrazzoli V, di Murro C, Picciani C, Catamo G, et al. Occurrence of Actinobacillus actinomycetemcomitans in patients wearing orthodontic appliances. A cross-sectional study. J Clin Periodontol 1996;23:112-8.  Back to cited text no. 15
Dowell VR Jr., Hawkins TM. Laboratory Methods in Anaerobic Bacteriology: CDC Laboratory Manual. Atlanta, GA: USDHEW, CDC; 1974.  Back to cited text no. 16
Naranjo AA, Triviño ML, Jaramillo A, Betancourth M, Botero JE. Changes in the subgingival microbiota and periodontal parameters before and 3 months after bracket placement. Am J Orthod Dentofacial Orthop 2006;130:275.e17-22.  Back to cited text no. 17
Paolantonio M, Festa F, di Placido G, D'Attilio M, Catamo G, Piccolomini R. Site-specific subgingival colonization by Actinobacillus actinomycetemcomitans in orthodontic patients. Am J Orthod Dentofacial Orthop 1999;115:423-8.  Back to cited text no. 18
Asikainen S, Alaluusua S, Kari K, Kleemola-Kujala E. Subgingival microflora and periodontal conditions in healthy teenagers. J Periodontol 1986;57:505-9.  Back to cited text no. 19
Kloehn JS, Pfeifer JS. The effect of orthodontic treatment on the periodontium. Angle Orthod 1974;44:127-34.  Back to cited text no. 20
Hamp SE, Lundström F, Nyman S. Periodontal conditions in adolescents subjected to multiband orthodontic treatment with controlled oral hygiene. Eur J Orthod 1982;4:77-86.  Back to cited text no. 21
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. 22
Choi DS, Cha BK, Jost-Brinkmann PG, Lee SY, Chang BS, Jang I, et al. Microbiologic changes in subgingival plaque after removal of fixed orthodontic appliances. Angle Orthod 2009;79:1149-55.  Back to cited text no. 23
Silva TC, Valarelli TM, Sakai VT, Tessarolli V, Machado MA. Oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children. Braz J Oral Sci 2013;12:132-7.  Back to cited text no. 24


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4]


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