|Year : 2016 | Volume
| Issue : 1 | Page : 3-9
Effect of fixed space maintainers and removable appliances on oral microflora in children: An in vivo study
Ritesh Kundu1, Abhay M Tripathi1, Jagdish Narain Jaiswal1, Ujjala Ghoshal2, Madhuchanda Palit1, Sonali Khanduja1
1 Department of Pedodontics and Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Web Publication||2-Feb-2016|
Department of Pedodontics and Preventive Dentistry,Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Raibarelly Road, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Oral habits and caries if left untreated may result in crowding and arch length discrepancy in developing dentition. Therefore, appliances are used to maintain the arch length and for proper relationship between dental arches. However, its insertion may cause an increase in bacterial concentration. Aim: This study aimed to evaluate the growth of Streptococcus mutans, Lactobacillus sp., and Candida albicans in saliva during the first 6 months of orthodontic therapy. Materials and Methods: Twenty children in the age group of 6-15 years were selected with regard to indication of orthodontic treatment, and subsequently grouped as patients to be treated with fixed space maintainers or removable appliances. Unstimulated saliva was collected in a sterile container at baseline and at 1-month, 3-month, and 6-month recalls for every selected child. Samples collected were processed for bacterial culture in different culture media at different time intervals. Statistical Analysis: SPSS (Statistical Package for Social Sciences) Version 15.0 statistical software was carried for bacterial counts. Chi-square test and t-test were performed to know the effects of each variable and to reveal the statistical significance. Result: Bacterial counts of Streptococcus mutans, Lactobacillus sp., and Candida albicans were found to be statistically significant (P 0≤ 0.001), (P < 0.05), and (P < 0.001), respectively in both the groups at all intervals. Conclusion: At different time intervals, the total numbers of bacterial count of Streptococcus mutans were comparatively higher, followed by Lactobacillus sp. and Candida albicans.
Keywords: Microbiological analysis, Orthodontic appliances, Streptococcus mutans
|How to cite this article:|
Kundu R, Tripathi AM, Jaiswal JN, Ghoshal U, Palit M, Khanduja S. Effect of fixed space maintainers and removable appliances on oral microflora in children: An in vivo study. J Indian Soc Pedod Prev Dent 2016;34:3-9
|How to cite this URL:|
Kundu R, Tripathi AM, Jaiswal JN, Ghoshal U, Palit M, Khanduja S. Effect of fixed space maintainers and removable appliances on oral microflora in children: An in vivo study. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2020 May 31];34:3-9. Available from: http://www.jisppd.com/text.asp?2016/34/1/3/175498
| Introduction|| |
Oral habits as well as early loss of primary teeth due to caries, if left untreated, may be the main reason of malocclusion in permanent dentition, which may result in crowding and arch length discrepancy in developing dentition.  Therefore, removable and fixed orthodontic treatment and space maintainers are used to maintain the arch length and to promote a proper relationship between dental arches.  However, often these orthodontic treatments may cause plaque accumulation and gingival inflammation, if oral hygiene is not properly maintained. ,, The insertion of these appliances may cause an increase in bacterial concentration, decrease in the buffer capacity, pH, and salivary flow rate in the oral environment. 
It has been seen that Streptococcus mutans has been associated in particular with early demineralization, while lactobacilli are implicated more with lesion progression and cavitation. Thus, high salivary counts seemed to reflect conditions that enhanced the risk of colonization of Streptococcus mutans.  Nevertheless, the changes observed in the bacterial count between pretreatment and during treatment vary among studies.  In contrast to the findings reporting an increase in Streptococcus mutans count during orthodontic therapy, some investigators found no change.  Likewise, there has been disharmony between studies indicating an elevation in Lactobacillus sp. levels in some and no difference being revealed after the insertion of orthodontic appliances in some others.  The inconsistencies between these findings remain unclear and should be studied further. 
In recent years, there has been an increasing interest in the relationship between oral fungal flora and dental caries, which has resulted in a volume of research data that have focused attention on the possible direct role of Candida albicans in the caries etiology of healthy subjects. However, the number of similar studies remains limited in children.  Hence, there is a need to examine the prevalence of oral fungal species in the oral mucous membrane of children with dental caries.  Multiple studies about the effect of orthodontic appliances on oral microbiota have been well established, but very few studies are available about the effect of microorganisms on removable appliances and fixed space maintainers worn by children of mixed dentition age. 
Hence, the aim of the present study was to evaluate the growth of Streptococcus mutans, Lactobacillus sp., and Candida albicans in saliva during the first 6 months of orthodontic therapy.
| Materials and Methods|| |
The present study was conducted at the Department of Pedodontics and Preventive Dentistry at Sardar Patel Post Graduate Institute of Dental and Medical Sciences in collaboration with the Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, (SGPGI) Lucknow.
Twenty children in the age group of 6-15 years were selected. Subjects were examined intraorally and radiologically with regard to indication of treatment and subsequently grouped as patients to be treated with removable appliances (Group I) or fixed space maintainers (Group II). Before the insertion of appliances, all participants received oral hygiene instructions and guidance on proper tooth brushing techniques. All participants' oral hygiene was considered to be ideal at the beginning of the therapy based on Decayed Missing Filling Teeth Index and Simplified Oral Hygiene Tndex.
Signed consent forms were collected from parents prior to the commencement of the study, followed by clearance from the ethical committee. Previously, a pilot study was carried out in the same department for an overview of the proper study design and to take care of possible constraints during the main study.
Children were selected on the basis of:
- Those who needed treatment, depending on.
- Prolonged oral habits causing malocclusion.
- Early loss of primary teeth leading to space loss and malocclusion.
- Having improper arch length and improper relationship between the dental arches.
- Those who were free of systemic diseases.
Patients were excluded if:
- They had used any kind of medication or antibacterial agents such as mouth rinse and/or steroids during the last three months.
Patient were withdrawn if:
- They had migrated and did not turn up.
- They got ill and started taking antibiotics.
- They were uncooperative and discontinued wearing the appliances.
- They had any kind of accidental trauma because of which they could not wear the appliance.
For every selected child, unstimulated saliva was collected in a sterile container in the midafternoon at the baseline and at 1-month, 3-month, and 6-month recalls. Participants were instructed to swallow the residual saliva present in the mouth before the beginning of collection, and then, with the head down at 45 degrees of flexion, with one hand holding the sterile container and mouth slightly open, saliva was allowed to drip from the lower lip into the sterile container.
For microbiological analysis, samples collected were immediately transported to SGPGI and processed within 30 min of collection. Each saliva sample of 1 mL was diluted with 9 mL of sterile saline in the ratio 1: 9 and was serially diluted to 10−5 and 10−6 dilution before plating. Diluted sample in 0.1 mL was spread evenly over Mueller Hinton Agar (MHA), Man-Rogosa-Sharpe Agar (MRS), and Sabouraud dextrose agar (SDA) with the help of a cotton swab to ensure an even distribution of the sample.
For Streptococcus mutan sand Lactobacillus sp.: MHA and MRS culture plates were inserted into the Anoxometer (Anoxomate Type A and 2CTS Mart Microbiology BV, Holand) and were incubated anaerobically (with 5% CO 2 ) for 48 h at 37°C in the incubator.
For Candida albicans: The SDA plates were incubated aerobically at 37°C for 48-72 h in the incubator.
Growth was observed on the plates and bacterial colonies were observed under a microscope and deduced on the basis of colony morphology, shape, and color. The number of colony-forming units (CFU) were counted using digital colony counter and transformed to CFU/sample by multiplying it with the dilution factor.
Statistical analysis was carried out using SPSS (Statistical Package for Social Sciences) Version 15.0 statistical software (IBM Corporation 2009) on bacterial counts, and was expressed as values of logarithm 10 (log 10 ) CFU). All the data were presented in tabular and bar diagram forms. Analysis with chi-square test, Student's t-test, and paired t-test was performed to know the effects of each variable and to reveal the statistical significance. The statistical comparison of pre- and posttreatment bacteria counts in saliva was done using the paired t-test. The confidence level of the study was kept at 95%, hence a P value <0.05 has been considered significant, P value <0.01 has been considered highly significant, and P value <0.001 has been considered very highly significant.
| Results|| |
In both the groups, there was seen an incremental trend in the number of bacterial counts on Streptococcus mutans and Lactobacillus sp. from baseline to follow-up visits at 1 month, 3 months, and 6 months, which was found to be statistically significant; (P ≤ 0.001) and (P < 0.05), respectively [Figure 1] and [Figure 2] and [Table 1] and [Table 2].
|Table 1: Intragroup change of Streptococcus mutans at different time intervals (paired t-test)|
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|Table 2: Intragroup change of Lactobacillus sp.at different time intervals (paired t-test)|
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|Figure 1: (a) Growth of Streptococcus mutans at 1 month (b) Growth of Streptococcus mutans at 3 months (c) Growth of Streptococcus mutans at 6 months|
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|Figure 2: (a) Growth of Lactobacillus sp. at 1 month (b) Growth of Lactobacillus sp. at 3 months (c) Growth of Lactobacillus sp. at 6 months|
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In both the groups, an incremental change in the number of bacterial counts on Candida albicans (SDA) from baseline to 1 month, 3 months, and 6 months was observed, with a sudden increase in the number of candidal counts between baseline to the end of the first month, which was found to be statistically significant (P < 0.001) between any two observations [Figure 3] and [Table 3].
|Table 3: Intragroup change of Candida albicans at different time intervals (paired t-test)|
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|Figure 3: (a) Growth of Candida albicans at 1 month (b) Growth of Candida albicans at 3 months|
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The difference in bacterial counts of the above microorganisms at all intervals was found to be statistically significant. Bacterial counts in MHA for Streptococcus mutans were found to be significantly higher than that of Lactobacillus sp. and Candida albicans in MRS and SDA, respectively, at all time intervals [Graph 1[Additional file 1]] and [Table 4].
|Table 4: Comparison of number of bacterial counts in different culture media at different time intervals|
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| Discussion|| |
The introduction of orthodontic appliances into the mouth greatly inhibits oral hygiene and increases the number of retentive areas, leading to an increase in plaque and biofilm formation. This in turn predisposes to increased carriage of microbes and progression of a gingival reaction that might result in increased periodontal involvement and damage, and subsequent infection. ,,,
As the plaque in orthodontic patient increases, there is also a greater concentration of bacteria and carbohydrate in each milligram of plaque.  Increased amounts of plaque and elevated levels of Streptococcus mutans and of Lactobacilli sp. are detected in the oral cavity after bonding orthodontic attachments. , These are identified as the main pathogens in dental caries, and their presence increases the risk of decalcification. 
In order to evaluate the growth of these microorganisms, 20 children in the age group of 6-15 years were included in the study. The reason for selecting children in this age group was because any preventive and interceptive orthodontic treatment should be addressed at the early mixed dentition stage (The American Academy of Pediatric Dentistry guidelines 2009). 
Unstimulated saliva of children of either gender as per the inclusion and exclusion criteria of the patients was collected in a sterile container in the midafternoon at the baseline and at 1-month, 3-month, and 6-month recalls. Saliva collection was done in the midafternoon according to Wu et al. (2008), who suggested that the unstimulated flow rate fluctuates with the circadian cycle and is at its maximum in the midafternoon.  The reason for considering the unstimulated whole saliva in the present study is that the test is simple and cost-effective. Furthermore, the extended use of unstimulated whole saliva may even lead to the earlier detection of hyposalivation in children and thereby in many cases may help in early detection of the underlying diseases. 
Questions concerning the reliability of saliva over plaque or oral swab samples can even be raised. Saliva was used as one of the sites for sample collection in this study as it is a fluid where all the other intraoral sites are immersed and thus contains dislodged oral microorganisms from various intraoral sites [Marcotteand Lavoie (1998)]. 
Chiappin (2007) stated that saliva specimens, after collection, should preferably be kept on ice, aliquoted, and frozen as soon as possible to maintain the sample integrity, as refrigeration prevents the degradation of some molecules in saliva and prevents further bacterial growth. Thus, to avoid degradation of salivary compounds, it has been proposed that specimens can often be stored at room temperature and carried immediately or within 30-90 min from collection for its analysis.  Hence, for microbiological analysis, samples collected were transported to the laboratory and processed within 30 min of collection.
Each saliva sample was serially diluted with 9 mL of sterile saline to tenfold dilution of 10 -6 before plating, and 0.1 mL of the diluted sample was plated on respective agar plates by sterile cotton swab in front of a bunsen burner inside a laminar air flow unit to ensure an even distribution of the sample. The samples were chosen to be withdrawn from the sixth dilution (10−6 ) because a readable count of bacteria can be seen on the agar surface at this dilution, as determined by the pilot study. Laminar air flow unit was used in the study as it is the most efficient operating room ventilating system by which air contamination can be reduced with an efficient ventilation system. 
The plates were sealed, placed in an anaerobic jar with anoxometer system in a microaerophilic environment, and incubated at 37°C for 48 h for Streptococcus mutans and Lactobacillus sp. and 72 h aerobically for Candida albicans, which is in accordance with Nikawa et al. (1998), who stated that the colonization of Streptococcus mutans isolates reaches its maximum after 48-h incubation and then declines, irrespective of the presence of saliva or serum. In contrast, the colonization of each isolate of Candida albicans gradually increases and plateaus after 72-h incubation. 
Bacterial colonies were observed under a light microscope and deduced under 1000× magnification on the basis of colony morphology, shape, and color; in case of doubt, confirmation for Streptococcus mutans and lactobacilli was done by Gram staining or catalase tests, respectively.
Colony counting was done under a digital colony counter by manually counting the colonies on plates illuminated by transmitted light, and the number of CFUs were multiplied by the number of times the original milliliter of sample was diluted (the dilution factor of the plate counted), which was expressed as the CFU/mL of saliva.
The present study suggested that the microscopic counts of Streptococcus mutans and Lactobacillus sp. increased significantly 6 months after the insertion of fixed space maintainers or removable appliances. This could be attributed to the fact that orthodontic treatment may affect the oral microbiota equilibrium: bands, brackets, wires, and acrylic resins increase the risk of retention of food particles and microorganisms. ,,
Minimal change was also observed between baseline and the third month and maximum change was observed between baseline and at the end of the sixth month for Streptococcus mutans and Lactobacillus sp. This could be related to the fact that initially the patients were very well motivated, received oral hygiene instructions, and maintained their oral hygiene using fluoride toothpaste. The recall periods were also shorter, which might have encouraged the patients to apply oral hygiene instructions routinely, therefore no changes in the bacteriological counts were observed. ,,
The present study stated that maximum growth was observed with Streptococcus mutans, followed by Lactobacillus sp. This could be related to the fact that lactobacilli are strongly associated with progression of dental caries, while streptococci are mainly associated with the onset of caries [Sanpei et al. (2010)].  Basaran et al. (2006) have even stated that Streptococcus mutans has been associated in particular with early demineralization, while Lactobacilli sp. are implicated more with lesion progression and cavitation. 
Kupietzky et al. (2005) stated that brackets similar to the proximal surfaces of teeth or margins of restorations may act as a substratum on which the pellicle formation may build allowing the attachments of Lactobacillus species. The lactobacilli usually takes advantage of pioneer bacteria's lattice networks, which is a form of mechanical retention and source of nutrients.  Chang et al. (1999) even stated that lactobacilli do not play a significant role in the initiation of caries.  Therefore it may be speculated that lactobacilli increased significantly as secondary invaders in the development of dental caries after the appliance placement following the initiation of Streptococcus mutans.
The present study revealed a change in the number of bacterial counts on Candida albicans (SDA) from baseline till the end of 6 months and there was an incremental trend in number of bacterial counts, which was found to be statistically significant. Recent studies have revealed that any appliances in the mouth seem to alter the microbiological environment by providing a suitable adherence surface for Candida.  This is in accordance with Pithon et al. (2012), where the authors stated that an acrylic structure may influence the proliferation of microorganisms, when it acts as a food deposit, depending on its size and smoothness.  Darwazeh et al. (2003) and Budtz-Jorgensen and Bertram (1970) also stated that an increased prevalence of oral candidal carriage after the wearing of removable appliances, which includes an acrylic plate, may be explained partly by the affinity of Candida species to plastic polymers. ,
A similar study was performed by Arendorf et al. (1985) where the authors found an apparent increased prevalence of candidal carriage in denture-wearing individuals, which may be accounted for by the highly significant increase in the density of Candida at mucosal sites. Nevertheless, the importance of a possible occlusive or protective effect of an appliance to the growth of Candida was also clearly demonstrated, with palatal sites showing the highest predilection for candidal recovery and almost the highest density. Under normal circumstances, the posterior of the tongue is the most prevalent and colonized site [Arendorf and Walker (1980)], perhaps because of the filiform papillae, which may favor the increased growth of Candida.
The present study revealed a sudden increase in the number of candidal counts between baseline and the end of the first month, and has shown an incremental growth thereafter. This could be attributed to the fact that a varying degree of gum inflammation and mucosal damage was seen within the first month of insertion of removable and fixed appliances, which could have decreased the local defense mechanism of the oral cavity. 
Among the three microorganisms evaluated the present study revealed that Candida albicans had the least growth. The difference between the microbial counts may be due to variations in the sampling techniques. The ideal sampling technique to ascertain overall candidal carriage is the oral rinse technique, whereas in the present study, saliva samples were considered.  Pathak et al. (2013) even stated that a low prevalence of Candida may be due to consideration of healthy individuals for the study, which is in accordance with the present study. 
| Conclusion|| |
The present study indicates that both favorable (salivary) and unfavorable (microbial) changes are elicited by the placement of orthodontic appliances. Further studies are also required to determine whether the trends seen in the present study occur throughout the duration of fixed appliances treatment, or reverse upon the removal of the appliances. Levels of cariogenic pathogens should be constantly reduced during orthodontic treatment by preventive measures such as oral hygiene maintenance and reducing the consumption of sugar.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]