Introduction
The objectives of the study were to evaluate and compare the effects of the systemic consumption of probiotic curd and the topical application of probiotic toothpaste on the Streptococcus mutans levels in the plaque of orthodontic patients.
Methods
The study consisted of 60 orthodontic patients divided into 3 groups of 20 each. Group 1 was the control group. The patients in group 2 were given probiotic curd, and those in group 3 were asked to brush twice daily with probiotic toothpaste (GD toothpaste; Dental Asia Manufacturing, Shah Alam, Selangor, Malaysia). Samples were collected at 2 times: before the study began and after 30 days. Plaque specimens were collected from the labial surfaces immediately surrounding the orthodontic brackets of the maxillary lateral incisors using a 4-pass technique. The presence of S mutans was evaluated using real-time polymerase chain reaction. Statistical analysis was performed, and comparisons were made using a 2-tailed chi-square test for categorical data ( P <0.05).
Results
At the end of the study, there were reductions in S mutans concentration in groups 2 and 3 that were statistically significant compared with group 1, but there was no statistically significant difference between groups 2 and 3.
Conclusions
The consumption of probiotic curd and the use of probiotic toothpaste cause a significant decrease in the S mutans levels in the plaque around brackets in orthodontic patients. Although the probiotic toothpaste was more effective than systemic consumption, this was not statistically significant.
Advances in orthodontics have improved the quality of appliances and treatment protocols, raising the standard of patient care. However, enamel demineralization is still a problem associated with orthodontic treatment, leading to the formation of white spot lesions; this is a grave concern to orthodontists and patients. The overall prevalence of white spot lesions among orthodontic patients has been reported to be between 4.9% and 84%. A white spot lesion is the precursor of enamel caries. White spot lesions develop as a result of an interrupted process with periods of remineralization and demineralization. When basic oral hygiene is poor, orthodontic appliances create areas of plaque stagnation, especially around brackets, bands, wires, and other attachments; this facilitates the development of white spot lesions. Levels of acidogenic bacteria, present in the plaque, notably Streptococcus mutans , are higher in orthodontic patients than in nonorthodontic patients. This causes demineralization around the brackets and leads to white spot lesions. They are most prevalent around the cervical region of bands in the posterior region, whereas in the anterior region, the lateral incisors in both arches, followed by the canines, are most commonly affected.
Various methods have been suggested to inhibit or reverse enamel demineralization. Fluoride delivery systems, casein phosphopeptide amorphous calcium phosphate, and enamel surface attenuation with an argon laser have proved to be useful. Continuous fluoride release from fluoride-containing sealants, elastomeric chains, primers, and adhesives in bonding brackets is also useful. Often, the application of fluoride not only requires frequent visits to the dentist but also causes discoloration of the teeth, leading to decreased esthetics.
Biological methods such as antibiotics, antimicrobial therapy with chlorhexidine, povidone iodine, fluoride, and penicillin have gained importance in recent years. The application of broad-spectrum antibiotics and antimicrobial therapy can suppress the caries infection but never totally eliminate it. None of these medicaments has been able to successfully preclude the regrowth of residual pathogens or reinfection from external sources; this means that antibiotic and antimicrobial therapies must be given at regular intervals for effective long-term results.
At the turn of the 20th century, Elie Metchnichkoff, a Nobel Prize-winning Russian, made the revolutionary discovery of probiotics . Probiotics are “live microbial food supplements which beneficially affect the host animal by improving its intestinal microbial balance.” Lactic acid bacteria and bifidobacteria are the most common types of microbes used as probiotics, but certain yeasts and bacilli can also be helpful. They act by competitively inhibiting the pathogenic bacteria because they have greater adhesion to the tissues. They inhibit pathogens but do not inhibit friendly bacteria. Studies have shown that once the pathogenic organisms are replaced the reintroduction of the pathogen does not occur easily.
Probiotics are commonly consumed as part of the diet in several cultures in the form of fermented foods such as yogurt and soy yogurt, or as dietary supplements with added active live cultures. They have proved to be beneficial in treating malnourishment, lactose intolerance, calcium availability, bowel problems such as constipation, urogenital infections, and atopic diseases such as antibiotic-induced diarrhoea, and in improving the immune system, alleviating chronic intestinal inflammatory diseases, and preventing and treating pathogen-induced diarrhea.
A few studies have evaluated the effects of local administration of probiotic agents such as mouthwashes, lozenges, tablets, straws, milk, cheese, ice cream, chewing gums, yogurt, and other supplements and have found that these have a beneficial effect on oral health. The benefits on oral health in preventing gingivitis, halitosis, and caries have been recognized, and thus probiotics have been incorporated into mouthwashes and dentifrices for popular consumption. Some studies have established that the level of S mutans in saliva is reduced after the use of probiotics; this would be beneficial in orthodontic patients also.
However, there are few studies in the literature on the effects of probiotics in orthodontic patients, since their use in our speciality is still in an infantile stage. S mutans concentration in plaque would be more representative of the caries-inducing potential in the anterior teeth where salivary clearance is less effective. Since the localized effect of probiotics on the plaque surrounding orthodontic brackets has not been studied, we conceived this study to evaluate whether probiotic systems are beneficial to orthodontic patients. It is desirable to establish which delivery system is more efficient, and thus this study was designed to compare the efficacy of systemic ingestion and topical applications.
Our aims were to evaluate and compare the effect of probiotic systems (systemic and local) on the S mutans levels in the plaque surrounding brackets in orthodontic patients.
Material and methods
The study was double blinded and randomized, consisting of 60 randomly selected patients having orthodontic treatment in the Department of Orthodontics and Dentofacial Orthopaedics at Sri Ramachandra University in India. The following enrollment criteria were used: (1) orthodontic treatment with the straight wire appliance (MBT, 0.022-in slot; 3M Unitek, Monrovia, Calif), (2) permanent dentition, (3) good general health (no significant medical history or drug use during the last month), (4) no anti-inflammatory or antibiotic medications taken in the month before the study, (5) no chewing gum or mouthwash used in the last week and during the study, (6) habit of brushing twice daily with fluoride toothpaste, and (7) age between 14 and 29 years (average, 20 years).
All subjects had good oral health with no open or untreated caries lesions or gingival inflammation, and they claimed to have daily tooth brushing habits. There were 42 female and 18 male subjects. They were divided into 3 groups of 20 each. Group 1 consisted of patients who received no probiotic treatment (control group). The patients in group 2 were given 200 mg of probiotic curd (Active Plus; Nestle, Chennai, India), instructed to eat it with their lunch for 30 days, and asked to brush twice daily with their regular fluoride toothpaste (Colgate Strong Teeth; Colgate-Palmolive Ltd, Solan, Himachal Pradesh, India). The patients in group 3 were asked to brush twice daily with probiotic toothpaste (GD; Dental Asia Manufacturing, Shah Alam, Selangor, Malaysia) only for 30 days and to discontinue using their normal toothpaste. The patients were asked to brush with an up-and-down motion on the front teeth and a circular motion on the back teeth for 2 minutes; this was demonstrated by the same operator (J.E.J.).
The patients were instructed to avoid chewing gums, mouthwashes, and antibiotics during the study. Samples were collected at 2 times: before the study began and after 30 days.
At each time interval, the elastomeric modules (Ormco, Orange, Calif) were carefully removed to disengage the archwires by the same operator. Plaque specimens were collected from the labial surfaces immediately surrounding the orthodontic brackets of the maxillary lateral incisors with a sterilized scaler using a 4-pass technique as suggested by Pellegrini et al. Four passes, each along the tooth at the bracket interface at the gingival, mesial, distal, and occlusal aspects, were used to prevent overloading the instrument tip.
The samples were placed into individual micropipette tubes with anonymous coding and sealed for transport for DNA isolation to the Medox Biotech India laboratory in Chennai, India. The coding of the specimens was not disclosed to the laboratory personnel and helped to minimize experimental bias.
The ultrapure genomic DNA Spin Miniprep (Medox Biotech India) kit was used for fast isolation of genomic DNA. The quality of DNA thus obtained was measured using a nano-drop technique in the Department of Biomedicine of Sri Ramachandra University. The primers and probes specific for S mutans were manufactured by Bangalore Genei (Shushruti Nagar, Bangalore, India). The oligonucleotide primers used were Sm F5 5′-AGC CAT GCG CAA TCA ACA GGT T and Sm R4 5′CGC AAC GCG AAC ATC TTG ATC AG. The samples were then taken to the Central Research Foundation, Sri Ramachandra University, where the real-time polymerase chain reaction was done (model 7900HT; Applied Biosystems, Invitrogen BioServices India Pvt Ltd, Whitefield, Bangalore, India) using SYBR green assay for relative quantification of the bacteria in the samples.
The polymerase chain reaction values were obtained in the form of a graph that was interpreted with the software from Applied Biosystems. The values were tabulated, and the statistical analysis was performed with SPSS software (version 2; SPSS, Chicago, Ill). Comparisons of the reduction in S mutans in the samples before the study and after 30 days, and also comparisons among the 3 groups, were done using a 2-tailed chi-square test for categorical data. P <0.05 was considered statistically significant.
In a real-time polymerase chain reaction assay, a positive reaction is detected by accumulation of a fluorescent signal. The cycle threshold is defined as the number of cycles required for the fluorescent signal to cross the threshold. Real-time assays undergo 40 cycles of amplification. If the sample does not reach this level even after 40 cycles, then it does not contain the organism and is called “undetermined.”
The real-time polymerase chain reaction shows the relative quantification of the bacteria in the samples. The quantification is done by evaluating the cycle threshold values, which are the threshold values at which there is expression of the bacterial genome. The cycle threshold value is inversely proportional to the amount of bacterial genome present. An increase in cycle threshold values indicates a decrease in bacteria, and a decrease in cycle threshold values indicates an increase in bacteria.
Results
Before the study began, 16 samples (80%) showed genomic expression in group 1, 15 samples (75%) showed genomic expression in group 2, and 14 samples (70%) showed genomic expression in group 3 ( Table I ).
There was no statistically significant difference in the S mutans concentrations among the 3 groups before the study ( P >0.05).
After 30 days, 15 samples (93.75%) showed genomic expression in group 1, 9 samples (60%) showed genomic expression in group 2, and 8 samples (57%) showed genomic expression in group 3. Five samples in group 1, 11samples in group 2, and 12 samples in group 3 were genetically indeterminate ( Table I ) (calculated by cycle threshold values greater than 40).
In group 1, 18% (3 of 16) of the samples showed a reduction in S mutans concentration, whereas in groups 2 and 3, all samples (15 of 15, and 14 of 14, respectively) showed reductions in S mutans concentrations (calculated by the increase in cycle threshold values). The samples with increases in the cycle threshold values after 30 days would have reductions in S mutans .
This showed no statistically significant reduction in S mutans in group 1, but there were statistically significant reductions in groups 2 and 3 ( Tables I and II ).
