|Year : 2020 | Volume
| Issue : 2 | Page : 40-46
Impact of Different Bedtime Oral Cleaning Methods on Dental-Damaging Microbiota Levels
Pranav Chhaliyil1, Kael F Fischer2, Bernd Schoel3, Pradheep Chhalliyil3
1 MSAE, Fairfield, IA, USA
2 Department of Pathology, University of Utah, Salt Lake City, Utah, USA
3 Genetic-ID NA INC, Fairfield, IA, USA
|Date of Submission||18-Jan-2020|
|Date of Decision||30-Jan-2020|
|Date of Acceptance||06-Mar-2020|
|Date of Web Publication||10-Jun-2020|
Sakthi Foundation, 4690 S. Lakeshore Dr, # 2072, Tempe, AZ 85282
Source of Support: None, Conflict of Interest: None
Introduction: Bedtime teeth cleaning is strongly recommended to limit overnight oral bacterial growth, but the impact of different cleaning methods on oral microbiota remains to be determined. Here we evaluated the efficiency of three oral cleaning methods in decreasing distinct subtypes of dental-damaging bacteria (DDB) using quantitative real-time PCR (qPCR) and 16s-based taxonomic profiling. Materials and Methods: This was a randomized, controlled study of 58 healthy subjects who performed three timed oral cleaning methods for two consecutive nights: tooth brushing with sodium fluoride-containing toothpaste followed by tongue cleaning (BT); cleaning of gums and teeth by rubbing with an index finger followed by tongue cleaning (GIFT); and GIFT with the addition of rice husk activated charcoal (CT). Saliva samples were collected the following morning for qPCR and metagenomics analysis. Results: All three oral cleaning methods resulted in a significant decrease (P < 0.006) in the quantity of DDB compared with no cleaning (NC) controls. Bonferroni post hoc analysis showed that GIFT and CT decreased Aggregatibacter actinomycetemcomitans and Streptococcus mutans levels compared to the BT method (P < 0.005). Metagenomics data also showed a more significant decrease in many pathogenic bacteria using the GIFT and CT methods compared to the BT method. Conclusion: BT and GIFT are effective oral cleaning methods and reduce DDB levels. The greater flexibility of a finger to reach all areas of the teeth, gums, and inner cheeks that are inaccessible to a toothbrush to disturb biofilms makes GIFT a better method than traditional toothbrushing for bedtime oral cleaning.
Keywords: Bedtime, biofilm, caries prevention, charcoal, dental cleaning, microbiota, sugar, tooth brushing, tongue cleaning, water swishing
|How to cite this article:|
Chhaliyil P, Fischer KF, Schoel B, Chhalliyil P. Impact of Different Bedtime Oral Cleaning Methods on Dental-Damaging Microbiota Levels. Dent Hypotheses 2020;11:40-6
| Introduction|| |
Oral hygiene is vital for good health, with regular tooth brushing and cleaning recommended by the World Health Organization (WHO). Regular toothbrushing twice daily with a fluoridated toothpaste is recommended for all age groups to prevent dental caries. With respect to oral cleaning methods, recent studies have shown that morning brushing with toothpaste should be supplemented with other oral cleaning methods such as tongue cleaning to reduce the oral bacterial flora., Microorganisms from the oral cavity have been shown to cause a number of oral diseases including caries (tooth decay), periodontitis (gum disease), endodontic (root canal) infections, alveolar osteitis (dry socket), and tonsillitis (infection of the tonsils). Caries, periodontitis, otitis media, and other infections are now recognized to be caused by a consortium of organisms in a biofilm rather than a single pathogen. 16s-based taxonomic profiling of bacteria from the oral cavity has revealed the microbial diversity in the mouth, which form a biofilm matrix with interactions between opportunistic pathogens and commensals driving the pathogenesis of dental caries.
The oral microbiome comprises over 600 bacterial species. Although distinct bacterial subsets predominate in different areas of the oral cavity, only a small percentage of the oral microbiome is thought to be harmful, with most being beneficial. Dental caries is a complex pathogenetic process, with bacteria as one of the contributory factors. Whilst the aim of oral hygiene is to reduce bacterial load, any remaining bacteria still have the potential to cause caries in conjunction with other factors. The number of oral bacteria varies significantly depending on oral hygiene. For example, a well-cleaned tooth can host up to 103–105 live bacteria, whereas a poorly cleaned tooth may have 108-109 bacteria., Calculations show that there are about 20 billion microbes in the entire oral cavity, which are estimated to grow to 50 billion during eight hours of overnight sleep. This increase may in part be due to the lack of saliva flushing during sleep, since saliva production is highest during the daytime and dramatically decreases during sleep. Therefore, bedtime brushing is strongly recommended to limit bacterial growth facilitated by reduced saliva levels. Furthermore, a number of studies have shown that bedtime toothbrushing is a protective factor for dental caries. However, little is known about the effects of various cleaning methods on oral microbiota, particularly when performed as part of bedtime oral healthcare maintenance.
In an ongoing study, we discovered a novel bedtime oral-cleaning technique, which we termed the “GIFT” method (cleaning of gums and teeth by rubbing with index finger, followed by tongue cleaning). The GIFT method was initially designed as a control group in which subjects were asked to use their flexible index finger to reach and rub all parts of their mouth, including their gums and teeth, without a toothbrush, toothpaste, or tooth powder. Interestingly, the group that used the GIFT method had significantly lower bacterial counts than any of the other methods tested. Here we used quantitative polymerase chain reaction (qPCR) and 16s rDNA sequencing to further determine whether the GIFT method is superior to toothbrushing and tongue cleaning for removing the harmful microbiota. Moreover, we assessed whether the efficiency of GIFT could be improved using adsorbing charcoal.
| Materials and Methods|| |
Study design and oral cleaning methods
Each of 58 healthy subjects (28 boys and 30 girls, 10–12 years old) performed one of three oral cleaning methods timed in minutes. Each subject was provided with a kit containing the cleaning materials and sample collection tubes. After dinner and just before going to bed, subjects ate cane sugar cubes (6 g) for two reasons: (1) to normalize the food factor in causing variations in the oral microbiota; and (2) although eating sugar increases the growth of oral bacteria, this allows the determination of the efficiency of bedtime cleaning. Thereafter, subjects did not eat or drink anything else until breakfast the next morning. On the first two nights, as a control, no cleaning (NC) of the oral cavity was performed after eating the cane sugar cubes. Then, the subjects randomly performed one of the following oral-cleaning methods for two consecutive nights after eating the cane sugar cubes:
- Brushing teeth with sodium fluoride-containing Colgate toothpaste, followed by tongue cleaning (BT). Subjects were instructed to brush with 500 mg toothpaste followed by tongue cleaning. Tongue cleaning was accomplished using a curved, stainless steel scraper to gently clean the tongue five times, followed by thorough rinsing of the oral cavity three times with water.
- Cleaning of gums and teeth by rubbing with index finger, followed by tongue cleaning (GIFT). Subjects were instructed to thoroughly rub their teeth and gums in all the oral cavity areas with their index fingers [Supplemental Figure S1], followed by tongue cleaning with water as described in the BT method above. Subjects performed water swishing 3-5 times to thoroughly clean the oral cavity.
- GIFT with the addition of rice husk-activated charcoal, followed by tongue cleaning (CT). Subjects were instructed to perform the GIFT method, as described above, using approximately 100 mg activated charcoal powder, followed by tongue cleaning as described in the BT method above.
Every morning following the bedtime oral-cleaning methods, subjects were asked to spit approximately 1 ml saliva into a vial containing lysis buffer (Fast-ID DNA extraction kit, Genetic-ID, Fairfield, IA). Samples were frozen until the DNA could be extracted in the laboratory in line with best practice to minimize changes to the microbiota. All subjects repeated the methods described above after a gap of ten days.
Saliva tubes were homogenized with 0.1 mm zirconium beads (Research Products International Corp., Mount Prospect, IL) using a BioSpec Mini-BeadBeater for 30 seconds at maximum speed. DNA was extracted using Fast ID DNA extraction kit. 200 ng of purified DNA samples were used for all molecular analysis.
For determination of the ratio of specific dental-damaging bacteria (DDB), quantitative real-time PCR (qPCR) was performed using Taqman universal bacterial, Aggregatibacter actinomycetemcomitans (A. a.), and Streptococcus mutans (S. m.) primers and probes and TaqMan™ Universal PCR (Applied Biosystems) mastermix. qPCR was performed using a BioRad CFX 96 with cycle conditions and primer-probe concentrations as previously described., The ratio of DDB to universal bacterial concentrations was calculated using BioRad CFX 96 software.
A metagenomic sequencing library was prepared by amplification of variable regions 3 and 4 (V3-V4) of the 16s rRNA gene. Illumina MiSeq was used for sequencing V3-V4 amplicons from both ends. Twenty random samples from five individuals were used for sequencing. QIIME (v1.9.1) and Greengenes databases were used to assign bacterial taxa.,
100 g of rice husk (Ambika Rice Mills, India) was heated at 150−250°C in an iron vessel for 20–30 minutes until the husk turned black. The charcoal was then finely powdered in a high-speed blender to reduce its abrasiveness. The surface area measurement of the charcoal was performed with a TriStar II (Micromeritics, Norcross, GA) based on the isothermal adsorption of nitrogen. A single-point or multipoint method was used to calculate the surface area using the Brunauer, Emmett, and Teller (BET) formula. The pore size distribution was measured using nitrogen adsorption/desorption isotherms at liquid nitrogen temperature and relative pressures (P/Po) ranging from 0.05 to 1.0. A high uptake of nitrogen at low P/Po indicates filling of the micropores (<20 angstroms).
Consent was obtained from each participant. Before beginning the study, each participant and the participant’s parent signed an informed consent form for study participation, to provide saliva samples, and for the use of personal data required for the study. The parents and participants were warned about: (i) the consumption of sugar before bedtime and the potential damage caused by it; (ii) the potential abrasiveness of the charcoal; and (iii) the avoidance of fluoride in oral cleaning during the experiments in the two groups. Parents also monitored the effectiveness of the cleaning procedures including tongue cleaning and GIFT. Adults supervisors completed follow-up questions every day to ensure the cleaning procedures were always performed correctly. Ethical approval was received from the School SRC committee, and all experiments were performed in accordance with relevant guidelines and regulations. Subjects and their parents were also instructed on how to perform the IRB-approved procedures, including how to use the activated charcoal.
ANOVA statistical analysis and Bonferroni adjustment for multiple comparisons was performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Error bars represent the standard error of the mean (SEM).
| Results|| |
The GIFT method efficiently reduces harmful bacteria
To study the efficiency of the cleaning methods in decreasing bacterial quantity, one of the most abundant DDBs, A. a., was analyzed. Of the various dental cleaning methods (BT, GIFT, CT, and NC), the amount of A. a. relative to total bacteria in the saliva samples was significantly reduced as shown by qPCR [Figure 1]A. ANOVA analysis showed significant reductions in A. a. of 20% by BT, 40% by GIFT, and 34% by CT compared with those in the NC control. Overall, the greatest reduction was found with the GIFT method.
|Figure 1 (A) Alteration in A. a. bacterial levels after oral cleaning. The percent of A. a. to total bacterial levels was significantly decreased by all three cleaning methods compared with NC control. The error bars represent SEM, and the P-values (ANOVA) were calculated relative to the NC group (BT, brushing followed by tongue cleaning, CT, GIFT method using charcoal followed by tongue cleaning, GIFT, gum and teeth rubbing using index finger followed by tongue cleaning, NC, no cleaning). (B) Post hoc analysis of the three oral-cleaning methods. A Bonferroni post hoc analysis showed that the percent of A. a. to total bacterial levels was significantly decreased by the GIFT and CT cleaning methods compared with the BT method. The error bars represent SEM values, and the P-values were calculated relative to the BT method (BT, brushing followed by tongue cleaning, CT, GIFT method using charcoal followed by tongue cleaning, GIFT, gum and teeth rubbing using index finger followed by tongue cleaning).|
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Next, a post hoc analysis was performed to determine which of the three oral-cleaning methods showed statistically significant decreases in A. a. levels. GIFT and CT cleaning methods showed significant decreases in A. a. levels compared with the BT cleaning method [Figure 1]B. These findings suggest that the GIFT method of oral cleaning is superior to other methods tested.
Metagenomic analysis verifies bacterial alterations after oral cleaning
To extend and further determine the changes in microbiota, sequencing analysis was performed, which further indicated that salivary bacterial abundance was different between the oral cleaning and NC control groups. These alterations were observed at all taxonomic levels [Figure 2], [Table 1] and [Table 2]. There was an overall decrease in levels of Actinobacteria, Bacteroides, and Firmicutes and an increase in Proteobacteria in the oral cleaning groups compared with the NC group [Figure 2]. At the phylum level, significantly altered groups relative to NC control group were Actinobacteria (most altered by GIFT and CT), Firmicutes (equally altered by BT and GIFT and most altered by CT), and Proteobacteria (most altered by GIFT and CT) [Table 1]. At the genus level, the commonly known dental-damaging genera such as Aggregatibacter and Actinobacteria, both of which are implicated in periodontal disease, were significantly reduced by GIFT and CT relative to NC control [Table 2]. These metagenomic analyses demonstrate that oral cleaning results in a significant change in the oral microbiota.
|Figure 2 Alterations in the distribution of bacterial phyla after oral cleaning. The distribution of bacterial phyla in the saliva samples (n=5) was altered by all three cleaning methods (BT, brushing followed by tongue cleaning, CT, GIFT method using charcoal followed by tongue cleaning, GIFT, gum and teeth rubbing using index finger followed by tongue cleaning, NC, no cleaning).|
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| Discussion|| |
Dysbiosis of microorganisms in the oral cavity has been shown to cause a number of oral and systemic diseases. Dental cleaning has been used for centuries to avoid oral dysbiosis and the subsequent development of various harmful inflammatory oral diseases. Cleaning the oral cavity twice a day reduces bacterial counts and prevents gum disease.,, Here, for the first time, we demonstrated using qPCR and 16s microbiota analysis that bedtime brushing is important for oral health. Our novel GIFT method, with and without charcoal, decreased the levels of Aggregatibacter actinomycetemcomitans (A. a.) compared to the no cleaning and toothbrushing groups. The GIFT and CT methods were superior to the other methods, significantly reducing the amounts of two of the most aggressive DDBs: Streptococcus mutans (Firmicutes) and A. a. (Proteobacteria) after GIFT cleaning.
Greater flexibility of a finger to reach all the areas of the teeth, gums, and inner cheeks that are not accessible to a toothbrush and disturbing the biofilms makes the GIFT method better than traditional toothbrushing. Since an undisturbed biofilm promotes plaque formation,,, use of the GIFT method over traditional dental cleaning is better for oral health because the GIFT method effectively disturbs biofilm formation and maturation.
Pathogenic bacteria such as Streptococcus mitis, S. mutans, S. sanguis, and S. milleri  and the fungus Candida adhere to plastic surfaces on toothbrush heads, even after short exposure times,, and a significant number of bacteria can be found in toothbrushes even after a day or week after brushing., Because contaminated toothbrushes can reintroduce microorganisms into the oral cavity and promote the transmission of oral disease and oral infection, the GIFT method helps to reduce these two major limitations of current approaches., Although highly significant reductions in bacterial numbers have been detected after the use of single-use and throw toothbrushes, using the GIFT method could significantly contribute to reducing recurring plastic waste.
Although mechanically brushing using toothpaste removed a significant quantity of bacteria, cleaning the tongue further enhanced the cleaning effects of brushing, suggesting that tongue cleaning is a critical component of maintaining good oral health. The tongue is not only a possible reservoir for bacteria to re(colonize) periodontal tissues, but is also a source of oral malodor. The removal of the tongue coating reduces bad breath with tooth brushing and also the bacterial load., Our studies showed that the BT and GIFT methods both effectively decreased the bacterial load and DDB bacteria with bedtime brushing.
Charcoal coats the tongue surface and adsorbs bacteria present on the tongue, suggesting that tongue cleaning followed by the use of charcoal may further enhance the reduction in bacterial counts in the oral cavity. Activated charcoal is well known for its high adsorption properties. Subjects using charcoal were shown to have less caries than those who used toothpaste and toothbrushing. The current study also shows that the CT method was very effective in decreasing DDBs. Our unpublished studies show that fine powdered charcoal (pore size < 8.8 nM) is less abrasive and safe for enamel, in contrast to coarse charcoal powder or some toothbrushes and toothpaste brands.
In conclusion, the greater flexibility of a finger to reach all the areas of the teeth, gums, and inner cheeks that are not accessible to a toothbrush makes GIFT an effective method for disturbing biofilm formation and maturation to plaque and caries. The GIFT method can complement or enhance tooth brushing for bedtime oral cleaning.
Financial support and sponsorship
Conflicts of interest
The authors declare no conflicts of interest with respect to the authorship and/or publication of this article.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]
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