Research 2014-15

Summary

 

Oral health is a fundamental component of overall health. Therefore, oral cleansing is vital for children and adults. Several research studies show that poor oral health is connected to cancer, diabetes, heart disease and other diseases. One of the simple ways of prevention is to keep the mouth clean. I was curious to know which cleaning method was the best. I tested with 8 different oral cleaning methods. The objective of this research study is to give awareness on Bed-Time oral cleaning because oral microbes grow from 20 to 33 billion within 8 hours of sleep without the flushing action of saliva. I divided my 88 subjects into 8 different groups were each group followed a specific procedure.

 

I then did DNA extraction to the samples and did qPCR analyses. I calculated the p value using Annova Single Factor Analysis. The qPCR analyses showed that upon oral cleaning, there was a decrease in the bacterial count for all of the cleaning methods. However, the most statistically significant groups were Brushing + Tongue Cleaning and Finger Rubbing + Water Swishing. I conclude that of these two, Finger Rubbing + Water Swishing is easier, more convenient and more cost effective, making it ideal for developing countries. I did further research on Finger Rubbing + Water Swishing by finding how much Aggregatibacter actinomycetemcomitans (a gram negative, dental damaging bacteria, associated with Periodontitis) is decreased. I wrote a children’s book bringing Dental Awareness on the importance of bed time mouth cleaning.

Why?

 

What ways can we improve the health of all people regardless of economic status?

 

In today’s world, there are so many diseases that people suffer from. Curing these diseases is not affordable to everyone and many people die because they can’t afford treatment. Several research studies show that poor oral health is connected to cancer, diabetes, heart disease and other diseases. Prevention may be the key. One of the simple ways of prevention is to keep the mouth clean. Which oral cleaning method is the most effective? My hypothesis was that brushing is the best method.

My research can help those suffering in poverty remain healthy even when...

 

  • Nearly 1/2 of the world’s population — more than 3 billion people — live on less than $2.50 a day. More than 1.3 billion live on less than $1.25 a day.

  • 1 billion children worldwide are living in poverty. According to UNICEF, 22,000 children die each day due to poverty.

  • 805 million people worldwide do not have enough food to eat.

  • 80% of the world population lives on less than $10 a day.

 

Importance of this Research

 

Everybody knows that cleaning the oral cavity is good for oral hygiene. However not everyone is aware that during night sleep when saliva does not do flushing action, bacteria grows a lot and causes heavy damage not only to teeth and gums, but also play a role in causing some deadly diseases like heart disease, cancer, diabetes, etc.  A few years ago, when I was lazy to brush my teeth in the morning, my dad had me spit into petri dishes just to see how much bacteria is in my mouth. I was amazed to see the bacteria in my mouth and then my interest grew to find the following research information:

 

  • Recent data from the NIH Human Microbiome Project (HMP) revealed that the oral microbiome has the largest core of commonly shared microbes among unrelated individuals compared to other habitats such as gut or skin (Costello et al., 2009; Li et al., 2013; Zhou et al., 2013).

  • There are 500 to 650 different bacterial species that live in the teeth, tongue, biofilms that cover the cheeks and oral mucosa.

  • There is an estimate of 20 billion oral microbes in our oral cavity. During overnight sleep of 8 hours, the bacteria multiplies to approximately to 33 billion microbes.

  • 1 ml of saliva contains 100 million microbes and so we swallow about 100 billion microbes in 24 hours influencing the gut microbiome and overall health.

  • Dental health not only affects our physical body but also our psychology. Oral discomfort in the mouth leads to less growth, bad conduct, and even less learning.

Dentists and parents suggest cleaning the oral cavity at least twice daily will reduce bacterial count and prevent gum diseases. However, children generally do not know the benefits of cleaning the oral cavity, and which cleaning method is the best. Midnight snackers and those who eat sweets and starchy food at dinner, do not know how much that increases the bacteria in their mouth.

Design & Testing

 

All the 88 subjects were divided into eight groups (Table 1).   

 

 

SALIVA COLLECTION PROCEDURE

 

Procedure (Days 1 and 2)

1. Before going to bed, one raw sugar cube was chewed well and then no solids or liquids were consumed. The mouth was also not cleaned.

2. After waking up in the morning, without drinking or eating anything, saliva was spit into the labelled collection tube containing lysis buffer to lyse the human and bacterial cells and also stops further growth of bacteria.

3. The tubes were closed and shaken four to five times to mix well and few drops of binding buffer was added. Again the tubes were shaken well.

Procedure (Days 3 and 4)

4. Before going to bed, one raw sugar cube was chewed well. They cleaned their mouth with their assigned cleaning agent and went to bed.

5. After waking up in the morning without drinking or eating anything, saliva was spit into the labelled collection tube containing lysis buffer to lyse the bacterial and human cells and also stops further growth of bacteria.

6. The tubes were closed and shaken four to five times to mix well and few drops of binding buffer was added. Again the tubes were shaken well.

The experiment was repeated twice after a gap of one week.

 

DNA EXTRACTION

 

To all the sample tubes, 50 µl of silica coated beads were added and mixed well and processed with the Thermo Scientific Kingfisher Automatic DNA Extractor. The beads were washed twice with wash buffer and twice with 70% ethanol and one time with 100% ethanol. Then 200µl elution buffer was added to the beads to elute the pure DNA. The qPCR Reaction Equal quantity of 200 ng DNA in 10 µl from all the samples was mixed with 12.5 µl of 2x Path-ID mastermix (Life Technologies). Real-time qPCR was carried out with an initial incubation of 10 min at 95ºC followed by 45 cycles consisting of denaturing at 95ºC for 10 seconds; annealing at 60ºC for 10 seconds for 45 cycles. Ten-fold serial dilutions of genomic DNA provided seven data points for standard curve generation. In each standard curve, the concentration of standard sample (known amount of DNA/DNA from known number of bacteria) was plotted against its crossing points. Quantification of the individual target bacteria and total bacteria from the experimental samples were calculated using the standard curves. Based on the concentration of known bacterial DNA, the amount of unknown bacterial DNA concentration was calculated by the PCR software. Similarly, another set of PCR reaction with human primers was done and human DNA concentration was calculated. The qPCR data of bacterial DNA concentration was normalized to human DNA concentration, while the qPCR data of Aggregatibacter actinomycetemcomitans DNA concentration was calculated  using the formula

 

Aggregatibacter DNA Concentration  

___________________________ X 100  

Total Bacterial DNA Concentration

 

ANNOVA STATISTICAL ANALYSIS

 

All statements of significance were based on alpha = 0.05, Annova  test (in Excel), and all Percent  reductions were calculated versus the control.

 

 

SALIVA COLLECTION PROCEDURE

 

Procedure (Days 1 and 2)

1. Before going to bed, one raw sugar cube was chewed well and then no solids or liquids were consumed. The mouth was also not cleaned.

2. After waking up in the morning, without drinking or eating anything, saliva was spit into the labelled collection tube containing lysis buffer to lyse the human and bacterial cells and also stops further growth of bacteria.

3. The tubes were closed and shaken four to five times to mix well and few drops of binding buffer was added. Again the tubes were shaken well.

Procedure (Days 3 and 4)

4. Before going to bed, one raw sugar cube was chewed well. They cleaned their mouth with their assigned cleaning agent and went to bed.

5. After waking up in the morning without drinking or eating anything, saliva was spit into the labelled collection tube containing lysis buffer to lyse the bacterial and human cells and also stops further growth of bacteria.

6. The tubes were closed and shaken four to five times to mix well and few drops of binding buffer was added. Again the tubes were shaken well.

The experiment was repeated twice after a gap of one week.

 

DNA EXTRACTION

 

To all the sample tubes, 50 µl of silica coated beads were added and mixed well and processed with the Thermo Scientific Kingfisher Automatic DNA Extractor. The beads were washed twice with wash buffer and twice with 70% ethanol and one time with 100% ethanol. Then 200µl elution buffer was added to the beads to elute the pure DNA. The qPCR Reaction Equal quantity of 200 ng DNA in 10 µl from all the samples was mixed with 12.5 µl of 2x Path-ID mastermix (Life Technologies). Real-time qPCR was carried out with an initial incubation of 10 min at 95ºC followed by 45 cycles consisting of denaturing at 95ºC for 10 seconds; annealing at 60ºC for 10 seconds for 45 cycles. Ten-fold serial dilutions of genomic DNA provided seven data points for standard curve generation. In each standard curve, the concentration of standard sample (known amount of DNA/DNA from known number of bacteria) was plotted against its crossing points. Quantification of the individual target bacteria and total bacteria from the experimental samples were calculated using the standard curves. Based on the concentration of known bacterial DNA, the amount of unknown bacterial DNA concentration was calculated by the PCR software. Similarly, another set of PCR reaction with human primers was done and human DNA concentration was calculated. The qPCR data of bacterial DNA concentration was normalized to human DNA concentration, while the qPCR data of Aggregatibacter actinomycetemcomitans DNA concentration was calculated  using the formula

Aggregatibacter DNA Concentration  

___________________________    X 100  

Total Bacterial DNA Concentration

 

ANNOVA STATISTICAL ANALYSIS

 

All statements of significance were based on alpha = 0.05, Annova  test (in Excel), and all Percent  reductions were calculated versus the control.

 

Design & Testing

 

A total of 88 subjects were used in the study (Table 1), who participated in my Dental Camp that I collaborated with a non-profit organization. The camp gave awareness on the importance of Bed-Time Brushing and disease prevention to these school children. In the camp, I conducted a survey regarding their dental health and dietary habits of the participating subjects.

There were 55 boys and 33 girls ranging from ages 10 to 13 years and only two out of the 88 subjects were vegetarians. Each subject served both in the control group (No Oral Cleaning) and their respective Oral Cleaning group.

Table 1. Survey of children's age, dietary habits and cleaning method groups.

 

 

Change in total bacterial count as determined by qPCR

 

As shown in Fig 1, 2 & 3 there was a  percentage change in the bacterial count with and without cleaning in the qPCR analyses.  Two cleaning methods decreased more than 50% bacteria: Toothpaste Brushing + Tongue Cleaning (63%) and Chlorhexidine (73%). However, the ratio of bacterial cells to human cells in the samples of Brushing, Brushing + Tongue cleaning and Finger Rubbing + Water Swishing showed only significant changes (p< 0.05) in the bacterial count after doing Annova Single Factor Analysis.

 

Figure 1. Change in total bacterial count as determined by qPCR

 

 

 

Figure 2 & 3.  Change in total bacterial counts in both the trials of all the cleaning methods.

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Cost analysis of each oral cleaning method per year.

 

Dental visits are very expensive and aren't affordable for all people, making it especially true for developing countries. Cost analysis was done to see how much a person would spend on these cleaning methods a year. Finger Rubbing was $0, making it the cheapest method.

 

Figure 5. Change in Aggregatibacter actinomycetemcomitans bacterial count as determined by qPCR

Several oral bacteria are found to be the causative in gum diseases. Since Finger Rubbing + Water Swishing is easy, cost effective and convenient, further research was done to find out how much it reduced the count of Aggregatibacter actinomycetemcomitans, a gram negative, dental damaging bacteria, that is associated with periodontitis. There was a 44.4% decrease of bacterial count after Finger Rubbing + Water Swishing.

Conclusion

 

Though there are several scientific studies published, this is a unique research quantifying the effect of bed-time oral cleaning on bacterial count using PCR method. This research study concludes that out of the eight cleaning methods, Brushing + Tongue Cleaning and Finger Rubbing + Water Swishing significantly decreases the bacterial count (p<0.05)  Out of these two methods, the easiest, most convenient and cost effective way of cleaning is simple Finger Rubbing + Water Swishing.

 

From the qPCR analyses, the percentage change in the bacterial count with and without cleaning was calculated. The ratio of bacterial cells to human cells in the samples Brushing + Tongue Cleaning and Finger Rubbing + Water Swishing showed significant changes (p<0.05) in the bacterial count (Fig 1). The percentage change in the ratio of bacterial cells to human cells in the samples of Neem Twig Rubbing, Oil Swishing, Chlorhexidine Mouthwash and the other two mouthwashes showed change in the bacterial count, but not statistically significant.

 

There was a decrease in all of the cleaning methods of both of the trials (Fig 2 & 3).This high variation in the data could be due to the inefficient usage of the cleaning agents. Maybe the subjects weren’t familiar in using mouthwashes and maybe they weren’t comfortable doing Neem Twig Brushing and Oil Swishing because of its unpleasant taste. The subjects may have done well in Brushing + Tongue Cleaning and Finger Rubbing + Water Swishing because of their familiarity in using them. The cost analysis showed that Finger Rubbing + Water Swishing was $0 because only your fingers and water are necessary for this method, making this the perfect method for those who are least able to afford dental care (Fig 5).

I conclude that the results of this study showed that when using Finger Rubbing + Water Swishing and Toothpaste Brushing + Tongue Cleaning as a part of regular oral hygiene routine, they will provide significantly greater oral microbial reductions. I also conclude that with this affordable cleaning method (Finger Rubbing + Water Swishing) anyone can prevent many deadly diseases and enjoy overall good health.

 

Children's eBook: I plan to travel in the future to many parts of the world and give Dental Awareness to children to prevent many diseases. To convey the importance of bed-time oral cleaning effectively, I made a Dental Awareness children's book that they can refer to everyday. Though the book is intended for children above preschool age, I am sure all ages will equally benefit. I am excited to publish this book so that everyone can enjoy their future with a great overall health.

Health & Safety

 

Registered Laboratory: Genetic ID, Fairfield, Iowa

 

Adult Mentor: Pradheep Chhalliyil

 

Potentially Hazardous Biological Agents 

 

There were no hazardous biological agents because the tubes where my subjects spit their saliva, contained lysis buffer which lysed the cells, not allowing any potentially hazardous biological agents to be active in causing any disease. All experimentation was conducted using DNA from the dead cells in the saliva samples.

Gloves was worn at all times during the experimentation as a safety precaution. The chemicals used were Lysis buffer, Opti buffer, Saline, TE buffer, and PCR master mixes. All these chemicals are suggested to be harmless by the kit manufacturers and will be disposed following OHSA.  

 

Acknowledgements & References

 

Acknowledgements

 

I would like to thank...

  • My teacher Barbara Hays for giving me advice and helping me edit my application and childrens' book.

  • My teacher Richard Incorvia for encouraging me to have my own live radio show which increased my communication skills.

  • Porayar Aandavar School Children who were the subjects of my study.

  • My parents Pradheep and Priya Chhalliyil who supported me and motivated me not to give up when doing DNA Extractions and qPCR Analysis which seemed hard at first.

  • Genetic ID for allowing me to use their lab facilities for my research.

 

References

 

  • Teles, Ricardo, Flavia Teles, Jorge Frias-Lopez, Bruce Paster, and Anne Haffajee. "Lessons Learned and Unlearned in Periodontal Microbiology."Periodontology 2000. U.S. National Library of Medicine, n.d. Web. 26 Apr. 2015.

  • Matsui, Miki, Naoyuki Chosa, Yu Shimoyama, Kentaro Minami, Shigenobu Kimura, and Mitsuo Kishi. "Effects of Tongue Cleaning on Bacterial Flora in Tongue Coating and Dental Plaque: A Crossover Study." BMC Oral Health. BioMed Central, n.d. Web. 26 Apr. 2015.

  • Loesche, Walter J. Microbiology of Dental Decay and Periodontal Disease. U.S. National Library of Medicine, n.d. Web. 26 Apr. 2015.

  • United Nations Development Programme. "Sustaining Human Progress: Reducing Vulnerabilities and Building Resilience." Human Development Report, 2014. Web Accessed February 25, 2015.

  • United Nations Inter-agency Group for Child Mortality Estimation (UN IGME). "UNICEF: Committing to Child Survival: A promise renewed." UNICEF, 2014. Web Accessed February 25, 2015.

  • FAO, IFAD and WFP. "The State of Food Insecurity in the World 2014. Strengthening the enabling environment for food security and nutrition." Food and Agriculture Organization of the UN, 2014. Web Accessed February 25, 2015.

  • Global Heritage Fund. "Poverty Facts." Global Heritage Fund | GHF. Accessed February 25, 2014, http://globalheritagefund.org/our_approach/poverty_facts.

 

 

 

Print Print | Sitemap
© Pranav Chhaliyil