A Quantitative Comparison of the Zone of Inhibition against Streptococcus mutans and Oral Bacterial Growth using Crest®, Colgate®, and Aquafresh® Toothpastes

Final Draft

Kevan Johanson

Senior Seminar 2

5-8-2007

Table of Contents

Page Numbers

1. Abstract & 1

2. Introduction 2

2. Methods 7

3. Results 13

4. Discussion 16

5. Consent Form – Appendix 1 19

6. Acknowledgements 21

7. Literature Cited 22

Abstract

This study evaluated the antibacterial efficiency of Crest®, Colgate®, and Aquafresh® toothpastes using in vivo and in vitro experiments. The in vivo experiment measured the optical density of bacteria samples grown from participants’ teeth by using a spectrophotometer. The in vitro experiment measured the zone of inhibition of the three toothpastes against Streptococcus mutans, with water as a control group. Statistical analyses showed no difference in optical density among Crest®, Colgate®, and Aquafresh® toothpastes. However, there was a significantly larger zone of inhibition for the Colgate toothpaste compared to Crest®, Aquafresh, and control groups (p = 0.0005 at a 98.95% confidence interval).

Introduction

Dental hygiene is an important field in human health. Pathogenic and non-pathogenic bacteria are constant residents of the human mouth. Scientific research has shown that the use of oral hygienic products plays a vital role in the reduction of human oral bacteria. One of toothpaste’s purposes is to prevent oral bacteria growth, in order to prevent pathogenic infection. Within the oral cavity, the most common cariogenic (caries-causing) of the Streptococci genus is Streptococcus mutans (Tortora et al., 2004). My study observed the antibacterial effectiveness of Colgate®, Crest®, and Aquafresh® toothpastes on oral bacteria and S. mutans. The reason I chose toothpaste for this study was because of its extensive use by humans, many varieties and brands, and easy availability. The overall purpose of most active ingredients of toothpaste is to maintain healthy teeth and gums by preventing bacterial growth in the oral cavity. The active ingredients of toothpastes can include antibacterial agents, anti-cavity agents, enzymes for amplifying saliva’s antibacterial properties, and enzymes that prevent decay. The antibacterial agent triclosan and the anti-caries (cavity) agent fluoride are common ingredients in toothpaste. For my study, I examined the antibacterial activity of triclosan and fluoride in three toothpastes.

Sullivan et al. (2003) tested toothpaste containing triclosan on resistant oral Streptococci and measured the in vitro sensitivity of Streptococci strains against triclosan. Triclosan is an antibacterial agent commonly found in soaps, mouthwash, toothpaste, and other hygiene items. Triclosan is a biphenolic derivative that kills gram positive bacteria such as Streptococci and Staphylococci. A biphenol is a derivative of phenol, which either helps reduce irritation or increases antibacterial activity. Triclosan inhibits enzymes that synthesize fatty acids, which compromise the bacterium’s plasma membrane (Brown, 2005). The Sullivan et al. (2003) study also analyzed the impact of triclosan on the normal oral microflora. Nine subjects used toothpaste containing 0.3% triclosan and sodium lauryl sulphate to brush their teeth twice a day for fourteen days. The nine subjects were in good health, and none had been administered antibacterial agents in the three months before the study. On the first day of the study, 5 saliva samples in 15 minutes were collected per subject. The concentration of the triclosan in saliva was 3.6 g/mL of saliva, which was determined by agar diffusion procedures (Sullivan et al. 2003). Once the saliva concentration had been determined, the saliva was added to and incubated on selective agar plates. Streptococci strains were grown into colonies on the plates and subsequently isolated into pure cultures by T-square methods. The purpose of the T-square technique was to isolate bacteria from mixed species colonies down to the genus level (Brown, 2005). Gram stain and biochemical tests were conducted to identify the bacterial species and colony forming units (CFU). As a result of triclosan use, there were reductions of Lactobacilli in the oral microflora; however, there was no change in the susceptibility of Streptococci towards triclosan. Lactobacilli showed a significant reduction in concentration from 5.5 CFU on day 0 to 5.2 CFU on day 14 with a p-value of less than 0.05 using an Analysis of Variance (ANOVA). S. mutans showed no increased susceptibility to triclosan. Sullivan et al. (2003) reported 3.4 CFU for S. mutans on day 0 and a CFU of 4.1 on day 14. Data analysis determined that using triclosan in toothpaste showed no statistically significant difference on the in vitro sensitivity of resistant oral Streptococci. My study observed the antibacterial activity of three toothpastes on S. mutans by measuring zones of inhibition. The study by Sullivan et al. (2003) influenced this study to test the effectiveness of triclosan in toothpaste against S. mutans and oral bacteria.

Battino et al. (2004) researched the in vitro antioxidant activity of 12 antioxidant-enriched toothpastes. Antioxidants in toothpaste may benefit the host’s antioxidant defenses in the case of periodontal diseases (PD). PD are chronic inflammatory conditions that arise from the interaction between pathogenic bacteria and the host immune responses. The toothpastes contained mixtures of sodium ascorbyl phosphate, alpha-tocopherol acetate, pycnogenol, allantoin, and methyl salycilate. The antioxidant activity was measured by a series of tests, such as, the ABTS-decolorization assay and the Comet assay (Battino et al., 2004). The results indicated that antioxidant activity depended on the solubility of the active ingredients. The toothpastes that contained sodium ascorbyl phosphate displayed clear I50 values (mg toothpaste / ml water) ranging from 50 to 80 mg of toothpaste per milliliter of water, which measure antioxidant concentration. The Comet assay showed limited antioxidant protection from sodium ascorbyl phosphate when the toothpaste was in the presence of hydrogen perioxide. However, Battino et al. (2004) found that sodium ascorbyl phosphate in toothpaste possessed antioxidant activity. This study was similar to mine; because I tested three toothpastes for their antibacterial properties as Battino et al. (2004) did with antioxidant properties.

De Leo et al. (1990) researched the prevalence of S. mutans and dental decay in children from Genoa, Italy. This study’s goal was to obtain information on the prevalence of dental decay and to find the proportional amount of Streptococcus mutans in plaque samples from subjects. Samples of saliva, bacteria, and plaque from the first molar in each quadrant were collected. After the saliva was spread on selective agar plates for growth, S. mutans in the samples was reported as present or absent. De Leo et al. (1990) reported S. mutans was present in 58 of the 105 subjects. Seventy-seven of the subjects were caries active (decayed teeth) and 28 subjects were caries-free. The presence of S. mutans in subjects with caries was 63%. S. mutans was found in only 31% of subjects with no caries. S. mutans was associated with dental decay, by their levels in plaque samples.

Clayton et al. (2000) tested the effects of penicillin on Endamoeba gingivalis from oral bacterial cultures (Clayton et al., 2000). They obtained E. gingivalis from each subject’s mouth. The bacteria were spread onto warmed culture media. After incubation, the bacteria displayed no decrease in stability. The penicillin concentrate had little or no effect on bacterial growth, because the pH remained at the bacterium’s optimum level. Clayton et al. (2000) ultimately found that their prescribed penicillin dosage was not enough to kill the bacteria in the log and lag phases of growth. This article brought to my attention that researching toothpastes’ ingredients was critical for testing their antibacterial efficacy against oral bacteria. I used well-known name-brand toothpastes containing the active ingredients triclosan and fluoride.

Romao et al. (1990) conducted tests on the effectiveness of human saliva as a cleaning agent. The use of saliva for cleaning has been practiced for centuries (Romao et al., 1990). They conducted solubility and resistance tests to measure the effect of saliva on different surfaces. By using thin-layer chromatography to analyze the lipids in dirt that had been removed by salvia (Romao et al. 1990), they found that saliva removed lipids from the dirt. The results reported that saliva was an effective surface cleaner. One concern was that the saliva degraded the surface of the object and attacked red and blue pigments, vermilion and azurite respectively. The vermilion and azurite colors were degraded due to the enzymatic activity of saliva. Their study was significant because saliva plays an important role in cleaning the human mouth. However, since saliva was nearly impossible to control in my study, I assumed it was constant among all subjects.

I compared the antibacterial activities of three toothpastes: Crest®, Colgate®, and Aquafresh®. Crest®, Colgate®, and Aquafresh® toothpastes all contained sodium fluoride for anti-cavity protection. Colgate® was the only toothpaste I used that contained triclosan. I had two separate hypotheses: one for the in vivo experiment and the other for the in vitro experiment. First, I hypothesized that bacterial growth would be the most inhibited by Colgate® toothpaste with a p-value less than 0.05, which would have had a lower optical density than the samples treated with Crest® and Aquafresh® toothpastes. I predicted triclosan and sodium fluoride together would be more effective in killing oral bacteria than sodium fluoride alone. The second hypothesis was Crest®, Colgate®, and Aquafresh® would show no significant difference in zone of inhibition measurements of S. mutans. This prediction of no difference between the different toothpastes’ antibacterial activity was because the in vitro sensitivity of Streptococcus mutans was resistant towards triclosan according to Sullivan et al. (2003). The data from the two experiments in this study were analyzed using ANOVA and Tukey multiple comparisons tests (Minitab®, 2005), to quantitatively compare antibacterial efficacy among the three toothpastes. This study provided statistical evidence regarding these toothpastes’ antibacterial efficacy in vivo and against S. mutans in vitro.

Methods

I conducted two different experiments with three toothpastes. The methods are presented separately for the two experiments and are independent of each other. The in vivo experiment tested the amount of oral bacteria present in subject after using toothpaste. Subjects participated by using toothpaste for a week. The swabbing samples collected on days 1 and 8, before and after brushing their teeth were incubated in nutrient broth and measured for absorbance using a spectrophotometer. The in vitro experiment measured the zone of inhibition from the toothpastes against S. mutans. After spreading a bacterial lawn, I applied filter discs from toothpaste slurries to the plates and incubated for 24 hours at 37°C. The zone of inhibition was conducted to determine the antibacterial efficacy of toothpaste in preventing growth of S. mutans.. During the experiments, I always wore goggles, gloves and lab coat and used sterile cotton swabs to protect the health of the participants and myself, along with maintaining a sterile environment to avoid contamination.

Subject Experiment using Toothpaste

For the first experiment using human subjects, I analyzed a total of 30 subjects divided evenly among three toothpastes. No subjects were used who were participating in oral studies that would drastically influence my studies’ aim. The subjects in the mouthwash study were valid as participants in my study because mouthwash is a regular hygienic task. The subjects were at least 18 years of age and recruited from Saint Martin’s University. Consent forms were approved by the Institutional Review Board (IRB), which reviews proposed studies using human subjects before approving them to use subjects ( Appendix 1), signed by each subject, and placed in a sealed envelope before any testing took place. Each subject was then evaluated from a questionnaire, in order to distribute the tobacco users evenly amongst the three tested toothpastes. The subjects used one of the three following toothpastes: Crest®, Colgate®, and Aquafresh®. I selected these toothpastes because they were similar when comparing their active ingredient, sodium fluoride (Battino et al., 2005) and because they are commonly used by consumers. I tested Colgate® because it additionally contained the antibacterial phenol, triclosan. The subjects were given toothbrushes and toothpaste and required to brush twice daily for 7 days. I dispensed the toothpastes into pill containers because it kept the toothpaste clean and it made it easy for subjects to use. I provided subjects with their assigned toothpaste, gave them new toothbrushes, and provided directions with contact information if they had any questions.

I swabbed each participant’s mouth twice on day 1 and 8 of the study. After subjects rinsed their mouth with water for 30 seconds, the first swab for bacteria was collected from their mouth. The subjects then brushed their teeth for two minutes and rinsed with water before a second swab was collected in the same area. Each time I collected bacteria, I thoroughly swabbed near the crown and gum-line area of the front of mandibular (bottom) teeth to ensure collection. The sterile swabbing precautions I used were important to maintain the safety and health of the subjects and researcher. Each swab was transferred to its own nutrient broth tube to incubate at 37˚C for 24 and 48 hours.

Preparation of Nutrient Broth Tubes

In preparation for the human experiment, I prepared 120 nutrient broth tubes (4 broth tubes per subject) by adding about 10 mL to each tube totaling 1.2 liters. I mixed 9.6 grams of Ward’s® nutrient broth powder with 1.2 liters of deionized water in an Erlenmeyer flask. Once the broth was completely dissolved and poured into tubes, the tube caps were loosely screwed on to be autoclaved. The tubes were sterilized in the Tuttnauer 2540E autoclave at 121°C and 15 psi for 15 minutes, tightly capped with labels, and placed in the refrigerator at 4°C.

Using the Spectrophotometer

The swabs from days 1 and 7 were submerged in separate nutrient broth tubes and incubated for growth at 37°C for 24 and 48 hours. After the broth was incubated for bacterial growth, a sample of each tube was transferred into individual cuvettes to prepare for the Spectronic Instruments D20+ spectrophotometer. The spectrophotometer reads the optical density of bacteria by measuring absorbance of light through the broth (Brown, 2005). The absorbance data were collected from the broth sample after the 24th and 48th hour of incubation. After the spectrophotometer had warmed up for 10 minutes, I tuned the wavelength to 686 and set the absorbance to zero (Brown, 2005). I then poured the sterile nutrient broth control into a cuvette and reset the absorbance at zero. The cuvette samples were placed in the spectrophotometer and absorbance data were recorded. The growth period of 24 hours was selected because it mimics brushing teeth once per day. The temperature during incubation was 37°C, because this is the optimum temperature for most bacteria in humans and the average temperature of the human body. The optical density measurements allowed me to compare the antibacterial activity of the three toothpastes. On day 1, I measured the instant impact of the toothpaste and on day 7 the data allowed me to compare the antibacterial activity of the three toothpastes after short-term use.