Kevin Cunningham, Donald Fisher, Jack Spreen

Kevin Cunningham, Donald Fisher, Jack Spreen

Professor Teh

Biology 3b

November 25, 2015

Oral Bacteria Inhibition With the Use of Listerine®

Introduction

Humans and various types of bacteria form symbiotic relationships virtually everywhere throughout our bodies. A notable symbiotic relationship occurs in the mouths of humans. Traditionally, humans brush their teeth to rid themselves of harmful bacteria that can cause bad breath and gingivitis. However, most dentists recommend also adding an oral antiseptic mouthwash to individuals’ daily mouth care regimen to reduce plaque and biofilms(Arweiler, et al., 2001). Also, the consistent use of oral antiseptics may greatly increase the health of gums and teeth as well as reduce the possibility of contracting oral disease. (Gunsolley, 2010) the clinical benefits of anti-plaque mouth rinse are similar to the benefits of oral hygiene instructions at six month recall appointments. One of the more prominent brands is Listerine®. Listerine® claims that brushing only rids you of twenty-five percent (25%) of the bacteria that attributes to tooth decay, gingivitis and bad breath. While adding Listerine® to an individual’s daily regimen, will aid in the removal of the remaining seventy-five percent (75%) of harmful bacteria. The goal of this experiment was to attain if Listerine® would in-fact remove significantly more bacteria in the mouth over only brushing without the use of Listerine®. The intellectual merit that can be obtained testing Listerine’s effectiveness as an antiseptic mouthwash can provide helpful data to those who suffer from oral diseases caused by bacterial growth within the mouth. (Yang, et al., 2015), alcohol rinse free solutions were shown to be effective in reducing plaque and bacteria levels immediately, and the same was shown in long term patients as well (Gaffer, et al., 1997). While using an oral antiseptic changes levels of bacteria in the mouth, there was not found to be a significant difference in product effectiveness (Strydonck, et al. 2005). The overall goal of this research is to aid in clarifying if Listerine® will infact reduce the amount of bacterial colonies found within the human mouth.

Methods

Ten (10) subjects were swabbed a total of four (4) times to gather a total of forty (40) swabs. Subjects were swabbed on the inside of the mouth swabbing over gums, teeth, and inside of cheek. Subjects were swabbed upon waking up in the morning, the first swab was taken without brushing or drinking water, the second swab was taken after subject had brushed for sixty (60) seconds. The third and fourth swabs were taken a day later. The third swab was taken before rising in the morning before the subject had brushed, the fourth and final swab was taken after subject had both brushed for sixty (60) seconds and swish with Listerine® for sixty (60) seconds. Each Swab was labeled in correspondence to their test number and subject initials, then isolated into bags , and kept chilled until brought into the lab. Test numbers were chronologically assigned in the same sequence as test were conducted. Swabs were placed in test tubes with sterile water. Dilutions were performed on each sample tube a total of two (2) times. One (1) ml of the final dilution was then mixed vigorously and added to petri dishes with nutrient agar using the pourplate method, aseptic technique and incubated for forty-eight (48) hours. Visual bacterial counts were then taken of developed bacterial colonies and entered into Microsoft Excel and statistical analysis performed using descriptive statistics and a paired one-tailed student's T-test.

Results

This experiment produced a lot of raw data that was collected and analyzed. The recorded amount of bacterial growth in colonies per each agar was multiplied by 121 to account for the solution dilutions for a more accurate representation of the actual amount of bacteria recorded per each subject. Pouring each agar plate directly after each solution was diluted would have happened in an ideal world. Due to lab and time limitations, each dilute solution waited approximately 72 hours until the actual pour which could have affected bacterial growth. After examining each agar plate, the mean was taken of all the subjects bacterial count per agar plate. The control pre-brush (Test 1) resulted in a higher mean bacterial count compared following post bush with no rinse (Test 2) . As expected, our results showed a similar trend between our second pre-brush control (Test 3) compared to the post brush with the antiseptic rinse (Test 4). Recording from each individual agar plates indicated a decrease in bacterial count was observed between test 1 and 2. A similar trend observed from test 1 and 2 was also recorded from test 3 and 4. The test recordings between test 1 -2 and 3-4 were very consistent in term of results. The two control groups for pre brush test contained higher bacterial count because brushing alone will decrease bacterial count. When oral antiseptic is added to brushing, the gap between bacterial amounts in each subject increases significantly compared to brushing alone. A visual representation of bacterial amounts observed is represented in Figure1.

Figure 1. Graph shows mean of bacterial colonies observed on agar nutrient plates. (P-value of 0.006 was found using a one-tailed student T-test. Error bars are ±SEM).

Note the decrease in bacterial amount between test 2 and 4. Another visual will demonstrate the change in bacteria count in terms of percentage to demonstrate the effect of oral cleaning technique practiced in this experiment.

Figure 2. Shows the reduction in bacterial colonies found using bacterial colony counts on Agar nutrient plates.

Discussion

The data found in the experiment resulted in showing that supplementing oral healthcare with a product such as Listerine® will decrease the amount of bacteria found within the mouth, shown in Figure 2, the resulting findings state that there was found to be a mean bacterial count of 181.5 colonies or a reduction of 42.3% of bacteria compared to the baseline of 100%, using the group without brushing or swishing. The control group, which consisted of only brushing, yielded 205.7 colonies or only a reduction of 34.6%. Johnson and Johnson argues on their campaign ad that brushing removes 25% of bacteria in the mouth, and that adding Listerine® will remove 99.9% of bacteria, this statement was found to be untrue. Continual use of the product is necessary to inhibit bacterial buildup and healthy gums. A factor during this test was the lack of a quick interchange between dilution and placing the samples into the agar nutrient plates to culture an accurate amount of bacteria. There was a delay of 48 hours between dilution and proper placement in agar plates which may have affected the accuracy of the results. However, the results did quantify the hypothesis that there would be a significant decrease in bacteria found in samples taken after swishing with Listerine®. Another pattern arose that certain individuals in the test groups were on different schedules. Subjects would brush their teeth prior to going to bed and swabs were taken when subjects first arose in the morning before ingestion of food or water, usually around 8AM. However, certain subjects in the group would not retire to bed until 4AM. Meaning bacteria only had roughly 4 hours to re-cultivate in the mouth from the previous days brushing. These individuals’ counts looked to be lower in both pre and post brushing samples than those that had 8 or more hours from their last brushing. A necessary adjustment to testing parameters would be to swab subjects only after an allotted time from the previous days brushing.A recent publication by Johnson and Johnson, owners of the product Listerine®, have recently begun a campaign stating that it takes 21 days of supplementation to remove all of the harmful bacteria. This would be found to be true seeing as it may take more time for the complete removal of the biofilms found in the mouth. The company states that during the first initial swishing of the product 99.9% of harmful bacteria are killed, but 0.1% remaining in the mouth can reproduce and come back again stronger than previously. This would have to do with the remaining bacteria’s genetic coding that gave it resistance to the antiseptic found in Listerine®. However, prolonged use of Listerine® will eventually lead to the removal and destruction of those bacterial cells. Another factor that affects bacterial counts in the inhibition of biofilms found on the teeth created by bacteria. Once the initial treatment with Listerine® occurs, individuals must continue to use the antiseptic to treat and control the bacteria, discontinuing usage of the antiseptic could lead to a greater bacterial response to treatment at a later time (Gordon, et al. 1985). However, different types of plaque and oral bacteria can be found within individuals based on dietary and various oral activities, involving smoking, drinking, or other activities involving the mouth, The individual response by oral bacteria to these stimuli could affect the strength of the antibacterial properties of some mouthwashes (Burt, B. A.. 1983).

This is beneficial to individuals over the long term. However, a secondary thought would be the removal of symbiotic bacteria found within the mouth that is beneficial to the host. Several strains of beneficial bacteria contain antimicrobial functions that remove unhealthy

Conclusion

This experiment yielded beneficial results showing that Listerine® will infact remove a greater percentage of the bacteria from the mouth. Though it does not remove the amount Johnson and Johnson has advertised and stated through their own scientifical research. Further testing should be conducted using long term effect analysis to validate the statement made by the company. However, the results quantify the hypothesis that there would be a drastic reduction of bacterial colonies found within the mouth.

Citations

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