ABSTRACT
Title: Efficacy of Alcohol-Free Chlorhexidine in Reducing the Levels of Streptococcus Mutans.
Purpose: To compare the antimicrobial properties of Alcohol-Free Chlorhexidine with other antimicrobial mouth-rinses.
Methods: Streptococcus mutans (GS5 strain), was grown on agars, and resuspended in a sterile culture medium trypticase soy broth (TSB) supplemented with yeast extract. Four unique experiments were conducted each adding the inoculums of the bacterial suspension to the test antimicrobial agents.
Results: Experiment #1: Growth of S. mutans was fully inhibited at 5% and 1% concentrations of alcohol-free chlorhexidine (AFC) and alcohol-containing chlorhexidine (CHX). Bacterial growth occurred in all other dilutions with the most notable in 1% Biotene® (OD max = 1.383 nm) followed by 0.01% Biotene® (OD max = 1.328). The greatest doubling time was 0.01% CHX at 2.1013 hr.
Experiment #2: Generation time and OD max were similar for all dilutions of the different reagents.
Experiment #3: 5 Minute short-term exposure of undiluted CHX inhibited the growth of S. mutans. AFC inhibited bacterial growth for 6 hours. The OD max and doubling time were 1.288 and 1.1213 hr respectively. The OD max and generation time for Biotene® were 0.891 and 1.5516 hr respectively.
Experiment #4: 5 minute short-term exposure of undiluted CHX and AFC inhibited the growth of S. mutans. Bacterial growth occurred in all other dilutions with the most notable in full strength Biotene® (OD max = 1.629) followed by 1% ethanol (OD max = 1.598). The greatest doubling time was 5% Ethanol at 1.706 hr.
Conclusions: Alcohol-free Chlorhexidine is effective in suppressing bacterial growth of S. mutans and, therefore, is a viable alternative to alcohol-containing Chlorhexidine.
INTRODUCTION
Antimicrobial mouth-rinse solutions are used in many clinical situations for both prophylactic and therapeutic purposes (1). More specifically, mouth-rinses have become an important adjunctive procedure to good mechanical oral hygiene or in the prevention of infections subsequent to bacteria from intraoral surgical procedures or immunosuppression due to radiation to the head/neck.1,4,5
Chlorhexidine gluconate (CHX), a broad-spectrum antibacterial agent, is one of the most widely used antiseptic agents in comprehensive dental care.5 Effective against both Gram-positive and Gram-negative microorganisms, fungi and some viruses, it has been shown to inhibit plaque formation, reduce gingival inflammation and prevent dental caries.3,5 Moreover, in oral use, CHX is free from systemic toxicity, and microbial resistance or alterations of the oral microbiota do not occur.8,11 However, the prolonged use of CHX is limited due to the development of extrinsic tooth and tongue discolourations, as well as alterations in taste leading to decreased patient compliance.2,3,5,7,9,11 Therefore, the majority of dental practitioners do not recommend long-term daily use of CHX as a mouth-rinse.11 Gingival desquamation and painful mucosa have also been reported in severe cases.1,5
Current literature supports the daily use of 0.12% or 0.2% CHX as antiseptic agents. However, an additional limiting factor is that routinely used chlorhexidine mouth-rinses contain alcohol, which may cause pain, irritation and be detrimental to patients with mucositis, patients undergoing head/neck radiation therapy, young children, immunocompromised patients, or recovering alcoholics. Potentially adverse effects of alcohol-containing CHX mouth-rinse led to the formulation of a new alcohol-free 0.12% CHX (AFC) mouth-rinse.1,2,11
Biotene® is an alcohol-free mouth-rinse that can improve many of the symptoms of radiation-induced xerostomia. It acts by producing an antimicrobial enzyme system in the oral cavity that penetrates the cell walls of plaque-forming bacteria, destroying them just gingival to the gumline.12 This enzyme system consists of innate human salivary defense proteins; lysozyme, lactoferrin and peroxidase. Lactoferrin is an iron-binding glycoprotein that displays bactericidal activity against a variety of bacteria in iron-free form, apo-LF, and bacteriostasis through iron sequestration.6,10
Both AFC and Biotene® are commercially available antimicrobial agents used to improve oral hygiene and prevent bacterial infection. However, few studies have focused on the efficacy of AFC as a viable antimicrobial mouth-rinse.
The object of this investigation was to compare the antimicrobial properties of Alcohol-Free Chlorhexidine Gluconate with other antimicrobial mouth-rinses.
METHODS AND MATERIALS
Species of Bacteria:
The bacteria selected for this study is found orally in saliva, soft tissue surfaces and within the dental biofilm; Streptococcus mutans (GS5 strain).
Optical Density Measurements:
Microorganism growth can be measured by the Bioscreen C Automated Growth Curve Analysis System (Fig. 1). Bioscreen C is a computer-controlled incubator/reader/ shaker. It has 8 filters which range from 405nm to 600nm including a wide-band filter and it uses a honeycomb microplate to measure changes in optical density versus time to produce microbiological growth curves.
As microorganisms grow, they increase the turbidity of their growth medium.
By measuring the turbidity of this medium over time, an optical density curve can be generated. The curve reflects the growth (increased concentration) of the organism of interest.
BIOSCREEN PREPARATION:
Four unique experiments were conducted testing 0.12% alcohol-free chlorhexidine (AFC), 0.12% Chlorhexidine Gluconate (CHX), and Biotene®, oral mouth-rinses. All microbial reagents were filtered with 0.22mm micropore filter. Distilled water was used as the negative control while a sterile culture medium, trypticase soy broth (TSB) supplemented with yeast extract, was used as the positive control. S. mutans (SM GS5 strain) was cultured on 1.5% of trypticase soy broth supplemented with 0.3% of yeast extract, 0.75% of Bacto-agar and 2.5% heat inactivated horse serum and incubated for 24 hours at 37°C. Fresh MTSB (900 uL) was inoculated with the bacteria (100 uL) and the suspensions were added to the test microbial agents and distilled water.
In the first experiment four dilutions, 5%, 1%, 0.1% and 0.01% in TSB, each of AFC, CHX and Biotene® were made. The same dilutions were made with distilled water as controls. The inoculums of the bacterial suspension were added to the diluted reagents and briefly vortexed.
The second experiment tested the short-term exposure of two dilutions, 5% and 1% in TSB, each of AFC, CHX and Biotene®. A 5% dilution was made with distilled water as a control. Inoculums (1000 uL) of the bacterial suspension were centrifuged for 1 minute at ~13 000 rpm. The testing reagents (1000 uL) were then added to the bacterial pellet and vortexed. All dilutions were left to set at room temperature for 30 minutes. Each was then centrifuged, followed by reagent removal, MTSB wash, vortex and centrifuge. The pellet formed was then suspended in 1000 uL MTSB and vortexed.
The reagents in the third experiment were undiluted (100%) solutions of AFC, CHX, Biotene® and distilled water. In the fourth experiment they were undiluted solutions of AFC, CHX, Biotene® and distilled water and four dilutions, 1%, 5%, 11.6% and 15%, each of alcohol. In both trials inoculums (1000 uL) of the bacterial suspension were centrifuged for 1 minute at ~13 000 rpm. The testing reagents (1000 uL)
were then added to the bacterial pellet, vortexed and all dilutions were left to set at room temperature for 5 minutes. Each was then centrifuged, followed by reagent removal, MTSB wash, vortex and finally centrifuged. This was then repeated, completing a second wash with MTSB, a final centrifuge and lastly, suspension in 1000 uL of MTSB.
The final bioscreen preparation in experiments 1, 2, 3, and 4 consisted of aliquots (200 uL) of the bacteria in combination with the oral mouth-rinses, AFC, CHX, Biotene®, distilled water and TSB being placed in honeycomb wells (Fig. 2) yielding 90, 40, 25 and 45 samples respectively. Each aliquot was dispensed five times,in different wells, to ensure uniformity and reproducibility. Mineral oil, (0.1 mL) drops were then dispensed into each well to prevent evaporation. The honeycomb wells were then placed into the Bioscreen Microbiological Growth Analyzer for 24 hours at a wide band wavelength range of 420 nm to 580 nm to monitor changes in optical density. Growth curves and doubling time were then calculated.
RESULTS
Experiment #1: Growth of S. mutans was fully inhibited at 5% and 1% concentrations of AFC and CHX (Figs.3 & 4). The bacteria were killed and no significant changes in optical density (OD) were noted. Under the same conditions, bacterial growth occurred in all other dilutions (Figs. 5 & 6). The most notable growth occurred in the 1% Biotene® dilution with an OD max = 1.383 and a doubling time (Td) = 1.4734 hr. This was followed by the 0.01% Biotene® dilution with an OD max = 1.328 and a Td = 1.3053 hr. The greatest doubling time was 0.01% CHX at 2.1013 hr. The dilutions of AFC and CHX mouth-rinses that were ineffective in inhibiting the bacterial growth had an OD of approximately 1.3 nm, which was similar to the OD of Biotene® and the controls.
Experiment #2: Generation time and OD max were similar for all dilutions of the different reagents. Growth began at approximately two hours for every dilution. There appears to have been some growth inhibition compared to the initial incubation experiment, however, the reagents were not effective, and bacteria still grew (Fig. 7).
Experiment #3: The controls, H2O and TSB, did not kill the bacteria and neither did Biotene® with an OD max and generation time of 0.891 and 1.5516 hr, respectively. Short-term exposure of undiluted CHX completely inhibited growth of S. mutans. AFC inhibited SM bacterial growth for six hours, at which time there appeared to be bacterial growth. The OD max and doubling time were 1.288 and 1.1213 hr, respectively. The OD max and generation time for Biotene® were 0.891 and 1.5516 hr, respectively (Fig.8).
Experiment #4: Five minute short-term exposure of undiluted AFC and CHX inhibited the growth of S. mutans. Bacterial growth occurred in all other reagents and dilutions of alcohol with the most notable growth occurring in undiluted Biotene® (OD max = 1.629) followed by 1% ethanol (OD max = 1.598). The greatest doubling time was 5% Ethanol at 1.706 hr (Fig.9).
DISCUSSION
The observations and results in this investigation suggest that alcohol-free chlorhexidine is an effective mouth-rinse in suppressing the growth of S. mutans. The first experiment tested the growth of bacteria in the presence of different dilutions (5%, 1%, 0.1%, 0.01%) of the tested reagents. From the first experiment, it was observed that as low as 1% AFC and CHX were effective in inhibiting the growth of the bacteria. Biotene® did not inhibit microbial growth and the outcome of the controls, distilled water and MTSB, were as expected, no killing occurred.
Following the first trial, the second consisted of investigating the outcome of exposing bacteria to 1% and 5% dilutions of antimicrobial mouthrinses for 30 minutes. Generation time and OD max were similar for both dilutions of the different reagents. Moreover, there appeared to have been some growth inhibition compared to the initial incubation experiment as the growth of the bacteria was delayed. However, the reagents were not effective, and the S. mutans still grew.
The intention for the third experiment was to simulate a more clinical situation. The S. mutans were exposed to the undiluted antimicrobial mouth-rinses, AFC, CHX, Biotene®, and distilled water for 5 minutes. The undiluted alcohol-containing CHX completely inhibited the growth of S. mutans. Conversely, the AFC inhibited bacterial growth for 6 hours, at which time there appeared to be growth of bacteria. At this point, contamination from other microbiota was suspected in the AFC mouth-rinse. For this reason, the experiment was repeated. In addition, whether or not alcohol was a confounding variable was to be determined. This fourth trial demonstrated that after 5-minute exposure to undiluted antimicrobial mouth-rinses and different dilutions of alcohol there was no bacterial growth in either the AFC reagent or the CHX reagent. Moreover, the alcohol dilutions did not inhibit the growth of the S. mutans.
Although the last trial demonstrated that there is no difference in the efficacy between the AFC and alcohol-containing CHX, further studies are necessary; (1) to determine the difference in the formulation and (2) the mechanisms causing the inhibition of the bacteria between the AFC and alcohol-containing CHX; and to clinically test and confirm the consistency of the results displayed in this study. Current data suggests that there is only one clinical trial demonstrating the efficacy of AFC with respect to plaque inhibition. The results of that study were consistent with this one.
In comparing Yokoyama’s study (2009), Biotene® was found to be effective in inhibiting the growth of Aggregatibacter actinomycetemcomitans, a different bacteria species. However, in this research project it was ineffective against S. mutans. Perhaps Biotene is effective against bacterial species that are primarily anaerobic and found subgingivally. If this was the case, one may postulate that Biotene would better be served as an antibacterial mouth-rinse for periodontal disease, whereas, CHX mouth-rinses should be primarily reserved for caries prevention.
CONCLUSION
1) Alcohol-Free Chlorhexidine is effective in suppressing bacterial growth of S. mutans and, therefore, is a viable alternative to alcohol-containing Chlorhexidine.
2) Alcohol does not contribute to the antimicrobial properties of Chlorhexidine.OH
Rebecca Cohen, DDS, Paed Cert, FRCD(C), Class of 2011; Faculty Advisor, Casey Chen, BDS, PhD, DDS.
Acknowledgements: Dr. Casey Chen for his encouragement and Jason Jee and Christine Hwang for their help and support. This research paper is submitted in partial fulfillment of the requirements for completion of the Advanced Pediatric Dentistry Program at the Herman Ostrow School of Dentistry of USC.
REFERENCES
1. Bolanowski SJ, Gescheider GA, Sutton SV. Relationship between oral pain and ethanol concentration in mouthrinses. J periodontal Res 30:192-197, 1995.
2. Eldridege et al. Efficacy of an alcohol free chlorhexidine mouthrinse as an antimicrobial agent. The journ of Pros Dent 80 (6):685-690, 1998.
3. Flotra L, Gjermo P, Rolla G, Waerhaung J. Side effects of chlorhexidine mouth washes. Scan J Dent Res 79:119-125, 1971.
4. Gunsolley JC. Clinical efficacy of antimicrobial mouthrinses. Journal of Dentistry 38:S6-S10, 2010.
5. Kocak MM, Ozcan S, Kocak S, Topuz O, Erten H. Comparison of the efficacy of three different mouthrinse solutions in decreasing the level of streptococcus mutans in saliva. Eur J Dent 3:57-61, 2009.
6. Lassiter MO, Newsome AL, Sams LD, Arnold RR. Characterization of lactoferrin interaction with Streptococcus mutans. J Dent Res 66:480-485, 1987.
7. Loe H, Schiott CR. The effect of mouthrinses and topical application of chlorhexidine on the development of plaque and gingiv
itis in man. J Perio Res 5:79-83, 1970.
8. Screenivasan P, Gaffar A. Antiplaque biocides and bacterial resistance: a review. J Clin Periodontol 29: 965-974, 2002
9. Smith RJ, Moran J, Addy M, Dohery F, Newcombe RG. Comparative staining in vitro and plaque inhibitory properties in vivo of 0.12% and 0.2% chlorhexidine mouthrinses. Journal of Clin Perio. 22(8):613-617, 1995.
10. Tenovuo, J. Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety. Oral Diseases 8:23-29, 2002.
11. Van Strydonck DAC, Timmerman MF, van der Velden U, van der Weijden GA. Plaque inhibition of two commervcally available chlorhexidine mouthrinses. J Clin Periodontol32: 305-309, 2005.
12. Warde et al. A Phase II study of Biotene in the treatment of postradiation xerostomia in patients with head and neck cancer. Suppor Care Cancer 8:203-208, 2000.
Bioscreen References
1. D’Arrigo M, Garcia de Fernando GD, Velasco de Diego R, Ordonez JA, George SM, Pin C. Indirect measurement of the lag time distribution of single cells of Listeria innocua in food. Appl Environ Microbiol 72:2533-8, 2006.
2. Francois K, Devlieghere F, Standaert AR, Geeraerd AH, Cool I, Van impe JF, Debevere J. Environmental factors influencing the relationship between optical density and cell count for Listeria monocytogenes. J Appl Microbiol 99:1503-1515, 2005.
3. Metris A, George SM, Baranyi J. Use of optical density detection times to assess the effect of acetic acid on single-cell kinetics. Appl Environ Microbiol 72:6674-6679, 2006.
4. Murakami C, Kaeberlein M. Quantifying yeast chronological life span by outgrowth of aged cells. JoVE. 27. http://www.jove.com/index/Details.stp? ID=1156,doi:10.3791/1156.
