Antimicrobial Effect of Mouthwashes: An In vitro study.

 

Manupati Prasanth* and Adarsh Krishna Capoor

School of Microbiology, Mahatma Gandhi National Institute of Research and Social Action (MGNIRSA), Hyderabad, Andhra Pradesh, India.

*Corresponding Author E-mail:- prasanthmanupati@gmail.com

 

Introduction and objective: Mechanical tooth cleaning, by means of a toothbrush, remains the most reliable and common method for controlling supra-gingival plaque. However, poor patient compliance has reduced the usefulness of such measures, as not all individuals are likely to follow the optimal oral hygiene regimen. Consequently, chemotherapeutic agents (e.g. Mouthwashes) are recommended for use as adjuncts to mechanical plaque control. Few of these mouthwashes, however, have undergone testing, as evidenced by the amount of information on their safety and efficacy in literature. Based on this scanty information, the present study was designed to evaluate anti-microbial effect of different mouthwashes by using standard agar well diffusion method and to determine their suitability for regular usage.

Materials and Methods: A total of eight mouthwashes was tested for  their  anti-microbial  activity against three oral pathogens namely, Streptococcus mutans (MTCC 890), Escherichia coli (MTCC 579) and Candida albicans (MTCC 854) by well agar diffusion assay.

Results: Triclosan containing mouthwash showed higher zones of inhibition against all the organisms tested  (p<0.05) as compared to the Chlorhexidine gluconate and Potassium nitrate formulations.

Conclusion: This study shows that mouthwash formulations containing triclosan are more effective in controlling the oral microflora compared to Chlorhexidine gluconate and Potassium nitrate formulations.

 

 

 

INTRODUCTION:

Oral disease is a major health problem throughout the world. Dental caries and periodontal diseases are among the most prevalent oral health problems in the human community, despite great improvements in management of oral hygiene.

 

Dental caries (tooth decay) is a complex biochemical process that involves a progressive localized destruction of susceptible dental hard tissues by acidic by-products resulting from the bacterial fermentation of dietary carbohydrates¹. Dental caries is one of the most prevalent human diseases, particularly in industrialized areas. According to the World Health Organization (W.H.O), 60-90% of school children worldwide have dental cavities². However, a marked decline in the prevalence of caries has been observed in developed countries. On the other hand it is on the rise in developing countries.

 

In India, with the increase in the consumption of highly refined sugars and inadequate exposure to fluorides, the menace is showing an upward trend; where the prevalence is reported to be about 50% – 60%³. There is substantial evidence suggesting that Streptococcus mutans is the chief bacterium that causes plaque and dental caries⁴. In addition, other microflora, like Escherichia coli and Candida albicans are also associated with active caries lesions. Candida is the most common yeast, found in the oral cavities. It is, by far, the most common fungal species infecting dental root canals.  Prevalence of Candida species infections has increased during the past decade⁵. Acrylic dentures add to increasing risk of Candida colonization. The species is resistant to internal medication. Poor oral hygiene is one of the reasons for the accumulation of these microbes and their harmful activities.

 

Periodontal disease is a continuum of oral diseases of polymicrobial origin, characterized by a bacterial plaque (biofilm) induced gingival inflammation which, if left untreated, may lead to chronic infection and tooth loss. The continuum of disease begins with gingivitis, which is inflammation of the gingiva in response to the bacterial biofilm on adjacent teeth. Periodontal disease is considered multifactorial in nature, with a number of factors contributing to its initiation and progression. These include poor oral hygiene, specific plaque bacteria, smoking, systemic conditions (e.g. Diabetes), aging and such other susceptible hosts⁶. Streptococci, spirochetes and bacteroides are found to be the possible pathogens responsible for the disease. The W.H.O estimates that a severe periodontal disease, which may result in tooth loss, is found in 5-20% of middle-aged adults worldwide². In India, the prevalence of periodontal diseases is reported to be about 67.7% in 15 year olds and 89.6% in adults in the age group of 35 to 44 years ⁷.

 

Oral diseases, however, are largely preventable and implementation of effective control measures will enable indivivuals to maintain good oral hygiene. However, the total cost of oral dental health care is high and not within the reach of the majority of the population in low income countries in the developing world. Hence there is an urgent need to promote such preventive measures which are acceptable, easily available and cost effective.

 

Mechanical tooth cleaning, by means of a toothbrush, remains the most reliable and common method for controlling supra-gingival plaque. However, poor patient compliance has reduced the usefulness of such measures, as not all individuals are likely to follow the optimal oral hygiene regimen. Consequently, chemotherapeutic agents (e.g. Mouthwashes) are recommended for use as adjuncts to mechanical plaque control⁸. These are proposed to be specifically effective in interdental areas. Several antimicrobial chemicals, incorporated into mouthwash, can be beneficial in the reducing the levels of oral bacteria, specifically Streptococcus mutans ⁹.

 

There are many brands of mouthwashes marketed in India. Each has its own composition and concentration of ingredients. Many mouthwashes claim to have antimicrobial properties, but in fact candid examination is needed to evaluate their effectiveness. The success of any mouthwash, in part, is determined by its ability to eliminate oral pathogenic microorganisms and decrease the risk of infection in the mouth. In this study, an attempt is made to examine eight brands of mouthwash marketed in India for their anti-caries property by using standard agar well diffusion method.

 

MATERIALS AND METHODS:

Test microorganisms:

Pure cultures of Streptococcus mutans (MTCC 890), Escherichia coli (MTCC 579) and Candida albicans (MTCC 854) were procured from Institute of Microbial Technology, Chandigarh, India. These were cultured on specific media, procured from HiMedia Laboratory Pvt. Ltd., Mumbai, India, recommended for different microorganisms such as brain heart infusion broth (S. mutans), and nutrient broth (E.coli and C.albicans) and incubated aerobically at 37°C for 24 h while Candida albicans was incubated for 48 h.

 

Antimicrobial assay:

Eight different brands of Mouthwashes, were procured from Hyderabad-Secunderabad market. The particulars about the ingredients included in each mouthwash formulation as mentioned by the manufacturers are noted (Table 1). The antimicrobial activity of each mouthwash formulation was determined using a modified agar well diffusion method ¹⁰. Growth from freshly sub-cultured isolates was suspended in 10 mL of sterile saline to obtain a turbidity of 0.5 McFarland standards. Nutrient agar and brain heart infusion agar plates were prepared and seeded with 0.5 mL of suspended cultures of each isolate (Nutrient agar was used for E.coli and C. albicans where as brain heart infusion agar was used for Streptococcus mutans strain). The mouthwash solutions were made by mixing the calculated amount of the mouthwashes (2.0 ml) in a measured volume (2 ml) of the sterile pyrogen-free distilled water to give 1:1 dilution. They were further diluted in sterile pyrogen-free distilled water and four different dilutions of 1:2, 1:4, 1:8 and 1:16 were made. Then wells of 8 mm diameter, were punched into the plate surface with equal distance from each other. Each well was then filled with 0.2mL diluted mouthwash solutions, while the same amount of sterile distilled water was also added as a control. The plates were then incubated at 37°C for 24 h (48 h for yeast species). The antimicrobial activity was evaluated by measuring the diameter of zones of inhibition in mm (Figure 1). All the plates were made in triplicate and the experiments repeated three times.

 

 

 

Table 1. Ingredients of various mouthwashes tested for antimicrobial potential.

 

 

 

STATISTICAL ANALYSIS:

Results were expressed as Mean ± Standard Deviation for illustration. Data were tested using a statistical package, namely, SPSS Windows version 19. Statistical analysis of the results was based on Kruskal–Wallis test. A P-value less than 0.05 was considered to be significant.

 

RESULTS:

The analysis exhibited a wide variation in the effectiveness among the sample mouthwashes, a feature that may have been largely due to their antimicrobial active ingredients (Table 1). The results of this investigation shows that mouthwash formulation E has maximum zones of inhibition against the test organism, Streptococcus mutans (p<0.012, Table 2) compared to all other mouthwash formulations. In E. coli and Candida albicans, the zones of inhibition are less in comparison to Streptococcus mutans but are significantly different at higher dilutions (1:8, 1:16, p<0.008, Tables 3 and 4) for mouthwash formulation E. Whereas in comparison to mouthwash formulation E, mouthwash formulation C showed maximum inhibition against E. coli (p<0.008, Tables 3). The mean values ± standard deviation of zones of inhibition are given for all the test organisms. Each experiment was repeated three times (n = 3).

 

 

Table 2. Antimicrobial activity of mouthwash formulations against Streptococcus mutans.        

 

Table 3. Antimicrobial activity of mouthwash formulations against Escherichia coli.

 

Table 4. Antimicrobial activity of mouthwash formulations against Candida albicans

 

Fig. 1. Zones of inhibition produced by mouthwash formulation E at 24 h against the three tested microorganisms at five different dilutions. (a) Streptococcus mutans (b) Escherichia coli (c) Candida albicans (d) Control

 

 

DISCUSSION:

A majority of oral diseases exhibit multifactorial       etiology^11,12. The interplay of host, agent and environmental factors ultimately determine the oral health status of an individual. Dental caries and periodontal diseases are two of the most prevalent chronic diseases worldwide ¹³.

 

Dental caries is an infectious microbiologic disease of the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic substance of the tooth. The infection results in loss of tooth minerals that begin on the outer surface of the tooth and can progress through the dentin to the pulp, ultimately compromising the vitality of the tooth. If left untreated, it may lead to pain, infection and even tooth loss. Periodontal diseases (gingivitis and periodontitis) are destructive inflammatory diseases of the gingiva and the supporting structures of the teeth, induced by a bacterial plaque (biofilm).

 

Extensive research during the last several decades has shown the crucial role played by dental plaque in the initiation and progression of dental caries and periodontal diseases. Dental plaque is considered to be the primary etiological factor for dental diseases¹². The complex microbial ecosystem of dental plaque along with the metabolic byproducts poses a profound challenge on the integrity of hard and soft tissues of the oral cavity. The importance of periodic removal of dental plaque cannot be ignored or over emphasized. Consequently, chemotherapeutic agents (e.g. Mouthwashes) are recommended for control and plaque removal ⁸.

In the present study, all the investigated mouthwashes exhibited wide variations in their effectiveness against the microorganisms tested. Among the formulations, formulation E emerged as the most effective antimicrobial agent, based on the mean diameter of the zone of microbial inhibition produced in agar well diffusion method, against all the pathogens tested. This might be due to the synergistic effect between the active ingredients of the formulation. Effective ingredients of formulation E are Triclosan, Sodium fluoride and Ethyl alcohol.

 

Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol] has been used for more than 25 years as a general antibacterial and antifungal agent in hospitals and personal health care products such as antimicrobial soap, toothpaste, mouthrinse and deodorants. Triclosan inhibits growth of microorganisms by inhibiting the enoyl – reductase enzymes of type II fatty acid synthesis in susceptible bacteria, damaging the bacterial cytoplasmic membrane, leading to leakage, thereby preventing the organism from functioning or reproducing. Systematic reviews of six-month clinical studies have concluded that triclosan when incorporated within a polymer, migrates to the surface where it is bound. Because it is not water – soluble, it does not leach out, and it continuously inhibits the growth of bacteria in contact with the surface using barrier or blocking action^{14 -16}. Today triclosan is the active ingredient in many oral hygiene formulations.

 

Fluoride has played a significant role in improvement of oral and dental health during the past 50 years. It has been frequently used in oral health products including toothpastes and mouthwashes. Many clinical studies have demonstrated the efficiency of sodium fluoride in the prevention of dental caries by promoting remineralisation with fluorapatite and fluorohydroxyapatite, thereby increasing enamel resistance to acid attack^17,18.

 

Ethyl alcohol, is a common ingredient in oral health products due to of its antiseptic properties. Formulation F was found to be less effective as compared to formulation E.

 

Next to triclosan formulations, chlorhexidine gluconate products, such as formulations A, B, C and D were found to have antimicrobial activities and these were statistically significant. However, formulation C shows highest microbial inhibition than triclosan formulations, in relation to E.coli; this observation adds information to the earlier study ¹⁹.

 

Chlorhexidine gluconate is a cationic biguanide with a single chlorine substitute in each phenol ring and also, it is most widely used chemotherapeutic agents in dentistry because of its outstanding bacteriostatic properties and substantivity. Its efficacy is mainly based on the cationic molecule binds to the negatively charged cell walls of the microbes, destabilizing their osmotic balance^20,21. Chlorhexidine, in the form of a salt, has been used since the 1950’s as an oral antiseptic in mouthwash, toothpaste, and chewing gum²². Despite being discovered in the 1950s, it is still considered one of the most effective anti–plaque agents in dentistry. Its long term use is limited by its disagreeable taste and propensity to stain teeth brown ²³.

 

Potassium nitrate and sodium fluoride products, such as formulations G and H were found to have lack of antimicrobial activity²⁴. It is known that a balance exists in each person’s oral microbial population. If this balance is gone astray, opportunistic microorganisms can multiply, enabling the initiation of disease processes. Therefore, the mouthwash formulation identified as having the maximum sensitivity to all three organisms at all concentrations and thus probably the strongest antimicrobial properties, may not be certainly superior to the other mouthwashes. Because, the formulation used in vivo is likely to be diluted by saliva, the level to which antimicrobial properties are buffered or lost in dilution in vitro of interest.²⁵

 

CONCLUSION:

This study shows that mouthwash formulations containing Triclosan are more effective in controlling the oral microflora compared to Chlorhexidine gluconate and Potassium nitrate formulations; as given in (Table 1) and the sample can be arranged in descending order of their effectiveness as given below.

Formulation E> Formulation F> Formulation A> Formulation B> Formulation C> Formulation D> Formulation G > Formulation H.

 

ACKNOWLEDGEMENT:

Sincere thanks Dr. N. Balakrishnan, Department of Biostatistics, National Institute of Nutrition, for carrying out the statistical analysis.

 

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Received on 28.03.2013          Modified on 15.04.2013

Accepted on 19.04.2013         © RJPT All right reserved