Evaluation of antimicrobial efficacy of Chlorhexidine, Listerine, and herbal mouthwashes in decontamination of toothbrushes-An invitro study

Toothbrushing and toothbrushes play an important role in oral hygiene. Disinfection, storage and changing of toothbrushes at regular intervals are essential. The aim of the present study is to evaluate the invitro ef icacy of Chlorhexidine, Listerine® and herbalmouthwash (HimalayaOro-TTM) against microorganisms as toothbrush disinfectants. A total of 20 participants were recruited. Brand new identical toothbrushes were given to each participant and they were asked to brush their teeth twice daily. They were instructed to use tap water to rinse the used toothbrushes. The toothbrushes were collected after a week. The bristles of each toothbrush were cut and kept in separate vials containing transport media. Then, they were sent for microbiological evaluation. The number of colony-forming units were measured. The vials containing bristles were divided into four groups, each group containing 5 vials: Group I: Chlorhexidine mouthwash; Group II: Listerine mouthwash; Group III: Herbal mouthwash; Group IV: Distilled water. The bristles were immersed in test-tube containing respective mouthwashes for one hour. And, colony-forming units were measured. In all three test groups, there was a signi icant difference in the reduction of microbes after sanitization withmouthwashes. The highest reductionwas seen in group I and the least reductionwas group IV. Disinfection of toothbrush may prevent the occurrence of oral infections. Chlorhexidine mouthwash was found to be more effective than other mouthwashes as a disinfecting agent in reducing the microbial load.

Chlorhexidine, Disinfection, Oral hygiene, Toothbrushing A Toothbrushing and toothbrushes play an important role in oral hygiene. Disinfection, storage and changing of toothbrushes at regular intervals are essential. The aim of the present study is to evaluate the invitro ef icacy of Chlorhexidine, Listerine ® and herbal mouthwash (Himalaya Oro-T TM ) against microorganisms as toothbrush disinfectants. A total of 20 participants were recruited. Brand new identical toothbrushes were given to each participant and they were asked to brush their teeth twice daily. They were instructed to use tap water to rinse the used toothbrushes. The toothbrushes were collected after a week. The bristles of each toothbrush were cut and kept in separate vials containing transport media. Then, they were sent for microbiological evaluation. The number of colony-forming units were measured. The vials containing bristles were divided into four groups, each group containing 5 vials: Group I: Chlorhexidine mouthwash; Group II: Listerine mouthwash; Group III: Herbal mouthwash; Group IV: Distilled water. The bristles were immersed in test-tube containing respective mouthwashes for one hour. And, colony-forming units were measured. In all three test groups, there was a signi icant difference in the reduction of microbes after sanitization with mouthwashes. The highest reduction was seen in group I and the least reduction was group IV. Disinfection of toothbrush may prevent the occurrence of oral infections. Chlorhexidine mouthwash was found to be more effective than other mouthwashes as a disinfecting agent in reducing the microbial load.

INTRODUCTION
Toothbrushing helps to maintain oral hygiene which is essential for good oral health. Inspite of the widespread use of toothbrushes, the awareness of proper storage of toothbrushes is lacking. The microorganisms may contaminate the toothbrushes to a great extent. Aerosols from toilet lushing may cross-contaminate and facilitate the bacterial growth in the used toothbrushes which are kept in the bathroom. The possible cause for recontamination of the oral cavity may be due to the retention and survival of microorganisms on the toothbrush after brushing. In 1920, Cobb, in his study, reported that contaminated toothbrush might cause mouth infection to recur and it may extend further to the throat (Cobb, 1920). Malmberg et al. reported that toothbrushes used by children had growth of molds and enteric yeasts. The coliforms which originate from the toilet have also been found in toothbrushes (Saleh, 2011). Kauffman gave a few techniques of sanitisation like drying toothbrushes under sunlight, immersion in formaldehyde and use of table salt on the toothbrush surface in order to absorb moisture from it. The other procedures include spraying antimicrobial solutions, washing the toothbrushes in a dishwasher, use of disinfecting solution. Microwave and ultraviolet light are the other methods (Kauffman, 1930).
Among the chemical agents, Chlorhexidine solution (CHX) has been proven to be an effective toothbrush disinfectant. It has also been reported that it can kill various microbial species (Bhat et al., 2003). It has been shown that Listerine presented an ef icient reduction of microorganisms. Neem and turmeric are some of the herbal products which were investigated for their effectiveness as antimicrobial solutions (Muller et al., 1989). Though various sanitization techniques have been tried, procedures for sustaining the cleanliness of toothbrushes are not yet described properly (Sato et al., 2005). Therefore, this study was undertaken to ind the effectiveness of Chlorhexidine, Listerine, and herbal mouthwashes for toothbrush disinfection.

MATERIALS AND METHODS
The patients visiting the department of periodontics, SRM Kattankulathur Dental College and Hospital were recruited for the study. This study was approved by and ethical clearance was obtained from the Institutional Ethical Committee (IEC/2020/564). The participants were informed about the procedure and were asked to sign the informed consent. 20 systemically healthy gingivitis patients aged between 22-28 years having minimum of 20 teeth were included in the study. Patients with periodontitis, patients using any form of partial dentures, patients who cannot follow brushing technique, patients who are edentulous, patients undergoing orthodontic treatment, patients with systemic conditions predisposing to periodontal disease were excluded from the study.
New identical multi-tufted nylon toothbrushes with soft bristles were given to each participant, before the start of the study. The participants were instructed to brush twice daily with the toothbrush that has been given and to use running tap water to rinse the used toothbrushes. They were collected from all the study participants after one week. The bristles of each toothbrush were cut and kept in separate vials containing 1ml of transport media (thioglycolate medium). Then, they were sent for microbiological evaluation (Maratha Mandal's Central Research Laboratory, Belgaum). Along with that, two unused packed toothbrushes were also cultured to assess bacterial growth, if any.

Pre-sanitization
The bristles were transferred from each vial to the test tube containing distilled water. Then, the microbial analysis was done using distilled water on blood agar by spread plate technique. The incubation period was 24-48 hours at 37 • C to facilitate bacterial growth. Then, the colonies were counted using a digital colony counter and measured as colony forming units (CFU).

Post-sanitization
After measuring the colonies, the vials containing bristles were divided into four groups, each group containing ive vials; then, the bristles were immersed in the respective mouthwashes for one hour. Then, they were vortexed for 2 mins. The microbial analysis was done similarly and colony-forming units were counted.

RESULTS AND DISCUSSION
Microorganisms present in the mouth contaminate the toothbrush during oral use. After the use of a toothbrush, it is rinsed with plain water and stored in a place near or in the bathroom, where the moist conditions will enhance bacterial growth (Balappanavar, 2009).

Figure 1: Microbial analysis in fresh toothbrushes showed no colony formation
The microorganisms can remain on the toothbrushes for a certain period and can colonize it. After 24 hours of use of a toothbrush, it gets colonized to a great extent which was observed by Svanberg (1978). Streptococci harboured on children's toothbrushes may cause pharyngitis or tonsillitis, which has been shown in some studies (Fischer, 1999). Furthermore, improperly rinsed toothbrushes may lead to bacteremia (Muller et al., 1989).Therefore, the decontamination of toothbrushes is essential to maintain good oral health. Hexidine mouthwash, Hydrogen peroxide, Dettol and Cetylpyridinium chloride are some of the chemical disinfecting solutions that have been tried. Neem and turmeric are the herbal products which were explored for their ef icacy as antimicrobial solutions. Some studies suggest that herbal products and chemical disinfection are effective in decontamination of toothbrushes (Muller et al., 1989).

Figure 2: Colonies formed after sanitization of toothbrushes
The present study was undertaken to evaluate the ef icacy of three types of mouthwash, namely Chlorhexidine, Listerine and herbal mouthwashes as a disinfecting solution. For this purpose, 20 systemically healthy patients were asked to brush twice daily and wash it under running tap water. Then, the brushes were collected in 7 days which was in accordance with the studies done by Sogi et al. (2002); Bhat et al. (2003).
On microbial analysis, fresh toothbrushes showed no colonies, which is represented in Figure 1. The colony-forming units of pre-sanitization procedure were in the range of 300-500 CFUs in all four groups. Before intervention (pre-sanitization), there was no signi icant difference in the CFUs among the groups.
In all the three test groups, there was a signi icant difference in the reduction of microbes after sanitization with respective mouthwashes. Table 1 shows the comparison of colony-forming units before and after sanitization. Figure 2 shows colonies formed after sanitization of toothbrushes. The highest reduction in colony-forming units among test groups was seen in group I (Chlorhexidine mouthwash) followed by group II (Listerine), then group III (herbal mouthwash) and least reduction was the group IV (distilled water).
In the present study, the stored toothbrushes showed different species of bacteria after oral use. Previous studies which found similar outcomes have indicated that toothbrushes will have a risk of colonization by bacteria after daily use (Fischer, 1999;Sconyers et al., 1973;Schlein et al., 1991).
CHX solution results in the present study are in accordance with the results of Balappanavar (2009); Bhat et al. (2012) in which bacterial count reduction was seen with CHX solution. CHX has a broadspectrum antimicrobial effect and it is has been established as the gold standard. In contaminated toothbrushes, this solution has shown to inhibit bacterial count effectively, is cost effective and easily available. Therefore, CHX, as a disinfecting solution is recommended.
Results in Listerine group reveals a more effective reduction in the total viable bacterial count when compared to herbal mouthwash. A disinfecting solution like herbal mouthwash which was used in the present study also showed a reduction in the number of colony-forming units. This shows that it has an antimicrobial property. Storage conditions of toothbrushes play the main role in bacterial survival (Dayoub et al., 1977).
Signi icant differences were found before and after the sanitization of toothbrushes. This implies that a proper and de inite decontamination procedure is required for the toothbrushes after oral use. The limitations of the study include small sample size, no direct observation of the study participants and no identi ication of speci ic microorganism. Further research is needed to understand the disinfection of toothbrushes to come up with an effective protocol.

CONCLUSIONS
Toothbrushes kept inside the bathroom can be a source of contamination with microbes; its disinfection may prevent the occurrence of oral infections. It can be concluded that chlorhexidine mouthwash is more effective than other mouthwashes as a disinfecting agent in reducing the microbial counts. Further investigations are required to analyse different herbal products as disinfecting solution at different concentrations.