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ORIGINAL ARTICLE
Year : 2021  |  Volume : 19  |  Issue : 4  |  Page : 315-319

Qualitative and quantitative evaluation of bacterial contamination of toothbrushes kept in different sanitary settings: A comparative study


1 Depatment of Pedodontics and Preventive Dentistry, Himachal Dental College, Sundar Nagar, Himachal Pradesh, India
2 Depatment of Microbiology, Himachal Dental College, Sundar Nagar, Himachal Pradesh, India

Date of Submission10-Mar-2021
Date of Decision10-Apr-2021
Date of Acceptance09-Nov-2021
Date of Web Publication15-Dec-2021

Correspondence Address:
Meenakshi Thakur
(Consultant Pedodontist) Klsm Rotary Eye and ENT Hospital, Udhampur, Jammu and Kashmir - 182 101
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_36_21

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  Abstract 


Background: Oral health is an integral part of general health and toothbrushes are the most commonly used oral hygiene aid. Unfortunately, proper care of toothbrush is often neglected and is kept in bathrooms which are a good place to harbor millions of microorganisms. Aim: To assess the bacterial contamination of toothbrushes for Streptococcus mutans, Staphylococcus aureus, Candida albicans, and Lactobacillus when kept in different sanitary settings before and after the use of disinfectant. Materials and Methods: A total of 60 volunteers were randomly selected between the age of 6–16 years. Toothbrushes were grouped into two categories: Group 1: Outside the bathroom. Group 2: Within the bathroom with attached toilet. Group 2 was further divided into two subgroups: Group 2 (i): Kept individually in separate containers. Group 2 (ii) kept with that of family members and siblings within the same container. The present study was carried for 3 months in two phases with the same participants both before and after the use of disinfectant. Results: Microbial contamination was highest in Streptococcus and least in Lactobacillus before and after the use of disinfectant when toothbrushes were kept outside the bathroom and microbial contamination was similar in both Streptococcus and Lacctobacilus when kept inside the bathroom with attached toilet in separate container and same container both before and after the use of disinfectant. Conclusion: A high level of pathogenic microorganisms were seen in toothbrushes kept in the bathroom for 3 months which were reduced by dipping them in 0.12-% chlorhexidine gluconate.

Keywords: Disinfectant, microbial contamination, sanitary settings, toothbrush contamination


How to cite this article:
Thakur M, Mittal S, Sharma AK, Sharma DK, Gupta KK, Pathania V. Qualitative and quantitative evaluation of bacterial contamination of toothbrushes kept in different sanitary settings: A comparative study. J Indian Assoc Public Health Dent 2021;19:315-9

How to cite this URL:
Thakur M, Mittal S, Sharma AK, Sharma DK, Gupta KK, Pathania V. Qualitative and quantitative evaluation of bacterial contamination of toothbrushes kept in different sanitary settings: A comparative study. J Indian Assoc Public Health Dent [serial online] 2021 [cited 2022 Jan 23];19:315-9. Available from: https://www.jiaphd.org/text.asp?2021/19/4/315/332534




  Introduction Top


Oral health is an essential part of general health. The maintenance of oral hygiene becomes a crucial factor as it directly and indirectly reflects the overall well-being of an individual.[1] Oral diseases are controlled by decreasing the bacterial load in the oral cavity and this can be achieved by maintaining good oral hygiene.[2] Oral hygiene was in practice as early as 3000 B.C. by the Sumerians.[3] Brushing teeth is the basic mode of oral hygiene practice. Miswak, Neem, and Babool chewing sticks were the sole oral hygiene aids used by different populations in earlier days.[1] Cleaning teeth with powder of gull nut and pepper was recommended by the Arabians. The Chinese were the earliest people to use the “Chewstick” made of plant limbs or roots with one end beaten into a soft fibrous condition used for scrubbing and brushing the teeth.[3]

Later, toothbrush writhe in as the main component among all oral hygiene methods, as a result of civilization. To promote oral health and prevent dental diseases toothbrushes are the most commonly used oral hygiene aid.[1]

Toothbrush is taken in consideration as a niche for many microbes.[4] The toothbrush can harbor different types of microorganisms such as bacteria, fungi, and viruses, facilitating translocation and transmission of these organisms.[5]

The average life span of a manual toothbrush is approximately 3 months.[1],[6] Hence, ADA recommends changing toothbrushes once in 3–4 months depending on fraying of toothbrush bristles as it reduces the cleaning effectiveness.[1] Attempts were made to lower bacterial survival time, deter colonization, and biofilm inhibition by toothbrushes containing antibacterial agents and sterilization methods of brushes were devised.[7]

It is of uttermost importance to educate the public about proper storage, replacement, and disinfection of toothbrushes as there is a complete lack of awareness among the public regarding toothbrush maintenance. There are numerous procedures described to reduce the microbiological load of toothbrushes, such as continuous brush exchange, submerging the brush into microbicide solutions, spraying antiseptic solutions or using ozone, all of which have been successful in decontaminating the brushes but are not always inexpensive or easy to perform,[8] hence the objective of our study is to evaluate the effect of storage condition on microbial contamination of toothbrush head between the bristle tufts both before and after the use of disinfectant.


  Materials and Methods Top


The present cross-sectional study was carried out on 60 randomly selected volunteers between the age group of 6–16 years attending outpatient Department of Pedodontics and Preventive Dentistry, Himachal Dental College Sundernagar. Stratified random sampling method was used. Ethical Clearance has been taken from the Institutional Ethical Committee (HDC/Ethical/Pedo./2016/12) review board. The medical history of the participants was obtained to rule out any existing oral and systemic diseases.

Study design

In the present study, oral hygiene kit containing toothbrush and toothpaste was provided to all the participating children (without any oral lesion or cavity, if cavities were there, they were restored) after proper oral hygiene instructions and brushing demonstrations instructed to both children and their parents. After brushing, depending on different sanitary conditions, toothbrushes were kept and were grouped into two categories:

  • Group 1: Toothbrushes were kept outside the bathroom
  • Group 2: Toothbrushes were kept within the bathroom with attached toilet.


Group 2 was further divided into two subgroups:

  • Group 2 (i): Toothbrushes were kept individually in separate container
  • Group 2 (ii): Toothbrushes were kept with that of family members and siblings within the same container.


The present study was carried out in two Phases with the same participants: (Phase I and Phase II). 20 participants were present in Group 1and 40 participants were in Group 2 as it was further divided into two subgroups with 20 participants in each subgroup.

Phase I

Volunteers had followed the following instructions:

  • Brushing twice daily
  • No use of mouthwashes
  • No use of disinfectant/antimicrobial for toothbrush.


Phase II

Same volunteers of Phase I had participated in Phase II.

Volunteers had followed the following instructions:

  • Brushing twice daily
  • No use of mouthwashes
  • Dipping the toothbrush in the chlorhexidine disinfectant (0.12%) for 20 s.


Toothbrushes were collected from all the participants after 3 months of regular use in a sterile container, after drying the toothbrush naturally. Maintaining aseptic measures and adhering to the strict protocol, handles of toothbrushes were cut off using heat sterile scissors. The bristles of toothbrushes were trimmed using scissor. After that with the help of sterile toothpick, a streak of deposit between the bristle tufts were taken and inoculated into the glucose broth, which supports the growth of microorganisms and further incubated for 24 h.

Sterilized agar plates were selected and hard deposit growth was inoculated having different agar media with the help of inoculation loop following incubation and colony-forming units (CFU) were counted manually. Identification of microorganisms such as Streptococcus mutans, Streptococcus aureus, Lactobacillus, and Candida albicans were done using standard microbiological protocols.

Different growth media used for bacterial growth

Mitis salivarius agar media was used for the growth of Streptococcus mutans, Sabourauds dextrose agar was media used for the growth of Candida, Rogosa SL agar media was used for the growth of lactobacilli, and Mannitol salt agar was used for the growth of Staphylococcus aureus. Serial dilution was done for CFU count.

Bacterial load was checked by calculating microorganisms per ml from bacterial suspensions.

Bacterial load/ml = number of colonies/amount plated × dilution factor

The statistical analysis was performed using SPSS 21 software (SPSS Inc., Chicago, IL,USA). ANOVA and t-test were used for comparative analysis. The P ≤ 0. was considered statistically significant.


  Results Top


In the present study along with these bacteria, Mycobacterium species (Cornybacterium diptheriae) growth was also seen in toothbrushes which were kept outside the bathroom which is in accordance with a study done by Orogu and Ehiwario.[9]

[Table 1] depicts the microbial contamination load of the toothbrushes when kept outside the bathroom in individual containers (Phase [i] and Phase [ii]). The maximum load was obtained for Streptococcus strain and minimum for Lactobacillus in both the subgroups who did not use disinfectant and who used disinfectant. The change in the contamination load before and after the use of disinfectant for the different bacterial strains was assessed by paired t-test. The change in the reduction of bacterial load for Streptococcus, Staphylococcus, and Candida before and after use of disinfectant was statistically significant.
Table 1: Comparison of different levels of microbial contamination of toothbrushes kept outside the bathroom before and after using disinfectant

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[Table 2] depicts the microbial contamination load of the toothbrushes when kept inside the bathroom with attached toilet in separate container with and without disinfectant (Phase [i] and Phase [ii]). The maximum load was obtained for Streptococcus strain and minimum for Lactobacillus both before after the use of disinfectant.
Table 2: Comparison of different microbial contamination of toothbrushes kept inside the bathroom with attached toilet in separate containers before and after using disinfectant

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[Table 3] depicts the microbial contamination load of the toothbrushes when kept inside the bathroom in the same container with and without disinfectant. The maximum load was obtained for Streptococcus strain and minimum for Lactobacillus both before after the use of disinfectant. The change in the reduction of bacterial load for Streptococcus, Staphylococcus, and Candida before and after use of disinfectant was statistically significant while for Lactobacillus showed a minimal load decrease only and are statistically nonsignificant.
Table 3: Comparison of different microbial contamination of toothbrushes kept inside the bathroom in the same container before and after using disinfectant

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Two separate analyses were done to evaluate whether the use of disinfectant decreases microbial load across all storage conditions.

The percent decrease of the microbial load was calculated for all different types of bacterial strain except Lactobacillus in all three storage conditions as the contamination of Lactobacillus was very less. Next, the change for each type of microbial load across three storage conditions was analyzed using one-way ANOVA. The results are presented in [Table 4]. The ANOVA revealed that the percentage decrease of all three different microbes did not vary significantly across different storage conditions.
Table 4: ANOVA results for percent decrease in contamination load for different microbes separately across three different storage conditions

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  Discussion Top


The present cross-sectional study was carried out to evaluate the microbial contamination of toothbrushes used by children between the age group of 6–16 years in different sanitary settings. The present study was done in the Department of Pedodontics and Preventive Dentistry, Himachal Dental College, Sundernagar.

Toothbrush head between the bristle tufts was selected to assess the microbial contamination as it not only harbors microorganisms but also provides a favorable environment for their growth. Toothbrush contamination could pose a significant risk of dissemination of infection for certain patients such as immune suppressed, cardiopathic, organ transplant recipients.[1]

Toothbrush often become contaminated with microorganisms which originate not only from the oral cavity but also from the environment in which they are stored. Dispersed aerosols from toilet flushing, wet environment of the bathroom, and contaminated finger contact contribute to toothbrush contamination. Most of the families generally store their toothbrushes in a common container which can lead to cross-infection. There is a possibility of re-infection when the individual uses the contaminated toothbrush. In 1920, Cobb was the first investigator to report the recurrence of infection in mouth in patient using contaminated toothbrush.[8]

Taking this aspect into consideration, the present study was undertaken to evaluate the presence of microorganisms in the toothbrushes and the effect of different sanitary settings and disinfectants to decontaminate them.

In our study, S. Mutans was the most common bacteria found on the used toothbrushes and the results were similar to studies done by Raiyani et al.,[2] Saini and Kulkarni,[10] Quirynen et al.,[11] Nascimento et al.,[12] Svanberg[13] and Karibasappa et a1.[1] Talib et al.[14] stated that the Staphylococci was one of the mostly found microorganisms on many toothbrushes which was contrary to the present study. Staphylococci are nonfastidious organisms that grow well on a range of selective media; their presence may be related to the fact that most of the individuals used their fingers during post brushing rinsing of their toothbrushes.

In the present study along with these bacteria, Mycobacterium species (Cornybacterium Diptheriae) growth was also seen in toothbrushes which were kept outside the bathroom which is in accordance with a study done by Orogu and Ehiwario.[9]

S. mutans, S. aureus, Candida, and Lactobacilli were observed after 3 months from toothbrushes which were used twice daily and kept with family members in a same container. Healthy participants participated in this study with no oral lesions like oral candidiasis and dental caries. If there were any dental caries present, tooth was restored. The predominant microorganism which was highest in number were S. mutans followed by other microorganisms. Orogu et al. observed Lactobacillus presence in a comparative study of bacteriological examination of daily used toothbrushes stored in the bathroom[12] whereas no growth of Lactobacillus was detected on any of the Rogosa plates which may be due to the inclusion of the small number of subjects in their study.[9]

In accordance with one analysis, the maximum load was obtained for Streptococcus strain and minimum for Lactobacillus, both before and after the use of disinfectant. Streptococcus, Staphylococcus, and candida contamination showed a significant decrease after the use of disinfectant. Paulo Nelson-Filho et al. stated that immersion of the toothbrush in 0.12%chlorhexidine gluconate is an efficient method for toothbrush disinfection which showed similarity to our study.[15]

In our evaluation in outside storage conditions, maximum decrease was observed for Candida contamination (53%), followed by Streptococcus (44%) and least reduction was observed for Staphylococcus (35%). However, this difference was not significant and was in accordance with a similar study conducted by Sukhabogii et al.,[6] Nanjunda-Swamy et al.,[16] Ankola et al.[17] Contamination of Lactobacillus was nonsignificant that is why it was not included in this intracomparison analysis.

In separate storage conditions, our investigation revealed maximum decrease for Candida contamination (57%), intermediate for Staphylococcus (47%), and minimum for Streptococcus (38%), however, these differences were not significant (F = 0.660, P = 0.5255, df 2, 26) as showed in a study done by Sukhabogii et al.[6]

In the same storage condition, the maximum decrease was observed for Candida (51%) and least for Staphylococcus (37%) and intermediate reduction for Streptococcus (42%) in the present study, and these differences were not significant (F = 1.008, P = 0.378, df 2, 27).

Limitations

  • Only one disinfectant was used, i.e., chlorhexidine gluconate which showed the best antimicrobial efficacy, but few studies also showed that other disinfectants like EDTA, 50% White vinegar proved to be effective disinfectant which is cost-effective and easy to access. Nilofer et al.,[18] Basman et al.[19]
  • Toothbrushes used for only 3 months duration that were examined for microbial contamination was the deficit in this study. Further studies should have been conducted to assess microbial contamination of toothbrush head used for 15 days, 1.5, 2, and 2.5 months, to gain knowledge about the pathogenicity of microorganisms and to evaluate the occurrence of any infections in the subjects following the use of contaminated toothbrushes.



  Summary and Conclusion Top


The present comparative study was conducted to evaluate qualitatively and quantitatively bacterial contamination of toothbrushes in different sanitary settings.

The following conclusions can be drawn:

  • A high level of pathogenic microorganisms were seen in toothbrushes which were kept in the bathroom for 3 months and hence keeping the toothbrush in the room is better as compared to those kept in the bathroom with attached toilet
  • There was a reduction of microbial contamination by dipping toothbrushes in 0.12% chlorhexidine gluconate after brushing in all three settings.


There is a need for disinfection methods that are rapidly effective, nontoxic, and easily implemented. Even though we have basic knowledge regarding disinfection procedures for our instruments and environment, certain things are practically not implemented such as decontamination of toothbrushes. Some of the diseases which could be transmitted through contaminated toothbrushes have been unnoticed. Therefore, there is a necessity to concentrate on disinfection of toothbrushes, thereby preventing infections, reinfections, or cross infections.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Karibasappa GN, Nagesh L, Sujatha BK. Assessment of microbial contamination of toothbrush head: An in vitro study. Indian J Dent Res 2011;22:2-5.  Back to cited text no. 1
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Raiyani CM, Arora R, Bhayya DP, Dogra S, Katageri AA, Singh V. Assessment of microbial contamination on twice a day used toothbrush head after 1-month and 3 months: An in vitro study. J Nat Sci Biol Med 2015;6 Suppl 1:S44-8.  Back to cited text no. 2
    
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Contreras A, Arce R, Botero JE, Jaramillo A, Betancourt M. Toothbrush contamination in family members. Rev Clin Periodoncia Implantol Rehabil 2010;3:24-6.  Back to cited text no. 5
    
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Orogu JO, Ehiwario NJ. Comparative study of bacteriological examination of daily use toothbrushes stored in the bathroom and room. E3 J Sci Res 2016;4:037-46.  Back to cited text no. 9
    
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Saini R, Kulkarni V. Toothbrush. A favorable media for bacterial growth. Int J Exp Dent Sci 2013;2:27-8.  Back to cited text no. 10
    
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Quirynen M, de Soete M, Pauwels M, Goossens K, Teughels W, van Eldere J, et al. Bacterial survival rate on tooth- and interdental brushes in relation to the use of toothpaste. J Clin Periodontol 2001;28:1106-14.  Back to cited text no. 11
    
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Nascimento AP, Faria G, Watanabe E, Ito IY. Efficacy of mouthrinse spray in inhibiting cariogenic biofilm formation on toothbrush bristles. Braz J Oral Sci 2008;7:1489-92.  Back to cited text no. 12
    
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Svanberg M. Contamination of toothpaste and toothbrush by Streptococcus mutans. Scand J Dent Res 1978;86:412-4.  Back to cited text no. 13
    
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Talib RA, Alnaimi RJ, Mustafa EA. The microbial contamination of toothbrushes and their disinfection by antimicrobial solutions. Al Rafidain Dent J 2008;8:144-50.  Back to cited text no. 14
    
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Nelson Filho P, Macari S, Faria G, Assed S, Ito IY. Microbial contamination of toothbrushes and their decontamination. Pediatr Dent 2000;22:381-4.  Back to cited text no. 15
    
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Nanjunda-Swamy K, Madihalli AU, Prashanth MB. Evaluation of Streptococcus mutans contamination of tooth brushes and their decontamination using various disinfectants – An in vitro study. J Adv Oral Res 2011;2:23-30.  Back to cited text no. 16
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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