Int J Oral-Med Sci 22（1）:23–34, 2023

23

Original Article
Low - environmental - impact Bamboo Toothbrushes Demonstrate Comparable Hygienic Condition to That of Plastic and Biomass Plastic Toothbrushes after Useand Storage
Yoshimasa Ishii,1 Itaru Suzuki,2 Haruka Sakazume - Suzuki,2 Yukitoshi Kurakawa,3 Toshikazu Uchiyama,3Yuichiro Okada,4 Teruaki Nagashima,5 Chieko Taguchi,2 and Kazumune Arikawa2
1

Department of Dental and Oral Surgery, Zama General Hospital, Zama, Kanagawa 252 - 0011, Japan
Department of Community Oral Health, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271 - 8587,
Japan
3
Department of Operative Dentistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271 - 8587, Japan
4
Department of Histology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271 - 8587, Japan
5
Nihon University Graduate School Dentistry at Matsudo, Community Oral Health, Matsudo, Chiba 271 - 8587, Japan
2

Article History

Abstract

Received 26 October 2023

Brushing with a toothbrush is effective for removing oral biofilms. However, tooth-Accepted 15 November 2023

brushes can be a source of infectious diseases after use. Recently, the environmental
impacts of plastics, such as marine pollution, have become an issue. This includes thedisposal of plastic toothbrushes. Consequently, bamboo toothbrushes, with a low environmental impact, have become widely - used as alternatives. Although there are many
reports on the hygiene of plastic toothbrushes after use, there are few reports on bamboo toothbrushes. Thus, we compared the hygiene of bamboo toothbrushes with thoseof plastic and biomass plastic toothbrushes after use and storage. Three types of toothbrushes were immersed in bacterial cultures and washed, and the number of bacteriaon the toothbrushes after 12 and 24 h of storage was examined. The number of bacteriaon the bamboo toothbrushes immediately after immersion was significantly higher thanon the other two types, but this number was 95 – 99％ lower after 12 – 24 h of storage.
No differences were observed for different bacterial species（Actinomyces oris and Streptococcus mutans）
or different storage conditions（wet and dry）. However, pre - wetting
before immersion significantly reduced the number of bacteria on the bamboo tooth-

Keywords:

brushes. These results indicate that the hygienic conditions of bamboo toothbrushes after 12 – 24 h of storage were comparable to those of plastic or biomass toothbrushes.

bamboo toothbrush, plastic, SDGs,

Furthermore, pre - wetting the bamboo toothbrushes before use improved hygiene im-

Actinomyces oris,

mediately after use. Bamboo toothbrushes are a better choice as they provide equiva-

Streptococcus mutans

lent hygiene to that of plastic toothbrushes and also have a low environmental impact.

Introduction

by the attachment of initial colonizers to the tooth sur-

The formation of oral biofilms is thought to be import-

face, followed by the attachment and aggregation of a

ant in the pathogenesis of oral infections such as dental

wide variety of oral bacteria to form mature biofilms（1）.

caries and periodontal disease. Oral biofilms are formed

Although brushing with a toothbrush can remove oral

Correspondence to: biofilms（2, 3）, various bacteria have been reported to ad-

Itaru Suzuki
E-mail: suzuki.itaru@nihon-u.ac.jp
doi：10.5466/ijoms.22.23

here to toothbrushes following use. These include Enterobacteriaceae and Micrococcaceae（4）,Streptococcus, Actinomyces, Porphyromonas, and Fusobacterium（5）, which are

24:23–34, 2023
Int J Oral-Med Sci 22（1）

Table 1．Characteristics of the test toothbrushes

associated with oral infections, Staphylococcus aureus（S.

capita in Japan in 2014 was the second largest in the

aureus）, which is associated with nosocomial infections,world（16）. In addition to discarded trash, microplastics

Candida albicans（C. albicans）, which is associated withare also considered a problem（17）, and microplastics areoral candidiasis（6）, Klebsiella pneumoniae, which is asso-

released by the daily use of plastic toothbrushes（18）. In

ciated with the development of respiratory tract infec-

Japan, the “Law Concerning the Promotion of Resource

tions, and Streptococcus pyogenes, which is associated with

Recycling of Plastics” was enacted in 2021, which stipu-

the development of pharyngitis（7）. Moreover, Ryan et al.
reported that microflora from the environment may ad-lates that the emission of microplastics must be controlled by manufacturers of specific plastic - using prod-

here to toothbrushes after storage（8）. Interestingly, her-ucts（19）.

pes simplex virus type 1 has also been detected（9）.

Given this, several alternatives to plastic toothbrushes

In a study that investigated toothbrush storage meth-

are available, including bamboo toothbrushes. There is an

ods after use, microorganisms grew easily on toothbrushes that retain moisture, forming a self - contained ecosys-

abundance of bamboo in Japan, so it has been used fortem（10）. Therefore, toothbrushes may become a source

generation（20）. Bamboo toothbrushes have been shownof infection depending on how they are used and stored

to have a lower life cycle impact assessment than plastic（11, 12）. Currently, toothbrushes of various shapes andor electric toothbrushes（21, 22）. In other words, they are

sizes are being marketed, influenced by various research

considered to have a lower environmental impact.

various functions, including and a fuel for biomass power

reports and changes in consumer brushing objectives（3）.

There have been numerous reports on toothbrush hy-

In 2015, the United Nations adopted “Transforming

giene. However, to the best of our knowledge, there have

Our World: The 2030 Agenda for Sustainable Develop-

been no reports on the hygiene of bamboo toothbrushes.

ment,” which includes the Sustainable Development Goals

Therefore, the purpose of this study was to investigate（SDGs）（13）. One of the SDGs was “By 2025, prevent andthe hygienic conditions of bamboo toothbrushes after use

significantly reduce all forms of marine pollution, includ-and storage in an in vitro experimental system using bac-ing marine sedimentation and eutrophication, especiallypollution from land - based activities.” This goal describes

teria that have been reported to be present in the oral

the marine pollution caused by marine plastics and thefective endocarditis.

cavity and are potential causes of oral infections and in-concerns about the impact on ecosystems caused by theuptake and ingestion of marine plastics by marine organ-

Materials and Methods

isms（14）.

Test toothbrushes

The contribution of plastic toothbrushes to marine pol-Three types of toothbrushes were used in this study:

lution has been reported in many studies. Okano et al. re-bamboo toothbrushes（Astep Inc.）, biomass plastic tooth-

ported five plastic toothbrushes per 100 m in a survey ofbrushes（Company A）, and plastic toothbrushes（Company

persistent drifting debris ashore in Japan（15）. AccordingA）. The characteristics of the toothbrushes are presented

to a report by the United Nations Environment Pro-in Table 1. All toothbrushes were commercially available,

gramme（UNEP）, the total amount of plastic waste per

not the type used in medical institutions. New and un-

Int J Oral-Med Sci 22（1）:23–34, 2023

opened toothbrushes were used in the experiments.

25s to remove adhering bacteria and used as samples forquantification.

Test strains
Actinomyces oris
（A. oris）
strain MG1（23）, an initial colonizer, and Streptococcus mutans（S. mutans）
strain UA159（24）, a cariogenic bacterium, were used in this study.

Evaluation of the number of adherent bacteria
Next, the number of bacteria adhering to the toothbrushes was quantitatively evaluated. Each sample containing adherent bacteria was serially diluted 10 to 1000

Immersion of toothbrushes in bacterial culture

times using sterile PBS, and 100 µl of each sample con-

The method of immersing each toothbrush in the bac-taining adherent bacteria was seeded on BHI agar or Mi-

terial culture was based on several reports with sometis Salivarius（MS）agar in triplicate. BHI agar medium

modifications（25, 26, 27）. First, each strain was incubated

was used to quantify A. oris, and MS agar was used toin brain heart infusion（BHI）
liquid medium at 37°C for
16 – 18 h under 5 ％ CO2. After centrifugation of the cul-quantify S. mutans. The number of colonies formed onture medium at 3000×g for 10 min at 4°C, the pellet washerent bacteria was calculated.

the agar was measured and the average number of ad-

washed with sterile phosphate buffer solution（PBS）andcentrifuged again under the same conditions. After cen-

Statistical analysis

trifugation, the supernatant was removed and the pellet

Three independent experiments were performed. One -was suspended in sterile PBS and used for subsequent

way analysis of variance（ANOVA）was used for the sta-experiments. The bacterial culture was then adjusted totistical analysis of differences in the number of adherent

OD600=0.1 in sterile PBS and placed in a sterilized beaker.

bacteria among the three types of toothbrushes and at

Each unopened toothbrush was immersed in the adjusted bacterial solution for 3 min such that the head（25different storage times, followed by multiple comparisonsusing the Tukey or Games – Howell method. Student’smm）and neck（10 mm）
were immersed. In an additionalt - test was used for statistical analysis of the differences

experiment, bamboo toothbrushes were immersed inin the number of adherent bacteria between the dry andsterile distilled water（DW）
for 5 s before immersion inwet bamboo toothbrushes. The significance level for eachthe bacterial culture to reproduce wet conditions before

test was set at 0.05. Excel（Microsoft Excel 2019; Micro-

toothbrush use.

soft Co., Albuquerque, NM, USA）
and SPSS（IBM SPSS

After immersion, each toothbrush was removed fromthe beaker and rinsed with sterile DW for 3 s to remove

Statistics 28; IBM Corporation, Armonk, NY, USA）wereused for data analysis.

the excess bacterial culture. This method mimicked thewashing of each toothbrush under running water and

Results

was based on the method described by Kim et al.（28）.

Differences in the amount of bacteria on each toothbrush

After washing, each toothbrush was placed in a dry orin a dry environment

wet environment for 12 or 24 h. The storage environment

First, we investigated differences in the number of oral

in this study was established according to Katsuno etbacteria on each toothbrush when stored in a dry envi-al.（29）. Each toothbrush was placed upright in a disin-ronment. For A. oris, the highest number of adherent

fected box in a room（room temperature 27°C, humidity
40 – 50％）and covered with a lid to prevent falling bacte-

bacteria was observed on the bamboo toothbrush at base-ria from adhering to it. The box was not sealed to ensure

toothbrushes; however, the differences were not statisti-

constant ventilation. On the other hand, the wet environ-cally significant. The number of adherent bacteria after

ment was conducted in a sealed box with the humidity

12 h and 24 h of storage was lower than the number ofadjusted to 70 - 80%. At 0 h（immediately after immersion

adherent bacteria at 0 h for all toothbrushes; however,［baseline］）, and after 12 and 24 h of storage, the tooth-the differences were only statistically significant for thebrushes were collected, disassembled into bristles andbamboo toothbrush. In addition, the number of adherent

bodies directly or with sterile pliers, and immersed in 10

bacteria after 24 h of storage was lower than the number

ml of sterile PBS. The bristles were then vortexed for 30

of adherent bacteria after 12 h of storage for all tooth-line（0 h）, followed by the biomass plastic and plastic

:23–34, 2023
Int J Oral-Med Sci 22
（1）

26

a

b

Fig. 1．
（a）Number of Actinomyces oris on toothbrushes stored under dry conditions.
（b）
Number of Streptococcus mutans bacteria
on toothbrushes stored under dry conditions. The vertical axis indicates the number of CFU per 100 µl and the horizontal
axis indicates the storage time（0 h, 12 h, 24 h）. Black bars indicate bamboo toothbrushes, white bars indicate biomass
plastic toothbrushes, and grey bars indicate plastic toothbrushes. Data are expressed as mean ± standard deviation（SD）
for the three independent experiments. Statistical analysis of the differences in the number of adherent bacteria between
the three toothbrushes and storage time is performed using one - way ANOVA, followed by multiple comparisons using
either the Tukey or the Games – Howell method. ＊P < 0.05, ＊＊P < 0.01.
Table 2．Percentage reduction of bacteria on toothbrushes stored under dry conditions.

brushes; however, no statistically significant differences

the highest number of S. mutans, followed by the biomass

were observed（Fig. 1a）.
We calculated the percentage decrease in the number

plastic and plastic toothbrushes. The amount of S. mutans on each toothbrush was approximately 2 – 3 timesof A. oris on each toothbrush from baseline（0 h）to 12 hhigher than of the amount of A. oris, but the patternsof storage and from baseline（0 h）
to 24 h of storage. The

were similar. The number of S. mutans decreased be-

reduction rates of A. oris on the bamboo toothbrush were

tween 0 h and 12 h and between 0 h and 24 h on all

98.49％ and 99.82％ after 12 h and 24 h of incubation, re-toothbrushes with a statistically significant decrease forspectively. The reduction rate of A. oris on the biomass

the bamboo and biomass plastic toothbrushes. No statisti-

plastic toothbrush was 91.41％ and 100％ after 12 h andcally significant reduction in bacteria was observed be-

24 h of storage, respectively. The percentage reduction of

tween 12 h and 24 h on any toothbrush（Fig. 1b）. The

A. oris on the plastic toothbrush was 92.11％ and 100％

percentage decrease in the amount of S. mutans on each

after 12 h and 24 h of storage, respectively（Table 2）.

toothbrush from baseline（0 h）
to 12 h of storage and from

Next, the amount of S. mutans on each toothbrush wasbaseline（0 h）
to 24 h of storage was examined. The reduc-examined. At baseline（0 h）, the bamboo toothbrush hadtion rates of S. mutans on the bamboo toothbrushes wereInt J Oral-Med Sci 22（1）:23–34, 2023

a

27

b

Fig. 2．
（a）Number of Actinomyces oris on toothbrushes stored under wet conditions.
（b）
Number of Streptococcus mutans bacteria
on toothbrushes stored under wet conditions. The vertical axis indicates the number of CFU per 100 µl, and the horizontal axis indicates the storage time（0 h, 12 h, 24 h）. Black bars indicate bamboo toothbrushes, white bars indicate biomass
plastic toothbrushes, and grey bars indicate plastic toothbrushes. Data are expressed as the mean ± standard deviation（SD）in the three independent experiments. Statistical analysis of the differences in the number of adherent bacteria between the three toothbrushes and storage time is performed using one - way ANOVA, followed by multiple comparisonsusing either the Tukey or the Games – Howell method. ＊P < 0.05, ＊＊P < 0.01.
Table 3．Percentage reduction of bacteria on toothbrushes stored under wet conditions.

95.51 ％ after 12 h of storage and 98.46 ％ after 24 h ofsignificant decrease in the number of bacteria after 24 h

storage. The reduction rates for biomass plastic tooth-of storage compared to after 12 h of storage for all tooth-brushes were 96.63％ and 95.59％ after 12 h and 24 h ofbrushes（Fig. 2a）. The reduction rates of A. oris after 12

storage, respectively. The reduction rate of S. mutans on

h and 24 h of storage were 98.74％ and 97.64％ , respec-

plastic toothbrushes was 99.56％ after 12 h of storage andtively for the bamboo toothbrush. The reduction rates for

96.69％ after 24 h of storage（Table 2）.

the biomass plastic toothbrushes were 93.16％ and96.53％ after 12 h and 24 h of storage, respectively. The

Differences in the amount of bacteria adhering to eachreduction rates of the plastic toothbrush were 97.07％

toothbrush in a wet environment

and 95.02 ％ after 12 h and 24 h of storage, respectively

We investigated differences in the number of oral bac-（Table 3）.

teria on each toothbrush when stored in a wet environ-

The number of S. mutans cells on each toothbrush wasment. Compared to baseline（0 h）, the number of A. oris

determined. Compared to baseline（0 h）, all toothbrushes

bacteria was reduced after 12 h and 24 h of immersion

showed a decrease in the number of adherent bacteria af-

for all toothbrushes with statistically significant differenc-

ter 12 h and 24 h of storage with a statistically significant

es for the bamboo toothbrush. There was no statisticallydecrease for the bamboo toothbrush only. As in the wet

28:23–34, 2023
Int J Oral-Med Sci 22
（1）

a

b

Fig. 3．（a）Number of Actinomyces oris on toothbrush bristles stored under dry conditions.
The vertical axis shows the number of CFU per 100 µl and the horizontal axis shows the storage time
（0 h, 12 h, 24 h）. Blackbars indicate the nylon 610（bamboo toothbrush）, white bars indicate（biomass plastic toothbrush（PBT）, and grey bars indicate
the PBT（plastic toothbrush）. Statistical analysis of the differences in the number of adherent bacteria between the three bristle
materials and storage times is performed using one - way ANOVA, followed by multiple comparisons using either the Tukey orGames – Howell method. ＊＊P < 0.01.
（b）Number of Actinomyces oris on the head and neck of toothbrushes stored under dry conditions.
The vertical axis shows the number of CFU per 100 µl and the horizontal axis shows the storage time
（0 h, 12 h, 24 h）. The black,white, and grey bars indicate bamboo（bamboo toothbrushes）, plastic（biomass plastic toothbrushes）, and plastic（plastic toothbrushes）, respectively. Data are presented as the mean ± standard deviation（SD）for three independent experiments. Statisticalanalysis of the differences in the number of adherent bacteria between the three toothbrush body materials and storage times isperformed using a one - way ANOVA, followed by multiple comparisons using either the Tukey or the Games – Howell method.
＊＊P < 0.01.
Table 4a．Percent reduction of A. oris on toothbrush bristles after storage under dry conditions

condition, there was no statistically significant decrease

ment

in the number of bacteria between 0 h and 12 h of stor-

We examined the number of A. oris on the bristles,

age and between 0 h and 24 h of storage for any of thehead, and neck of each toothbrush. The bamboo tooth-

toothbrushes（Fig. 2b）
. The decrease in the number of S.

brush bristles used in this study were made of nylon 610,

mutans on each bamboo toothbrush after 12 h and 24 h ofand the biomass plastic and plastic toothbrush bristles

storage was 97.18％ and 96.83％ , respectively. The reduc-were made of polybutyleneterephthalate（PBT）. The

tion rates for the biomass plastic toothbrush were 95.45％

number of bacteria on the nylon 610 bristles at baseline（0

and 94.60％ after 12 h and 24 h of storage, respectively.

h）was significantly higher than that on the PBT bristles.

The plastic toothbrush showed a 95.59％ reduction after

However, there was a significant decrease between base-

12 h of storage and a 97.25％ reduction after 24 h of stor-line（0 h）and 12 h and between baseline（0 h）and 24 h ofage（Table 3）.

storage（Fig. 3a）. The reduction in the number of adherent bacteria was 94.96％ after 12 h of storage and 94.46％

Differences in the amount of A. oris adhering to the bris-

after 24 h of storage（Table 4a）. Biomass plastic and plas-tles, head, and neck of each toothbrush in a dry environ-tic toothbrushes—both with bristles made of PBT—Int J Oral-Med Sci 22（1）:23–34, 2023

29

Table 4b．Percent reduction of A. oris on toothbrush body after storage under dry conditions

showed similar trends. The number of bacterial cells on
the bristles at baseline（0 h）
did not differ significantly between the two types of toothbrush. However, after 12 h
and 24 h of storage, the decreases were statistically significant（Fig. 3a）. The reduction in the amount of adherent bacteria was 99.92 – 99.93％ after 12 h of storage and100％ after 24 h of storage（Table 4a）.
The amount of A. oris on the head and neck of the
toothbrushes was also examined. For the bamboo toothbrushes, the amount of A. oris that adhered to the headand neck of the toothbrush at baseline（0 h）was significantly higher than that of the biomass plastic and theplastic toothbrushes. However, after 12 h and 24 h ofstorage, the number of A. oris decreased significantly（Fig. 3b）. Interestingly, the number of A. oris on thebamboo toothbrush before decomposition was higher
than that on the toothbrush after decomposition（Fig. 1a）.
The reduction rates for A. oris were 98.69％ and 99.27％
after 12 h and 24 h of storage, respectively（Table 4b）.
The number of bacteria on the biomass plastic tooth-Fig. 4．N umber of Actinomyces oris bacteria on bamboo
toothbrushes pre - wetted with water.
The vertical axis shows the number of CFU per 100 µl, andthe horizontal axis shows the conditions and storage time（0 h,12 h）. Data are presented as the mean ± standard deviation（SD）for the three independent experiments. Statistical analysis of the difference in the number of adherent bacteria between the two conditions is performed using the Student’st - test. ＊P < 0.05, ＊＊, P < 0.01.
Table 5．Percent reduction of A. oris on wet or not wet bamboo toothbrushes

brush at baseline（0 h）
was significantly higher than thaton the plastic toothbrush. After 12 h and 24 h of storage,
the decrease was statistically significant（Fig. 3a）. The reduction rate of A. oris was 92.95％ after 12 h of storage
and 99.05％ after 24 h of storage（Table 4b）. The numberof A. oris in the plastic was significantly reduced after 12h and after 24 h of storage（Fig. 3a）. The reduction rates

Next, we conducted an experiment in which bamboo

for A. oris were 98.91％ and 98.73％ after 12 h and 24 h

toothbrushes were immersed in sterile DW for 5 s imme-of storage, respectively（Table 4b）.

diately before immersion in the bacterial solution to reproduce the prewet conditions of the bamboo toothbrush-

Amount of A. oris on bamboo toothbrushes when pre - wet-es immediately before brushing. Immediately after

ted with water
The results showed that at baseline（0 h）, the number

immersion（0 h）, the number of A. oris was significantly
reduced in the pre - wetted condition compared with thatof attached bacteria on the dry bamboo toothbrushes wasin the non - wetted condition. In the pre - wetted condition,

significantly higher than that on toothbrushes made ofother materials（Figs. 1 – 3）.

the number of A. oris on the bamboo toothbrushes after
12 h of storage was significantly reduced compared to30:23–34, 2023
Int J Oral-Med Sci 22（1）that at baseline（Fig. 4）
. The number of adherent bacteria

A difference in the number of bacteria was observed im-

was 99.82％（Table 5）.

mediately after toothbrushes were immersed in the bacterial solution. This suggests that differences between

Discussion
In recent years, toothbrushes made of various materi-

tooth surfaces and toothbrush materials may influenceadhesion ability.

als have been manufactured and marketed as alternatives

After immersion of the bamboo toothbrushes in theto plastic toothbrushes. There have been several reports

bacterial solution for 12 h and 24 h, the number of adher-of bacteria and viruses have been detected on tooth-ing bacteria was reduced by more than 98 ％（Table 2）.

brushes after use（5 - 12）. However, few studies have fo-

Furthermore, the number of adherent bacteria was com-cused on toothbrushes made of alternatives to plastics.

parable to that of the plastic and biomass plastic tooth-

Therefore, we investigated the differences in the number

brushes（Fig. 1a, b）. Bamboo possesses antimicrobialof bacteria adhering to conventional plastic toothbrushes,

properties against S. mutans, S. aureus, and C. albicans

bamboo toothbrushes, and biomass plastic toothbrushes.

（35, 36）. The main components are furans and phenolic

A. oris and S. mutans—the strains used in this study—

compounds（36）. This suggests that the antimicrobial ac-

can cause infective endocarditis（30, 31）. A. oris and S.

tivity of bamboo significantly reduces the number of at-mutans are oral bacteria associated with oral infections.

tached bacteria. Spolidorio et al. reported that when S.

A. oris is an early adherent, adhering to tooth surfaces

mutans was deposited on plastic toothbrushes and al-

and serving as a scaffold for the formation of mature oral

lowed to stand, the amount of deposited bacteria in-biofilms, contributing to dental caries and periodontal dis-creased up to 2 h after immersion, but was not detected

ease. On the other hand, S. mutans contribute to caries

after 8 h（34, 37）. Furthermore, Wetzel et al. found thatdevelopment by forming glucans. It has also been report-

the number of adherent S. mutans, Lactobacilli, or C. al-ed to be detected on toothbrushes used in the actual oralbicans increased 2 h after children had used them; how-cavity（4, 5）. Therefore, our experiments were conducted

ever, the authors reported a decrease in the number ofusing A. oris and S. mutans. The amount of A. oris and S.

attached bacteria after 8 h（38）. Bunetel et al. also report-mutans adhering to the bamboo toothbrushes was theed a significant decrease in the number of adherent S.
mutans 24 h after immersion in a S. mutans - containing

highest when stored in a dry environment in this study（Fig. 1a, b）. Bamboo has many holes in its cross - section

fluid（26）. In other words, the results were consistentowing to the sparseness of its fibers, which makes it awith those of the plastic and biomass plastic experiments.

highly water absorbent material（32）. On the other hand,

These results suggest that bamboo toothbrushes stored

biomass plastic is a plastic partly comprising biomass as

in a dry environment are as hygienic as plastic or biomass - based plastic toothbrushes after 12 h.

the raw material, and its properties are not significantly
different from those of conventional plastics（31, 33）. InInterestingly, similar results were obtained when tooth-

other words, the number of adhering bacteria was higher

brushes were stored in a wet environment（Fig. 2b）. A

on bamboo toothbrushes compared to plastic and biomass

study using interdental brushes reported that storage inplastic toothbrushes owing to water absorption by thehumid environments affected bacterial growth（29）. Onbacterial solution and adhesion of the bacteria between

the other hand, it has been reported that there is no cor-

the bamboo fibers. On the other hand, more adherent

relation between the amount of microorganisms on tooth-bacteria were observed when S. mutans was used com-brushes after use and the storage method（39）. Based onpared to when A. oris was used. S. mutans utilizes gluco-

the results of this study, it was concluded that high or

syltransferase（GTF）
in the presence of sucrose to pro-

low humidity had no effect on the number of bacteria onduce extracellular polysaccharides, contributing to thetoothbrushes. However, it is possible that the humidity information of a strong biofilm on the tooth surface（34）.

the boxes decreased during the experiment, which may

The experimental conditions of the present study in-have affected the results.

volved adhesion to materials that are quite different to

A typical toothbrush consists of the head, neck, andthat of tooth surfaces, and the experiment was conducted

handle. The toothbrushes used in this study had a similar

in an environment in which no sugar source was present.

structure. The materials of the toothbrush body and bris-

Int J Oral-Med Sci 22（1）:23–34, 2023

31

tles were different. Therefore, to clarify whether differ-

was comparable to that of plastic and biomass plastic

ences in the bristles affect the amount of bacteria ad-

toothbrushes. However, the number of bacteria on bam-hered to the toothbrushes, toothbrushes were

boo toothbrushes immediately after immersion in thedeconstructed into the bristle and toothbrush body, andbacterial solution tended to increase more than that on

the amount of bacteria adhered to each was quantitative-

plastic and biomass plastic toothbrushes.

ly determined. The bristles of the bamboo toothbrushes

From these results, we concluded that immediately af-

used in this study were made of nylon 610, whereas thoseter use, bamboo toothbrushes were less hygienic thanof the biomass plastic and plastic toothbrushes wereother toothbrushes. Therefore, we investigated a method

made of PBT. On nylon 610, the amount of A. oris thatto reduce the number of bacteria on bamboo toothbrush-adhered to the bristles immediately after immersion ines immediately after immersion. The bamboo toothbrushes were pre - wetted with water prior to use. The number

the bacterial solution was significantly higher than thaton PBT（Fig. 3a）. In other words, the bristles of the bamboo toothbrush used in this study had a higher water ab-of A. oris on bamboo toothbrushes immediately after immersion was significantly reduced under pre - wet condi-

sorption capacity than the bristles of the other two types

tions compared with non - wet conditions. Under pre - wetof toothbrush, suggesting that it may be more susceptible

conditions, the number of A. oris on the bamboo tooth-to bacterial attachment. The reduction in the number ofbrushes after 12 h of incubation was significantly reduced

A. oris on the nylon 610 bristles was approximately 94％
after 12 – 24 h of storage（Table 4a）. In PBT, the bristles（Fig. 4）. The reduction in adherent A. oris was 99.81 ％of the biomass and plastic toothbrushes, the amount of A.

sorbent, it is possible that the concentration of bacteria

oris adhered to the bristles was significantly reduced af-

on the toothbrushes was reduced by the water. In otherwords, pre - wetting bamboo toothbrushes with water im-

ter 12 and 24 h of storage（Fig. 3a）. The reduction in thenumber of A. oris after 12 – 24 h of incubation was approximately 100％
（Table 4a）. Nylon 610 and PBT are excellent drying materials, and it has been reported that

（Table 5）. Because bamboo toothbrushes are water ab-

mediately before use may lead to improved hygiene immediately after use. Sluijs et al. reported no significant
difference in plaque scores between dry and pre - wetThis suggests that storing the bristles in a dry environ-

toothbrushes（41）. In other words, there is a small possibility that pre - wetting the toothbrush just prior to use

ment may have thoroughly dried them, making it difficult

may have no effect on plaque removal. Therefore, studies

for the bacteria to exist as live cells. In contrast, quantifi-

on the effects of wet bamboo toothbrushes on plaque re-cation of the number of A. oris on the head and neck ofmoval are required.

the drying rate is almost 100％ after 100 minutes（40）.

the toothbrushes showed that the number of bacteria on

The use of a toothbrush is considered the most effec-

the bamboo toothbrushes immediately after immersion

tive way to remove oral biofilms that are responsible forwas significantly higher than that on the biomass plastic

the development of dental caries and periodontal diseases

and plastic toothbrushes（Fig. 3b）. Interestingly, the num-（2）
. Several reports have been published on toothbrush

ber of A. oris on the bamboo toothbrush before decompo-

replacement periods. Raiyani et al. reported that tooth-sition was higher than that on the plastic toothbrush（Fig.

brushes should be replaced after 3 to 4 weeks because

1a and 3b）. This result suggests that the number of A.

toothbrushes used for 3 months were significantly moreoris on the bamboo toothbrushes was higher than that on
the pre - degradation toothbrushes, because the bacteria

contaminated than toothbrushes used for 1 month（42）.

were detected in the holes where the bristles had been

toothbrush that had been used 3 times a day for 3 min

implanted. The reason for the statistically significant de-each time for 10 weeks became 2.5％ thinner, which may

crease in the number of A. oris on all toothbrushes after
12 – 24 h of storage was similar, possibly due to the anti-

lead to a decrease in plaque removal effectiveness
（43）
. In

bacterial properties of bamboo and drying time
（Fig. 3b）.

the number of toothbrushes used is also expected to in-

These results indicated that the number of bacteria on

Suda et al. reported that the fibers of the bristles of aother words, as the frequency of toothbrushing increases,
crease.

the bamboo toothbrush after immersion in the bacterial

Currently, plastic toothbrushes are widely used; howev-

solution decreased significantly after 12 h of storage ander, in view of the environmental impact of plastics, tooth-

:23–34, 2023
Int J Oral-Med Sci 22（1）

32

brushes with a lower environmental impact should be se-

4.

Zinn MK, Schages L, Bockmühl D: The toothbrush

lected（13 - 19）. In this study, the hygienic conditions of

microbiome: impact of user age, period of use andbamboo toothbrushes with a lower environmental impact
after 12 – 24 h of storage were comparable to those ofbristle material on the microbial communities of
toothbrushes. Microorganisms, 8: 1379, 2020. doi:

plastic or biomass toothbrushes. Furthermore, pre - wetting the bamboo toothbrushes with water immediately

10.3390/microorganisms8091379.
5.

before use improved hygiene immediately after use. Intion of toothbrushes using Illumina MiSeq. Oral Biolother words, the hygienic conditions of plastic, biomass
plastic, and bamboo toothbrushes after storage were

Lee SY, Lee SY: Assessment of bacterial contaminaRes, 43: 180 - 188, 2019.

6.

Shang Q, Gao Y, Qin T, Wang S, Shi Y, Chen T: In-

comparable; therefore, the use of bamboo toothbrushes

teraction of oral and toothbrush microbiota affects

with a lower environmental impact may be effective inoral cavity health. Front Cell Infect Microbiol, 10: 17,

achieving the SDGs from the perspective of the dental
field.

2020. doi: 10.3389/fcimb.2020.00017.
7.

Ferreira CA, Savi GD, Panatto AP, Generoso JD, Bar-

This research had several limitations. First, the tooth-ichello T: Microbiological evaluation of bristles of fre-brushes tested in this study were only assessed after one

quently used toothbrushes. Dental Press J Orthod, 17:

exposure to bacteria, simulating a single use only. Second,
the number of bacteria present on the toothbrushes was

72 - 76, 2012.
8.

Blaustein RA, Michelitsch LM, Glawe AJ, Lee H, Hut-not assessed between 0 h and 12 h. Third, the levels oftelmaier S, Hellgeth N, Ben Maamar S, Hartmannbacteria were not assessed in actual human oral cavities.

EM: Toothbrush microbiomes feature a meeting

These three factors may limit the strength of the results.

ground for human oral and environmental microbiota.

In future studies, we plan to examine the use of bamboo
toothbrushes after multiple uses, 2 – 3 hours after immer-

Microbiome, 9: 32, 2021. doi: 10.1186/s40168 - 020 -sion, and in the actual human oral cavity.

00983 - x
9.

Frazelle MR, Munro CL. Toothbrush contamination: areview of the literature. Nurs Res Pract, 2012: 420630,

Acknowledgments

2012. doi: 10.1155/2012/420630

This research was supported by a research incentive

10. Saini R, Saini S: Microbial flora on toothbrush - at

donation from Astep Corporation. We would like to thank
Editage.jp（www.edit - age.jp）
for the English language ed-

greater risk. Ann Niger Med, 4: 31, 2010. doi:iting.
Conflict of Interest
The authors declare that they have no conflicts of interest associated with this manuscript.

10.4103/0331 - 3131.73882
11. Neal PR, Rippin JW: The efficacy of a toothbrush disinfectant spray - - an in vitro study. J Dent, 31: 153 157, 2003.
12. Asumang P, Inkabi SE, Inkabi S: Toothbrush bristles,
a harbor of microbes and the risk of infection. Int J
Oral Sci, 9: 25 - 27, 2019.

References
1.

2.

Kolenbrander PE, Palmer RJ Jr, Periasamy S, Jakubo-

world: the 2030 Agenda for Sustainable Development.

vics NS: Oral multispecies biofilm development andthe key role of cell - cell distance. Nat Rev Microbiol,

UN Doc. A/RES/70/1（September 25, 2015）.
14. Otsuka Y, Takada H, Nihei Y, Kameda Y, Nishikawa

8: 471 - 480, 2010.

K: Current status and issues of microplastic pollution

Freire PL de L, Campos PH, Carvalho FG, Rodrigues

research. JSWE, 44: 35 - 42, 2021.

JA, Diniz MB: Is pomegranate peels infusion effective
for disinfection of toothbrushes? Revista De Odontologia Da UNESP, 45: 253 - 257, 2016.
3.

13. United Nations General Assembly: Transforming ourWatanabe T, Kobayashi K: Recent toothbrush circumstances - Why is it wide flocking toothbrush
now? - . JSP, 60: 87 - 94, 2018.

15. Okano T, Yasumoto M, Andou S: Properties and categories of non - biodegradable waste items found inbeach litter. JSMCWM, 21: 94 - 105, 2010.
16. Valentina T, Ancuta M, Diana VZ, Marius MM, Dragos S: The influence of the plastic bags on the environment. Mater Plast, 55: 595 - 599, 2018.

Int J Oral-Med Sci 22（1）:23–34, 2023

33

17. Hartmann NB, Hüffer T, Thompson RC, Hassellöv M,

27. Shah S, Kunte S, Jagtap C, Jajoo S, Patel A, Shah P:

Verschoor A, Daugaard AE, Rist S, Karlsson T,

Efficacy of two different mouth rinses in inhibition of

Brennholt N, Cole M, Herrling MP, Hess MC, Ivleva

the growth of Streptococcus mutans on toothbrush

NP, Lusher AL, Wagner M: Are we speaking thesame language? Recommendations for a definition

bristles. JDRR, 8: 261 - 266, 2021.
28. Bélanger - Giguère K, Giguère S, Bélanger M: Disinfec-

and categorization framework for plastic debris. En-

tion of toothbrushes contaminated with Streptococcus

viron Sci Technol, 53: 1039 - 1047, 2019.

mutans. Am J Dent, 24: 155 - 158, 2011.

18. Fang C, Gopalan S, Zhang X, Xu L, Niu J, Naidu R:

29. Katsuno Y, Sato T, Aiba C: The relationship between

Raman imaging to identify microplastics released

the bacterial contamination of interdental brushes

from toothbrushes: algorithms and particle analysis.

and interdental brush cleaning and storage condi-

Environ Pollut, 337: 122510, 2023. doi: 10.1016/j.en-

tions. JNOHS, 9: 58 - 62, 2019.

vpol.2023.122510

30. Phichaphop C, Apiwattanakul N, Wanitkun S, Boon-

19. “Law for Recycling Plastic Materials” in Japan, e - gov

sathorn S: Bacterial endocarditis caused by Actino-

portal, Ministry of the Environment. Available from:https://elaws.e - gov.go.jp/document?lawid=503AC000

myces oris: first reported case and literature review.

0000060, cited 14 Oct 2023.

2324709620910645, 2020. doi: 10.1177/2324709

20. Kodani J, Esaki K: Effect of the duration of abandonment on development of understory vegetation in

J Investig Med High Impact Case Rep, 8:620910645
31. Nomura R, Matayoshi S, Otsugu M, Kitamura T, Ter-bamboo forests. Jan J For Environ, 54: 19 - 28, 2012.

amoto N, Nakano K: Contribution of severe dental

21. Lyne A, Ashley P, Saget S, Porto CM, Underwood B,
Duane B: Combining evidence - based healthcare withcaries induced by Streptococcus mutans to the patho-

environmental sustainability: using the toothbrush asa model. Br Dental J, 229, 303 - 309, 2020.
22. Guinée JB, Heijungs R, Huppes G, Zamagni A, Masoni

genicity of infective endocarditis. Infect Immun, 88:e00897 - 19, 2020.
32. Sato K, Fujikawa T, Koga C: Improved effect of thehigh water content clay using the water absorptivity

P, Buonamici R, Ekvall T, Rydberg T: Life cycle as-of bamboo. Geosynth Eng Pap, 29: 191 - 196, 2014.

sessment: past, present, and future. Environ Sci Tech-

33. Funabashi M, Kunioka M: Evaluation of biobased content of biobased polymer by measuring carbon - 14 us-nol, 45: 90 - 96, 2011.
23. Suzuki I, Shimizu T, Senpuku H: Role of SCFAs forfimbrillin - dependent biofilm formation of Actinomyces

ing accelerator mass spectrometry. Netsu Sokutei, 39:oris. Microorganisms, 6: 114, 2018. doi: 10.3390/micro-

34. Ooshima T, Matsumura M, Hoshino T, Kawabata S,

143 - 150, 2012.

24. Senpuku H, Nakamura T, Iwabuchi Y, Hirayama S,

Sobue S, Fujiwara T: Contributions of three glucosyltransferases to sucrose - dependent adherence of

Nakao R, Ohnishi M: Effects of complex DNA andStreptococcus mutans. J Dent Res, 80: 1672 - 1677,

organisms6040114

MVs with GTF extracted from Streptococcus mutans
on the oral biofilm. Molecules, 24: 3131, 2019. doi:
10.3390/molecules24173131
25. Schmidt JC, Bux M, Filipuzzi - Jenny E, Kulik EM,
Waltimo T, Weiger R, Walter C: Influence of time,

2001.
35. Ramful R, Sunthar TPM, Kamei K, Pezzotti G: Investigating the antibacterial characteristics of Japanesebamboo. Antibiotics（Basel）, 11: 569, 2022. doi:
10.3390/antibiotics11050569

toothpaste and saliva in the retention of Streptococcus

36. Teraoka F, Takahashi J: Antibacterial activity ofmutans and Streptococcus sanguinis on different tooth-bamboo extract and its application in clinical dentist-brushes. J Appl Oral Sci, 22: 152 - 158, 2014.

ry part 1. Relationship between concentration of bam-

26. Bunetel L, Tricot - Doleux S, Agnani G, Bonnaure - Mallet M: In vitro evaluation of the retention of threeboo extract and antibacterial activity. JSDMD, 14:219 - 224, 1995.

species of pathogenic microorganisms by three differ-

37. Spolidorio DM, Goto E, Negrini TD, Spolidorio LC: Vi-

ent types of toothbrush. Oral Microbiol Immunol, 15:

ability of Streptococcus mutans on transparent and313 - 316, 2000.

opaque toothbrushes. J Dent Hyg, 77: 114 - 117, 2003.

34:23–34, 2023
Int J Oral-Med Sci 22（1）

38. Wetzel WE, Schaumburg C, Ansari F, Kroeger T,

41. van der Sluijs E, Slot DE, Hennequin - Hoenderdos NL,

Sziegoleit A: Microbial contamination of toothbrushes

van der Weijden GA: Dry brushing: Does it improvewith different principles of filament anchoring. J Am

plaque removal? A secondary analysis. Int J Dent

Dent Assoc, 136: 758 - 806, 2005.

Hyg, 16: 519 - 526, 2018.

39. Oh JH, Lee MR, Seo JH, Chang YS: Micro - organism
according to storage method of the tooth - brush.
IJCPD, 9: 193 - 197, 2013.
40. Shinohara Y, Koshimune K: The experiment of moisture transfer on clothes: wetting and drying of fabrics: the development of elementary school home economics curriculum based on ESD. Bull Jpn Curric
Res Dev, 37: 1 - 11, 2014.

42. 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 - monthand 3 months: an in vitro study. J Nat Sci Biol Med, 6:
S44 - S48, 2015.
43. Suda Y, Suzuki S, Miyashita H, Hasegawa K: Dimensional changes of filament taken from the worn - out
tooth brush. JSP, 22: 122 - 129, 1979.