Received: 14 December 2021 | Revised: 24 September 2022 | Accepted: 10 December 2022

DOI: 10.1111/idh.12652

ORIGINAL ARTICLE

Bacterial decontamination of toothbrushes by immersion

in a mouthwash containing 0.05% chlorhexidine and 0.05%
cetylpyridinium chloride: A randomized controlled trial

Gerard Àlvarez | Agnès Soler-­Ollé | Sergio Isabal | Rubén León | Vanessa Blanc

Department of Microbiology, DENTAID

Research Center, Cerdanyola del Vallès,
Spain
Correspondence
Vanessa Blanc, Department of
Microbiology, DENTAID Research Center,
Cerdanyola del Vallès, Spain.
Email: blanc@dentaid.es
Abstract
Objective: Toothbrushes are colonized by microorganisms, implying a risk of infec-
tion. That risk can be reduced by decreasing the microbial contamination of the fila-
ments. Therefore, this study aimed to determine the antiseptic efficacy of a 0.05%
chlorhexidine + 0.05% cetylpyridinium chloride mouthwash on toothbrushes.
Methods: A total of twelve toothbrushes used three times/day for 14 days by orally
and systemically healthy people were randomly split into two groups, and their heads
were immersed for 2 h in PBS (control) or Perio·Aid Active Control (treatment). The
microorganisms were recovered, and their number was calculated by culture, quanti-
tative PCR, and viability PCR. Statistical differences were first assessed with a two-­
way mixed ANOVA and subsequently with Student's t-­test.
Results: The results showed no statistical differences in the total number of cells
for the treatment (mean ± CI95% of 7.27 ± 1.09 log10 bacteria/ml) and the control
(7.62 ± 0.64 log10 bacteria/ml) groups, but a significantly lower number of live cells in
the treatment group (4.58 ± 0.61 log10 viable bacteria/ml and 2.15 ± 1.42 log10 cfu/ml)
than in the control group (6.49 ± 1.39 log10 viable bacteria/ml and 5.04 ± 0.93 log10
cfu/ml).
Conclusions: Based on our findings, sanitization of toothbrushes with this mouthwash
reduces the number of live microorganisms adhered to the filaments. Such decrease
of the bacterial load could include bacteria from the oral cavity, from the environ-
ment, and from nearby toothbrushes since the quantification was not limited to any
bacterial taxon.

KEYWORDS
microbial decontamination, prevention, propidium monoazide, toothbrushing, viability PCR

1 | I NTRO D U C TI O N
The oral cavity can harbor hundreds of microbial species.1,2 Under
certain circumstances, microbial communities shift from a symbiotic
relation with the host (eubiosis) to a pathogenic state (dysbiosis),
which may lead to oral diseases. 2,3 One way of disease prevention is
the use of toothbrushes, which helps maintain oral health by hinder-
ing the accumulation of dental plaque.4,5 However, shortly after the
first use, toothbrushes can be colonized by oral and environmental
microorganisms,4,6–­8 which can remain viable on the filaments for

© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Int J Dent Hygiene. 2023;21:357–364.  wileyonlinelibrary.com/journal/idh | 357

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358
between 1 day and 1 week using food and epithelial debris, as well as
components of oral secretions such as saliva and gingival crevicular
fluid, as their nutrient.1,4,8,9 Hence, toothbrushes become a potential
source of oral and non-­oral infection, as well as disease vectors be-
tween household members.1,4,8
Regular toothbrush sanitization could prevent an excess of vi-
able pathogens on filaments.7 Several disinfection methods have
shown to decrease the number of bacteria on filaments. 6,7,10–­16
Although, according to the results, both chlorhexidine (CHX) and
1,14,16
cetylpyridinium chloride (CPC) are among the most effective.
CHX and CPC are two broad-­spectrum antiseptics that are widely
used in clinical medicine.7,17 In dentistry, CHX is considered the
gold standard antiseptic7 and it is commonly used as an adjuvant
17,18
of periodontal therapy. CPC is effective in reducing microbial
colonization and dental biofilm formation and in preventing gin-
givitis.17,19,20 Moreover, CPC has shown a virucidal effect against
several coronaviruses, including SARS-­C oV-­2 and influenza
21–­2 5
virus.
Regarding methodology, all of the toothbrush decontamina-
tion studies reviewed but two1,26 were based on culture methods.
However, between 30 and 50% of oral bacteria are not yet cultur-
2,27
able, and others exhibit strict culture requirements and long in-
cubation times. 28 Culture-­independent methods, on the other hand,
are not limited by culturability and allow for the detection of spe-
cific microorganisms, even when the numbers in samples are low.
Moreover, in studies with antimicrobials, only the live cells in a sam-
ple can be quantified by exposing samples to propidium monoazide
(PMA) before quantitative PCR (qPCR), a method known as viability
PCR (vPCR). 29,30
To our knowledge, the efficacy of CHX as a toothbrush disin-
fectant has only been studied using concentrations of 0.12%14 and
0.2%,7,16 but not low-­concentration CHX mouthwashes. Such prod-
ucts can include other antiseptics, like CPC, and are recommended
during the maintenance phase after periodontal treatment due to
their effectiveness and reduced side effects.18 Therefore, the aim
of our work, which was designed as a proof-­of-­concept study, was
to assess the efficacy of a 0.05% CHX + 0.05% CPC formulation in
reducing bacterial load on toothbrushes, using culture, qPCR, and
vPCR as quantification methods.
2 | St u d y p o p u l at i o n a n d m e t h o d o l o g y
2.1 | Experimental design
This was a proof-­
of-­
concept study designed (following the
CONSORT guidelines) as a parallel, randomized controlled trial.
Based on previous studies, 6,31 the sample size was set to 12 tooth-
brushes provided by individuals with good oral health that vol-
unteered for this study. All of them were from northeast Spain
and employees at an oral health company. The inclusion criteria
included systemic health, retention of at least 20 natural teeth,
no diagnosis of periodontitis, no antibiotic intake in the previous
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ÀLVAREZ et al.
6 months, and no mouthrinse usage in the previous week. All
volunteers signed their consent after being informed about the
experiment, which neither involved any invasive procedures nor
posed a risk for their health. All participants received the same oral
care products, consisting of a medium-­s trength toothbrush and a
fluoride paste without antiseptics. Also, they were instructed to
brush their teeth three times a day after each meal for 14 days,
storing their toothbrush head up and with the cap on, and to avoid
any other type of oral hygiene. No particular instructions were
given about the brushing technique. On the morning of the 15th
day, they used their toothbrushes for the last time and delivered
them in person to the microbiology laboratory at the DENTAID
Research Center. The toothbrushes were randomly divided into
two groups of six and immediately entered the treatment step.
The allocation process was performed by the author VB: each vol-
unteer received a number from 1 to 12. Then, six were assigned
to the treatment group using the R command sample (x = c (1:12),
size = 6, replace = FALSE), which returned six different random
numbers from 1 to 12. The remaining six were allocated to the
control group. The investigators that carried out the experiments
were blinded until the statistical analysis phase.
2.2 | Treatment
In a laminar flow cabinet, the toothbrushes were decapitated, and
the heads were individually immersed in sterile tubes containing
15 ml of phosphate-­buffered saline (PBS; control group) or Perio·Aid
Active Control (Dentaid S.L.; composition: 0.05% chlorhexidine di-
gluconate and 0.05% cetylpyridinium chloride; treatment group).
After 2 h, each head was transferred to a new sterile tube containing
15 ml of PBS. Then, to harvest the bacterial cells from the bristles,
the tubes were vortexed for 3 min and centrifuged 10 min at 6000
x g and 15°C. The supernatant was removed, and the cells were sus-
pended in 1 ml PBS. We assumed that very few cells would be re-
leased during the immersion because they adhere so strongly to the
bristles,13 and therefore the bacteria were not collected before the
treatment.
2.3 | Determination of bacterial load
Bacteria in both the control and the treatment groups were quan-
tified by culture (colony-­forming units per ml: cfu/ml), qPCR (bac-
teria/ml) and vPCR (live bacteria/ml). For plate culture, the cell
suspensions were serially diluted and spread on blood agar plates. 30
After 7 days of incubation at 37°C under anerobic conditions, the
cfu/ml were calculated without regard to colony morphology. For
qPCR and vPCR, 250 μl of the bacterial suspension from each of
the brush heads was placed in each of two 1.5-­ml microcentrifuge
tubes, one for each of the PCR methods. The tubes used for vPCR
analysis were treated with PMA as in Àlvarez et al., 30 but with
some modifications: the tubes were incubated 5 min in the dark

ÀLVAREZ et al.
with 50 μM PMAxx dye (Biotium) and then exposed to a 650 W
halogen light for 3 min. These two steps were conducted twice
in a row. Thereafter, both qPCR and vPCR tubes followed the
same protocol: the cells were pelleted at 12,000 x g for 4 min, and
DNA extraction and real-­t ime PCR were conducted as previously
described. 30 The so-­c alled universal oligonucleotides and probe
for the quantification of all bacteria were located in conserved
regions of the 16S rRNA gene. 32 After testing several concentra-
tions, the optimized reaction mix contained 0.6 μM of forward
primer, 0.7 μM of reverse primer, and 0.2 μM of TaqMan probe.
The standard curve was also prepared as in Àlvarez et al., 30 using
DNA from Streptococcus gordonii ATCC 49818, Veillonella parvula
NCTC 11810, Actinomyces naeslundii DSM 17233, Fusobacterium
nucleatum DSM 20482, and Porphyromonas gingivalis DSM 20709
as templates.
2.4 | Mortality index estimate
The mortality index was calculated within each group as log10 (qPCR/
vPCR). This is the ratio between the total (live and dead) number of
cells (qPCR) and the live cells (vPCR), which is roughly the proportion
of dead/live cells on a log10 scale.
2.5 | Non-­culturable index estimate
The non-­culturable index was calculated within each group as
log10 (vPCR/culture). This is the ratio between the number of live
bacteria (culturable or not) and the culturable cells, which is
roughly the proportion of viable but non-­culturable/culturable on
a log10 scale.
2.6 | Statistics
Based on the aim of this study, the most important determination to
be made was whether the number of live cells (measured directly by
vPCR and culture) was significantly different in the treatment than
in the control group.
The statistical analysis was conducted in R v4.0.4. 33 Prior
to any analysis, the data were transformed to a log10 scale. The
Shapiro–­W ilk normality test and the Bartlett's test assessed the
normal distribution of the data and the homogeneity of the vari-
ances, respectively. Next, the differences between groups (con-
trol and treatment) and methods (qPCR, vPCR, and culture) were
evaluated with a two-­way mixed ANOVA. Then, the groups were
compared in pairs by independent sample Student's t-­tests and
the methods by paired sample Student's t-­tests. The mortality
and non-­culturable indices were examined by independent sample
Student's t-­test. In all tests, alpha was set at 0.05, and p values
were adjusted for multiple comparisons with the Bonferroni cor-
rection when necessary.
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359
F I G U R E 1 Number of bacteria recovered from the
toothbrushes. Counts from qPCR and vPCR have been transformed
to cfu/ml by means of a standard curve. Bar height and whiskers
represent the mean and the confidence interval at 95%,
respectively. Control and treatment groups were compared by
independent sample Student's t-­test and methods within each
group by paired sample Student's t-­test and adjusted for multiple
comparisons with the Bonferroni correction. Pairs of bars with
statistically significant differences are highlighted with a bracket
and the corresponding p value.
3 | R E S U LT S
3.1 | qPCR standardization
The number of bacteria adhered to the toothbrush bristles was
measured by qPCR (live and dead), vPCR (live) and culture. All re-
sults were expressed as cfu/ml on a log10 scale. In the case of both
qPCR and vPCR, a standard curve had to be constructed to relate
the crossing points with the number of cfu/ml. This standard curve
ranged from 5.47 × 101 to 1.51 × 109 cfu/ml, showing an efficiency
of 1.822.
3.2 | Determination of bacterial load
The number of bacteria could be quantified by all three methods
(qPCR, vPCR, and culture) for all 12 toothbrushes after 2 h of immer-
sion in PBS (control) or 0.05% CHX + 0.05% CPC mouthwash (treat-
ment). The total number of bacteria (qPCR) on the toothbrush heads
was 7.62 ± 0.64 (mean ± CI95%; control) and 7.27 ± 1.09 log10 cfu/ml
(treatment) (Figure 1, Table 1), the number of live bacteria (vPCR)
was 6.49 ± 1.39 (control) and 4.58 ± 0.61 (treatment) log10 cfu/ml,
and the quantity of culturable bacteria was 5.04 ± 0.93 (control) and
2.15 ± 1.42 (treatment) log10 cfu/ml.
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360 ÀLVAREZ et al.

TA B L E 1 Measuring of bacteria
Control group Treatment group
recovered from the toothbrushes
(mean ± CI 95%) (mean ± CI 95%) p Values

qPCR (total bacteria/ml) 7.62 ± 0.64 7.27 ± 1.09 0.52a

0.053b
0.0036c
vPCR (viable bacteria/ml) 6.49 ± 1.39 4.58 ± 0.61 0.019a
0.08d
0.014 e
Culture (cfu/ml) 5.04 ± 0.93 2.15 ± 1.42 0.0026a
Mortality index (≈dead/live) 1.13 ± 0.93 2.69 ± 1.15 0.022a
Non-­culturable index 1.45 ± 1.35 2.43 ± 1.42 0.229a
(≈non-­culturable/culturable)
a
Comparison: control group vs. treatment group within each method.
b
Comparison: qPCR vs. vPCR within the control group.
c
Comparison: qPCR vs. vPCR within the treatment group.
d
Comparison: vPCR vs. culture within the control group.
e
Comparison: vPCR vs. culture within the treatment group.

in the number of bacteria in the control group between the mea-

sures by vPCR and culture (p = 0.08) or between qPCR and vPCR
(p = 0.053). All other comparisons were statistically significant: num-
ber of live bacteria (vPCR) between the control and the treatment
groups (p = 0.019), culturable bacteria between the control and the
treatment (p = 0.0026), bacteria in the treatment group between
qPCR and vPCR (p = 0.0036), and bacteria in the treatment group
between vPCR and culture (p = 0.014).

3.3 | Mortality index estimate

The mortality index was 1.13 ± 0.93 (mean ± CI95%) and 2.69 ± 1.15
in the control and the treatment groups, respectively (Figure 2,
Table 1). Both indices were significantly different (p = 0.022). This
index shows that the ratio of dead/live bacteria was 12.49 in the
control and 488.78 in the treatment group.

3.4 | Non-­culturable index estimate

F I G U R E 2 Mortality and non-­culturable indices observed in the
toothbrushes. Mortality (dead/live bacteria) and non-­culturable
(viable but non-­culturable/culturable) indices are labeled as qPCR–­
vPCR and vPCR–­culture, respectively. The central line in each
box represents the median. Control and treatment groups were
compared by independent sample Student's t-­test. Pairs of boxes
with statistically significant differences are highlighted with a
bracket and the corresponding p value.
The non-­culturable index was 1.45 ± 1.35 (mean ± CI 95%) and
2.43 ± 1.42 in the control and the treatment groups, respectively
(Figure 2, Table 1). This means that the ratio of non-­culturable/cul-
turable bacteria was 27.18 in the control and 268.15 in the treatment
group. Nonetheless, these indices were not significantly different
(p = 0.229).

The two-­way mixed ANOVA test confirmed that there were sta-
tistically significant differences between the control and treatment
groups (p = 0.005) and between the three methods (p = 1.18 × 10−9).
Afterward, the groups and methods were compared in pairs to es-
tablish which cases were statistically different (Figure 1, Table 1). No
significant differences were observed in the total number of bacteria
(qPCR) between the control and the treatment groups (p = 0.52), or
4 | DISCUSSION
Toothbrushes are an essential tool for daily oral hygiene.4 However,
regardless of the health status of the user, regular use of tooth-
brushes causes filaments to accumulate a great number of oral and
environmental bacteria, archaea, fungi, and viruses.4,8 Therefore,

ÀLVAREZ et al.
toothbrushes become a risk of infection and transmission between
1,7,8
subjects, and their pathogenic load could be higher in people
with oral disease or systemic illness, in environments with high pres-
ence of pathogens such as hospitals, etcetera.4
Patients with oral disease showed a significant decrease in
symptoms when they replaced their toothbrushes. Although
dentists and dental hygienists recommend replacing your tooth-
brush every 3 months based on filament deformation, changing
your toothbrush more frequently could diminish the risk of cross-­
contamination in the oral cavity. In fact, weekly replacement has
been suggested for contaminated environments (e.g., hospitals)
and diseased people. 6 However, this is neither practical nor en-
vironmentally sustainable. Other strategies to reduce microbial
4
contamination, like the use of a cap, filaments with antibacterial
8 14
coating, or rinsing toothbrushes in tap water, have been shown
ineffective. Conversely, exposure of toothbrushes to any of a num-
ber of antimicrobial agents (e.g., green tea,7,10 microwaves,12 UV
10,12 34 13 6,14
light, white vinegar, essential oils, parabens, CPC
and CHX 7,14,16) has been seen to reduce bacterial load. 35 As far
as we know, the current study is the first to evaluate a 0.05%
CHX + 0.05% CPC mouthwash as a toothbrush disinfectant. To
this end, the participants of this study (all of them healthy) were
supplied with a new toothbrush and were instructed to use it three
times a day for 2 weeks. After this period, the toothbrushes were
expected to carry high loads of bacteria.7 The samples were then
treated with PBS or the mouthwash and then analyzed by qPCR
to determine the number of bacteria (live and dead), by vPCR to
quantify only the live bacteria and by culture. The latter was used
to estimate the number of strict and facultative anerobes, which
includes most oral bacteria (main interest of this work), including
early colonizers like Streptococcus spp. and Actinomyces spp., peri-
odontopathogens, etc. 36
The total number of bacteria/ml recovered from the bristles
6,8
was in the range observed in other studies (Figure 1, qPCR),
and the recoveries were not significantly different between the
control and the treatment group, i.e., there were no statistical
differences between groups in the microbial load adhered to the
toothbrushes. The latter confirmed our assumption (see methods)
that the treatments with PBS and the mouthwash would produce
none or little release of cells due to the strong adherence of the
bacteria to the filaments.13 It also excluded a possible bias in the
study related to differences in the brushing technique, storage, or
oral bacterial load. Moreover, both groups showed a lower num-
ber of live bacteria/ml (vPCR) than total bacteria/ml (qPCR), but
that difference (which relates to the presence of dead cells) was
only statistically significant in the treatment group (Figure 1). The
number of live bacteria/ml recovered in the treatment group was
significantly lower (≈81-­fold) than in the control group (Figure 1,
vPCR). Furthermore, the mortality index (comparison between
qPCR and vPCR within each group; Figure 2, qPCR-­vPCR) was, on
average, significantly higher in the treatment group (≈489 dead
bacteria/live bacteria) than in the control (≈12 dead bacteria/live
bacteria) group. This was supported by the culture method, which
|
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361
also detected a significantly lower number of culturable cells in
the treatment than in the control group (≈776-­fold). Therefore,
we found that a single treatment with the 0.05% CHX + 0.05%
CPC mouthwash significantly reduced the bacterial load on the
toothbrushes.
As seen in the literature, exposure to 0.12% 37 or 0.2% CHX
solutions7,10 reduced bacterial loads by 4.60-­to 12.52-­fold when
comparing the control and treatment groups. Regarding 0.05%
CPC solutions, Chandrdas et al. described an 11.28-­fold difference
using an aqueous 0.05% CPC solution,10 and Sato et al. detected a
significant reduction in the number of black-­pigmented anerobes
and in Gram-­n egative aerobic bacilli.15 Moreover, do Nascimento
et al. found a high antimicrobial effect with 0.05% CPC, but 0.12%
CHX was effective only in some of the species analyzed.1 The
current study, which has been able to detect live bacteria using
culture and culture-­independent methods, has described ≈776-­
and ≈81-­fold reductions by culture and by vPCR, respectively.
1,10,15
Therefore, we believe that the 0.05% CHX + 0.05% CPC formula-
tion has shown similar or better effects than 0.12% or 0.2% CHX.
Nevertheless, caution must be taken when comparing our results
with those of other studies due to methodological differences.
While many studies used selective media for specific groups of
bacteria, 6,7,10,13,14 our culture method, like others,15,37 included a
non-­s elective medium for routine plating of oral bacteria. The rea-
son was that this proof-­of-­concept study aimed to determine the
decontamination efficacy of the mouthwash. CHX and CPC are
broad-­spectrum antiseptics,17 and therefore, it was reasonable to
assume that there would be an overall decrease in bacterial load,
which could imply a decreased risk of a wide range of infections.
Future studies could recruit patients with different oral diseases
in order to examine pathogens of interest. Though, given the reli-
ability of CHX and CPC proven in clinical trials and through real-­
world evidence in the treatment of periodontal diseases and the
fact that biofilm in toothbrushes may be partially unstructured, it
is likely that a 0.05% CHX + 0.05% CPC mouthwash could also tar-
get most pathogens. Do Nascimento et al.1,26 used checkerboard
DNA–­D NA hybridization to specifically analyze 38 bacterial spe-
cies and five Candida spp., although the suitability of this method
in studies with antimicrobials is debatable because it cannot dis-
criminate between live and dead cells. Besides, do Nascimento
et al.1 and Sato et al.15 exposed their toothbrushes to CPC after
each use, while in the present study and in others7,10 a single ex-
posure was performed at the end of the experiment. The period
of use of each toothbrush, which in the current study was 15 days,
ranged from 5 h to 3 months,7 and sanitization time, which in our
study was 2 h, ranged from 5 min to 24 h.7 Therefore, an additional
comparative study would be needed to determine whether the an-
timicrobial capacity of the 0.05% CHX + 0.05% CPC formulation
was greater than that of other formulations, with a higher concen-
tration of CHX or different active ingredients.
In general, the vPCR method tends to quantify higher num-
bers of live bacteria than culture because vPCR, unlike culture,
can detect viable but non-­
culturable cells, ghosts (non-­
v iable
 |
362
cells that have still not lost their membrane integrity), 29,38 etc. In
this study, only the treatment group showed a significant higher
number by vPCR than by culture (Figure 1). Furthermore, the non-­
culturable index (ratio between the vPCR and culture measures;
Figure 2, vPCR-­culture) was higher in the treatment group (≈268-­
fold non-­culturable/culturable) than in the control group (≈27-­fold
non-­culturable/culturable), and the control/treatment group ratio
of live bacteria was higher by culture (≈776-­fold) than by vPCR
(≈81-­fold). These numerical differences between groups are hard
to explain based on the difference between the two methods: be-
cause CHX and CPC act by disrupting cell membranes, 29 all cells
killed by the 0.05% CHX + 0.05% CPC solution would be perme-
able to PMA and, consequently, the non-­culturable index should
not be higher in the treatment group than in the control group. We
believe that CHX and CPC in the mouthwash may have remained
bound to the bacterial surfaces 39,40 and continued acting as an-
tiseptics after the treatment. This could be measured by culture
but not by vPCR, where cells are exposed to PMA just after the
treatment. Although, despite the numerical differences explained
above, the non-­culturable index was not found to be statistically
different between the control and the treatment groups (Figure 2,
vPCR-­culture), which may be due to the high variation in the quan-
tification by culture in the treatment group (Figure 1, culture).
A future study that includes this issue as a secondary objective
would require a larger sample size. However, the sample size of
the current proof-­of-­concept study was calculated to have enough
statistical power (≥0.8) to answer the aim (to determine the effi-
ciency of the 0.05% CHX + 0.05% CPC mouthwash in reducing the
bacterial load in toothbrushes).
It is worth mentioning that oral viruses residing in the oral cav-
ity22 can contaminate toothbrushes4 and remain stable for several
days.6 Therefore, toothbrushes may be a source of viral infection
and transmission.4,6,8 As a matter of fact, the spread of SARS-­CoV-­2
virus among cohabitants was significantly higher when they shared
a toothbrush holder or a tube of toothpaste, among others.41 Both
CHX and CPC are effective in vitro against several lipid-­enveloped
viruses. 24 Moreover, CPC has been shown to reduce the symptoms
24
of influenza patients, and to have an antiviral effect in vitro against
SARS-­CoV-­2. 22 The current study has not determined the antiviral
effect of the 0.05% CHX + 0.05% CPC mouthwash; however, based
on the evidence, we believe that the combined effect of CHX and
CPC could reduce the infectivity of some of the viral species that
may be present in toothbrushes.
The 0.05% CHX + 0.05% CPC formulation tested in the present
work is recommended to be used during the maintenance phase
18
after periodontal treatment. During this long-­term treatment, we
believe that the health of the patients would benefit from using the
same product to disinfect their toothbrush. Furthermore, healthy
people or individuals with other diseases could also benefit from the
sanitization since toothbrushes can carry oral and non-­oral bacterial
and viral pathogens,1,4,6,24,41 against which the 0.05% CHX + 0.05%
CPC mouthwash could be effective due to the antimicrobial and
antiviral activity shown for CHX and CPC in the present and other
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ÀLVAREZ et al.
studies. Nonetheless, future studies should determine the periodic-
ity and time of exposure to this formulation to establish the optimal
disinfection regimen.
5 | CO N C LU S I O N
In this study, we have proved that the 0.05% CHX + 0.05% CPC
mouthwash is effective in reducing the number of live bacteria ad-
hered to the toothbrushes. The results were supported by both cul-
ture and culture-­independent methods. Nonetheless, the difference
in the number of live cells when comparing control and treatment
was about 10-­fold higher with culture than with vPCR. Further re-
search would be necessary to assess this difference.
6 | C LI N I C A L R E LE VA N C E
6.1 | Scientific rationale for study
Toothbrushes are colonized by oral and environmental microorgan-
isms, which fosters repeated self-­contamination of the mouth, cross-­
infection between household members, etc. Here, we have studied
the reduction in the bacterial load in toothbrushes after exposure to
an antiseptic.
6.2 | Principal findings
The number of live microorganisms adhered to the filaments was
significantly reduced after a 2-­hour exposure to a 0.05% chlorhex-
idine + 0.05% cetylpyridinium chloride mouthwash.
6.3 | Practical implications
The use of an antiseptic to reduce the number of viable patho-
gens adhered to toothbrushes may cut down on cross-­infections,
improve the response to treatments, and reduce disease recovery
times.
AU T H O R C O N T R I B U T I O N S
Conceptualisation: Vanessa Blanc; Methodology: Vanessa Blanc
and Rubén León, with contributions by Agnès Soler-­Ollé, Sergio
Isabal, and Gerard Àlvarez; Investigation: Agnès Soler-­Ollé, Sergio
Isabal, and Gerard Àlvarez; Formal analysis: Gerard Àlvarez;
Visualization: Gerard Àlvarez; Writing –­ original draft preparation:
Gerard Àlvarez; Writing –­review and editing: Vanessa Blanc, Rubén
León, Agnès Soler-­Ollé and Gerard Àlvarez.
AC K N O​W L E D
​ G E ​M E N T S
We would like to thank Ms. Ann Bangle for her help in reviewing the
language.
 |

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ÀLVAREZ et al. 363

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How to cite this article: Àlvarez G, Soler-­Ollé A, Isabal S, León
R, Blanc V. Bacterial decontamination of toothbrushes by
immersion in a mouthwash containing 0.05% chlorhexidine
and 0.05% cetylpyridinium chloride: A randomized controlled
trial. Int J Dent Hygiene. 2023;21:357-364. doi:10.1111/
idh.12652