Article

In vitro efficacy of five pediatric toothpastes in the

inhibition of Streptococcus mutans ATCC 25175.
Eficacia in vitro de cinco pastas dentales pediátricas en la inhibición
de Streptococcus mutans ATCC 25175.
José Miñano.1,2 Abstract: The present in vitro study compared the inhibitory action of five
Maria V. Espinoza.2 pediatric toothpastes against Streptococcus mutans ATCC 25175. Materials

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and Methods: Cross-sectional, comparative and experimental study. The
Affiliations: microorganism Streptococcus mutans ATCC 25175 was inoculated onto solid

in
1
Práctica Privada,Perú. culture medium of Müeller-Hinton supplemented with blood, then the plates
2
Universidad Privada Antenor Orrego. were inoculated with five pediatric toothpastes: Oral B Stages, Colgate Smiles,
Trujillo, Perú. Aquafresh My Little Teeth, Dentito and Denture Kids. Samples were incubated

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at 37°C for 48 hours. Subsequently the inhibition halos were measured; the
Corresponding author: María V. Espinoza. experiment was repeated 20 times for each sample. Statistical analysis was
Avda. América Sur 3145, Trujillo #13008, performed with ANOVA complemented with Tukey's test. Results: Oral B

of
Perú. Phone:56-9 4387 1082. E-mail: Stages yielded a mean inhibitory halo of 23.2mm, Colgate Smiles an average
mespinozas@upao.edu.pe
of 21.7mm, Aquafresh My Little Teeth of 13.6mm, Dentito of 18.5mm, and
Receipt : 04/08/2019 Revised : 07/23/2019
Denture Kids a mean of 23.0mm. When performing the ANOVA test, it
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Acceptance : 09/03/2019 was found that there was a significant difference in the inhibitory action of
pediatric toothpastes (p<0.005) and when using Tukey's multiple comparison
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test, Oral B and Denture Kids presented similar inhibitory action. Conclusion:
All toothpastes presented inhibitory action against Streptococcus mutans ATCC
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25175. A significant difference between their effectiveness was observed.

Oral B Stages showed the greatest degree of inhibition.
Keywords: Toothpaste; Streptococcus mutans; culture media; child; cross-
sectional studies; analysis of variance.
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Resumen: El presente estudio in vitro comparo la eficacia inhibitoria de cinco

pastas dentales pediátricas frente a la bacteria Streptococcus mutans ATCC25175.
Material y Métodos: El estudio fue transversal, comparativo y experimental. Se
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inoculó Streptococcus mutans ATCC 25175 en un medio de cultivo Müeller-Hinton

complementado con sangre, luego a las placas cultivadas se le inocularon cincopastas
dentales pediátricas: Oral B Stages, Colgate Smiles, Aquafresh My LittleTeeth,
Dentito y Denture Kids. Se incubó a 37ºC por 48 horas y posteriormente semidió
los halos de inhibición, se replicó el experimento 20 veces para cada uno. El análisis
estadístico se realizó con el test de ANVA complementado con el test de Tukey.
Resultado: Oral B Stages generó una media inhibitoria de 23,2mm, ColgateSmiles
una media de 21,7mm, Aquafresh My Little Teeh de 13,6mm, Dentitode 18,5mm
y finalmente Denture Kids una media de 23,0mm. Al realizar laprueba ANOVA
se encontró que hay diferencia significativa en la acción inhibitoriade las pastas
Cite as: dentales pediátricas (p<0.005) y al emplear la prueba Tukey (comparación de
Miñano J & Espinoza MV. múltiples) la pasta Oral B y Denture Kids presentaron acción inhibidora similar.
In vitro efficacy of five pediatric toothpastes Conclusión: Todas las pastas presentaron acción inhibitoria sobre Streptococcus
in the inhibition of Streptococcus mutans mutans ATCC 25175 existiendo diferencia significativa entre la efectividad de
ATCC 25175. estas, con la pasta Oral B Stages demuestrando mayor acción inhibitoria.
J Oral Res 2020; 9(1):29-35. Palabra Clave: Pastas de dientes; Streptococcus mutans; medios de cultivo; niño;
Doi:10.17126/joralres.2020.005 estudios transversales; análisis de variación.
ISSN Print 0719-2460 - ISSN Online 0719-2479. www.joralres.com/2020 29
 Miñano J & Espinoza MV.
In vitro efficacy of five pediatric toothpastes in the inhibition of Streptococcus mutans ATCC 25175.
J Oral Res 2020; 9(1):29-35. Doi:10.17126/joralres.2020.005

INTRODUCTION. childhood development and those with active caries

There is currently a growing interest in improving the have psychosocial behavioral problems since they have
oral health of children from an early age. The World higher rates of lack of sleep, attention deficit, depression,
Health Organization (WHO) defines oral health as the anxiety, aggressiveness and hyperactivity.14
absence of caries, periodontal disease, orofacial pain, loss There is a high prevalence of childhood caries in Peru,
of teeth and other diseases or disorders that hamper the related to multiple factors such as high sugar diet, acid-
ability to chew, talk and smile in humans, damaging their producing bacteria, poor oral hygiene, and socioeconomic
psychosocial well-being. Caries is the most prevalent oral status. Oral health procedures should be supervised by
disease in preschool and school age children, in contrast parents and they should also favor those toothpastes
to periodontal or pulp diseases. According to the data that contain antimicrobial agents such as xylitol, fluoride
provided by the WHO, 60 to 90% of children worldwide or others. Little is known about the inhibitory action

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suffer from caries.1 According to the ministries of health against Streptococcus mutans of pediatric toothpastes
of Latin American countries, the prevalence of caries is available in the Peruvian market. As this microorganism

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high in countries such as Brazil with 80%, 2 Colombia with is related to the presence of dental caries, being able
52.2%, 3 Chile with 70.4%, 4 and Argentina with 87.4%.5 to identify the components of toothpastes and their

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According to an epidemiological study conducted by the properties that can help children deal with dental caries.
Peruvian Ministry of Health (MINSA) from 2012 to 2014, The aim of the present study is to determine the in vitro
the prevalence of caries in children under 12 years of efficacy of five pediatric toothpastes in the inhibition of
age was 85.6%. 6
Caries at early ages is a significant aggravating
factor in the oral health of children, since they will be
of Streptococcus mutans ATCC 25175.

MATERIALS AND METHODS.

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more likely to develop caries in mixed and permanent Cross-sectional, comparative and experimental study,
dentition.7 The oral health of children depends on the carried out in the Medical Microbiology Laboratories of
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constant supervision of parents or guardians. 8 There is the School of Human Medicine at Universidad Nacional
sometimes difficulty because parents are not aware of de Trujillo - La Libertad, Perú. All biosafety and laboratory
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correct brushing techniques and which toothpastes or standards were met. Five pediatric toothpastes available
mouthwashes are suitable for their children.9 in the Peruvian market were used: Aquafresh My Little
The main risk factors for caries are the high prevalence Teeth15, Colgate Smiles,16 Dentito,17 Denture Kids,18 and
of Streptococcus mutans and lactobacilli in the oral Oral B Stages.19 They were placed in a sterile tuberculin
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cavity, teeth with weaken enamel susceptible to an syringe (Figure 1) in order to be inoculated in the form
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acidic medium and with poor mineralization capacity, a of discs onto the plates where strains of Streptococcus
cariogenic diet based on sugar consumption, and poor mutans ATCC 25275 had been previously inoculated.
oral hygiene associated with cognitive, economic and The strain of Streptococcus mutans ATCC 25175 was
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social factors.10 cultured in thioglycolate broth, then incubated for 48

The most prevalent microorganism in incipient caries hours at 37°C. Two days later the growth of the strain
and enamel lesions is Streptococcus mutans. It plays a was confirmed when a strong turbidity was observed in
very important role at the early stages of the disease but the test tubes.
not in its progression.11 Streptococcus mutans is native to Thirty Petri dishes of Mueller Hinton medium enriched
humans and becomes pathogenic when there is a change with 5% rabbit blood were prepared. Inoculation of the
in the oral environment, that is, high consumption of plates was performed, and then the inoculated plates
refined sugars (such as sucrose).12 were perforated with a microbiological punch making
The use of toothpastes for the oral hygiene of children four holes, 6mm in diameter, in each plate. Then five
has increased in recent times and their composition has control plates were made by filling two holes with 0.1ml
changed. Currently, many types of toothpaste contain of each toothpaste using of tuberculin syringes, and
antimicrobial agents with the aim of preventing and in the two other holes the negative control (distilled
controlling oral diseases associated with common water) and positive control (0.12% chlorhexidine)
microorganisms of the oral cavity.13 were inoculated. Then, 25 repeat plates were made by
The presence of caries in children affects their early inoculating directly in the four holes 0.1ml of toothpaste

30 ISSN Print 0719-2460 - ISSN Online 0719-2479. www.joralres.com/2020

 Miñano J & Espinoza MV.
In vitro efficacy of five pediatric toothpastes in the inhibition of Streptococcus mutans ATCC 25175.
J Oral Res 2020; 9(1):29-35. Doi:10.17126/joralres.2020.005

(Figure 2). Finally, each plate was labeled with the name of incubating the plates, the inhibition halos generated
of the corresponding toothpaste and the repeat number. by each pediatric toothpaste were measured (Figure 3).
Plates were incubated in a micro anaerobic environment A 0.01mm precision digital Vernier caliper was used to
at a temperature of 37°C for 48 hours. After 48 hours obtain the diameter of the halos.

Figure 1. Pediatric toothpastes in aliquoted into tuberculin syringes.

A B C D E

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in
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of
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A: Aquafresh, My Little Teeth B: Colgate Smiles. C: Dentito. D: Denture Kids. E: Oral B Stages.
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Figure 2. Repeat plates of the five pediatric toothpastes.
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A
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B
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E
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Repeat plates of toothpastes: A: Aquafresh, My Little Teeth B: Colgate Smiles. C: Dentito. D: Denture Kids. E: Oral B Stages.

ISSN Print 0719-2460 - ISSN Online 0719-2479. www.joralres.com/2020 31

 Miñano J & Espinoza MV.
In vitro efficacy of five pediatric toothpastes in the inhibition of Streptococcus mutans ATCC 25175.
J Oral Res 2020; 9(1):29-35. Doi:10.17126/joralres.2020.005

Figure 3. Halos of bacterial inhibition of the five pediatric toothpastes.

1 3 5

2 4 6

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in
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of
1: Halos of inhibition of control plate Denture (C1: Distilled water, C2: chlorhexidine). 2: Halos of inhibition of Aquafresh My Little Teeth. 3:
Halos of inhibition of Colgate Smiles. 4: Halos of inhibition of Dentito. 5: Halos of inhibition of Denture Kids. 6: Halos of inhibition Oral B Stages.

RESULTS. mean halo of 29.0mm.

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The inhibitory effect of each pediatric toothpaste was The analysis of variances or ANOVA (Table 2) reveals
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determined quantitatively by the value of the means of a significant difference in the inhibitory action of each
the inhibition halos that each toothpaste generated on the of the pediatric toothpastes against Streptococcus
culture plates of Streptococcus mutans ATCC 25175. mutans ATCC 25175. After the ANOVA test, a multiple
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The inhibition halos of Oral B Stages were the largest, comparison test was performed, in this case, the Tukey's
with an average of 23.2mm (Table 1). The negative HSD test (Table 3).
control (distilled water) resulted in no inhibition halo, It showed 94.1% similarity in the inhibitory action of
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while the positive control (chlorhexidine) resulted in a Oral B Stages and Denture Kids.
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Table 1. Measures of inhibition halos for Streptococcus mutans ATCC 25175 of the five pediatric toothpastes.
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N Mean (mm) S.D (mm) Lowest (mm) Highest (mm)

Aquafresh My Little Teeth 20 13.6 1.0 11.8 15.7
Colgate Smiles 20 21.7 1.1 18.9 22.9
Dentito 20 18.5 1.6 16.3 21.7
Denture Kids 20 23.0 0.7 21.8 24.3
Oral B Stages 20 23.2 0.9 22.1 24.9
S.D: Standard deviation.

Table 2. Analysis of Variance between groups of pediatric toothpastes.

Sum of squares df Mean sum of squares F-Statistic p-value

Between groups 10223.18 5 1703.86 1715.45 0.000
Within groups 132.10 133 .99
Total 10355.28 139
p-value: p<0.05. df: degrees of freedom. F: F-statistic

32 ISSN Print 0719-2460 - ISSN Online 0719-2479. www.joralres.com/2020

 Miñano J & Espinoza MV.
In vitro efficacy of five pediatric toothpastes in the inhibition of Streptococcus mutans ATCC 25175.
J Oral Res 2020; 9(1):29-35. Doi:10.17126/joralres.2020.005

Table 3 . Pediatric toothpaste comparison test.

Tukey's HSDa
Type of toothpaste N Subset for alpha = 0.05
1 2 3 4
Aquafresh My Little Teeth 20 13.554
Dentito 20 18.508
Colgate Smiles 20 21.726
Denture Kids 20 22.986
Oral B Stages 20 23.231
p-value 1.000 1.000 1.000 .941

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The means for the groups are displayed in the homogeneous subsets. a: It uses the sample size of the harmonic mean=20.000.

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DISCUSSION. ficant inhibitory action in this experiment. It is the

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Dental caries in children is a serious and a recurrent only toothpaste that contains xylitol (10%), an artificial
oral health problem. This condition begins at very early sweetener used in toothpastes as a bactericidal agent
ages of life and has a tendency to become a chronic against Streptococcus mutans25 strains. It also has methyl

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problem even in adulthood. 20 This disease is associated and propyl paraben that is used as preservatives for their
with early colonization with cariogenic microorganisms bacteriostatic properties against Gram positive bacteria
such as Streptococcus mutans.3 and as an antifungal agent. 26
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One of the oral hygiene measures to control these Xylitol is a substance of natural origin that prevents
microorganisms is the use of toothpaste, as it contains the appearance of caries, helps to decrease the amount
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antimicrobial agents such as fluoride and others. 21 The of bacterial plaque, and improves halitosis and the
present study found that Aquafresh My Little Teeth quality of oral soft tissues. In addition this toothpaste
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is the toothpaste with the lowest inhibitory effect, contains calcium lactate which, when combined with
possibly because it does not contain extra antimicrobial xylitol, provides calcium to the developing teeth.18
substances such as xylitol, containing only fluoride in the Finally, Oral B Stages toothpaste showed the greatest
form of sodium fluoride. inhibitory action. This may be due this toothpaste
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The addition of xylitol in fluoridated dentifrices is containing other components such as benzyl alcohol,
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currently being promoted for the prevention and control which is an antimicrobial preservative effective against
of dental caries, and its use is supported by the American Gram positive bacteria and some fungi. 27
Academy of Pediatric Dentistry. 22 It also contains thymol, which is a flavoring substance
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The Peruvian toothpaste Dentito has a slightly derived from thyme, which has antimicrobial and
higher inhibitory power compared to Aquafresh My fungicidal properties and is used in some dental rinses
Little Teeth. This may be because it contains sodium and toothpastes. 28 Streptococcus mutans is a facultative
benzoate, which is a salt used as a preservative that has anaerobic microorganism and its virulence is related to
antiseptic, antifungal and bacteriostatic properties. 23 its ability to metabolize large amounts of sugars.
Colgate toothpaste presented a greater inhibitory action It also produces lactic acid and lives in an acidic
than the previous two, maybe due to the inclusion of environment conditioned by it. It has a high capacity of
cinnamaldehyde and eugenol, which are used to give adherence to enamel walls due to the synthesis of glucans
this toothpaste the smell and taste of cinnamon and and fructans from glucosyltransferase. Streptococcus
cloves. This result is consistent with the study by Erazo mutans has a spherical, ovoid round shape (cocci), grows
et al.,24 who found that these aromatic substances are in chains or in pairs, and does not present mobility nor
able to inhibit Streptococcus mutans Denture Kids is a form spores. 29
toothpaste with an increased presence and use in the It should be noted that the statistical analysis found
market in recent years. that Denture Kids has an inhibitory action 94.1% similar
This toothpaste generated the second most signi- to Oral B Stages. They are the only toothpastes that

ISSN Print 0719-2460 - ISSN Online 0719-2479. www.joralres.com/2020 33

 Miñano J & Espinoza MV.
In vitro efficacy of five pediatric toothpastes in the inhibition of Streptococcus mutans ATCC 25175.
J Oral Res 2020; 9(1):29-35. Doi:10.17126/joralres.2020.005

presented statistically comparable inhibitory action in the

experiment. They could be recommended to parents for Conflict of interests: The authors declare no conflict
the oral health care of their children, although achieving of interest or affiliation with any organization or entity,
good oral health does not only depend on choosing the nor any economic interests in the subject addressed in
right toothpaste but also on other relevant factors. this manuscript.
The differences shown between toothpastes regarding Ethics approval: Ethics Committee of the Universidad
their inhibitory efficacy against Streptococcus mutans Peruana de Ciencias Aplicadas (CEI/152–12-16).
could be related to the addition of extra components that Funding: Self-funded.
could have synergy with fluoride, the antibacterial and Authors’ contributions: All the authors wrote and
anticariogenic active ingredient present in all toothpastes, approved the final manuscript.
in the form of sodium or sodium monofluorophosphate. Acknowledgements: The authors would like to thank

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In Peru, most in vitro studies related to the inhibition Dr. Elva Mejía Delgado of the Microbiology Laboratory,
of Streptococcus mutans are carried out with homemade School of Medicine of Universidad Nacional de Trujillo

in
toothpastes, which are not yet commercialized, so it – Perú, for the processing of samples.
becomes relevant to study the products available in

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the market. A common question by parents is “Which
toothpaste is the best?”, and the answer must be based
on scientific evidence. For many parents it probably does
not matter which brands are used for oral hygiene, as all
they perceive all toothpastes to be good as long as the
brushing technique and the frequency of the brushing
of
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are adequate.
However, it is known that tooth decay is a multifactorial
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disease and more efficient measures are needed to

help prevent the onset of this disease. Therefore it is
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important to choose the toothpastes that best inhibit

pathogens such as Streptococcus mutans or those whose
inhibitory effects last longer in the mouth.
The present study does not define which toothpaste is
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the best due to the natural limitations of an in vitro study.

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For drawing such conclusion, it is necessary to study

other factors such as their effectiveness preventing
biofilm formation, how they counteract acid salivary
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pH, or the antimicrobial inhibitory action against other

cariogenic microorganisms.

CONCLUSION.
The in vitro antimicrobial inhibitory action that
pediatric toothpastes have against Streptococcus mutans
ATCC 25175 has been demonstrated. Oral B Stages,
Colgate Smiles and Denture Kids are the toothpastes
that inhibited this microorganism most effectively.

34 ISSN Print 0719-2460 - ISSN Online 0719-2479. www.joralres.com/2020

 Miñano J & Espinoza MV.
In vitro efficacy of five pediatric toothpastes in the inhibition of Streptococcus mutans ATCC 25175.
J Oral Res 2020; 9(1):29-35. Doi:10.17126/joralres.2020.005

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