UNIVERSIDADE FEDERAL FLUMINENSE
INSTITUTO DE SAÚDE DE NOVA FRIBURGO
FACULDADE DE ODONTOLOGIA
THAMYRIS PY DOMINGOS FAIAL SANTOS
ATIVIDADE ANTIMICROBIANA DE ANTISSÉPTICOS BUCAIS
COMERCIAIS EM BIOFILMES: UM ESTUDO EX VIVO.
NOVA FRIBURGO
2017
ii
THAMYRIS PY DOMINGOS FAIAL SANTOS
ATIVIDADE ANTIMICROBIANA DE ANTISSÉPTICOS BUCAIS
COMERCIAIS EM BIOFILMES: UM ESTUDO EX VIVO.
Dissertação apresentada à Faculdade de
Odontologia da Universidade Federal
Fluminense / Instituto de Saúde de Nova
Friburgo, como parte dos requisitos
exigidos para a obtenção do título de
Mestre em Odontologia, na Área de
concentração em Biologia e Patologia
Buco-Dental.
Orientadora: Profa. Dra. Natalia Iorio Lopes Pontes
Orientador: Prof. Dr. Helvécio Cardoso Corrêa Póvoa
NOVA FRIBURGO
2017
iii
FICHA CATALOGRÁFICA
iv
THAMYRIS PY DOMINGOS FAIAL SANTOS
ATIVIDADE ANTIMICROBIANA DE ANTISSÉPTICOS COMERCIAIS
EM BIOFILMES: UM ESTUDO EX VIVO.
Dissertação apresentada à Faculdade de
Odontologia da Universidade Federal
Fluminense / Instituto de Saúde de Nova
Friburgo, como parte dos requisitos
exigidos para a obtenção do título de
Mestre em Odontologia, na Área de
concentração em Biologia e Patologia
Buco-Dental.
Aprovado em :
BANCA EXAMINADORA
Profa. Dra. Lívia Azeredo Alves Antunes
Profa. Dra. Natalia Iorio Lopes Pontes Póvoa
Profa. Dra. Paula Alvarez Abreu
NOVA FRIBURGO
2017
v
AGRADECIMENTOS
Agradeço aos meus pais, Edson e Sueli, por sempre me darem suporte e terem investido em
minha educação.
Agradeço aos meus filhos, Manuella e Bruno, por me incentivarem a ser sempre melhor, seja
com uma palavra amiga, ou com um simples sorriso sem dente.
Agradeço ao meu marido Bruno Quaresma, por me fazer sempre acreditar em mim, por todo
amor e paciência.
Agradeço à minha orientadora Natalia Iorio, por tanto ter se dedicado aos meus projetos, por
toda a compreensão, por tudo o que me ensinou, por ser meu exemplo e uma grande amiga.
Agradeço ao Helvécio, por ter se dedicado a este trabalho.
Agradeço à Pâmela Ornellas e a Caroline Corrêa, por terem me ajudado na execução deste
trabalho.
Agradeço às professoras membros da banca de qualificação, Andréa Videira Assaf e Ângela
Scarparo Caldo-Teixeira, pelas sugestões apresentadas.
Por fim, agradeço a Deus pela oportunidade, e sempre abençoar os meus projetos, a mim e a
minha família.
vi
RESUMO
Antissépticos bucais apresentam grande variedade comercial e seus princípios ativos
frequentemente são: Cloreto de Cetilpiridinio (CPC); Clorexidina (CLX); Fluoreto de Sódio
(FS) e Timol (THY). O objetivo desse estudo foi avaliar a atividade de 7 antissépticos
comerciais em biofilmes orais, ex vivo, por 7 dias consecutivos. Os biofilmes foram formados
sobre membranas de acetato celulose, a partir de um “pool” de saliva não estimulada de 7
voluntários saudáveis, sem cárie e doença periodontal e com os dentes naturais. Os
antissépticos avaliados foram: Oral-B® (CPC); Cepacol ® (CPC); Periogard sem álcool®
(CLX); Noplak Max® (CLX+CPC); Listerine Cool Mint® (THY; Listerine Zero® (THY);
Plax Fresh Mint® (CPC+FS), além de um controle positivo (CLX 0,12%) e um negativo
(água destilada). Os biofilmes formados foram avaliados através da quantificação das
Unidades Formadoras de Colônias (UFCs) de microrganismos totais, Lactobacillus, Candida
albicans, Candida tropicalis, Streptococcus totais e Estreptococos do grupo mutans (EGM),
após o primeiro, quarto e sétimo dia de tratamento com os antissépticos. Os antissépticos
contendo clorexidina apresentaram o melhor desempenho na redução de todos os
microrganismos avaliados, sendo o Periogard sem álcool® o mais efetivo na redução de
microrganismos totais, Lactobacillus, Candida e Streptococcus totais, mesmo quando
comparado ao controle positivo. O antisséptico Oral B® apresentou o melhor desempenho
dentre os antissépticos que continham cloreto de cetilpiridínio. Nenhum antisséptico foi capaz
de eliminar todos os microrganismos. Antissépticos contendo clorexidina constituem uma
terapia complementar efetiva na redução de microrganismos, entretanto diferentes
componentes em sua formulação interferem em seu desempenho.
Palavras-chave: Antissépticos Bucais, Biofilme, Cárie Dentária
vii
ABSTRACT
Mouthwashes have a wide commercial variety and their active principles are often:
Cetylpyridinium chloride (CPC); Chlorhexidine (CLX); Sodium Fluoride (FS) and Thymol
(THY).The aim of this study was to evaluate the activity of 7 commercial mouthwashes in
oral biofilms, ex vivo, for 7 consecutive days. Biofilms were formed on cellulose acetate
membranes from an unstimulated saliva pool of 7 healthy volunteers, without caries and
periodontal disease and with natural teeth. The mouthwashes evaluated were: Oral-B®
(CPC); Cepacol ® (CPC); Alcohol-free Periogard® (CLX); Noplak Max® (CLX+CPC);
Listerine Cool Mint® (THY); Listerine Zero® (THY); Plax Fresh Mint® (CPC+SF), and a
positive control (CLX 0.12%) and a negative (distilled water). The biofilms formed were
evaluated by quantification of Colony Forming Units (CFUs) of total microorganisms,
Lactobacillus spp., Streptococcus spp., Candida albicans, Candida tropicalis, and mutans
group streptococci (MGS), after the first, fourth and seventh days of mouthwash treatment.
The mouthwashes containing chlorhexidine presented the best performance in reducing all the
microorganisms evaluated, being the alcohol-free Periogard® the most effective in reducing
total microorganisms, Lactobacillus spp., Candida and Streptococcus spp., even when
compared to the positive control. The mouthwash Oral B® presented the best performance
among the mouthwashes containing cetylpyridinium chloride. No mouthwash was able to
eliminate all microorganisms. Mouthwashes containing chlorhexidine are an effective
complementary therapy in the reduction of microorganisms, however different components in
its formulation interfere significantly in its performance.
Keywords: Mouthwashes, Biofilms, Dental Caries.
viii
SUMÁRIO
1. INTRODUÇÃO 1
2. OBJETIVOS 4
3. CAPÍTULO 5
3.1. Artigo 5
3.1.1. Abstract 6
3.1.2. Introduction 7
3.1.3. Results 8
3.1.4. Discussion 12
3.1.5. Materials and methods 14
3.1.7. Acknowledgments 18
3.1.8. References 19
3.1.9. Table and Figures 25
4. CONSIDERAÇÕES FINAIS 32
5. REFERÊNCIAS 33
6. ANEXOS 37
Anexo 1. Parecer do comitê de ética em pesquisa com seres humanos 37
Anexo 2. Normas – Antimicrobial Agents and Chemotherapy 42
1
1. INTRODUÇÃO
A cárie é um processo patogênico que ocorre na superfície dentária, decorrente do
acúmulo de biofilme. Essa é uma das doenças infecciosas mais frequentes entre os humanos,
que resulta na desmineralização e até na formação de cavidade na superfície dentária
(ESCRIBANO et al., 2005).
A microbiota da cavidade bucal é um ecossistema complexo, formado por uma
grande variedade de microrganismos, como: Bacilos Gram positivos e negativos,
Espiroquetas, Candida, Streptococcus spp. e Staphylococcus spp. Estes encontram-se em
diversos nichos dentro da cavidade bucal, apresentando preferências por alguns. Sendo os
grupos mais prevalentes Streptococcus spp. e os bacilos Gram-positivos (HAMADA e
SLADE, 1980; LINOSSIER et al., 2011).
O biofilme oral é composto por espécies de bactérias que sintetizam matriz
extracelular, que representa de 75 a 80% do biofilme (COSTERTON et al.,1999;
KOLENBRANDER, 2000). Este biofilme, a princípio, atua como uma barreira, impedindo a
colonização por bactérias patogênicas. Um biofilme saudável pode ser formado por mais de
700 espécies microbianas, sendo menos de 1% destas patogênicas (ESCRIBANO et al.,
2005). Porém, em casos de desequilíbrio nas populações microbianas, ocorre a proliferação de
espécies patogênicas acidúricas e acidogênicas podendo resultar na perda de minerais e a
formação de uma lesão cariosa (MILICICH, 2008; ESCRIBANO et al., 2005).
O gênero bacteriano Streptococcus é composto por microrganismos cocos Gram
positivos, não móveis, catalase negativos, produtores de ácido láctico, propiônico, acético e
fórmico. Os microrganismos desse gênero apresentam capacidade de alterar o pH do meio de
7,0 para 4,2 em aproximadamente 24 horas, por meio de ácidos que são produzidos através da
fermentação de carboidratos como: sacarose, glicose e frutose (HAMADA e SLADE, 1980).
A espécie S. mutans, principal representante do Estreptococcus do Grupo Mutans (EGM),
apresenta uma grande variedade de estruturas que possibilitam a colonização e adesão à
superfície dental, que incluem fímbrias e fibrilas, polissacarídeos extracelulares insolúveis,
adesinas e mecanismos de aderência dependentes ou independentes de sacarose (HOJO et al.,
2009; DURSO et al. 2014).
A espécie Streptococcus mutans era considerada o principal agente etiológico da
cárie (LOESCHE et al., 1975), mas ao longo do tempo, outras espécies foram isoladas de
lesões cariosas, incluindo Lactobacillus spp. (BADET e THEBAUD, 2008), outras espécies
de Streptococcus e Candida (SIMON-SORO e MIRA, 2014).
2
Espécies do gênero Lactobacillus podem estar presentes em alimentos, plantas,
animais e humanos (BLAIOTTA et al., 2008). Nos seres humanos, a cavidade bucal, o trato
gastrintestinal e o genito-urinário são os nichos nos quais esses microrganismos são
comumente encontrados. A maioria das espécies não adere diretamente à superfície dentária,
necessitando de sítios retentivos para sua colonização, como aparelhos ortodônticos, próteses,
dentes em erupção e lesões cariosas (SIGURJONS et al., 1995). A produção de ácido e a
capacidade em sobreviver em meio ácido são as principais características cariogênicas desse
gênero (ADAMS e MARTEAU, 1995).
Os fungos do gênero Candida spp. apresentam muitos fatores de virulência, como
propriedades de aderência aos tecidos e as superfícies. Há várias espécies de Candida com
capacidade de colonização e infecção em humanos, sendo a Candida albicans a mais comum.
As espécies desse gênero podem causar lesões superficiais ou profundas, agudas ou crônicas
em órgãos internos, bem como na pele, garganta, língua e boca (TORRES et al., 2002).
A chave para a prevenção da cárie se faz pelo controle do biofilme, de forma
mecânica ou química. O controle mecânico é realizado pela escovação e utilização de fio
dental, e o controle químico, através de agentes antimicrobianos, os antissépticos bucais.
Estes são utilizados principalmente em paciente com dificuldade de controle mecânico
(GEBRAN e GEBERT, 2002).
Com a crescente popularização dos antissépticos bucais, e a diversidade desses
produtos no mercado, estudos vêm sendo realizados para testar a eficácia dos mesmos
(BUGNO et al., 2006; KOBAN et al., 2011; e GARCIA-GODOY, 2014).
A clorexidina (CLX) é considerada o mais potente princípio ativo presente nos
antissépticos bucais. Trata-se de uma bisguanina catiônica, que se liga diretamente à
superfície bacteriana (SARMENTO e MONTEIRO, 2014), favorecendo a lise da parede
celular, por conta do aumento da permeabilidade de membrana. Adicionalmente, também
reduz o metabolismo e a expressão de adesinas microbianas (TORRES et al., 2002). Esse
princípio ativo é comercializado principalmente na forma de sais de digluconato de
clorexidina e é considerado o padrão-ouro no controle químico de biofilmes bucais
(BALAGOPAL e ARJUNKUMAR, 2013), pois apresenta elevada substantividade sobre
biofilme, propriedade esta que consiste na retenção no local de ação, onde é liberado
lentamente, e evita que seu efeito seja rapidamente neutralizado pelo fluxo salivar. Entretanto
o uso em longo prazo pode causar manchas nos dentes (TORRES et al., 2002; GARCÍA-
CABALLERO et al., 2013).
3
O cloreto de cetilpiridínio (CPC) é um princípio ativo frequentemente encontrado
nos antissépticos bucais, trata-se de um quaternário de amônio, monovalente, catiônico e
tensoativo (MENDES et al., 1995), que aumenta a permeabilidade da parede celular
bacteriana, favorecendo sua lise (GEBRAN e GEBERT, 2002), interferindo no metabolismo e
na habilidade dos microrganismos em aderir às superfícies dentária (TORRES et al., 2000).
Esse agente possui maior poder de retenção inicial quando comparado à clorexidina,
entretanto apresenta menor substantividade. Seu uso prolongado pode causar sensação de
queimação, descoloração dos dentes e ulcerações recorrentes (TORRES et al., 2000).
O timol (THY), um fenol monoterpeno, geralmente isolado de Thymus vulgaris e
Origanum vulgare, popularmente conhecidos tomilho e orégano, respectivamente. Possui
atividade bactericida, fungicida e inseticida (PAVELA, 2014). Esse princípio ativo possui um
grupo hidroxila-fenólico em sua estrutura, que é conhecida por exibir potente atividade
antioxidante por absorção e neutralização de radicais livres (YANISHLIEVA et al, 1999).
Entretanto, apresenta baixa substantividade e pode causar sensação de queimação, gosto
amargo e manchas nos dentes (TORRES et al., 2000; GEBRAN e GEBERT, 2002).
O fluoreto de sódio (FS), através dos cristais de fluorhidroxiapatita, dificulta o
processo de desmineralização e acelera o processo de remineralização (BUZALAF et al.,
2011). Porém, apresenta baixa substantividade, pode causar fluorose e provocar manchas nos
dentes (GEBRAN e GEBERT, 2002). A incorporação de fluoreto de sódio em formulações de
antissépticos bucais pode potencializar o controle de cárie, especialmente pós-escovação
(MÜLLER, 2017).
O objetivo deste estudo foi analisar as mudanças na microbiota presente em um
biofilme oral ex vivo durante 7 dias de tratamento com diferentes antissépticos bucais.
4
2. OBJETIVOS
2.1 OBJETIVO GERAL
Verificar a atividade antimicrobiana de antissépticos comerciais em biofilmes, ex vivo,
formados a partir de um “pool” de saliva.
2.2 OBJETIVOS ESPECÍFICOS
- Quantificar as Unidades Formadoras de Colônias (UFC) de microrganismos totais presentes
nos biofilmes após 1, 4 e 7 dias de tratamento com cada antisséptico;
- Quantificar as UFCs de Streptococcus spp. presentes nos biofilmes após 1, 4 e 7 dias de
tratamento com cada antisséptico;
- Quantificar as UFCs Lactobacillus spp. presentes nos biofilmes após 1, 4 e 7 dias de
tratamento com cada antisséptico;
- Quantificar as UFCs de Candida albicans presentes nos biofilmes após 1, 4 e 7 dias de
tratamento com cada antisséptico;
- Quantificar as UFCs de Candida tropicalis presentes nos biofilmes após 1, 4 e 7 dias de
tratamento com cada antisséptico;
- Quantificar as UFCs de EGM presentes nos biofilmes após 1, 4 e 7 dias de tratamento com
cada antisséptico;
- Comparar os resultados de cada grupo microbiano após o tratamento com os diferentes
antissépticos;
- Comparar os resultados de cada antisséptico de acordo com os períodos de tratamento.
5
3. CAPÍTULO 1
3.1 ARTIGO: Trabalho a ser submetido para o periódico “Antimicrobial Agents and 2
Chemotherapy” 3
Antimicrobial Activity of Commercial Mouthwashes Against Biofilms: An ex vivo Study 4
5
6
Thamyris Py Domingos Faial Santos,a Caroline Corrêa da Silva,
a Pâmela de Oliveira 7
Ornellas,a Andréa Fonseca-Gonçalves,
b Helvécio Cardoso Corrêa Póvoa,
a Natalia Lopes 8
Pontes Póvoa Iorioa#
9
10
Department of Basic Sciences, Health Institute of Nova Friburgo, Fluminense Federal 11
University, Nova Friburgo, RJ, Brazila; Department of Pediatric Dentistry and Orthodontics, 12
Dental School, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazilb 13
14
Running Head: Mouthwashes Activity Against ex vivo Boifilms 15
16
#Address correspondence to Natalia L. P. P. Iorio, [email protected] 17
Universidade Federal Fluminense, Nova Friburgo, RJ, Brazil 18
Rua Doutor Silvio Henrique Braune, 22 – Centro, Nova Friburgo, Rio de Janeiro Brazil, CEP- 19
28625-650 20
Fax: +55-22-25287168 21
22
23
24
25
26
6
ABSTRACT 27
The aim of this study was to evaluate the activity of 7 commercial mouthwashes in oral 28
biofilms, ex vivo, for 7 consecutive days. Biofilms were formed on cellulose acetate 29
membranes from an unstimulated saliva pool of 7 healthy volunteers, without caries and 30
periodontal disease and with natural teeth. The mouthwashes evaluated were: Oral-B® 31
(Cetylpyridinium chloride); Cepacol ® (Cetylpyridinium chloride); Alcohol-free Periogard® 32
(Chlorhexidine); Noplak Max® (Chlorhexidine + Cetylpyridinium chloride); Listerine Cool 33
Mint® (Thymol); Listerine Zero® (Thymol); Plax Fresh Mint® (Cetylpyridinium chloride + 34
Sodium Fluoride), plus a positive control (chlorhexidine 0.12%) and a negative (distilled 35
water). The biofilms formed were evaluated by quantification of Colony Forming Units 36
(CFUs) of total microorganisms, Lactobacillus, Candida, Total Streptococcus and 37
Streptococcus mutans (EGM), after the first, fourth and seventh days of mouthwash treatment. 38
The mouthwashes containing chlorhexidine presented the best performance in reducing all the 39
microorganisms evaluated, being the alcohol-free Periogard® the most effective in reducing 40
total microorganisms, Lactobacillus, Candida and Streptococcus total, even when compared to 41
the positive control. The mouthwash Oral B® presented the best performance among the 42
mouthwashes containing cetylpyridinium chloride. No mouthwash was able to eliminate all 43
microorganisms. There was growth of yeasts, Gram negative bacilli and Gram negative cocci 44
in selective culture media for EGM, Lactobacillus and Streptococcus total. Mouthwashes 45
containing chlorhexidine are an effective complementary therapy in the reduction of 46
microorganisms, however different components in its formulation interfere significantly in its 47
performance. 48
Keywords: Mouthwashes, Oral Biofilms, Microbiota, ex vivo, Dental Caries. 49
50
51
7
INTRODUCTION 52
Dental plaque is a multispecies biofilm of microorganisms can lead inflammatory 53
processes, gingivitis, responsible by the began of periodontal disease, and dental caries 54
(Timmerman and Weijden, 2006). 55
Streptococcus mutans has long been considered the main etiological factor of dental 56
caries (Loesche et al., 1975), but overtime, other species have been isolated from carious 57
lesions, including Lactobacillus spp. (Badet and Thebaud, 2008), Streptococcus and Candida 58
(Simón-Soro and Mira, 2014). 59
Mechanical cleaning by tooth brushing and flossing are used to control the dental 60
biofilm (Parashar, 2015), but the concept of oral rinsing as a hygiene assistant has been used 61
since ancient times but the mouthwashes have changed a lot throughout history (Fishman, 62
1997). Nowadays, the main antimicrobial active principles in mouthwashes are chlorhexine 63
(CLX), cetylpyridinium chloride (CPC) and thymol (THY) (Rosing et al., 2017; Gunsolley 64
2010; Van Leeuwen et al, 2015). 65
CLX is a cationic bisbiguanide having broad-spectrum antibacterial activity (Lang, 66
1982) that has been the mouthwash of choice owing to its therapeutic effect (Eley, 1999). 67
When associated to oral procedures, the reduction of biofilm and gingivitis is approximately 68
in 33% and 26%, compared to controls hygiene (Strydonck et al., 2012). 69
CPC is an amphiphilic quaternary compound with a long history of safe and effective 70
use when incorporated into oral hygiene products (Haps et al., 2008) and has antimicrobial 71
activity (Van Leeuwen et al, 2015). 72
THY acts on cell membrane disruption, leakage of intracellular substances, and 73
subsequent changes in transmembrane potential (Shapiro and Guggenheim, 1995). 74
8
The sodium fluoride (SF) act reducing demineralization and increasing 75
remineralization of the tooth structure (Pizzo et al., 2007), and are noncytotoxic. The usage of 76
oral rinses supplemented with fluoride is safe (Müller, 2017). 77
Mouthwashes can change on the oral microbiota over time (Van Leeuwen et al., 78
2015). The aim of this study was to analyze the changes in the microbiota present in an ex 79
vivo model of oral biofilm, during 7 days of treatment with different mouthwashes. 80
81
RESULTS 82
Sterility of the membranes and media used was proved by blank control, in which no 83
turbidity of the wells was observed. The means and standard deviation in log10 to biofilm, at 84
the 1st, 4
th and 7
th days of treatment with each mouthwashes, of total microorganisms, 85
Lactobacillus spp., Streptococcus spp., C. albicans and C. tropicalis were presented in Table 86
1. The statistical differences between the mouthwashes treatment and between the treatment 87
periods for each microbial group could be observed in Fig. 1, Fig 2. Fig 3, Fig 4 and Fig 5, 88
respectively. 89
Total microorganisms count to biofilm after treatments 90
The mean for cultivable total microorganism counts to biofilm varied according 91
treatment and exposition time ranging in log10 from 10.09 (7th
day of treatment with G6) to 92
0.94 (7th
day of treatment with G8) (Table 1). At the 1st day, comparing with G1 treatment 93
(negative control), the THY groups (G2 and G3) did not showed significant differences, but 94
statistical differences were observed for all three mouthwashes containing CLX (G7, G8 and 95
G9). for the CPC groups (G4, G5 and G6) only G4 presented significant differences 96
comparing with G1 (water). While comparing with G9 (positive control), G4 (CPC) and G7 97
(CLX + CPC) presented similar results and G8 (CLX) showed the lowest count (p<0.05) (Fig 98
1). 99
9
At 4th
day, the mouthwashes of THY (G2 and G3) and one of CPC (G5) group were 100
similar to the control group (G1), if comparing with positive control (G9) only G8 (CLX) was 101
statistically similar (Fig 1). 102
At the 7th
day, the THY (G2 and G3) and two mouthwashes from CPC (G4 and G5) 103
group were similar to control group (G1). One representative of CPC group (G6) presented 104
the highest count. Comparing with control group (G9), the mouthwashes from CLX group 105
presented statistical differences, G7 with high count and G8 with low count (Fig 1). 106
The total microorganisms count was constant during 7 days for G1 (water) and G8 107
(CLX) treatments. The counts after THY mouthwashes (G2 and G3) increased in the fourth 108
day, it was also observed for two CPC members (G4 and G5). The other group from CPC 109
(G6) increased in each analyzed day. The mouthwashes containing CLX (G7, G8 and G9) 110
showed constant results in the first and seventh days (Fig 1). 111
Lactobacillus spp. count to biofilm after treatments 112
The counts of Lactobacillus spp. to biofilm varied according treatment and exposition 113
time, the mean ranging in log10 from 8.22 (7th
day of treatment with G1) to 0 (4th
and 7th
day 114
of treatment with G7 and G9). At the 1st day of treatment, the groups from THY (G2 and G3) 115
showed a similar count to G1 (water). The CLX (G7, G8 and G9) and G4 (CPC) did not 116
present statistical differences. Two groups of CPC (G5 and G6) showed upper counts to G9 117
(CLX control) (p<0.05) and nether to G1 (water) (p<0.05) (Fig 2). 118
The counts at the 4th
day presented the same profile that ones found at the first day, but 119
at the 7th
day the similar counts were observed to CLX group (G7, G8 and G9) and two 120
mouthwashes of CPC group (G4 and G6). G3 (THY) and G5 (CPC) were statistically 121
different when comparing with G1 (water) and G9 (CLX) (Fig 2). 122
At the 1st, 4
th and 7
th days of treatment, a constant count of Lactobacillus spp. to CLX 123
group (G7, G8 and G9), G4 (CPC), G5 (CPC) was observed. The other CPC mouthwash (G6) 124
10
decreased the Lactobacillus spp. count at the 7th
day, while G1 (water) and THY group (G2 125
and G3) increased during the treatment (Fig 2). 126
Streptococcus spp. count to biofilm after treatments 127
The count means to biofilm of Streptococcus spp. ranging in log10 from 9.01 (4th
day 128
of treatment with G2) to 0.43 (4th
day of G9) (Table 1). At the 1st day the mouthwashes 129
containing THY (G2 and G3) and two with CPC (G5 and G6) did not showed statistical 130
differences in its mean counts comparing with negative control group (G1-water). Whereas 131
G4 (CPC), G7 (CLX) and G8 (CLX) were statistically similar comparing with positive 132
control group (G9-CLX) (Fig 3). 133
At the 4th
day, THY (G2, G3) and CPC (G4, G5 and G6) mouthwashes presented 134
statistically similar count comparing with negative control group (G1). G8 (CLX) did not 135
showed statistical differences comparing with positive control group (G9-CLX), and G7 136
(CLX) had differences with G1 (water) and G9 (CLX) (Fig 3). 137
G1 (water), G2 (THY), G3 (THY) and G5 (CPC) presented statistically similar count 138
at the 7th
day of treatment, G8 (CLX) showed low count comparing with G9 (p<0.05) (Fig 3). 139
The analyses of G4, G6 and G7 at the 7th
day could not be performed for Streptococcus spp. 140
to biofilm because in the possible count dilution were not observed Gram-positive cocci. 141
During the days of treatment G1 (water), G4 (CPC), G6 (CPC) and G7 (CLX) 142
presented a tendency to increase the Streptococcus spp. count to biofilm. G2 (THY) as well 143
G9 (CLX) showed constant count in the first and seventh days. No tendency to increase of 144
decrease was observed in the counts of one representative of each mouthwash group (G3-145
THY, G5-CPC and G8-CLX) in the analyzed days (Fig 3). 146
C. albicans count to biofilm after treatments 147
The highest count mean of C. albicans to biofilm in log10 was 8.11 (1st day of 148
treatment with G1) and the lowest was 0 (4th
and 7th
day of G8 and G9) (Table 1). G2 (THY) 149
11
was the only group that presented a similar result comparing with the water (G1), the others 150
except two (G3-THY, G4-CPC, G6-CPC and G7-CLX) were similar if compared with G9 151
(CLX). G5 (CPC) and G8 (CLX) were statistically different of G9 (CLX) with a high and a 152
low count, respectively, at first day of treatment (Fig 4). 153
At the 4th
day, THY (G2 and G3) and CPC (G4, G5 and G6) groups, and G7 (CLX) 154
showed similar results comparing with G1 (negative control). G8 (CLX) and G9 (CLX) did 155
not present statistical differences. This results profile could also be observed at 7th
day, but G2 156
(THY) had the high statistic count comparing to G1 (water) (Fig 4). 157
It was observed a tendency to decrease C. albicans count to biofilm for the two 158
treatment controls (G1-water and G9-CLX), whereas an increase tendency was observed for 159
one representative of each group of mouthwashes (G3-THY, G4-THY and G7-THY). G2 160
(THY), G5 (CPC), G6 (CPC) and G8 (CLX) showed constant counts in the analyzed days 161
(Fig 4). 162
C. tropicalis count to biofilm after treatments 163
The log10 C. tropicalis count ranged from 7.28 (4th
day of treatment with G5) to 0 (1st, 164
4th
and 7th
day of G7, G8 and G9). The CLX groups (G7, G8 and G9) eliminated this yeast 165
from the first day of treatment (Table 1). At the 1st day, the THY (G2 and G3) and CPC (G4, 166
G5 and G6) groups presented significant differences comparing to negative (G1-water) and 167
positive (G9-CLX) controls (Fig 5). 168
At the 4th
day THY (G2 and G3) and CPC (G4, G5 and G6) groups presented similar 169
results comparing with negative group (G1-water), but at the 7th
day G3 (THY), G4 (CPC) 170
and G5 (CPC) changed this profile and came back to present statistical differences comparing 171
with G1 (water) (Fig 5). 172
12
The only one treatment that reduced the C. tropicalis count during the days was water 173
(G1), the others keep themselves constant (G2, G3, G4, G5, G6, G7, G8 and G9), but the CPC 174
group (G4, G5 and G6) had it count high at the 4th
day (Fig 5). 175
Mutans Group Streptococci (MGS) count to biofilm after treatments 176
It was only possible to count MGS to biofilm in the CLX group (G7, G8 and G9) and 177
these mouthwashes showed efficacy to eliminate this microorganism. For the others groups 178
of mouthwashes, it was not observed Gram-positive cocci in the possible count dilution. 179
180
DISCUSSION 181
The use of oral rinses has been widely applied as mechanical tools that may help oral 182
hygiene (Tartaglia et al., 2016) as an adjunct treatment for gingival health (James et al., 2017) 183
to improve periodontal health during pregnancy (Jiang et al., 2016), for the prevention of 184
ventilator-associated pneumonia (Hua et al., 2016; Zand et al., 2017) and for the reduction of 185
oropharyngeal colonization (Zand et al., 2017). This study evaluated the antimicrobial activity 186
of commercial mouthwashes against an ex vivo biofilm model with human saliva, evaluating 187
microorganisms associated with bacterial aggregates in subgingival biofilm (Lactobacillus 188
spp.) and that form corncob structures in supragingival plaque (Streptococcus spp. and C. 189
albicans) (Zijnge et al., 2010). 190
Studies have analyzed antimicrobial proprieties of mouthwashes containing essential 191
oils (EO) in its composition (Jain and Jain, 2016; Müller et al., 2017, Paulone et al., 2017). In 192
the present study, THY mouthwashes (G2 and G3) not presented statistical different results 193
comparing with G1 (water) in most cases. An in vivo study, statistical decrease of S. mutans 194
in saliva after essential oil rinse twice daily for 1 week was not observed (Jain and Jain, 195
2016). Paulone et al. (2017) showed that a THY mouthwash did not have effect against C. 196
albicans by disk diffusion assay. 197
13
Among the four mouthwashes contain EO, Müller et al., (2017) verified 198
heterogeneous (none, moderate and severe) antimicrobial results against bacteria planktonic 199
cells, but to the EO alone this effect was not observed. The authors credited the antimicrobial 200
effects to other ingredients, supplementing of active additives. 201
This study showed that biofilms treated with THY formulation without alcohol (G3) 202
showed decrease count for all microorganisms studied compared to the formulation with 203
alcohol (G2); this differences to Lactobacillus spp. at the 7th
day and C. tropicalis at 1th
and 204
7th
were statistically significant. In all cases, THY mouthwashes presented statistical greatter 205
counts than the positive control (G9-CLX). Müller et al. (2017) also observed better 206
antibacterial activity to CLX mouthwahses compared to EO ones. 207
The mouthwashes containing CPC exhibited varied antimicrobial effects, according to 208
the microorganisms and treatment periods evaluated. In 2017, a study evaluated two 209
mouthwashes contained CPC, one of them presented a moderate and the other a potent 210
activity against bacteria (Müller et al., 2017). This widely variations can be attributed to other 211
ingredients, which are able to act in synergic or antagonic way. 212
All three CPC mouthwashes tested by Schaeffer et al. (2011) were able to kill more 213
than 99.9% of planktonic cells of A. actinomycetemcomitans and S. mutans. In the present 214
study this percentage was only observed for the Lactobacillus spp. count at the first day, 215
probably because this study was realized with non-planktonic cells. 216
Two mouthwashes that contain CPC (G4 and G6) were responsible to the lower count 217
than that ones presented by THY mouthwashes (G2 and G3). In most cases, at least one CPC 218
representative showed statistical reduction compared with THY formulation without alcohol 219
(G3), especially to Lactobacillus spp. and Candida counts. Similar results was found in a 220
recently study, in which the four CPC mouthwashes had similar or low antimicrobial activity 221
compared to THY ones (Müller et al., 2017). 222
14
The effects of CPC test rinses on the viability of biofilms formed by salivary bacteria 223
were also tested before, the viability of aerobes/facultative anaerobes, total anaerobes and 224
Gram-negative anaerobes was also assessed by viable counting after four days of twice daily 225
treatment and CPC mouthwash exhibited significant viability reductions in all groups 226
compared to negative control (Latimer et al., 2015). At the 4th
day in the present study, the 227
majority CPC mouthwashes presented reduction counts comparing to water, but few 228
reductions were statistically significant, this fact can be justified by one daily treatment. 229
Fluoride aids in enamel remineralization (Premaraj et al., 2017). Mouthwashes with 230
this component have been recommended as an adjunct to oral hygiene as prophylactic 231
measure in fixed orthodontic patients to prevent white spot lesions (Khoroushi and Kachuie, 232
2017). In 2015, Latimer and co-workers showed that CPC mouthrinses, with and without 233
fluoride, exhibited significant antibacterial efficacy against oral bacteria in planktonic and 234
biofilm modes (Latimer et al., 2015). In the present study CPC mouthwash with fluoride (G6) 235
had similar statistical result with at least one without fluoride (G4 and G5), except in only one 236
case (7th
day/Total microorganisms). These results suggest that fluoride does not interfere 237
with the antimicrobial effects against the microorganisms analyzed in the ex vivo model used. 238
It was observed that CPC mouthwashes exhibited any counts tendency during the 239
analyzed days. It can be explained by the significant differences in 240
substantivity/bioavailability of CPC commercialized mouthrinses (Garcia-Godoy et al., 241
2014). 242
In the majority CPC mouthwashes presented statistical greater counts than the positive 243
control (G9-CLX), better antibacterial activity to CLX compared with CPC mouthwahses was 244
previously observed (Müller et al., 2017). 245
The greater antimicrobial results were presented by CLX mouthwashes. This is 246
commonly observed in the literature to bacteria and yeast (Jain and Jain, 2016; Mostajo et al., 247
15
2017; Müller et al., 2017; Paulone et al., 2017). 248
In general, the G8 (CLX) exhibited better performance than others CLX mouthwashes, 249
in particular to total microorganisms, Streptococcus spp. and C. albicans. This can also be 250
associated with other components from the commercial mouthwashes that can act in the 251
synergic way. 252
In 2003, a study showed the action synergistically of CPC with CLX to increase the 253
antimicrobial activity of CLX mouthwashes in bacteria planktonic cells (Herrera et al., 2003). 254
However, Babu and Garcia-Godoy (2004) did not observed differences between two oral 255
rinses that contained CLX or CLX+CPC, as both had the greatest antibacterial activity on 256
planktonic and biofilm-grown organisms. However, the present study showed that among the 257
CLX, the high total microorganisms and C. albicans counts were observed to the 258
mouthwashes that contained CLX with CPC. 259
Several adverse effects and citotoxic effects were attributed to mouthwashes, 260
especially the CLX ones (Balloni et al., 2016; Müller et al., 2017). In general, oral rinses with 261
antimicrobial activity are also cytotoxic (Faria et al., 2007; Müller et al., 2017). In this point 262
of view, it is important the rational use of mouthwashes. 263
In general, this is the first report describing the antimicrobial effects of 7 264
mouthwashes during 7 days of treatment against total microorganisms, Lactobacillus spp., 265
Streptococcus spp., C. albicans and C. tropicalis. No antiseptic was able to eliminate all 266
microorganisms, and antiseptics containing chlorhexidine are an effective complementary 267
therapy in the reduction of microorganisms, however different components in its formulation 268
could be interfering in its performance. 269
MATERIAL AND METHODS 270
Mouhtwashes and controls 271
Seven mouthwashes and two controls were analyzed in this study, which were 272
16
organized in nine groups. G1: sterile ultrapure water (negative control); G2: Listerine Cool 273
Mint® (THY) (Pfizer, lot number L1955B14); G3: Listerine Zero® (THY) (Pfizer, São 274
Paulo, Brazil, lot number L2665B05); G4: Oral-B® (CPC) (Lab. Rety, Rio de Janeiro, Brazil, 275
lot number L42165395UB); G5: Cepacol® (CPC) (Aventis Pharma, São Paulo, Brazil, lot 276
number L543563); G6: Plax Fresh Mint® (CPC+SF) (Colgate-Palmolive, lot number 277
L4353BR121); G7: Noplak® (CLX+CPC) (Lab. Daudt Oliveira, Rio de Janeiro, Brazil, lot 278
number L160296); G8: Periogard without alcohol® (CLX) (Colgate-Palmolive, São Paulo, 279
Brazil, lot number 5245BR121A) and G9: Chlorhexidine gluconate (FGM, Santa Catarina, 280
Brazil, lot number 080414) diluted in ultrapure water at 0.12% (positive control). 281
Pool of human saliva to form biofilm on membrane disks 282
A pool of human saliva was used as an inoculum accordance with the methodology 283
proposed by Antonio et al. (2011). Unstimulated saliva was collected from seven adult 284
volunteers (2 men and 5 women) who had fasted for 1 h. Moreover, none of them received 285
antibiotic therapy or used mouthwashes within the previous 3 months. These volunteers 286
signed a term of free and informed consent, authorizing saliva collection. The research was 287
approved by the Local Research Ethics Committee (Protocol No. 1.417.101). All the 288
volunteers presented a good state of general and oral health. Thus, unstimulated saliva was 289
collected from each individual in a graded tube, while the individual were comfortably seated. 290
Their mean whole saliva flow rate (0.272 mL/min) was registered. The saliva (7 mL) from 291
each volunteer was placed into a same larger tube, which was mixed, resulting in a pool with 292
1.02x108
CFU of cultivable total microorganisms to mL. From this pool, 2.7x103, 2.1x10
7, 4.4 293
x102, 2x10
1 and 6x10
1 CFU/mL of Lactobacillus spp., Streptococcus spp., Mutans Group 294
Streptococci (MGS), Candida tropicalis and Candida albicans were identified, respectively. 295
Biofilm model 296
17
To producing biofilms the membrane disks of cellulose acetate with pore size of 0.2 297
µm and diameter of 0.13 mm (Sartorius Biolab Products, Göttingen, Germany) were used. An 298
inoculum of 100 µL of the saliva pool was added inside of each well into a 24-well of a 24-299
well tissue culture plates (Kasvi, Paraná, Brazil) with a membrane disk and a 900 µL of Brain 300
Heart Infusion (BHI) broth (Becton, Dickinson and Company, Sparks, MD, USA) 301
supplemented with 2% sucrose (Isofar, Rio de Janeiro, Brazil). The system was incubated at 302
37˚C for 24 hours in 5% CO2 for biofilm formation. 303
Biofilm trataments 304
After biofilm formation on the disks, nine different groups of treatment (7 305
mouthwashes and 2 controls) were performed (18 membranes for each group). Before 306
treatment with 1 mL on biofilm for 30 s, the medium from each well was removed. Then the 307
antiseptic was removed and each membrane was washed 3 times with sterile ultrapure water 308
in order to remove the mouthwashes and planktonic cells, after that a new BHI broth (Difco) 309
with sucrose (Isofar) was inserted into the wells. The treatments were performed once a day, 310
at the same hour, during seven days. Six membrane disks, which did not receive inoculum and 311
treatment, were considered the blank control. 312
Microbial Cellular Viability after Treatments 313
Microbial cellular viability present in the biofilms after the treatments were defined by 314
Colony Forming Units (CFU) count. At the end of the first, fourth and seventh days of 315
treatment, six membranes of each treatment, were placed in glass tubes containing 1 mL of 316
sterile saline solution with 10 beads glass and these system were submitted to a tube shaker 317
(Kasvi) for 30 s / 3300 rpm following of three cycles of ultrasonic vibration (Bio Wash STD - 318
Bio Art São Carlos, Brazil) for 8 min/42000 hz and agitation (Kasvi) for 30 s / 3300 rpm, in 319
order to detach the microbial cells from biofilm, and decimal dilutions were performed up to 320
10-7
. Aliquots of 50 μL were plated to evaluate the microorganisms viability, on the following 321
18
culture media: BHI agar (Becton, Dickinson and Company, Sparks, MD, USA) to total 322
microorganisms, Rogosa Agar (Becton, Dickinson and Company) to Lactobacillus spp., Mitis 323
Salivarius Agar (Becton, Dickinson and Company) to Streptococcus spp., Mitis Salivarius 324
Agar (Becton, Dickinson and Company) containing bacitracin, sucrose and glucose to MGS 325
and CHROMagar Candida (Becton, Dickinson and Company) to Candida spp.. The plates 326
were then incubated at 37oC / 5% CO2 for 48 hours, except to CHROMagar Candida (Becton, 327
Dickinson and Company) that were incubated in the aerobically atmosphere. The results were 328
expressed in CFU/biofilm. Cell morphology was evaluated by Gram staining for all colonies 329
types grown on the selective media and only that ones with typical characteristics were count. 330
Statistical Analysis 331
Data were analyzed by means of the statistical software program SPSS version 20.0 332
(SPSS Inc., Chicago, USA). The Shapiro-Wilk test was used to verify the distribution of 333
normality. ANOVA followed by Tukey or Man-Whitney test was used to verify whether there 334
was statistical difference among the groups of treatment. 335
336
ACKNOWLEDGMENTS 337
This study was supported by grants from: Fundação Carlos Chagas Filho de Amparo à 338
Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Pró-Reitoria de Pesquisa, Pós-339
Graduação e Inovação / Plano de Desenvolvimento Institucional/Universidade Federal 340
Fluminense (PROPPI/PDI/UFF). 341
The authors declare no conflicts of interest. 342
343
344
345
346
19
REFERENCES 347
Antonio, A. G., et al. "Inhibitory properties of Coffea canephora extract against oral bacteria 348
and its effect on demineralisation of deciduous teeth." Archives of oral biology 56.6 (2011): 349
556-564. 350
Babu, Jegdish P., and Franklin Garcia-Godoy. "In vitro comparison of commercial oral rinses 351
on bacterial adhesion and their detachment from biofilm formed on hydroxyapatite 352
disks." Oral health & preventive dentistry12.4 (2004): 365-371. 353
Balloni, Stefania, et al. "Cytotoxicity of three commercial mouthrinses on extracellular matrix 354
metabolism and human gingival cell behaviour." Toxicology in Vitro 34 (2016): 88-96. 355
Badet, C., and N. B. Thebaud. "Ecology of lactobacilli in the oral cavity: a review of 356
literature." The open microbiology journal 2 (2008): 38. 357
Eley, B. "Antibacterial agents in the control of supragingival plaque--a review." British dental 358
journal 186.6 (1999). 359
Faria, Gisele, et al. "Evaluation of chlorhexidine toxicity injected in the paw of mice and 360
added to cultured l929 fibroblasts." Journal of endodontics 33.6 (2007): 715-722. 361
Fischman, Stuart L. "The history of oral hygiene products: how far have we come in 6000 362
years?." Periodontology 2000 15.1 (1997): 7-14. 363
Garcia-Godoy, Malgorzata A. Klukowska, and Yanhui H. Zhang. "Comparative 364
bioavailability and antimicrobial activity of cetylpyridinium chloride mouthrinses in vitro and 365
in vivo." American journal of dentistry 27.4 (2014). 366
Gunsolley, John C. "Clinical efficacy of antimicrobial mouthrinses." Journal of Dentistry 38 367
(2010): S6-S10. 368
Haps, S., et al. "The effect of cetylpyridinium chloride‐containing mouth rinses as adjuncts to 369
toothbrushing on plaque and parameters of gingival inflammation: a systematic 370
review." International journal of dental hygiene 6.4 (2008): 290-303. 371
20
Herrera, David, et al. "Differences in antimicrobial activity of four commercial 0.12% 372
chlorhexidine mouthrinse formulations: an in vitro contact test and salivary bacterial counts 373
study." Journal of clinical periodontology 30.4 (2003): 307-314. 374
Hua, Fang, et al. "Oral hygiene care for critically ill patients to prevent ventilator‐ associated 375
pneumonia." The Cochrane Library (2016). 376
Jain, Isha, and Pankaj Jain. "Comparative evaluation of antimicrobial efficacy of three 377
different formulations of mouth rinses with multi-herbal mouth rinse." Journal of Indian 378
Society of Pedodontics and Preventive Dentistry 34.4 (2016): 315. 379
James, Patrice, et al. "Chlorhexidine mouthrinse as an adjunctive treatment for gingival 380
health." The Cochrane Library (2017). 381
Jiang, H., et al. "Use of antiseptic mouthrinse during pregnancy and pregnancy outcomes: a 382
randomised controlled clinical trial in rural China." BJOG: An International Journal of 383
Obstetrics & Gynaecology 123.S3 (2016): 39-47. 384
Khoroushi, Maryam, and Marzie Kachuie. "Prevention and treatment of white spot lesions in 385
orthodontic patients." Contemporary clinical dentistry 8.1 (2017): 11. 386
Lang, N. P., et al. "Effects of supervised chlorhexidine mouthrinses in children." Journal of 387
periodontal research 17.1 (1982): 101-111. 388
Latimer, Joe, et al. "Antibacterial and anti-biofilm activity of mouthrinses containing 389
cetylpyridinium chloride and sodium fluoride." BMC microbiology 15.1 (2015): 169. 390
Loesche, W. J., et al. "Association of Streptococcus mutans with human dental 391
decay." Infection and immunity 11.6 (1975): 1252-1260. 392
Mostajo, Mercedes Fernandez, et al. "Effect of mouthwashes on the composition and 393
metabolic activity of oral biofilms grown in vitro." Clinical oral investigations 21.4 (2017): 394
1221-1230. 395
21
Müller, Heinz-Dieter, et al. "Cytotoxicity and Antimicrobial Activity of Oral Rinses In 396
Vitro." BioMed research international 2017 (2017). 397
Parashar, Amit. "Mouthwashes and Their Use in Different Oral Conditions." Scholars Journal 398
of Dental Sciences (SJDS) 2 (2015): 186-191. 399
Paulone, Simona, et al. "Candida albicans survival, growth and biofilm formation are 400
differently affected by mouthwashes: an in vitro study." New Microbiol 40 (2017): 45-52. 401
Pizzo, Giuseppe, et al. "Community water fluoridation and caries prevention: a critical 402
review." Clinical oral investigations 11.3 (2007): 189-193. 403
Premaraj, T. S., et al. "In vitro evaluation of surface properties of Pro Seal® and Opal® 404
SealTM in preventing white spot lesions." Orthodontics & Craniofacial Research 20.S1 405
(2017): 134-138. 406
Rösing, Cassiano Kuchenbecker, et al. "Efficacy of two mouthwashes with cetylpyridinium 407
chloride: a controlled randomized clinical trial." Brazilian Oral Research 31 (2017). 408
Schaeffer, Lyndsay M., et al. "In vitro antibacterial efficacy of cetylpyridinium chloride-409
containing mouthwashes." Journal of Clinical Dentistry 22.6 (2011): 183. 410
Shapiro, S., and B. Guggenheim. "The action of thymol on oral bacteria." Molecular Oral 411
Microbiology 10.4 (1995): 241-246. 412
Simón-Soro, Áurea, Miriam Guillén-Navarro, and Alex Mira. "Metatranscriptomics reveals 413
overall active bacterial composition in caries lesions." Journal of oral microbiology 6.1 414
(2014): 25443. 415
Strydonck, Daniëlle AC, et al. "Effect of a chlorhexidine mouthrinse on plaque, gingival 416
inflammation and staining in gingivitis patients: a systematic review." Journal of clinical 417
periodontology 39.11 (2012): 1042-1055. 418
Tartaglia, Gianluca M., et al. "Mouthwashes in the 21st century: a narrative review about 419
active molecules and effectiveness on the periodontal outcomes." Expert opinion on drug 420
22
delivery (2016): 1-10. 421
Timmerman, M. F., and G. A. Weijden. "Risk factors for periodontitis." International journal 422
of dental hygiene 4.1 (2006): 2-7. 423
Van Leeuwen, M. P. C., et al. "Long‐term efficacy of a 0.07% cetylpyridinium chloride 424
mouth rinse in relation to plaque and gingivitis: a 6‐month randomized, vehicle‐controlled 425
clinical trial." International journal of dental hygiene 13.2 (2015): 93-103. 426
Zand, Farid, et al. "The effects of oral rinse with 0.2% and 2% chlorhexidine on 427
oropharyngeal colonization and ventilator associated pneumonia in adults' intensive care 428
units." Journal of Critical Care (2017). 429
Zijnge, Vincent, et al. "Oral biofilm architecture on natural teeth." PloS one 5.2 (2010): 430
e9321. 431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
23
FIGURE LEGENDS 446
Figure 1 Results of CFU of Total microorganisms in log10 to biofilm during 7 days of 447
treatment with differents mouthwashes. Groups: 1, Ultrapure water (negative control); 2, 448
Listerine Cool Mint® (THY); 3, Listerine Zero® (THY); 4, Oral-B® (CPC); 5, Cepacol® 449
(CPC); 6, Plax Fresh Mint® (CPC+SF); 7, Noplak® (CLX+CPC); 8, Periogard without 450
alcohol® (CLX); 9, 0.12% Chlorhexidine gluconate (positive control). Different lowercase 451
letters in the same day represent statistically significant results between the group, different 452
uppercase letters in the same group of treatment represent statistically significant results 453
between the treatment periods. 454
455
Figure 2 Results of CFU of Lactobacillus spp. in log10 to biofilm during 7 days of treatment 456
with differents mouthwashes. Groups: 1, Ultrapure water (negative control); 2, Listerine Cool 457
Mint® (THY); 3, Listerine Zero® (THY); 4, Oral-B® (CPC); 5, Cepacol® (CPC); 6, Plax 458
Fresh Mint® (CPC+SF); 7, Noplak® (CLX+CPC); 8, Periogard without alcohol® (CLX); 9, 459
0.12% Chlorhexidine gluconate (positive control). Different lowercase letters in the same day 460
represent statistically significant results between the group, different uppercase letters in the 461
same group of treatment represent statistically significant results between the treatment 462
periods. 463
464
Figure 3 Results of CFU of Streptococcus spp. in log10 to biofilm during 7 days of treatment 465
with differents mouthwashes. Groups: 1, Ultrapure water (negative control); 2, Listerine Cool 466
Mint® (THY); 3, Listerine Zero® (THY); 4, Oral-B® (CPC); 5, Cepacol® (CPC); 6, Plax 467
Fresh Mint® (CPC+SF); 7, Noplak® (CLX+CPC); 8, Periogard without alcohol® (CLX); 9, 468
0.12% Chlorhexidine gluconate (positive control). Different lowercase letters in the same day 469
represent statistically significant results between the group, different uppercase letters in the 470
24
same group of treatment represent statistically significant results between the treatment 471
periods. *, In the possible count dilution were not observed Gram-positive cocci. 472
473
474
Figure 4 Results of CFU of C. albicans in log10 to biofilm during 7 days of treatment with 475
differents mouthwashes. Groups: 1, Ultrapure water (negative control); 2, Listerine Cool 476
Mint® (THY); 3, Listerine Zero® (THY); 4, Oral-B® (CPC); 5, Cepacol® (CPC); 6, Plax 477
Fresh Mint® (CPC+SF); 7, Noplak® (CLX+CPC); 8, Periogard without alcohol® (CLX); 9, 478
0.12% Chlorhexidine gluconate (positive control). Different lowercase letters in the same day 479
represent statistically significant results between the group, different uppercase letters in the 480
same group of treatment represent statistically significant results between the treatment 481
periods. 482
483
Figure 5 Results of CFU of C. tropicalis in log10 to biofilm during 7 days of treatment with 484
differents mouthwashes. Groups: 1, Ultrapure water (negative control); 2, Listerine Cool 485
Mint® (THY); 3, Listerine Zero® (THY); 4, Oral-B® (CPC); 5, Cepacol® (CPC); 6, Plax 486
Fresh Mint® (CPC+SF); 7, Noplak® (CLX+CPC); 8, Periogard without alcohol® (CLX); 9, 487
0.12% Chlorhexidine gluconate (positive control). Different lowercase letters in the same day 488
represent statistically significant results between the group, different uppercase letters in the 489
same group of treatment represent statistically significant results between the treatment 490
periods. 491
492
493
494
25
TABLE 1 Results of CFU of Total microorganisms, Lactobacillus spp., Streptococcus spp., C. albicans and C. tropicalis in log10 to biofilm 495
during 7 days of treatment with differents mouthwashes. 496
497 Groups: G1, Ultrapure water (negative control); G2, Listerine Cool Mint® (THY); G3, Listerine Zero® (THY); G4, Oral-B® (CPC); G5, 498
Cepacol® (CPC); G6, Plax Fresh Mint® (CPC+SF); G7, Noplak® (CLX+CPC); G8, Periogard without alcohol® (CLX); G9, 0.12% 499
Chlorhexidine gluconate (positive control); SD, Standard deviation; X, In the possible count dilution were not observed Gram-positive cocci. 500
26
501 502 503
504 FIG 1 505
506
507
FIG 2 508
27
509 FIG 3 510
511
512
FIG 4 513
514
515
28
516
FIG5 517
29
4. CONSIDERAÇÕES FINAIS
Este é o primeiro trabalho que avalia a ação antimicrobiana de diversos antissépticos
bucais, frente à microrganismos totais, Lactobacillus spp., Streptococcus spp., C. albicans e
C. tropicalis, utilizando um modelo de biofilme ex vivo, comparando a dois grupos controles,
durante sete dias de tratamento. Nenhum antisséptico foi capaz de eliminar todos os
microrganismos, e os antissépticos que contém clorexidina em suas composições constituem
uma terapia complementar eficaz na redução de microorganismos. No entanto, diferentes
componentes nas formulações dos antissépticos podem interferir no seu desempenho.
Futuramente, pesquisas clínicas devem ser realizadas para avaliar o efeito do tratamento
diário com antissépticos bucais sobre a microbiota oral.
30
5. REFERÊNCIAS
1. Adams, M. R., & Marteau, P. On the safety of lactic acid bacteria from food. Int J
Food Microbiol. 1995; 27(2-3), 263-264.
2. Badet, C., & Thebaud, N. B. Ecology of lactobacilli in the oral cavity: a review of
literature. Open Microbiol J. 2008; 2, 38.
3. Balagopal, S., & Arjunkumar, R. Chlorhexidine: the gold standard antiplaque agent. J
Pharm Sci Res. 2013; 5(12), 270-4.
4. Baffone, W., Sorgente, G., Campana, R., Patrone, V., Sisti, D., & Falcioni, T.
Comparative effect of chlorhexidine and some mouthrinses on bacterial biofilm
formation on titanium surface. Curr Microbiol. 2011; 62(2), 445-451.
5. Blaiotta, G., Fusco, V., Ercolini, D., Aponte, M., Pepe, O., & Villani, F. Lactobacillus
strain diversity based on partial hsp60 gene sequences and design of PCR-restriction
fragment length polymorphism assays for species identification and
differentiation. Appl Environ Microbiol. 2008; 74(1), 208-215.
6. Bugno, A., Nicoletti, M. A., Almodóvar, A. A., Pereira, T. C., & Auricchio, M. T.
Enxaguatórios bucais: avaliação da eficácia antimicrobiana de produtos
comercialmente disponíveis. Rev Inst Adolfo Lutz (Impr). 2006; 65(1), 40-45.
7. Buzalaf M. A. R., Pessan J. P., Honário H. M., Ten Cate J.
M. Mechanisms of action of fluoride for caries control. Monogr Oral Sci. 2011; 22,
97-114.
8. Costerton, J. W., Stewart, P. S., & Greenberg, E. P. Bacterial biofilms: a common
cause of persistent infections. Sci. 1999; 284(5418), 1318-1322.
31
9. Durso, S. C., Vieira, L. M., Cruz, J. N. S., Azevedo, C. S., Rodrigues, P. H., &
Simionato, M. R. L. Sucrose substitutes affect the cariogenic potential of
Streptococcus mutans biofilms. Caries Res. 2014; 48(3), 214-222.
10. Escribano, M., Matesanz, P., & Bascones, A. Pasado, presente y futuro de la
microbiología de la periodontitis. Av Periodoncia Implantol oral. 2005; 17(2), 79-87.
11. García-Caballero, L., Quintas, V., Prada-López, I., Seoane, J., Donos, N., & Tomás, I.
Chlorhexidine substantivity on salivary flora and plaque-like biofilm: an in situ
model. PLoS One. 2013; 8(12), e83522.
12. Gebran, M. P., & Gebert, A. P. O. Controle químico e mecânico de placa
bacteriana. Tuiuti Cien Cult. 2002; 26(3), 45-58.
13. Hamada, S., & Slade, H. D. Biology, immunology, and cariogenicity of Streptococcus
mutans. Microbiol Rev. 1980 44(2), 331.
14. Hojo, K., Nagaoka, S., Ohshima, T., & Maeda, N. Bacterial interactions in dental
biofilm development. J Dent Res. 2009; 88(11), 982-990.
15. Koban, I., Holtfreter, B., Hübner, N. O., Matthes, R., Sietmann, R., Kindel, E.,
Kocher, T. Antimicrobial efficacy of non‐thermal plasma in comparison to
chlorhexidine against dental biofilms on titanium discs in vitro–proof of principle
experiment. J Clin Periodontol. 2011; 38(10), 956-965.
16. Kolenbrander, P. E. Oral microbial communities: biofilms, interactions, and genetic
systems. Annu Rev Microbiol. 2000; 54(1), 413-437.
17. Linossier, A. C., Valenzuela, C. Y., Soler, E. R., & Contreras, E. M. Colonización de
la cavidad oral por Streptococcus grupo mutans, según edad, evaluado en saliva por un
método semi-cuantitativo. Rev Chil Infectol. 2011; 28(3), 230-237.
18. Loesche, W. J., Rowan, J., Straffon, L. H., & Loos, P. J. Association of Streptococcus
mutans with human dental decay. Infect Immun. 1975; 11(6), 1252-1260.
32
19. Mendes, M. M. S. G., Zenóbio, E. G., & Pereira, O. L. Agentes químicos para controle
de placa bacteriana. Periodontia. 1995; 253-6.
20. Milicich, G. Caries: Una perspectiva de la enfermedad oral que nos esforzamos por
manejar. J Minim Interv Dent. 2008; 1(1), 25-34.
21. Müller, H. D., Eick, S., Moritz, A., Lussi, A., & Gruber, R. Cytotoxicity and
Antimicrobial Activity of Oral Rinses In Vitro. BioMed Res Int. 2017.
22. Pavela, R. Acute, synergistic and antagonistic effects of some aromatic compounds on
the Spodoptera littoralis Boisd.(Lep., Noctuidae) larvae. Ind Crops Prod. 2014; 60,
247-258.
23. Sarmento, D. J., & de Brito Monteiro, B. V. Antimicrobial Potential of the Popular
Antiseptics Anapyon®, Água Rabelo® and Malvatricin® against Oral
Microorganisms. Pesqui Bras Odontopediatria Clin Integr. 2014; 13(4), 309-314.
24. Sigurjons, H., Magnusdottir, M. O., & Holbrook, W. P. Cariogenic bacteria in a
longitudinal study of approximal caries. Caries Res. 1995; 29(1), 42-45.
25. Torres, S. R., Peixoto, C. B., Caldas, D. M., Silva, E. B., Akiti, T., Nucci, M., & De
Uzeda, M. Relationship between salivary flow rates and Candida counts in subjects
with xerostomia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002; 93(2),
149-154.
26. Watnick, P., & Kolter, R. Biofilm, city of microbes. J Bacteriol. 2000; 182(10), 2675-
2679.
27. Yanishlieva, N. V., Marinova, E. M., Gordon, M. H., & Raneva, V. G. Antioxidant
activity and mechanism of action of thymol and carvacrol in two lipid systems. Food
Chem. 1999; 64(1), 59-66.
33
6 ANEXOS
6.1 PARECER DO COMITÊ DE ÉTICA EM PESQUISA COM SERES HUMANOS
34
35
36
37
38
6.2 NORMAS PARA PUBLICAÇÃO NO “Antimicrobial Agents and Chemotherapy”
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
Top Related