ATIVIDADE ANTIMICROBIANA DE ANTISSÉPTICOS BUCAIS COMERCIAIS EM BIOFILMES: UM ESTUDO EX VIVO

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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

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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

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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

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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.

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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.

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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.

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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

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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

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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).

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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.

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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;

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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

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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

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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

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Running Head: Mouthwashes Activity Against ex vivo Boifilms 15

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#Address correspondence to Natalia L. P. P. Iorio, iorionlp@yahoo.com.br 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

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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

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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

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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, 4th and 7th 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

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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

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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, 4th and 7th 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

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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

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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

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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

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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

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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

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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

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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, 2.1x107,4.4 293

x102, 2x101 and 6x101 CFU/mL of Lactobacillus spp., Streptococcus spp., Mutans Group 294

Streptococci (MGS), Candida tropicalis and Candida albicans were identified, respectively. 295

Biofilm model

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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 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 didnot 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

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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

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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

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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

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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

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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

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Khoroushi, Maryam, and Marzie Kachuie. "Prevention and treatment of white spot lesions in 385

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Latimer, Joe, et al. "Antibacterial and anti-biofilm activity of mouthrinses containing 389

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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

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Müller, Heinz-Dieter, et al. "Cytotoxicity and Antimicrobial Activity of Oral Rinses In 396

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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

467

468

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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

477

478

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

486

487

same group of treatment represent statistically significant results between the treatment 490 periods. 491 492 493 494

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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

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501 502 503 504 FIG 1 505 506 507 FIG 2 508

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509 FIG 3 510 511 512 FIG 4 513 514 515

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516

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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.

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6 ANEXOS

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