IGOR RENAN ZEN Efeito de soluções ou dentifrícios contendo trimetafosfato de sódio, xilitol, eritritol e fluoreto, em diferentes associações, sobre biofilmes de interesse cariogênico Araçatuba 2022 IGOR RENAN ZEN Efeito de soluções ou dentifrícios contendo trimetafosfato de sódio, xilitol, eritritol e fluoreto, em diferentes associações, sobre biofilmes de interesse cariogênico Tese apresentada à Faculdade de Odontologia de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, como parte dos requisitos para a obtenção do título de Doutor em Ciência Odontológica – Área: Saúde Bucal da Criança. Orientador: Prof. Dr. Juliano Pelim Pessan Coorientadores: Prof. Tit. Alberto Carlos Botazzo Delbem Prof. Dr. Douglas Roberto Monteiro Araçatuba 2022 Catalogação-na-Publicação Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Zen, Igor Renan. Z54e Efeito de soluções ou dentifrícios contendo trimeta- fosfato de sódio, xilitol, eritritol e fluoreto, em diferentes associações, sobre biofilmes de interesse cariogênico / Igor Renan Zen. - Araçatuba, 2022 128 f. : il. ; tab. Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientador: Prof. Juliano Pelim Pessan Coorientador: Prof. Alberto Carlos Botazzo Delbem Coorientador: Prof. Douglas Roberto Monteiro 1. Fluoretos 2. Biofilmes 3. Fosfatos 4. Xilitol 5. Eritritol I. T. Black D27 CDD 617.645 Claudio Hideo Matsumoto – CRB-8/5550 Zen, IR. Efeito de soluções ou dentifrícios contendo trimetafosfato de sódio, xilitol, eritritol e fluoreto, em diferentes associações, sobre biofilmes de interesse cariogênico. 2022. 128 f. (Doutorado em Ciência Odontológica, área de Saúde Bucal da Criança) – Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, Araçatuba 2022. RESUMO GERAL Este estudo avaliou o efeito de dentifrícios ou soluções contendo trimetafosfato de sódio(TMP), xilitol(X), eritritol(E) e fluoreto(F), em diferentes associações, sobre cepas e biofilmes cariogênicos. Três subprojetos (SP1, SP2 e SP3) apresentaram os objetivos: SP1) Avaliar o efeito de dentifrícios contendo “TMP(0,25%)”, “X(16%)”, “E(4%)”, “F(200 e 1100 ppm)” sozinhos ou em diferentes associações, sobre cepas isoladas de Streptococcus mutans(SM), Lactobacillus casei(LC), Actinomyces israelii(AI) e Candida albicans(CA). SP2) Avaliar o efeito de soluções contendo “TMP”(0,075%), “X”(4,8%), “E”(1,2%), “F”(60 e 330 ppm) e saliva artificial pura, sozinhos ou em diferentes associações sobre biofilmes mistos de SM e CA. SP3) Avaliar o efeito das mesmas soluções de SP2 sobre biofilmes microcosmos patogênicos com a incorporação ou não de SM. No SP1, cepas de SM, LC, AI e CA foram incorporadas ao meio de BHI- ágar, vertidas em placas, realizados poços no ágar e diferentes diluições de slurries dos dentifrícios foram adicionados. Os halos foram medidos com paquímetro digital. A análise estatística se deu por ANOVA dois critérios, e teste de Tukey HSD (p<0,05). Para SM, o maior halo foi observado por “200F+TMP” em todas as diluições, seguido por “200F+X+E”. Para LC, a tendência mostrou inibição microbiana promovida pelos polióis, potencializado pela associação com os outros compostos. Para AI, observou-se uma tendência menos definida. Para CA, o dentifrício experimental “200F+X+E+TMP” foi mais eficaz que os outros. No SP2, as mesmas soluções e grupos do SP1 foram usados a uma concentração final de 30% do valor inicial dos dentifrícios. Biofilmes mistos de SM e CA foram cultivados na presença contínua desses ativos e avaliou-se a quantificação de células viáveis (UFCs), biomassa total, atividade metabólica e componentes da matriz extracelular. A análise estatística se deu por ANOVA um critério e teste de Tukey HSD (p<0,05). As contagens de UFCs foram afetadas pelo F, enquanto a biomassa e atividade metabólica pelo TMP. Adicionalmente, observou-se efeito sinérgico desses ativos. Os polióis tiveram efeitos mais pronunciados nos carboidratos da matriz extracelular, com pouca ou nenhuma ação nas demais variáveis. A associação dos quatro ativos promoveu aumento no efeito antibiofilme, e foi afetado por F e/ou TMP, com pouco efeito dos polióis isoladamente. No SP3, biofilmes microcosmos foram formados em um modelo de biofilme de alto rendimento com ou sem a incorporação da cepa de SM. As mesmas soluções e concentrações de SP2 estavam constantemente presentes no meio de cultura. Analisou-se as UFCs e produção de acido lático dos biofilmes. Os dados foram analisados por ANOVA ou Kruskal-Wallis, e Student- Newman-Keuls (p<0,05). O grupo “60F+TMP” produziu quantidades de ácido lático significativamente menor, e apresentou reduções na contagem total de UFCs em biofilmes microcosmos, incorporados ou não com SM, comparado ao grupo controle. O grupo experimental promoveu diminuições sobre os parâmetros analisados. A associação de “F+TMP” e o grupo experimental reduziram as contagens de UFCs total e de SM, e a produção de ácido lático por biofilmes microcosmos derivados de saliva. Os resultados permitiram concluir que a associação dos quatro compostos ativos e “F+TMP” apresentaram reduções em todos os parâmetros avaliados. Palavras-chaves: Fluoretos. Biofilmes. Fosfatos. Xilitol. Eritritol. Zen, IR. Effect of solutions or dentifrices containing sodium trimetaphosphate, xylitol, erythritol and fluoride, in different associations, on biofilms of cariogenic interest.128 f. [thesis]. Araçatuba: UNESP – São Paulo State University;2022. GENERAL ABSTRACT This study evaluated the effect of dentifrices or solutions containing sodium trimetaphosphate(TMP), xylitol(X), erythritol(E) and fluoride(F), in different associations, on cariogenic strains and biofilms. Three subprojects (SP1, SP2 and SP3) presented the objectives: SP1) To evaluate the effect of dentifrices containing “TMP(0.25%)”, “X(16%)”, “E(4%)”, “F( 200 and 1100 ppm)” alone or in different associations, on isolated strains of Streptococcus mutans(SM), Lactobacillus casei(LC), Actinomyces israelii(AI) and Candida albicans(CA). SP2) Evaluate the effect of solutions containing “TMP”(0.075%), “X”(4.8%), “E”(1.2%), “F”(60 and 330 ppm) and pure artificial saliva, alone or in different associations on mixed SM and CA biofilms. SP3) To evaluate the effect of the same SP2 solutions on pathogenic microcosm biofilms with or without the incorporation of SM. In SP1, SM, LC, AI and CA strains were incorporated into the BHI-agar medium, poured into plates, wells were made in the agar, and different dilutions of dentifrice slurries were added. The halos were measured with a digital caliper. Statistical analysis was performed by two-way ANOVA and Tukey HSD test (p<0.05). For SM, the largest halo was observed by “200F+TMP” in all dilutions, followed by “200F+X+E”. For LC, the trend showed microbial inhibition promoted by polyols, potentiated by the association with the other compounds. For AI, a less defined trend was observed. For CA, the experimental dentifrice “200F+X+E+TMP” was more effective than the others. In SP2, the same solutions and groups of SP1 were used at a final concentration of 30% of the initial value of the dentifrices. Mixed biofilms of SM and CA were cultured in the continuous presence of these actives, and the quantification of viable cells (CFUs), total biomass, metabolic activity and extracellular matrix components were evaluated. Statistical analysis was performed by one-way ANOVA and Tukey HSD test (p<0.05). CFU counts were affected by F, while biomass and metabolic activity by TMP. Additionally, a synergistic effect of these actives was observed. Polyols had more pronounced effects on extracellular matrix carbohydrates, with little or no action on other variables. The association of the four actives promoted an increase in the antibiofilm effect and was affected by F and/or TMP, with little effect of polyols alone. In SP3, microcosm biofilms were formed in a high-throughput biofilm model with or without the incorporation of the SM strain. The same SP2 solutions and concentrations were constantly present in the culture medium. The CFUs and lactic acid production of biofilms were analyzed. Data were analyzed by ANOVA or Kruskal-Wallis and Student- Newman-Keuls (p<0.05). The “60F+TMP” group produced significantly lower amounts of lactic acid and showed reductions in total CFU counts in microcosm biofilms, whether or not incorporated with SM, compared to the control group. The experimental group promoted decreases in the analyzed parameters. The association of “F+TMP” and the experimental group reduced total and SM CFU counts and lactic acid production by saliva-derived microcosm biofilms. The results allowed us to conclude that the association of the four active compounds and “F+TMP” showed reductions in all evaluated parameters. Key words: Fluorides. Biofilms. Phosphates. Xylitol. Erythritol. LISTA DE SIGLAS LISTA DE SIGLAS 1100F 1100 ppm F 200F 200 ppm F 200F+TMP 200 ppm F + trimetafosfato de sódio/sodium trimetaphoshate 200F+X+E 200 ppm F + xilitol + eritritol/ 200 ppm F + xylitol + erythritol AI/A. israelii Actinomyces israelii ANOVA Analise de Variância/Analysis of Variance AS Saliva artificial/Artificial saliva ATCC American Type Culture Collection BHI Brain Heart Infusion BPW Água Peptonada Tamponada/ Buffered Peptone Water CA/C.albicans Candida albicans Ca+ Cálcio/Calcium CaF2 Fluoreto de Cálcio/Calcium Fluoride Cm Centímetros/Centimeters CO2 Dióxido de Carbono/Carbon dioxide CV Cristal Violeta/Crystal Violet SDA Sabouraud Dextrose Agar E Eritritol/Erythritol e.g./exempli gratia Por exemplo/ For example EXP/200F+TMP+X+E Grupo experimental/Experimental Group / 200 ppm F + Trimetafosfato de sódio + Xilitol + Eritritol/ 200 ppm F + Sodium trimetaphosphate + Xylitol + Erythritol F Flúor/Fluoride g Gramas/Grams Gtfs Glicosiltransferases/Glycosyltransferase h Hora/Hour H2 Hidrogênio/Hydrogen HSD Diferença Significativa Honesta/Honestly Significant Difference i.e./id est Isto é/That is KCl Cloreto de Potássio/ Potassium Cloride L/l Litros/Liters LC/L.casei Lactobacillus casei Log10 Logaritmo na base 10/ Logarithm, base 10 Mg Miligramas/Miligrams Mg+ Magnésio/Magnesium Min Minutos/Minutes mL Mililitros/Mililiters mm Milímetros/Milimetres Mmol Milimol Modelo AAA Amsterdam Active Attachment Model n Número/Number N2 Nitrogênio/Nitrogen NaCl Cloreto de Sódio/Sodium Cloride NaF Fluoreto de Sódio/Sodium Fluoride NaOH Hidróxido de Sódio/Sodium Hidroxide NC Controle Negativo/Negative Control Nm Nanômetro/Nanometer ºC Graus Celsius/ Degrees Celcius p Probabilidade/Probability pH Potencial de Hidrogênio/Potential of Hydrogen PIPES Piperazine-N,N′-bis(2-ethanesulfonic acid) ppm Parte por milhão/Parts per million Rpm Rotação por minuto/Revolutions per minute SAB Ágar Sacarose Bacitracina/ Sucrose Agar Bacitracin SD Desvio Padrão/Standard Deviation SDB Sabouraud Dextrose Broth SM/S. mutans Streptococcus mutans TMP Trimetafosfato de sódio/Sodium trimetaphosphate TMP+X+E Trimetafosfato de sódio + xilitol + eritritol/ Sodium trimetaphosphate + xylitol + erythritol TSBA Ágar Sangue /Tryptic Soy Agar Blood UFC/CFU Unidades de Formação de Colônias/Colony-Forming Unit v/v Volume por volume/Volume per volume VU Vrije Universiteit W Watts X Xilitol/Xylitol X+E Xilitol + eritritol/ Xylitol + erythritol XTT 2,3-bis (2-methoxy-6 4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide μL Microlitros/microliters LISTA DE FIGURAS LISTA DE FIGURAS CAPÍTULO 2 Fig. 1. Logarithm of colony-forming units cm-2 for S. mutans (a) and C. albicans (b), and absorbance values cm-2 obtained for the total biomass (c) and metabolic activity (d) quantification assays. Error bars denote the SD of the means. Different lowercase letters symbolize statistical differences among the groups (p<0.05, n=9). NC: negative control (untreated biofilms), “60F” = 60 ppm F; “330F” = 330 ppm F; “X” = 4.8% Xylitol; “E” = 1.2% Erythritol. ............................ 56 CAPÍTULO 3 Fig. 1. A: AAA-model with glass cover lips attached to a stainless-steel lid using nylon clamps. B: The lid is fitted on top of flat-bottomed 24-well plates. ............. 75 Fig. 2. Logarithm of total colony-forming units for (A): saliva-derived microcosm biofilms and (B): saliva-derived microcosm biofilms supplemented with Streptococcus mutans (C180-2) (ANOVA and Student-Newman-Keuls' tests, p<0.05, n=9). Error bars denote the SD of the means. Different uppercase letters symbolize statistical differences among the groups. NC: negative control (untreated biofilms), “60F” = 60 ppm F; “330F” = 330 ppm F; “TMP” = 0.075% Sodium trimetaphosphate; “X” = 4.8% Xylitol; “E” = 1.2% Erythritol.................... 76 Fig. 3. Logarithm of colony-forming units of Streptococcus mutans from saliva- derived microcosm biofilms supplemented with Streptococcus mutans (C180-2). (Kruskal-Wallis and Student-Newman-Keuls' tests, p<0.05, n=9). Bars denote the interquartile ranges. Different uppercase letters symbolize statistical differences among the groups. NC: negative control (untreated biofilms), “60F” = 60 ppm F; “330F” = 330 ppm F; “TMP” = 0.075% Sodium trimetaphosphate; “X” = 4.8% Xylitol; “E” = 1.2% Erythritol. ............................................................................... 77 Fig. 4. Concentration of lactate (mM) produced by (A): saliva-derived microcosm biofilms and (B): saliva-derived microcosm biofilms supplemented with Streptococcus mutans (C180-2). (ANOVA and Student-Newman-Keuls’ tests, p<0.05, n=9). Error bars denote the SD of the means. Different uppercase letters symbolize statistical differences among the groups. NC: negative control (untreated biofilms), “60F” = 60 ppm F; “330F” = 330 ppm F; “TMP” = 0.075% Sodium trimetaphosphate; “X” = 4.8% Xylitol; “E” = 1.2% Erythritol.................... 78 Fig. 5. Mean (SD) pH of the spent medium of saliva-derived microcosm biofilms from the day-period (unbuffered McBain medium supplemented with 0.2% sucrose) and night-period (buffered McBain medium without sucrose). Distinct upper-case letters indicate significant differences between day and night within each treatment solution. Different lower-case letters represent significant differences among the groups within a day and night periods of A: saliva-derived microcosm biofilms and B: saliva-derived microcosm biofilms supplemented with S. mutans (C180-2). Distinct upper-case letters indicate significant differences between day and night within each treatment solution. Different lower-case letters represent significant differences among the groups, separately for day and night periods (two-way, repeated measures ANOVA and Student-Newman-Keuls' tests, p<0.05, n=9). NC: negative control (untreated biofilms), “60F” = 60 ppm F; “330F” = 330 ppm F; “TMP” = 0.075% Sodium trimetaphosphate; “X” = 4.8% Xylitol; “E” = 1.2% Erythritol. ............................................................................... 79 LISTA DE TABELAS LISTA DE TABELAS CAPÍTULO 1 Table 1. Zone of inhibition of toothpastes containing different active compounds, at 5 dilutions, on Streptococcus mutans ............................................................. 30 Table 2. Zone of inhibition of toothpastes containing different active compounds, at 5 dilutions, on Lactobacillus casei ................................................................... 31 Table 3. Zone of inhibition of toothpastes containing different active compounds, at 5 dilutions, on Actinomyces israelii ................................................................. 31 Table 4. Zone of inhibition of toothpastes containing different active compounds, at 5 dilutions, on Candida albicans ..................................................................... 33 CAPÍTULO 2 Table 1. Mean (standard deviation) of protein, carbohydrate and DNA of dual- species biofilms obtained after treatment with different concentrations of fluoride, sodium trimetaphosphate, xylitol or erythritol, alone or in different associations . 57 SUMÁRIO INTRODUÇÃO GERAL ....................................................................................... 19 CAPÍTULO 1 ....................................................................................................... 22 Abstract ........................................................................................................... 24 Introduction ..................................................................................................... 25 Material and methods ...................................................................................... 26 Results ............................................................................................................ 28 Discussion ....................................................................................................... 29 Conclusion ...................................................................................................... 32 References ...................................................................................................... 32 Tables ............................................................................................................. 30 CAPÍTULO 2 ....................................................................................................... 34 Abstract ........................................................................................................... 36 Introduction ..................................................................................................... 37 Materials and methods .................................................................................... 38 Results ............................................................................................................ 41 Discussion ....................................................................................................... 42 References ...................................................................................................... 47 Tables ............................................................................................................. 57 Figures ............................................................................................................ 56 CAPÍTULO 3 ....................................................................................................... 57 Abstract ........................................................................................................... 59 Introduction ..................................................................................................... 60 Materials and methods .................................................................................... 61 Results ............................................................................................................ 64 Discussion ....................................................................................................... 65 Conclusion ...................................................................................................... 68 References ...................................................................................................... 69 Figures ............................................................................................................ 75 ANEXOS ............................................................................................................. 81 INTRODUÇÃO GERAL 19 INTRODUÇÃO GERAL A cárie dentária é uma doença que afeta mais de 3,5 bilhões de pessoas durante a dentição permanente e mais de 620 milhões de crianças durante a dentição primária (Kassebaum et al., 2017; Phantumvanit et al., 2018). Esta doença é mediada por biofilmes formados por um consórcio de diferentes micróbios. Também é modulada pela dieta na presença de açúcares fermentáveis, não transmissível e é considerada multifatorial (Machiulskiene et al., 2020). Um dos principais agentes etiológicos da cárie dentária é a bactéria gram-positiva Streptococcus mutans (S. mutans), devido a sua capacidade de colonizar a superfície dental, metabolizar carboidratos e produzir ácido láctico, além de ter a capacidade de crescer e se multiplicar em ambiente ácido (Falsetta et al., 2014; Lemos et al., 2019), levando à formação do biofilme dentário, o qual é definido como uma comunidade microbiana organizada, aderida a superfícies vivas ou inertes e envolvida por uma matriz extracelular produzida pelas células (Bowen, Burne, Wu, & Koo, 2018). No biofilme, os microrganismos apresentam-se embebidos em uma matriz extracelular composta por glicoproteínas e polissacarídeos (Lemos et al., 2019). Inicialmente, várias adesinas das bactérias odontopatogênicas interagem com as glicoproteínas salivares da película adquirida na superfície dos dentes através de cátions bivalentes. Na presença de sacarose, as bactérias aderem-se firmemente à superfície do dente como resultado da produção de exopolissacarídeos (glucanos) por meio da atividade da glicosiltransferase (Gtfs). Sendo assim, o acúmulo de biofilme faz com que o S. mutans metabolize eficientemente a sacarose (açúcar ou polímeros) para produzir grande quantidade de ácido láctico, capaz de solubilizar o componente mineral do dente e iniciar o processo de cárie (Lemos et al., 2019). Além do S. mutans, a cavidade bucal é habitada por mais de 700 diferentes espécies de microrganismos, dentre esses, espécies bacterianas como de Lactobacillus, Actinomyces e fúngicas como Candida sp.. Candida albicans (C. albicans) é o fungo mais comumente encontrado na cavidade oral, que pode ser um fator de risco para o desenvolvimento de cárie dentária (Kilian et al., 2016). A presença de C. albicans é importante na cárie na infância, uma vez que contribui para a patogênese em crianças cárie-ativas (Menon, Scoffield, Jackson, & Zhang, 2022). 20 O uso dos dentifrícios contendo fluoreto (F) atrelado a escovação dos dentes é considerado o melhor método preventivo da cárie dentária, visto que associa a remoção ou desorganização periódica do biofilme dental com as propriedades cariostáticas do F (Walsh, Worthington, Glenny, Marinho, & Jeroncic, 2019). Sendo assim, o íon flúor tem seu efeito por meio da manutenção da concentração de flúor na saliva devido ao uso frequente dos dentifrícios, e pela formação de produtos da reação esmalte-dentina com fluoreto, formando o mineral fluoreto de cálcio (CaF2), que, depositado no biofilme em lesões iniciais de cárie, é capaz de evitar a progressão da mesma (Buzalaf, Pessan, Marques Honório, & Ten Cate, 2011). A suplementação com sais de fosfato tem sido uma possibilidade para aumentar a efetividade dos dentifrícios fluoretados. Estudos in vitro e in situ demonstraram que dentifrícios com concentração reduzida de F suplementados com trimetafosfato de sódio (TMP) apresentam efetividade semelhante a de um dentifrício convencional (1.100 µg F/g) (Takeshita, Danelon, Castro, Cunha, & Delbem, 2016; Takeshita, Danelon, Castro, Sassaki, & Delbem, 2015). Além disso, a concentração de F e cálcio no biofilme formado na presença destes dentifrícios foi semelhante à observada para um dentifrício convencional (Takeshita et al., 2015). No entanto, o mecanismo pelo qual soluções contendo TMP, associados ou não ao F, atuam no biofilme ainda é incerto. Além desses sais de fosfato, existem produtos naturais que são fontes de agentes terapêuticos, e que podem ser utilizados na prevenção da cárie dentária, pois apresentam efeito sobre os microrganismos do biofilme. O xilitol e o eritritol são polióis utilizados como substitutos do açúcar em muitos produtos de cuidados bucais, pois não são cariogênicos e apresentam efeitos favoráveis sobre a saúde bucal (Kauko Mäkinen, 2000; Runnel et al., 2013). A literatura mostra que o xilitol diminui a acidogenicidade e volume do biofilme, e é capaz de inibir o crescimento de S. mutans (Kauko Mäkinen, 2000). Da mesma forma, o eritritol parece apresentar mecanismos semelhantes ao do xilitol e promove a redução dos microrganismos patogênicos relacionados à cárie dentária (S. mutans) (Park et al., 2014). Diante de todo contexto supracitado, e tendo em vista a eficácia dos ativos sozinhos, os objetivos do presente estudo foram: 1) Avaliar o efeito de dentifrícios contendo trimetafosfato de sódio (TMP), xilitol (X), eritritol (E) e fluoreto (F), em diferentes associações, sobre cepas isoladas de Streptococcus mutans, Lactobacillus 21 casei, Actinomyces israelii e Candida albicans na formação de halos de inibição. 2) Avaliar o efeito de soluções contendo trimetafosfato de sódio (TMP), xilitol (X), eritritol (E) e fluoreto (F), em diferentes associações sobre biofilmes mistos de Streptococcus mutans e Candida albicans. 3) Avaliar o efeito de soluções contendo trimetafosfato de sódio (TMP), xilitol (X), eritritol (E) e fluoreto (F), em diferentes associações sobre biofilmes microcosmos (polimicrobianos) suplementados ou não com Streptococcus mutans. Os dentifrícios foram testados no capítulo 1 e as soluções nos capítulos 2 e 3, sendo respectivamente: Dentifrícios: Trimetafosfato de sódio (0,25%), F (200 ppm), xilitol (16%) e eritritol (4%), 1100 ppm F (controle positivo) e placebo (sem adição de nenhum composto ativo). Soluções: Trimetafosfato de sódio (0,075%), F (60 ppm), xilitol (4,8%) e eritritol (1,2%), 330 ppm F (controle positivo) e Saliva artificial ou McBain puro (controle negativo). Para alcançar os objetivos propostos, o presente estudo foi dividido em três capítulos, como detalhado abaixo:  Capítulo 1: “Antimicrobial effect of low-fluoride toothpastes containing polyphosphate and polyols” (artigo submetido ao periódico Saudi Dental Journal);  Capítulo 2: “Evaluation of solutions containing fluoride, sodium trimetaphosphate, xylitol and erythritol on dual-species biofilms” (artigo submetido ao periódico Biofouling);  Capítulo 3: “Assessment of the effects of fluoride, sodium trimetaphosphate, xylitol and/or erythritol on saliva-derived microcosm biofilms” (artigo formatado nas normas do periódico Archives of Oral Biology). As referências da Introdução Geral encontram-se no anexo A. 22 CAPÍTULO 1 23 Antimicrobial effect of low-fluoride toothpastes containing polyphosphate and polyols Igor Zena igorzen@gmail.com Alberto Carlos Botazzo Delbema alberto.delbem@unesp.br Thayse Yumi Hosidaa thosida@hotmail.com Caio Sampaioa caiosampaio.o@hotmail.com Leonardo Antônio de Moraisa leo.a.morais@gmail.com Tamires Passadori Martinsa tamires.passadori@unesp.br Douglas Roberto Monteiroa douglasrmonteiro@hotmail.com Juliano Pelim Pessana juliano.pessan@unesp.br a Department of Preventive and Restorative Dentistry, School of Dentistry, Araçatuba, São Paulo State University (Unesp), Brazil *Corresponding author Juliano Pelim Pessan, DDS, MSc, PhD Rua José Bonifácio, 1193, 16015-050 Araçatuba – SP, Brazil Phone: +55 18 3636 3314 E-mail: juliano.pessan@unesp.br Este artigo segue as normas do periódico Saudi Dental Journal (Anexo B) 24 Antimicrobial effect of low-fluoride toothpastes containing polyphosphate and polyols Abstract This study evaluated the antimicrobial effect of toothpastes containing 200 ppm fluoride (200F), xylitol (X, 16%), erythritol (E, 4%), and sodium trimetaphosphate (TMP, 0.25%), alone or in different associations, against Streptococcus mutans (SM), Lactobacillus casei (LC), Actinomyces israelii (AI) and Candida albicans (CA). Suspensions of the microorganisms were added to BHI Agar medium. Five wells were made on each plate to receive toothpaste suspensions at different dilutions. Toothpastes containing no actives (Placebo) or 1100 ppm F (1100F) were used as negative and positive controls. 2-way ANOVA and Tukey’s HDS test were used (p<0.05). For SM, the largest halo was for 200F+TMP at all dilutions, followed by the 200F+X+E toothpaste. For LC, the overall trend showed that the polyols effectively inhibited microbial growth, and the association with the other compounds enhanced such effects. For AI, a less defined trend was observed. For CA, the experimental toothpaste (200F+X+E+TMP) was consistently more effective than the other treatments and followed by 200F+X+E. The association of polyols and TMP in a low- fluoride toothpaste effectively reduced the growth of cariogenic microorganisms (SM, CA, and LC), suggesting that this formulation could be an interesting alternative for children due to its low fluoride content. KEYWORDS: Toothpastes; Sugar Alcohols; Fluorides; Polyphosphates Total words: 2.497 Declarations of interest: none 25 Introduction Dental caries is a chronic, biofilm-modulated disease, which has increasingly affected children worldwide (Kassebaum et al., 2017), being associated with an increase in fermentable carbohydrates intake, and deficient removal or disorganization of dental biofilm. It is a sucrose-dependent biofilm disease, whose onset is related to the interaction of acidogenic and aciduric microorganisms in the oral cavity (Machiulskiene et al., 2020). Among the main microorganisms involved, Streptococcus mutans stand out for their ability to colonize dental surfaces, metabolize carbohydrates, and produce lactic acid (Klein et al., 2015). Other bacteria are related to the disease's onset and progression, including Lactobacillus and Actinomyces (Hamada & Slade, 1980). In addition, the fungus Candida albicans has been reported to contribute to the formation and development of cariogenic biofilms, especially in dentine cavities, as its proteolytic enzymes are able to break down collagen molecules (Falsetta et al., 2014; Menon et al., 2022). The association of mechanical and chemical methods is an effective method to reduce biofilm formation (Sampaio et al., 2020; Valkenburg, Else Slot, & Van der Weijden, 2020; Walsh et al., 2019). Among them, toothpastes present in their composition active agents that have mineralizing and/or antimicrobial action, such as Fluoride (F) (Valkenburg et al., 2020; Walsh et al., 2019). Furthermore, the addition of phosphate salts to F-containing toothpastes has been shown to boost the effects of F. In vitro and in situ studies have shown that low-F toothpastes containing sodium trimetaphosphate (TMP) have similar effects to those of a conventional formulation (1100 ppm F) on enamel de- and re-mineralization (Takeshita et al., 2016, 2015), and on F and calcium levels in the dental biofilm (Takeshita et al., 2015). In addition to the strategies above, products of natural origin, such as polyols, are used as sugar substitutes, which have ability to reduce the growth and attachment of S. mutans to surfaces and reduce the volume of dental biofilm (E. Söderling & Pienihäkkinen, 2022). Literature shows a synergism between xylitol and F on the inhibition of acid production by S. mutans, with significantly higher effects than the actives alone (Maehara, Iwami, Mayanagi, & Takahashi, 2005; Kauko K. Mäkinen, 2011). Furthermore, erythritol has a similar effect to xylitol and its action 26 seems to cause direct changes in the metabolism of caries-related microorganisms (De Cock et al., 2016; Janus et al., 2017). Considering the advantages of the above-mentioned strategies used alone, and the need to develop safer and more effective formulations for children, this study assessed in vitro the antimicrobial effect of toothpastes containing F at low concentration (200 ppm), xylitol, erythritol, and TMP, alone or in different associations on the inhibition of Streptococcus mutans (S. mutans), Lactobacillus casei (L. casei), Actinomyces israelii (A. israelii) and Candida albicans (C. albicans) growth. The study's null hypothesis was that the antimicrobial effects of the toothpastes associating two or more compounds would not be significantly different from the toothpastes containing the isolated actives. Material and methods The toothpastes were produced by Xlear Incorporation, American Fork, UT, USA, and contained in their composition the following actives: xylitol (X: 16%), erythritol (E: 4%), sodium trimetaphosphate (TMP: 0.25%), and sodium fluoride at 200 ppm F, alone or in different combinations. Formulations without any actives (placebo) or containing 1100 ppm F were also manufactured as controls. a) Placebo (without any active compound; negative control toothpaste); b) 1100 ppm F (positive control toothpaste); c) 200 ppm F (200F); d) TMP (0.25%); e) 200F+TMP; f) Xylitol (16%); g) Erythritol (4%); h) Xylitol+erythritol; i) 200F+xylitol+erythritol; j) TMP+xylitol+erythritol; k) 200F+TMP+xylitol+erythritol (experimental toothpaste). 27 2.1.1 Preparation of toothpaste slurries Toothpaste slurries were produced by dispersing 10 g of each toothpaste in 10 mL of sterile deionized water (1:1). Serial dilutions of the slurry were made using sterile deionized water, to obtain four additional dilutions at 1:2, 1:4, 1:8, and 1:16 (Malhotra & Shashikiran, 2017). 2.1.2 Antimicrobial assays Agar diffusion assays were performed with the reference strains of the American Type Culture Collection (ATCC): C. albicans (ATCC 10231), S. mutans (ATCC 25175) A. israelii (ATCC 10048) and L. casei (ATCC 393) (Malhotra & Shashikiran, 2017). These were reactivated from their original cultures in Sabouraud Dextrose Agar (SDA; Difco, Le Pont de Claix, France) for C. albicans, and in Brain and Heart Infusion Agar (BHI Agar; Difco) for S. mutans, A. israelli, and L. casei, and incubated in 5% CO2 at 37 ºC, during 24 hours. Following, five colonies of each species were individually added to BHI broth and incubated at 37 ºC for 18-24 hours. Aliquots of 300 μL of each bacterial suspension (optical density of 0.6, and absorbance of 550 nm - 107 CFU/ml for C. albicans and 108 CFU/ml for S. mutans, A. israelii and L. casei) was homogenized with 15 mL of BHI-agar at 45 ºC. Subsequently, equidistant wells (n = 5) were made on agar using sterilized cylinders (4 mm in diameter) and filled with 80 µL of the slurries of each toothpaste, at dilutions of 1:1, 1:2, 1:4, 1:8 and 1:16 (Malhotra & Shashikiran, 2017). The plates were kept for 2 hours at room temperature to allow the solutions to diffuse, and then incubated at 37 ºC for 24 hours. The experiments were performed in triplicate. For each inhibition halo, three independent measures were performed with the aid of a digital caliper (accuracy 0.01 mm) (MitutoyoCD-15B) (Malhotra & Shashikiran, 2017). 2.1.3 Statistical analysis Data were analyzed using the STATISTICA software (version 8.0). The data passed normality and homogeneity tests and were submitted to two-way Analysis of Variance (ANOVA), considering as variation factors toothpastes and dilutions, followed by the Tukey’s HSD test, adopting a significance level of 5%. 28 Results Overall, for S. mutans the largest inhibition halo was observed for 200F+TMP in all dilutions. Nonetheless, the effects of the polyols used together, as well as their association with 200 ppm F promoted larger inhibition halos compared with the other groups, with sustained effects for all the dilutions (Table 1). For L. casei, the general pattern was that the polyols, alone or coadministered, were effective in inhibiting microbial growth, and their association with the other actives potentiated the antimicrobial effects (Table 2). In this sense, larger inhibition halos were observed for toothpastes containing both polyols and F (at most all the dilutions), both polyols and TMP (for the dilutions 1:2, 1:4 and 1:8), and for the experimental toothpaste (at 1:1, 1:2 and 1:4 dilution). For A. israelii, no defined trend was observed for treatments at 1:1, 1:2, and 1:4 dilutions (Table 3). For the remaining dilutions (1:8 and 1:16), toothpaste containing 200 ppm F promoted the largest halos compared with the other treatments. Conversely for C. albicans, the experimental toothpaste (containing all actives) promoted a consistently higher inhibitory effect than the other treatments for all dilutions (except for 1:8) and followed by 200F+X+E (Table 4). 29 Discussion In this study, the antimicrobial effect of toothpastes containing 200 ppm F, xylitol (16%), erythritol (4%), and TMP (0.25%), isolated or in different associations on S. mutans, A. israelii, L. casei and C. albicans, was determined using an in vitro screening model. The results show that the toothpastes containing two or more actives promoted significantly larger inhibition halos than the isolated compounds for most of the pairwise comparisons, whose effects depended on the strain analyzed. Thus, the null hypothesis was partially rejected. S. mutans was one of the strains chosen due to its direct involvement in dental caries onset and progression. For this strain, while toothpastes containing TMP alone or F at low concentrations promoted only modest antimicrobial effects, the largest inhibition halos were achieved by the association between 200 ppm F+TMP, for all dilutions (Table 1). These findings show the synergism between the two compounds, which is in agreement with CFU data of C. albicans and S. mutans (Cavazana et al., 2019). Furthermore, this association was shown to reduce the total biofilm biomass, extracellular matrix components, and also promoted the highest pH values both before and after cariogenic challenges (Cavazana et al., 2019, 2020). In vitro and in situ studies have also shown that the association between F and TMP leads to synergistic effects on de- and re-mineralization (Manarelli, Delbem, Lima, Castilho, & Pessan, 2014; Takeshita et al., 2016, 2015), reason by which such combination was assessed in the present study. On the other hand, xylitol and/or erythritol promoted modest reductions in S. mutans growth. Literature reports that xylitol affects S. mutans cells by inhibiting glycolytic enzymes, which leads to reduction of growth and acid production (Söderling & Hietala-Lenkkeri, 2010). For erythritol, it appears to suppress the maturation of oral biofilms and prevent the development of dysbiosis (Janus et al., 2017). Despite the reasons for the small effects of the polyols on S. mutans in the present study are not apparent, it is possible that the effect of these actives on this bacterium would be related to their antibiofilm action (e.g., extracellular polysaccharides production) rather an inhibitory effect. As for Lactobacillus casei, conflicting evidence on the effects of xylitol is available from clinical studies. While some authors showed that xylitol's continued use promoted reductions in Lactobacillus counts in saliva (Mäkinen et al., 1995; 30 Mäkinen et al., 2008; Mäkinen et al., 1996), others showed that the use of xylitol- containing gum (Söderling et al., 2011) or toothpaste (Maden, Altun, Ozmen, & Basak, 2018) did not reduce Lactobacillus counts compared with control groups. The different methodologies, vehicles of administration, and study duration hinder a direct comparison between studies, as these variables may have impacted the results. In the present study, the largest inhibition zone was observed for the experimental toothpaste (at 1:1 dilution), followed by 1100F and 200F+X+E. Such effects might have resulted from xylitol’s action on this strain following a similar mechanism to that described for S. mutans, in which is related to the phosphotransferase system that inhibits the conservation of the bacteria and further growth (Hausman, Thompson, & London, 1984; Helanto, Aarnikunnas, Palva, Leisola, & Nyyssölä, 2006; London & Hausman, 1982; Radmerikhi, Azul, Fajardo, & Formantes, 2013). Regarding Actinomyces israelii, no striking pattern was observed among the groups on the inhibition halos compared with the placebo toothpaste, for any of the tested dilutions. This is in accordance with previous results showing that xylitol in the presence of glucose did not inhibit the growth of Actinomyces (Vadeboncoeur, Trahan, Mouton, & Mayrand, 1983). This fact can be explained by the low acidogenicity and acid tolerance of this strain (Tanzer, Livingston, & Thompson, 2001). They also reflect the status of non-cariogenic microorganisms compared with Streptococcus mutans and Lactobacillus casei (Tanzer et al., 2001; Yadav & Prakash, 2017). In contrast, Candida albicans has recently been used in in vitro biofilm protocols to reproduce conditions that better resemble those of cariogenic biofilms in vivo (Cavazana et al., 2019, 2020) as this fungus has been identified in biofilms collected from individuals presenting cavitaded caries lesions (Menon et al., 2022). In the present study, the highest inhibition halos were promoted by the experimental toothpaste (at 1:1, 1:2, and 1:4 dilutions; table 4), so that the simultaneous action of all compounds could explain such effects. Previous studies have shown that xylitol presents an inhibitory effect on C. albicans growth (Talattof, Azad, Zahed, & Shahradnia, 2018), which is due to an impaired nutrient absorption required to maintain yeast viability. The inability of C. albicans cells in catabolizing or excreting xylitol products accumulated in the cytoplasm also explains such effects, as such products lead to an increased osmotic strength and cell swelling (Ameglio, Di Giorgio, Terzaroli, & Gandolfo, 1990; Leepel, Sastra, Puspitawati, & Bachtiar, 2012). 31 Regarding erythritol, this compound was shown to increase the effect of benzethonium chloride against C. albicans, leading to the assumption that erythritol can help to disperse biofilms, favoring the penetration of fungicidal agents and weakening the microbial bond to surfaces (Ichikawa, Yano, Fujita, Kashiwabara, & Nagao, 2008). However, the literature on this topic is scarce, pointing to the need for more comprehensive studies on the relationship between erythritol and this fungus. A previous study showed that the association of F (500 ppm) and TMP on a biofilm of S. mutans and C. albicans was shown to promote alterations on the extracellular matrix and biofilm architecture compared with 1100 ppm F without TMP (Cavazana et al., 2019). Such effects were attributed to the interaction of these compounds, which directly acted on the inhibition of bacterial enzymes and the reduction of intra- and extra-cellular polysaccharides production (Hyun Koo, Sheng, Nguyen, & Marquis, 2006; Pandit, Kim, Jung, Chang, & Jeon, 2011). Based on this rationale, it could be hypothesized that the inhibitory effect for both S. mutans and C. albicans in the present study would result from the interaction (in different associations) of xylitol, erythritol, TMP, and/or F, through different mechanisms. Previous studies reported that these compounds act on cell metabolism, affecting the cytoplasm (Talattof et al., 2018), DNA and RNA (Janus et al., 2017), proteins and carbohydrates of the extracellular matrix (Cavazana et al., 2019), and adherence to surfaces (Leepel et al., 2012). In general, our data point to a significantly higher effect of the experimental toothpaste on the growth inhibition of most of the microorganisms assessed. This supports the idea of simultaneous administration of polyols, polyphosphate, and F at a reduced concentration (200 ppm F) as an alternative to manage cariogenic biofilms, especially for young children, as it would reduce systemic fluoride exposure, while sustaining its mineralizing effects (from F and TMP) and enhancing the antimicrobial effects (from all the actives) compared with a conventional (1100 ppm F) toothpaste. However, any extrapolations to clinical conditions would be extremely premature given the preliminary nature of the data obtained, and limitations inherent to the study protocol. Future studies with biofilms, especially formed on dental substrates, could bring important data in this regard. 32 Conclusion The association of TMP, F, xylitol, and erythritol was shown to be effective in reducing the growth of isolated cariogenic microorganisms under most of the conditions studied (i.e., microorganisms and dilutions), being, therefore, a promising alternative to control biofilms related to dental caries. References Ameglio, F., Di Giorgio, C., Terzaroli, P., & Gandolfo, G. M. (1990). “Giant cell” production by C. albicans cultured in xylitol. Microbiologica, 13(4), 343—346. Retrieved from http://europepmc.org/abstract/MED/2087203 Bamford, C. V., D’Mello, A., Nobbs, A. H., Dutton, L. C., Vickerman, M. M., & Jenkinson, H. F. (2009). Streptococcus gordonii modulates Candida albicans biofilm formation through intergeneric communication. Infection and Immunity, 77(9), 3696–3704. https://doi.org/10.1128/IAI.00438-09 Barbosa, J. O., Rossoni, R. D., Vilela, S. F. G., de Alvarenga, J. A., Velloso, M. dos S., Prata, M. C. de A., … Junqueira, J. C. (2016). Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans. PLOS ONE, 11(3), e0150457. Retrieved from https://doi.org/10.1371/journal.pone.0150457 Bowen, W. H., Burne, R. A., Wu, H., & Koo, H. (2018). Oral Biofilms: Pathogens, Matrix, and Polymicrobial Interactions in Microenvironments. Trends in Microbiology, 26(3), 229–242. https://doi.org/10.1016/j.tim.2017.09.008 Buzalaf, M. A. R., Pessan, J. P., Marques Honório, H., & Ten Cate, J. (2011). Mechanisms of action of fluoride for. Monogr Oral Sci., 22, 97–114. Cavazana, T. P., Hosida, T. Y., Pessan, J. P., Sampaio, C., Monteiro, D. R., & Delbem, A. C. B. (2019). Activity of sodium trimetaphosphate, associated or not with fluoride, on dual-species biofilms. Biofouling, 35(6), 710–718. https://doi.org/10.1080/08927014.2019.1653455 Cavazana, T. P., Pessan, J. P., Hosida, T. Y., Sampaio, C., Amarante, V. D. O. Z., Monteiro, D. R., & Delbem, A. C. B. (2020). Effects of Sodium 33 Trimetaphosphate, Associated or Not with Fluoride, on the Composition and pH of Mixed Biofilms, before and after Exposure to Sucrose. Caries Research, 54(4), 358–368. https://doi.org/10.1159/000501262 De Cock, P., Mäkinen, K., Honkala, E., Saag, M., Kennepohl, E., & Eapen, A. (2016). Erythritol Is More Effective Than Xylitol and Sorbitol in Managing Oral Health Endpoints. International Journal of Dentistry, 2016. https://doi.org/10.1155/2016/9868421 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry, 28(3), 350–356. https://doi.org/10.1021/ac60111a017 Eskandarian, T., Motamedifar, M., Arasteh, P., Eghbali, S. S., Adib, A., & Abdoli, Z. (2017). Comparison of antimicrobial effects of titanium tetrafluoride, chlorhexidine, xylitol and sodium fluoride on streptococcus mutans: An in-vitro study. Electronic Physician, 9(03), 4042–4047. https://doi.org/10.19082/4042 Falsetta, M. L., Klein, M. I., Colonne, P. M., Scott-Anne, K., Gregoire, S., Pai, C. H., … Koo, H. (2014). Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infection and Immunity, 82(5), 1968–1981. https://doi.org/10.1128/IAI.00087-14 Fernandes, R. A., Monteiro, D. R., Arias, L. S., Fernandes, G. L., Delbem, A. C. B., & Barbosa, D. B. (2016). Biofilm formation by Candida albicans and Streptococcus mutans in the presence of farnesol: a quantitative evaluation. Biofouling, 32(3), 329–338. https://doi.org/10.1080/08927014.2016.1144053 Flemming, H. C., & Wingender, J. (2010). The biofilm matrix. Nature Reviews Microbiology, 8(9), 623–633. https://doi.org/10.1038/nrmicro2415 Freire, I. R., Pessan, J. P., Amaral, J. G., Martinhon, C. C. R., Cunha, R. F., & Delbem, A. C. B. (2016). Anticaries effect of low-fluoride dentifrices with phosphates in children: A randomized, controlled trial. Journal of Dentistry, 50, 37–42. https://doi.org/10.1016/j.jdent.2016.04.013 Giertsen, E., Arthur, R. A., & Guggenheim, B. (2011). Effects of xylitol on survival of mutans streptococci in mixed-six-species in vitro biofilms modelling supragingival plaque. Caries Research, 45(1), 31–39. https://doi.org/10.1159/000322646 Hamada, S., & Slade, H. D. (1980). Biology , Immunology , and Cariogenicity of Streptococcus mutanst. Microbiol Rev., 44(2), 331–384. 34 Hashino, E., Kuboniwa, M., Alghamdi, S. A., Yamaguchi, M., Yamamoto, R., Cho, H., & Amano, A. (2013). Erythritol alters microstructure and metabolomic profiles of biofilm composed of Streptococcus gordonii and Porphyromonas gingivalis. Molecular Oral Microbiology, 28(6), 435–451. https://doi.org/10.1111/omi.12037 Hausman, S., Thompson, J., & London, J. (1984). Futile Xylitol Cycle in Lactobacillus casei. J Bacteriol, 160(1), 211–215. https://doi.org/10.1128/jb.160.1.211- 215.1984 Helanto, M., Aarnikunnas, J., Palva, A., Leisola, M., & Nyyssölä, A. (2006). Characterization of genes involved in fructose utilization by Lactobacillus fermentum. Arch Microbiol (2006), 186(1), 51–59. https://doi.org/10.1007/s00203-006-0120-x Ichikawa, T., Yano, Y., Fujita, Y., Kashiwabara, T., & Nagao, K. (2008). The enhancement effect of three sugar alcohols on the fungicidal effect of benzethonium chloride toward Candida albicans. Journal of Dentistry, 36(11), 965–968. https://doi.org/10.1016/j.jdent.2008.07.013 Janus, M. M., Volgenant, C. M. C., Brandt, B. W., Buijs, M. J., Keijser, B. J. F., Crielaard, W., … Krom, B. P. (2017). Effect of erythritol on microbial ecology of in vitro gingivitis biofilms. Journal of Oral Microbiology, 9(1). https://doi.org/10.1080/20002297.2017.1337477 Kassebaum, N. J., Smith, A. G. C., Bernabé, E., Fleming, T. D., Reynolds, A. E., Vos, T., … Yonemoto, N. (2017). Global, Regional, and National Prevalence, Incidence, and Disability-Adjusted Life Years for Oral Conditions for 195 Countries, 1990-2015: A Systematic Analysis for the Global Burden of Diseases, Injuries, and Risk Factors. Journal of Dental Research, 96(4), 380–387. https://doi.org/10.1177/0022034517693566 Kilian, M., Chapple, I. L. C., Hannig, M., Marsh, P. D., Meuric, V., Pedersen, A. M. L., … Zaura, E. (2016). The oral microbiome - An update for oral healthcare professionals. British Dental Journal, 221(10), 657–666. https://doi.org/10.1038/sj.bdj.2016.865 Klein, M. I., Hwang, G., Santos, P. H. S., Campanella, O. H., & Koo, H. (2015). Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms. Frontiers in Cellular and Infection Microbiology, 5(FEB), 1–8. https://doi.org/10.3389/fcimb.2015.00010 Klompmaker, S. H., Kohl, K., Fasel, N., & Mayer, A. (2017). Magnesium uptake by 35 connecting fluid-phase endocytosis to an intracellular inorganic cation filter. Nature Communications, 8(1). https://doi.org/10.1038/s41467-017-01930-5 Koo, H., Falsetta, M. L., & Klein, M. I. (2013). The Exopolysaccharide Matrix: A Virulence Determinant of Cariogenic Biofilm. Journal of Dental Research, 92(12), 1065–1073. https://doi.org/10.1177/0022034513504218 Koo, Hyun, Sheng, J., Nguyen, P. T. M., & Marquis, R. E. (2006). Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspenysion cultures and biofilms. FEMS Microbiology Letters, 254(1), 134–140. https://doi.org/10.1111/j.1574-6968.2005.00018.x Kościelniak, D., Gregorczyk-Maga, I., Jurczak, A., Staszczyk, M., Kołodziej, I., Magacz, M., … Jamka-Kasprzyk, M. (2019). Low concentration of xylitol improves children tooth protection against Streptococcus mutans biofilm formation. Oral Health, 4, 1–10. Lamfon, H., Porter, S. R., McCullough, M., & Pratten, J. (2003). Formation of Candida albicans biofilms on non-shedding oral surfaces. European Journal of Oral Sciences, 111(6), 465–471. https://doi.org/https://doi.org/10.1111/j.0909- 8836.2003.00084.x Lee, R. M., Hartman, P. A., Stahr, H. M., Olson, D. G., & Williams, F. D. (1994). Antibacterial mechanism of long-chain polyphosphates in Staphylococcus aureus. Journal of Food Protection, 57(4), 289–294. https://doi.org/10.4315/0362-028X-57.4.289 Leepel, L., Sastra, S., Puspitawati, R., & Bachtiar, B. (2012). Effect of Xylitol with Various Concentration and Duration on the Growth of Candida albicans (In Vitro study). Journal of Dentistry Indonesia, 16. https://doi.org/10.14693/jdi.v16i1.12 Lemos, J. A., Palmer, S. R., Zeng, L., Wen, Z. T., Kajfasz, J. K., Freires, I. A., … Brady, L. J. (2019). The Biology of Streptococcus mutans . Microbiology Spectrum, 7(1), 1–26. https://doi.org/10.1128/microbiolspec.gpp3-0051-2018 Loimaranta, V., Mazurel, D., Deng, D., & Söderling, E. (2020). Xylitol and erythritol inhibit real-time biofilm formation of Streptococcus mutans. BMC Microbiology, 20(1), 1–9. https://doi.org/10.1186/s12866-020-01867-8 London, J., & Hausman, S. (1982). Xylitol-Mediated Transient Inhibition of Ribitol Utilization by Lactobacillus casei. J Bacteriol, 150(2), 657–661. https://doi.org/10.1128/jb.150.2.657-661.1982 36 Machiulskiene, V., Campus, G., Carvalho, J. C., Dige, I., Ekstrand, K. R., Jablonski- Momeni, A., … Nyvad, B. (2020). Terminology of Dental Caries and Dental Caries Management: Consensus Report of a Workshop Organized by ORCA and Cariology Research Group of IADR. Caries Research, 54(1), 7–14. https://doi.org/10.1159/000503309 Maden, E., Altun, C., Ozmen, B., & Basak, F. (2018). Antimicrobial Effect of Toothpastes Containing Fluoride, Xylitol, or Xylitol-Probiotic on Salivary Streptococcus mutans and Lactobacillus in Children. Niger J Clin Pract, 21, 134–138. https://doi.org/10.4103/njcp.njcp_320_16 Maehara, H., Iwami, Y., Mayanagi, H., & Takahashi, N. (2005). Synergistic inhibition by combination of fluoride and xylitol on glycolysis by mutans streptococci and its biochemical mechanism. Caries Research, 39(6), 521–528. https://doi.org/10.1159/000088190 Mäkinen, K K, Bennett, C. A., Hujoel, P. P., Isokangas, P. J., Isotupa, K. P., Pape, H. R. J., & Makinen, P. L. (1995). Xylitol chewing gums and caries rates: a 40- month cohort study. Journal of Dental Research, 74(12), 1904–1913. https://doi.org/10.1177/00220345950740121501 Mäkinen, Kauko. (2000). The rocky road of xylitol to its clinical application. Journal of Dental Research, 79(6), 1352–1355. https://doi.org/10.1177/00220345000790060101 Mäkinen, Kauko K. (2010). Sugar Alcohols, Caries Incidence, and Remineralization of Caries Lesions: A Literature Review. International Journal of Dentistry, 2010(1), 1–23. https://doi.org/10.1155/2010/981072 Mäkinen, Kauko K. (2011). Sugar alcohol sweeteners as alternatives to sugar with special consideration of xylitol. Medical Principles and Practice, 20(4), 303–320. https://doi.org/10.1159/000324534 Mäkinen, KK., Alanen, P., Isokangas, P., Isotupa, K., Söderling, E., Mäkinen, P. L., … Zhang, B. (2008). Thirty-nine-month xylitol chewing-gum programme in initially 8-year-old school children: A feasibility study focusing on mutans streptococci and lactobacilli. International Dental Journal, 58(1), 41–50. https://doi.org/10.1111/j.1875-595X.2008.tb00175.x Mäkinen, KK, Chen, C., Mäkinen, P., Bennett, C., Isokangas, P., Isotupa, K., & Pape, H. (1996). Properties of whole saliva and dental plaque in relation to 40- month consumption of chewing gums containing xylitol, sorbitol or sucrose. 37 Caries Res, 30(3), 180–188. Malhotra, R., & Shashikiran, N. (2017). Comparison of Antimicrobial Activity of Child Formula Dentifrices at different Concentrations: An in vitro Study. International Journal of Clinical Pediatric Dentistry, 10(2), 131–135. https://doi.org/10.5005/jp- journals-10005-1422 Manarelli, M. M., Delbem, A. C. B., Lima, T. M. T., Castilho, F. C. N., & Pessan, J. P. (2014). In vitro remineralizing effect of fluoride varnishes containing sodium trimetaphosphate. Caries Research, 48(4), 299–305. https://doi.org/10.1159/000356308 Manarelli, Michele M., Delbem, A. C. B., Báez-Quintero, L. C., de Moraes, F. R. N., Cunha, R. F., & Pessan, J. P. (2017). Fluoride varnishes containing sodium trimetaphosphate reduce enamel demineralization in vitro. Acta Odontologica Scandinavica, 75(5), 376–378. https://doi.org/10.1080/00016357.2017.1318448 Matsumoto-Nakano, M. (2018). Role of Streptococcus mutans surface proteins for biofilm formation. Japanese Dental Science Review, 54(1), 22–29. https://doi.org/10.1016/j.jdsr.2017.08.002 Menon, L. U., Scoffield, J. A., Jackson, J. G., & Zhang, P. (2022). Candida albicans and Early Childhood Caries. Frontiers in Dental Medicine, 3. https://doi.org/10.3389/fdmed.2022.849274 Monteiro, D. R., Gorup, L. F., Silva, S., Negri, M., de Camargo, E. R., Oliveira, R., … Henriques, M. (2011). Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata. Biofouling, 27(7), 711–719. https://doi.org/10.1080/08927014.2011.599101 Monteiro, D. R., Silva, S., Negri, M., Gorup, L. F., de Camargo, E. R., Oliveira, R., … Henriques, M. (2013). Silver colloidal nanoparticles: Effect on matrix composition and structure of Candida albicans and Candida glabrata biofilms. Journal of Applied Microbiology, 114(4), 1175–1183. https://doi.org/10.1111/jam.12102 Pandit, S., Kim, J. E., Jung, K. H., Chang, K. W., & Jeon, J. G. (2011). Effect of sodium fluoride on the virulence factors and composition of Streptococcus mutans biofilms. Archives of Oral Biology, 56(7), 643–649. https://doi.org/10.1016/j.archoralbio.2010.12.012 Park, Y. N., Jeong, S. S., Zeng, J., Kim, S. H., Hong, S. J., Ohk, S. H., & Choi, C. H. (2014). Anti-cariogenic effects of erythritol on growth and adhesion of 38 Streptococcus mutans. Food Science and Biotechnology, 23(5), 1587–1591. https://doi.org/10.1007/s10068-014-0215-0 Pessan, J. P., Sampaio, C., Zen, I., Deng, D., Exterkate, R., Delbem, A. C. B., & Monteiro, D. R. (2021). Use of phosphate-based nanoparticles to enhance the effects of fluoride against dental caries and erosion. Nanotechnology for Dentistry Applications. https://doi.org/10.1088/978-0-7503-3671-0ch1 Phantumvanit, P., Makino, Y., Ogawa, H., Rugg-Gunn, A., Moynihan, P., Petersen, P. E., … Ungchusak, C. (2018). WHO Global Consultation on Public Health Intervention against Early Childhood Caries. Community Dentistry and Oral Epidemiology, 46(3), 280–287. https://doi.org/10.1111/cdoe.12362 Radmerikhi, S., Azul, E., Fajardo, K. R., & Formantes, B. (2013). Antimicrobial effect of different xylitol concentrations on Streptococcus mutans and Lactobacillus acidophilus count. Journal of Restorative Dentistry, 1, 95. https://doi.org/10.4103/2321-4619.118907 Raja, M., Hannan, A., & Ali, K. (2010). Association of Oral Candidal Carriage with Dental Caries in Children. Caries Research, 44(3), 272–276. https://doi.org/10.1159/000314675 Runnel, R., Mäkinen, K. K., Honkala, S., Olak, J., Mäkinen, P. L., Nõmmela, R., … Saag, M. (2013). Effect of three-year consumption of erythritol, xylitol and sorbitol candies on various plaque and salivary caries-related variables. Journal of Dentistry, 41(12), 1236–1244. https://doi.org/10.1016/j.jdent.2013.09.007 Sampaio, C., Delbem, A. C. B., Paiva, M. F., Zen, I., Danelon, M., Cunha, R. F., & Pessan, J. P. (2020). Amount of Dentifrice and Fluoride Concentration Influence Salivary Fluoride Concentrations and Fluoride Intake by Toddlers. Caries Research, 54(3), 234–241. https://doi.org/10.1159/000503780 Silva, S., Henriques, M., Martins, A., Oliveira, R., Williams, D., & Azeredo, J. (2009). Biofilms of non-Candida albicans Candida species: Quantification, structure and matrix composition. Medical Mycology, 47(7), 681–689. https://doi.org/10.3109/13693780802549594 Silva, V. M., Massaro, C., Buzalaf, M. A. R., Janson, G., & Garib, D. (2021). Prevention of non-cavitated lesions with fluoride and xylitol varnishes during orthodontic treatment: a randomized clinical trial. Clinical Oral Investigations, 25(6), 3421–3430. https://doi.org/10.1007/s00784-021-03930-8 Söderling, E., Hirvonen, A., Karjalainen, S., Fontana, M., Catt, D., & Seppä, L. 39 (2011). The Effect of xylitol on the composition of the oral flora: A pilot study. European Journal of Dentistry, 5(1), 24–31. https://doi.org/10.1055/s-0039- 1698855 Söderling, E. M., & Hietala-Lenkkeri, A. M. (2010). Xylitol and erythritol decrease adherence of polysaccharide-producing oral streptococci. Current Microbiology, 60(1), 25–29. https://doi.org/10.1007/s00284-009-9496-6 Söderling, E., & Pienihäkkinen, K. (2022). Effects of xylitol chewing gum and candies on the accumulation of dental plaque: a systematic review. Clinical Oral Investigations, 26(1), 119–129. https://doi.org/10.1007/s00784-021-04225-8 Takeshita, E. M., Danelon, M., Castro, L. P., Cunha, R. F., & Delbem, A. C. B. (2016). Remineralizing Potential of a Low Fluoride Toothpaste with Sodium Trimetaphosphate: An in situ Study. Caries Research, 50(6), 571–578. https://doi.org/10.1159/000449358 Takeshita, E. M., Danelon, M., Castro, L. P., Sassaki, K. T., & Delbem, A. C. B. (2015). Effectiveness of a toothpaste with low fluoride content combined with trimetaphosphate on dental biofilm and enamel demineralization in situ. Caries Research, 49(4), 394–400. https://doi.org/10.1159/000381960 Talattof, Z., Azad, A., Zahed, M., & Shahradnia, N. (2018). Antifungal activity of Xylitol against Candida albicans: An in vitro study. Journal of Contemporary Dental Practice, 19(2), 125–129. https://doi.org/10.5005/JP-JOURNALS-10024- 2225 Tanzer, J. M., Livingston, J., & Thompson, A. M. (2001). The Microbiology of Primary Dental Caries in Humans. J Dent Educ., 65(10), 1028–1037. https://doi.org/10.1002/j.0022-0337.2001.65.10.tb03446.x Vaara, M. (1992). Agents that increase the permeability of the outer membrane. Microbiological Reviews, 56(3), 395–411. https://doi.org/10.1128/mmbr.56.3.395-411.1992 Vadeboncoeur, C., Trahan, L., Mouton, C., & Mayrand, D. (1983). Effect of xylitol on the growth and glycolysis of acidogenic oral bacteria. J Dent Res., 62(8), 882– 884. https://doi.org/10.1177/00220345830620080601 Valkenburg, C., Else Slot, D., & Van der Weijden, G. A. (2020). What is the effect of active ingredients in dentifrice on inhibiting the regrowth of overnight plaque? A systematic review. International Journal of Dental Hygiene, 18(2), 128–141. https://doi.org/10.1111/idh.12423 40 Vieira, A. P. M., Arias, L. S., de Souza Neto, F. N., Kubo, A. M., Lima, B. H. R., de Camargo, E. R., … Monteiro, D. R. (2019). Antibiofilm effect of chlorhexidine- carrier nanosystem based on iron oxide magnetic nanoparticles and chitosan. Colloids and Surfaces B: Biointerfaces, 174(October 2018), 224–231. https://doi.org/10.1016/j.colsurfb.2018.11.023 Walsh, T., Worthington, H. V., Glenny, A. M., Marinho, V. C. C., & Jeroncic, A. (2019). Fluoride toothpastes of different concentrations for preventing dental caries. Cochrane Database of Systematic Reviews, 2019(3). https://doi.org/10.1002/14651858.CD007868.pub3 Xiao, J., Moon, Y., Li, L., Rustchenko, E., Wakabayashi, H., Zhao, X., … Kopycka- Kedzierawski, D. T. (2016). Candida Albicans carriage in children with severe early childhood caries (S-ECC) and maternal relatedness. PLoS ONE, 11(10), 1–16. https://doi.org/10.1371/journal.pone.0164242 Yadav, K., & Prakash, S. (2017). Dental Caries: A Microbiological Approach. Journal of Clinical Infectious Diseases & Practice, 02(01), 1–15. https://doi.org/10.4172/2476-213x.1000118 Zeng, L., Chen, L., & Burne, R. (2018). crossm Induction of Lactose Catabolism by Streptococcus mutans. Appl Environ Microbiol, 84(14), 1–17. https://doi.org/10.1128/AEM.00864-18. 30 Tables Table 1. Zone of inhibition (in mm) of toothpastes containing different active compounds, at 5 dilutions, on Streptococcus mutans TOOTHPASTE DILUTIONS (IN DEIONIZED WATER) GROUPS D1 D2 D4 D8 D16 PLA 10.7 ± 0.3 Aa (11.8 – 13.6) 10.4 ± 0.4 Bab (9.4 – 11.5) 10.5 ± 0.8 Ba (8.7 – 12.4) 10.5 ± 0.5 Bab (9.3 – 11.6) 10.3 ± 0.5 Bab (9.1 – 11.6) X 10.8 ± 0.6 Ab (9.2 – 12.3) 10.4 ± 0.5 Aab (9.0 – 11.7) 10.2 ± 0.4 Aa (9.2 – 11.2) 10.1 ± 0.3 Ab (9.4 – 10.8) 10.0 ± 0.3 Aab (9.5 – 10.7) E 10.2 ± 0.2 Ab (9.8 – 10.6) 10.5 ± 0.5 Aab (9.2 – 11.6) 10.3 ± 0.4 Aa (9.1 – 11.4) 10.5 ± 0.5 Aab (9.4 – 11.5) 9.8 ± 0.2 Ab (9.3 – 10.1) TMP 10.8 ± 0.2 Ab (10.3 – 11.1) 10.5 ± 0.5 Bab (9.3 – 11.7) 10.3 ± 0.4 Ba (8.6 – 11.9) 10.6 ± 0.4 Bab (9.4 – 11.7) 10.4 ± 0.3 Bab (9.4 – 11.0) 200F 10.2 ± 0.6 Ab (12.2 – 11.5) 12.2 ± 0.6 Bb (11.5 – 12.6) 11.7 ± 0.1 BCb (11.7 – 11.8) 11.7 ± 0.4 BCc (11.6 – 12.0) 11.9 ± 0.4 Cc (11.8 – 14.1) 1100F 11.1 ± 0.4 Ab (10.0 – 11.9) 11.2 ± 0.2 Aab (10.6 – 11.5) 11.1 ± 0.4 Aa (10.8 – 11.4) 11.6 ± 0.3 Aac (11.4 – 11.7) 11.3 ± 0.3 Aa (10.7 -11.9) X+E 11.4 ± 0.8 Aab (9.3 – 13.3) 10.2 ± 0.5 Aa (8.9 – 11.4) 11 ± 0.5 Aa (9.9 – 12.0) 10.5 ± 0.5 Aab (9.4 – 11.5) 10.2 ± 0.2 Aab (9.7 – 10.5) 200F+TMP 21.1 ± 0.2 Ac (20.9 – 21.2) 20.3 ± 0.4 Bc (19.2 – 21.5) 19.9 ± 0.3 Bc (19.7 – 20.0) 19.5 ± 0.5 Bd (19.4 – 19.9) 19.2 ± 0.2 Bd (19.1 – 19.4) 200F+X+E 11.4 ± 0.4 Aab (10.3 – 12.4) 11.4 ± 0.3 Ab (11.2 – 12.6) 11.3 ± 0.4 Cb (11.1 – 11.5) 11.3 ± 0.3 Cc (11.0 – 11.4) 11.2 ± 0.3 Cc (11.1 – 11.3) TMP+X+E 11.1 ± 0.6 Ab (9.5 – 12.6) 11.0 ± 0.5 Aab (9.8 – 12.1) 10.7 ± 0.5 Aa (9.5 – 11.8) 10.6 ± 0.5 Aab (9.4 – 11.7) 10.4 ± 0.6 Aab (8.8 – 12.0) EXP 10.7 ± 0.4 Ab (9.3 – 12.0) 10.2 ± 0.2 Aa (9.3 – 11.0) 10.2 ± 0.6 Aa (9.9 – 11.1) 10.1 ± 0.4 Aab (10.0 – 10.4) 10.0 ± 0.5 Ab (10.0 – 10.5) Results are presented as means ± SD (95% Confidence Intervals). Distinct upper-case letters indicate statistical significance for comparisons among dilutions within each group. Distinct lower-case letters indicate statistical significance for comparisons among treatments with each dilution (two-way ANOVA and Tukey’s HSD test, p <0.05; n = 9). PLA: Placebo, X: Xylitol (16%), E: Erythritol (4%), TMP: Sodium Trimetaphosphate (0.25%), 200F: 200 ppm Fluoride, 1100F: 1100 ppm Fluoride, XE: Xylitol (16%) + Erythritol (4%), 200F+TMP: 200 ppm Fluoride + Sodium Trimetaphosphate (0.25%), 200F+X+E: 200 ppm Fluoride + Xylitol (16%) + Erythritol (4%), TMP+X+E: Sodium Trimetaphosphate (0.25%) + Xylitol (16%) + Erythritol (4%), EXP: 200 ppm of Fluoride + Xylitol (16%) + Erythritol (4%) + Sodium Trimetaphosphate (0.25%). 31 Table 2. Zone of inhibition (in mm) of toothpastes containing different active compounds, at 5 dilutions, on Lactobacillus casei TOOTHPASTE DILUTIONS (IN DEIONIZED WATER) GROUPS D1 D2 D4 D8 D16 PLA 13.4 ± 0.3 Aab (12.6 – 14.2) 12.0 ± 0.1 Ba (11.8 – 12.1) 12.0 ± 0.4 Bab (10.8 – 13.4) 11.2 ± 0.2 Bab (10.5 – 11.8) 6.5 ± 0.4 Ca (5.4 – 7.4) X 15.4 ± 0.2 Ad (14.7 – 16.0) 12.9 ± 0.1 Bab (12.6 – 13.1) 12.0 ± 0.4 Babc (11.1 – 13.0) 11.9 ± 0.2 Bb (11.5 – 12.1) 6.5 ± 0.2 Ca (5.9 – 7.1) E 13.1 ± 0.3 Aab (12.9 – 13.2) 13.4 ± 0.4 Ab (13.4 – 13.8) 11.4 ± 0.4 Ba (10.2 – 12.5) 11.6 ± 0.3 Bab (11.4 – 11.7) 11.8 ± 0.3 Bb (11.5 – 11.8) TMP 12.8 ± 0.3 Abc (12.6 – 12.9) 12.6 ± 0.1 ABab (12.4 – 12.7) 13.2 ± 0.2 Ac (13.0 – 13.3) 11.5 ± 0.9 Bab (9.2 – 13.6) 6.5 ± 0.3 Ca (5.8 – 7.1) 200F 12.0 ± 0.1 Ac (11.5 – 12.3) 12.3 ± 0.5 Aab (11.1 – 13.5) 11.5 ± 0.5 Aa (11.0 – 11.6) 6.7 ± 0.2 Bc (6.2 – 7.1) 6.8 ± 0.2 Ba (6.3 – 7.1) 1100F 13.9 ± 0.2 Aa (13.4 – 14.4) 12.9 ± 0.2 ABab (12.7 – 13.0) 11.9 ± 0.6 Ba (11.0 – 12.7) 10.7 ± 0.8 Ca (8.8 – 12.6) 6.8 ± 0.2 Da (6.6 – 6.9) X+E 13.2 ± 0.1 Aab (13.0 – 13.3) 13.2 ± 0.1 Ab (13.0 – 13.3) 11.9 ± 0.1 Ba (11.6 – 12.2) 10.8 ± 0.2 Bab (10.2 – 11.4) 6.1 ± 0.7 Ca (4.4 – 7.7) 200F+TMP 11.9 ± 0.1 Ac (11.8 – 12.1) 12.1 ± 0.2 Aa (11.9 – 12.2) 11.9 ± 0.3 Aa (11.0 – 12.6) 11.0 ± 0.2 ABab (10.8 – 11.1) 9.9 ± 0.2 Bc (9.7 – 10.0) 200F+X+E 13.9 ± 0.2 Aab (13.4 – 14.4) 13.4 ± 0.4 Bc (13.1 – 13.6) 13.3 ± 0.4 Ac (13.2 – 13.5) 11.5 ± 0.1 Cab (11.3 – 11.6) 6.2 ± 0.3 Da (5.2 – 7.1) TMP+X+E 13.1 ± 0.2 Aab (12.9 – 13.2) 14.8 ± 0.3 Bd (14.5 – 14.8) 12.4 ± 0.9 Aabc (10.2 – 14.5) 12.2 ± 1.0 Bd (12.6 – 17.7) 6.5 ± 0.3 Ca (5.9 – 7.1) EXP 14.2 ± 0.2 Aa (14.0 – 14.4) 13.5 ± 0.3 Bab (13.1 – 13.9) 13.1 ± 0.3 Bbc (13.0 – 13.2) 11.0 ± 0.2 Cab (10.8 – 11.1) 9.3 ± 0.4 Dc (9.1 – 9.5) Results are presented as means ± SD (95% Confidence Intervals). Distinct upper-case letters indicate statistical significance for comparisons among dilutions within each group. Distinct lower-case letters indicate statistical significance for comparisons among treatments with each dilution (two-way ANOVA and Tukey’s HSD test, p <0.05; n = 9). PLA: Placebo, X: Xylitol (16%), E: Erythritol (4%), TMP: Sodium Trimetaphosphate (0.25%), 200F: 200 ppm Fluoride, 1100F: 1100 ppm Fluoride, XE: Xylitol (16%) + Erythritol (4%), 200F+TMP: 200 ppm Fluoride + Sodium Trimetaphosphate (0.25%), 200F+X+E: 200 ppm Fluoride + Xylitol (16%) + Erythritol (4%), TMP+X+E: Sodium Trimetaphosphate (0.25%) + Xylitol (16%) + Erythritol (4%), EXP: 200 ppm of Fluoride + Xylitol (16%) + Erythritol (4%) + Sodium Trimetaphosphate (0.25%). Table 3. Zone of inhibition (in mm) of toothpastes containing different active compounds, at 5 dilutions, on Actinomyces israelii 32 TOOTHPASTE DILUTIONS (IN DEIONIZED WATER) GROUPS D1 D2 D4 D8 D16 PLA 5.9 ± 0.1 Aa (5.8 – 6.1) 5.6 ± 0.2 Aa (5.3 – 6.0) 5.8 ± 0.3 Aa (5.2 – 6.5) 6.1 ± 0.2 Aa (5.6 – 6.4) 6.1 ± 0.1 Aa (5.8 – 6.4) X 5.9 ± 0.2 Aa (5.5 – 6.3) 5.9 ± 0.1 Aa (5.7 – 6.1) 6.1 ± 0.2 Aa (5.7 – 6.5) 5.9 ± 0.1 Aa (5.6 – 6.2) 6.0 ± 0.3 Aab (5.1 – 6.9) E 6.2 ± 0.1 Aa (6.2 – 6.2) 6.1 ± 0.2 Aa (5.7 – 6.4) 6.0 ± 0.2 Aa (5.7 – 6.4) 5.9 ± 0.1 Aa (5.6 – 6.2) 6.0 ± 0.1 Aab (5.5 – 6.3) TMP 5.9 ± 0.2 Aa (5.8 – 6.3) 6.0 ± 0.4 Aa (5.6 – 6.4) 5.9 ± 0.3 Aa (5.7 – 6.0) 6.0 ± 0.2 Aa (5.4 – 6.6) 5.9 ± 0.3 Aab (5.6 – 6.1) 200F 6.2 ± 0.1 Aa (6.0 – 6.3) 5.8 ± 0.1 Aa (5.7 – 6.0) 5.7 ± 0.1 Aa (5.2 – 6.1) 6.7 ± 0.2 Bb (6.2 – 7.1) 6.7 ± 0.4 Bc (6.2 – 6.9) 1100F 5.7 ± 0.3 Aa (5.5 – 6.0) 6.0 ± 0.3 Aa (5.8 – 6.3) 5.8 ± 0.2 Aa (5.4 – 6.2) 5.9 ± 0.2 Aa (5.3 – 6.6) 6.0 ± 0.2 Aab (6.0 – 6.2) X+E 6.0 ± 0.1 Aa (5.8 – 6.1) 5.7 ± 0.2 Aa (5.2 – 6.2) 5.8 ± 0.4 Aa (5.6 – 6.1) 5.7 ± 0.3 Aa (5.4 – 6.0) 5.9 ± 0.2 Aab (5.5 – 6.4) 200F+TMP 5.7 ± 0.2 Aa (5.2 – 6.3) 5.7 ± 0.2 Aa (5.2 – 6.2) 5.6 ± 0.2 Aa (5.2 – 6.1) 5.8 ± 0.3 Aa (5.3 – 6.3) 5.5 ± 0.4 Ab (5.1 – 6.0) 200F+X+E 5.7 ± 0.2 Aa (5.3 – 6.2) 5.8 ± 0.3 Aa (5.0 – 6.6) 5.7 ± 0.3 Aa (5.5 – 6.0) 5.9 ± 0.1 Aa (5.7 – 6.0) 5.1 ± 0.2 Aab (5.6 – 6.1) TMP+X+E 6.0 ± 0.2 Aa (5.8 – 6.1) 5.9 ± 0.1 Aa (5.7 – 6.2) 5.8 ± 0.3 Aa (5.6 – 6.0) 5.9 ± 0.3 Aa (5.7 – 6.0) 5.9 ± 0.3 Aab (5.7 – 6.0) EXP 5.8 ± 0.1 Aa (5.6 – 5.9) 6.0 ± 0.3 Aa (5.6 – 6.4) 5.9 ± 0.1 Aa (5.8 – 6.1) 5.9 ± 0.3 Aa (5.5 – 6.3) 6.1 ± 0.3 Aa (5.8 – 6.4) Results are presented as means ± SD (95% Confidence Intervals). Distinct upper-case letters indicate statistical significance for comparisons among dilutions within each group. Distinct lower-case letters indicate statistical significance for comparisons among treatments with each dilution (two-way ANOVA and Tukey’s HSD test, p <0.05; n = 9). PLA: Placebo, X: Xylitol (16%), E: Erythritol (4%), TMP: Sodium Trimetaphosphate (0.25%), 200F: 200 ppm Fluoride, 1100F: 1100 ppm Fluoride, XE: Xylitol (16%) + Erythritol (4%), 200F+TMP: 200 ppm Fluoride + Sodium Trimetaphosphate (0.25%), 200F+X+E: 200 ppm Fluoride + Xylitol (16%) + Erythritol (4%), TMP+X+E: Sodium Trimetaphosphate (0.25%) + Xylitol (16%) + Erythritol (4%), EXP: 200 ppm of Fluoride + Xylitol (16%) + Erythritol (4%) + Sodium Trimetaphosphate (0.25%). 33 Table 4. Zone of inhibition (in mm) of toothpastes containing different active compounds, at 5 dilutions, on Candida albicans TOOTHPASTE DILUTIONS (IN DEIONIZED WATER) GROUPS D1 D2 D4 D8 D16 PLA 5.6 ± 0.2 Aa (4.9 – 6.4) 5.5 ± 0.1 Aa (5.1 – 5.9) 5.6 ± 0.3 Aa (5.3 – 5.8) 5.8 ± 0.7 Aa (5.7 – 6.0) 5.6 ± 0.3 Aab (5.5 – 5.8) X 5.7 ± 0.1 Aa (5.6 – 5.9) 5.7 ± 0.3 Aa (5.3 – 6.2) 5.7 ± 0.3 Aa (4.9 – 6.4) 5.9 ± 0.5 Aa (5.7 – 6.8) 5.4 ± 0.4 Aa (5.2 – 5.5) E 5.9 ± 0.3 Aa (5.2 – 6.5) 5.8 ± 0.3 Aa (5.4 – 6.2) 5.9 ± 0.5 Aab (5.2 – 6.7) 5.8 ± 0.3 Aa (5.0 – 6.6) 5.9 ± 0.3 Aab (5.2 – 6.7) TMP 6.2 ± 0.2 Aab (5.5 – 6.9) 5.1 ± 0.1 ABa (5.1 – 6.0) 5.1 ± 0.2 ABab (5.0 – 6.1) 5.4 ± 0.2 Ba (5.2 – 6.5) 5.7 ± 0.5 ABab (5.3 – 6.1) 200F 5.8 ± 0.1 Aa (5.5 – 6.1) 5.5 ± 0.1 Aa (5.1 – 5.9) 5.6 ± 0.9 Aa (5.3 – 5.9) 5.8 ± 0.1 Aa (5.4 – 6.2) 5.6 ± 0.7 Aab (5.4 – 5.7) 1100F 6.1 ± 0.2 Aab (5.5 – 6.7) 5.9 ± 0.2 Aab (5.7 – 6.2) 6.1 ± 0.3 Aab (5.4 – 6.8) 5.8 ± 0.2 Aa (5.4 – 6.3) 5.8 ± 0.1 Aab (5.5 – 6.0) X+E 6.0 ± 0.5 Aab (5.8 – 6.3) 5.1 ± 0.3 Aa (5.1 – 6.0) 5.1 ± 0.2 Aa (5.1 – 5.4) 5.1 ± 0.3 Aa (5.0 – 5.7) 5.0 ± 0.9 Aab (5.0 – 5.4) 200F+TMP 5.8 ± 0.1 Aa (5.5 – 6.1) 5.5 ± 0.4 Aa (5.1 – 5.9) 5.6 ± 0.2 Aa (5.3 – 5.9) 5.8 ± 0.1 Aa (5.4 – 6.2) 5.6 ± 0.7 Aab (5.4 – 5.7) 200F+X+E 6.6 ± 0.1 ABb (6.4 – 6.7) 6.6 ± 0.2 Bb (6.3 – 6.9) 5.9 ± 0.4 ACab (5.6 – 6.2) 5.7 ± 0.3 Ca (5.3 – 6.1) 5.9 ± 0.2 ACab (5.3 – 6.5) TMP+X+E 5.7 ± 0.2 Aa (5.4 – 5.9) 5.7 ± 0.2 Aa (5.2 – 6.2) 5.8 ± 0.2 Aa (5.5 – 6.0) 5.8 ± 0.1 Aa (5.5 – 6.5) 5.7 ± 0.7 Aab (5.2 – 6.2) EXP 7.9 ± 0.4 Ac (6.7 – 9.1) 7.6 ± 0.3 Ac (6.6 – 8.5) 6.6 ± 0.2 Bb (6.1 – 7.1) 6.0 ± 0.7 Ba (5.4 – 6.7) 6.0 ± 0.1 Bb (5.9 – 6.5) Results are presented as means ± SD (95% Confidence Intervals). Distinct upper-case letters indicate statistical significance for comparisons among dilutions within each group. Distinct lower-case letters indicate statistical significance for comparisons among treatments with each dilution (two-way ANOVA and Tukey’s HSD test, p <0.05; n = 9). PLA: Placebo, X: Xylitol (16%), E: Erythritol (4%), TMP: Sodium Trimetaphosphate (0.25%), 200F: 200 ppm Fluoride, 1100F: 1100 ppm Fluoride, XE: Xylitol (16%) + Erythritol (4%), 200F+TMP: 200 ppm Fluoride + Sodium Trimetaphosphate (0.25%), 200F+X+E: 200 ppm Fluoride + Xylitol (16%) + Erythritol (4%), TMP+X+E: Sodium Trimetaphosphate (0.25%) + Xylitol (16%) + Erythritol (4%), EXP: 200 ppm of Fluoride + Xylitol (16%) + Erythritol (4%) + Sodium Trimetaphosphate (0.25%). 34 CAPÍTULO 2 35 Evaluation of solutions containing fluoride, sodium trimetaphosphate, xylitol and erythritol on dual-species biofilms Igor Zena, Alberto Carlos Botazzo Delbema, Tamires Passadori Martinsa, Leonardo Antonio de Moraisa, Caio Sampaioa, Thayse Yumi Hosidaa, Douglas Roberto Monteirob, Juliano Pelim Pessan*a aDepartment of Preventive and Restorative Dentistry, School of Dentistry, Araçatuba, São Paulo State University (Unesp), Brazil, bPostgraduate Program in Health Sciences, University of Western São Paulo (Unoeste), Presidente Prudente, Brazil Igor Zen1 https://orcid.org/0000-0002-2524-4365 Alberto Carlos Botazzo Delbem1 http://orcid.org/0000- 0002-8159-4853 Thayse Yumi Hosida1 http://orcid.org/0000-0001- 7007-330X Caio Sampaio1 http://orcid.org/0000-0002-6861-7205 Leonardo Antônio Morais1 https://orcid.org/0000-0003-1894-0087 Tamires Passadori Martins1 https://orcid.org/0000-0003-4153-1548 Douglas Roberto Monteiro1,2 http://orcid.org/0000-0001- 5229-5259 Juliano Pelim Pessan1* http://orcid.org/0000-0002- 1550-3933 *Corresponding author Juliano Pelim Pessan, DDS, MSc, PhD Associate Professor in Pediatric Dentistry Department of Preventive and Restorative Dentistry School of Dentistry, Araçatuba, São Paulo State University (Unesp) Rua José Bonifácio, 1193 16015-050 Araçatuba – SP, Brazil Phone: +55 18 3636 3314 E-mail: juliano.pessan@unesp.br Este artigo segue as normas do periódico Biofouling (Anexo C). 36 Evaluation of solutions containing fluoride, sodium trimetaphosphate, xylitol and erythritol on dual-species biofilms Abstract The aim of this study was to assess the effect solutions containing fluoride, sodium trimetaphosphate (TMP), xylitol and erythritol on dual-species biofilms of S. mutans and C. albicans. Biofilms were grown in the continuous presence these actives alone or in different associations. Quantification of Colony-Forming unity (CFU), metabolic activity, biomass, and extracellular matrix components were evaluated. CFU counts were mostly affected by F, while biomass and metabolic activity were more susceptible to TMP. A synergistic effect of these actives was observed. As for the polyols, their effect was more pronounced on the carbohydrate content of the extracellular matrix, with lesser or no action on the other variables. Overall, the association of the four actives led to increased antibiofilm effects. The dual-species biofilms analyzed were mostly affected by F and/or TMP, with little effects of the polyols administered alone. Co-administration of the four actives was effec4tive in reducing biofilm’s virulence. Keywords: Biofilms, Streptococcus mutans, Candida albicans, phosphate, sugar alcohols, fluoride Word count: 3.835 The authors report there are no competing interests to declare 37 Introduction Dental caries is considered a dynamic disease, biofilm-mediated and diet modulated, resulting in loss of the mineral dental tissues (Machiulskiene et al., 2020). One commensal member of all microorganisms related onset of this disease is Streptococcus mutans, which is found in caries lesions of both adults and children (Menon et al., 2022). Besides, Candida albicans is a commensal fungi found in 96% of children with dental caries (Raja, Hannan, & Ali, 2010), and oral cavities already colonized by this fungus may have nearly five times a greater risk of developing early childhood caries (Xiao et al., 2016). Furthermore, S. mutans can produce anchored proteins that allow and facilitate the binding to C. albicans, leading to a more virulent environment in the biofilm (Bamford et al., 2009). Fluoride (F) products play an important role in preventing, controlling, and arresting dental caries. Among the most used formulations, there is strong evidence on the clinical efficacy of toothpastes containing ~1100 ppm F or above in reducing the progression of the disease (Sampaio et al., 2020; Walsh et al., 2019), with a lower number of trials attesting the efficacy of formulations with lower F content. However, a there is a strong body of evidence from in vitro, in situ, and clinical studies showing that the addition of sodium trimetaphosphate (TMP) to low-F toothpastes promotes synergistic effects on de- and re-mineralization, and on the progression of caries lesions in children (Freire et al., 2016; Takeshita et al., 2016, 2015). Moreover, TMP presents effects over dual-species biofilms of S. mutans and C. albicans, acting directly on the metabolic activity and production of biofilm biomass, in addition to effects on protein, carbohydrate and DNA content of the extracellular matrix (Cavazana et al., 2019). In addition to the actives above, natural products such as xylitol and erythritol have demonstrated marked effects on biofilms (Cannon et al., 2020; Loimaranta, Mazurel, Deng, & Söderling, 2020). Both polyols have been shown to decrease biofilm acidogenicity and volume, and to inhibit the growth of S. mutans. The effects of these polyols over the extracellular matrix have also shown to decrease the microorganism’s adherence to enamel surface and to decrease the expression of genes involved in the metabolism of sucrose (Park et al., 2014). Given the different mechanisms of action of F, TMP, xylitol and erythritol on cariogenic biofilms, it would be possible that the association of these actives could 38 increase the antibiofilm effects of topically-applied formulations. Thus, this study aimed to assess the effects of F, TMP, xylitol or erythritol, alone or in different combinations, on dual-species biofilms of S. mutans and C. albicans, using different biofilm quantification assays (cultivable cells, total biomass, metabolic activity, and extracellular matrix composition). The study’s null hypothesis was that the effects of the actives alone would not be significantly different from those observed by different associations on the variables analyzed. Materials and methods Artificial saliva The culture medium used for biofilm formation was sucrose-containing artificial saliva (AS), and its composition for 1.0 l of deionized water was based on the protocol described by Lamfon et al. (2003) with modifications: 4 g of sucrose (Sigma-Aldrich, St Louis, MO, USA), 2 g of yeast extract (Sigma-Aldrich), 5 g of bacteriological peptone (Sigma-Aldrich), 1 g of mucin type III (Sigma-Aldrich), 0.35 g of NaCl (Sigma- Aldrich), 0.2 g of CaCl2 (Sigma-Aldrich), and 0.2 g of KCl (Sigma-Aldrich). The pH of the solution was adjusted with NaOH to 6.8. Preparation of the test solutions The solutions were prepared by weighing and diluting the following compounds: xylitol (Sigma-Aldrich), erythritol (Sigma-Aldrich), trisodium trimetaphosphate (Sigma-Aldrich), or sodium fluoride (NaF, Sigma-Aldrich), alone or in different associations, in order to achieve final concentrations of 4.8%, 1.2%, 0.075%, and 60 ppm F, respectively. The experimental groups in this study were: Pure AS (negative control = “NC”), NaF 330 ppm F (positive control – “330F”), Xylitol 4.8% (“X”), Erythritol 1.2% (“E”), Sodium trimetaphosphate 0.075% (“TMP”), NaF 60 ppm F (“60F”), “X+E”, “60F+TMP”, “TMP+X+E”, “60F+X+E”, and experimental solution containing 60F+TMP+X+E (“EXP”). All compounds were filter-sterilised to obtain an aseptic condition. The concentrations were adopted to achieve 30% of the initial concentrations used in a previous study, administered as toothpastes (Marcato et al., 2021). 39 Strains and growth conditions Two strains from the American Type Culture Collection (ATCC) were included in this study: C. albicans ATCC 10231 and S. mutans ATCC 25175. For C. albicans, colonies previously cultured on Sabouraud Dextrose Agar (SDA; Difco, Le Pont de Claix, France) were suspended in 10 ml of Sabouraud Dextrose Broth (Difco) and aerobically incubated overnight at 120 rpm and 37 ºC. Simultaneously, S. mutans colonies grown on Brain Heart Infusion agar (BHI Agar; Difco) were suspended in 10 ml of BHI broth (Difco) and statically incubated overnight in 5% CO2 at 37 ºC. Afterwards, fungal and bacterial cells were recovered by centrifugation (8,000 rpm, 5 min), and the cell pellets washed twice with 10 ml of 0.85% NaCl. The number of C. albicans cells was adjusted to 1x107 cells ml-1 in AS using a Neubauer counting chamber, while the number of bacterial cells was spectrophotometrically (640 nm) adjusted to 1x108 cells ml-1. Biofilm formation and growth in the presence of solutions Dual-species biofilms were formed in flat-bottom 96-well microtiter plates (Costar, Tewksbury, USA). For this, 100 µL of each microbial suspension (2x107 cells ml-1 for C. albicans, 2x108 cells ml-1 for S. mutans) were added to the wells and the plates were incubated in 5% CO2 at 37 ºC for 2 h. Next, AS was removed, and the wells were washed once with 0.85% NaCl. Each test solution was added to AS to achieve a final solution of 4 mL. These compounds were then pipetted in the walls containing adhered cells, and the plates were incubated for 24 h in 5% CO2 at 37 ºC in order to allow biofilm formation (Vieira et al., 2019). The medium was renewed every 24 h by removing 100 ml and adding an equal volume of fresh test solution. After completing 96 h biofilm formation, the test solutions were removed from the wells, and the resulting biofilms were washed once with 0.85% NaCl to eliminate planktonic cells. Biofilm quantification assays Cultivable cells The number of cultivable cells was assessed by enumeration of Colony-Forming Units (CFUs), as previously detailed (Fernandes et al., 2016). Briefly, the resulting biofilms after treatment were resuspended in 0.85% NaCl and scraped from the wells. Biofilm suspensions were then serially diluted (in 0.85% NaCl) and plated on CHROMagar Candida (Difco), and BHI agar supplemented with 7 µL ml-1 40 amphotericin B (Sigma-Aldrich). Agar plates were incubated for 24–48 h at 37 ºC, and the number of CFUs was expressed as log10 CFU cm-2. Total biofilm biomass Biofilm biomass was quantified by the crystal violet (CV) staining assay (Monteiro et al., 2011). Biofilms were fixed for 15 min at room temperature with 99% methanol (Sigma-Aldrich), stained for 5min with 1% CV (Sigma-Aldrich), and de-stained through exposure 33% acetic acid (Sigma-Aldrich). Absorbance values were read at 570 nm and represented as a function of the area of the wells (absorbance cm-2). Wells containing AS without microbial cells were used as blanks. Metabolic activity The evaluation of metabolic activity of biofilm cells was performed by the 2,3-bis (2- methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT; Sigma-Aldrich) reduction method (Fernandes et al., 2016). In summary, XTT and phenazine methosulphate (Sigma-Aldrich) solutions were combined and pipetted into the wells. The microliter plates were incubated (37 ºC, 120 rpm) for 3 h, protected from light, and the absorbance values were measured at 490 nm (absorbance cm-2). Blanks were processed as described for the total biomass assay. Analysis of extracellular matrix composition For this assay, dual-species biofilms were grown in the six-well plates (Costar) containing 4 ml of the microbial suspension, as described above. After the 96 h with solutions, the biofilms were resuspended in 0.85% NaCl, scraped from the wells, and the liquid phase of the extracellular matrix was extracted by sonication (for 30 s at 30W), as detailed elsewhere (S. Silva et al., 2009). The bicinchoninic acid method (Kit BCA; Sigma-Aldrich) was performed for protein determination of the extracellular matrix, using bovine serum albumin as the standard (S. Silva et al., 2009), while the carbohydrate content was measured using the method devised by Dubois et al. (1956), with glucose as the standard. For DNA content, a volume of 1.5 µl of the liquid phase of the extracellular matrix was spectrophotometrically analyzed (at 260 and 280 nm) in a Nanodrop Spectrophotometer (EONC Spectrophotometer of EONC, Biotek, Winooski, VT, USA) (Monteiro et al., 2013). Protein, carbohydrate and DNA values were expressed as mg g-1 dry weight of biofilm. 41 Statistical analysis All microbiological experiments were conducted in biological triplicate, on three different days. The normality of the data was verified by Shapiro–Wilk’s test, followed by one-way ANOVA and Tukey’s HSD post hoc test (SigmaPlot 12.0 software, Systat Software Inc., San Jose, CA, USA). All analyses were performed with a significance level of 5%. Results Regarding the number of viable cells of S. mutans, all test groups had significantly lower values compared to the NC, except for TMP alone (Figure 1a). Overall, the largest reductions were observed for 330F, followed by 60F+TMP, EXP and 60F, with a synergistic effect observed between TMP and 60F. For C. albicans, all test groups had significantly lower numbers of viable cells compared to the NC, with no clearly defined trend among groups containing the actives (Figure 2b). Solutions containing TMP, TMP+X+E, or 60F+TMP promoted significantly larger reductions in the total biofilm mass compared to the other groups (Figure 1c). On the other hand, solutions containing X, E, or X+E did not promote significant reductions compared to the NC. A similar trend was observed for the metabolic activity of the biofilms assessed (Figure 1d). All test solutions were able to reduce the protein and carbohydrate contents of the extracellular matrix of the biofilms compared to the NC (Table 1), with the largest effects seen for 330F and 60F (dose-response), TMP and 60F+TMP. As for DNA content, a similar pattern was observed, except that the polyols alone or associated with 60F or TMP did not promote significant reductions compared to the NC. 42 Discussion The high consumption of fermentable sugars in the diet, allied with the absence of proper hygiene of the oral cavity, create favorable conditions for the growth and development of cariogenic biofilms, especially at stagnation sites (Machiulskiene et al., 2020). To overcome this issue, consolidated F-based strategies have been tested in association with new preventive/therapeutic agents, which have been shown to promote significant effects on enamel de- and re- mineralization, as well as in reducing biofilm formation and, consequently, dental caries (Pessan et al., 2021). Within this context, the present study aimed to evaluate the effects of solutions containing F, TMP, X and E, alone or in different combinations, demonstrating that most of the associations affected several variables related to dual-species cariogenic biofilms of S. mutans and C. albicans, thus leading to partial rejection of the null hypothesis. Among the more than 700 different species of microbes that inhabit the oral cavity, S. mutans is one of the main cariogenic pathogens, which is related to the onset of caries lesions (Kilian et al., 2016; Matsumoto-Nakano, 2018). The present findings showed that the major effect on S. mutans CFU counts was promoted by F, in a dose-response manner (Figure 1a). Such effects were somehow expected considering the well-known bacteriostatic action of F on this bacterium (Buzalaf et al., 2011), what might be potentiated by the constant presence of this active during initial biofilm formation and growth. Interestingly, such bacteriostatic effects seem to have affected all the other variables analyzed, despite at a lesser extent and not following a dose-response trend. As for TMP, it was noteworthy that despite this polyphosphate promoted no reduction in S. mutans CFU counts when administered alone (compared with the negative control), it potentiated the effects of F. In fact, similar results were found by Cavazana et al. (2019) using the same biofilm model, but administering the actives at higher concentrations and in short-term exposure times (instead of its constant presence during biofilm formation). The synergism between the actives F and TMP has been extensively reported for enamel de- and re-mineralization when administered as solutions, toothpastes, mouthwashes, gels and varnishes (Cavazana et al., 2019; Danelon, Takeshita, Peixoto, Sassaki, & Delbem, 2014; Freire et al., 2016; Michele M. Manarelli et al., 2017; Takeshita et al., 2016, 2015). Collectively, these studies demonstrate that 43 TMP’s mechanism of action is related to its ability to adsorb onto the enamel surface, what hinders acid diffusion and decreases enamel demineralization. In our study, however, the absence of tooth enamel in the model suggests that the positive effects of F + TMP on the biofilms assessed might mostly be attributed to the inhibition of acidic niche formation and cell adhesion and aggregation due to the action of TMP (Flemming & Wingender, 2010). This hypothesis is supported by a previous study using the same dual-species model as in the present work, showing an enhanced buffering effect of F + TMP in comparison with the actives alone (Cavazana et al., 2020). The aforementioned synergism of F + TMP on CFU counts of S. mutans were, in general, reflected on all the other parameters assessed, including biofilm biomass, metabolic activity and, to a lesser extent, on the protein content of the extracellular matrix. Interestingly, however, the highest effects on biofilm biomass and metabolic activity were not promoted by F, but instead by TMP, and this polyphosphate was shown to potentiate the effects also of the polyols on both variables analyzed (Figure 1c and 1d). Regarding the extracellular matrix, TMP and F (administered alone or in association) also promoted significant reductions in protein, carbohydrate and DNA contents. Following a similar rationale to that described for F, it seems plausible that the effects of TMP on biofilm biomass and metabolic activity resulted in lower contents of the extracellular matrix components, with a synergistic effect with F on its protein content (Table 1). These findings are in line with previous data obtained by the use of F + TMP as treatment solutions, which demonstrated significant reductions in total biomass values, metabolic activity, and extracellular matrix components in mixed biofilms of S. mutans and C. albicans (Cavazana et al., 2019). Also, the administration of toothpastes containing these actives on polymicrobial biofilms formed in situ resulted in lower production of extracellular polysaccharides compared with F alone (Takeshita et al., 2015). A possible explanation for these reductions is related to a slight chelating ability of TMP (Vaara, 1992), which allows its binding to the cell wall of gram-positive bacteria, specifically through Ca+ and Mg+ ions, thus leading to changes in bacterial metabolism (Klompmaker, Kohl, Fasel, & Mayer, 2017; H. Koo, Falsetta, & Klein, 2013; Lee, Hartman, Stahr, Olson, & Williams, 1994). This reduction may also be associated with the bioavailability of nutrients in the culture medium, since possible interactions between TMP and ions 44 present in the medium may occur, causing a reduction in biofilm metabolism (Cavazana et al., 2019; Lee et al., 1994). As for xylitol and erythritol, the data obtained analyzed together point out to very modest effects of these polyols on the biofilms compared with the negative control. Although at first glance the trend observed was not expected, the sucrose content in the culture medium and the maturation stage of the biofilms likely played major roles on the results obtained. Given that sucrose was not administered as pulses (to mimic cariogenic challenges), but instead it was constantly present in the culture medium, bacteria would predominantly metabolize fructose and glucose (from sucrose) over sugar-alcohols, so it is seems unlikely that the polyols would be metabolized in the presence of hexoses (Chan et al., 2020; Zeng, Chen, & Burne, 2018). Similar results have been previously reported for single- and dual-species biofilms of S. mutans and C. albicans at mature formation stages (Eskandarian et al., 2017; Giertsen, Arthur, & Guggenheim, 2011). Conversely, biofilms at early stages (8h-24h) were shown to more susceptible to treatment with xylitol and erythritol compared with mature biofilms (Kościelniak et al., 2019; Loimaranta et al., 2020). Therefore, the time for biofilm formation in the present study (96 h) might also have influenced the results obtained concerning the effect of xylitol and erythritol. Despite the small effects of the polyols on CFU counts, biofilm biomass, and on protein and DNA contents of the extracellular matrix, these actives promoted reductions of ~30% in the carbohydrate concentrations of the biofilms (Table 1). This is important from a clinical perspective, as this parameter is directly related with the biofilms virulence (Klein et al., 2015). Data on the metabolic activity also deserve comment, since the results from biofilms treated with the polyols were as high as those observed for the negative control. Analyzed together, these two parameters are in line with the well-known futile cycle of the polyols (E. Söderling et al., 2011), given that the presence of the sugar alcohols led to highly active biofilms (also resulting in high biofilm biomass), but significantly lower reserve of extracellular carbohydrates in the biofilm matrix. The negligible effect of the actives over C. albicans cells (Figure 1b) must be addressed. Treatment with F and/or TMP has already been shown to be ineffective on this fungus (Cavazana et al., 2019, 2020), and the effects of polyols have usually b