Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Ciências Farmacêuticas Desenvolvimento de filmes orodispersíveis potencialmente probióticos para promoção da saúde oral Virgínia Barreto Lordello Dissertação apresentada ao Programa de Pós- Graduação em Alimentos e Nutrição para obtenção do título de Mestre. Área de Concentração: Ciência dos Alimentos Orientadora: Prof. Dra. Daniela Cardoso Umbelino Cavallini Coorientadora: Prof. Dra. Carla Raquel Fontana Araraquara 2019 Desenvolvimento de filmes orodispersíveis potencialmente probióticos para promoção da saúde oral Virgínia Barreto Lordello Dissertação apresentada ao Programa de Pós- Graduação em Alimentos e Nutrição para obtenção do título de Mestre. Área de Concentração: Ciência dos Alimentos Orientadora: Prof. Dra. Daniela Cardoso Umbelino Cavallini Coorientadora: Prof. Dra. Carla Raquel Fontana Araraquara 2019 AGRADECIMENTOS: O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (Capes) – Código de Financiamento 001. À Profa. Dra. Daniela Cardoso Umbelino Cavallini, por todo conhecimento compartilhado, pelas horas dedicadas a mim e ao meu trabalho, por toda paciência, confiança e carinho. À Prof. Dra. Carla Raquel Fontana por ter aceitado ser minha coorientadora, por todos os ensinamentos e por sua disponibilidade em me aceitar em seu laboratório. Ao Prof. Dr. Marlus Chorilli por ter aberto as portas do laboratório de farmacotécnica e pelos seus direcionamentos. À Andreia e aos alunos Amanda, Bruno e Franciele pela colaboração com a realização dos experimentos. Ao Prof. Marcelo Orlandi e ao técnico Diego Tita do laboratório de microscopia MEV- FEG do IQ-UNESP. Ao Departamento de Alimentos e Nutrição da FCFAr-UNESP e aos docentes, pela oportunidade, pelo aprendizado e experiência concedidos durante o curso de mestrado. Às empresas, Gelita, Labonathus, Biovital, Firmenich, e TIC GUMS, que colaboraram com o fornecimento de matérias-primas para a realização da pesquisa. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pela concessão de bolsa do mestrado. Aos membros da banca examinadora pelas correções e contribuição com esse trabalho. Aos funcionários da Seção de Pós-Graduação e da Biblioteca da FCFAr-UNESP. A todos que, de alguma forma, contribuíram para a realização deste trabalho. AGRADECIMENTOS PESSOAIS: Agradeço primeiramente a Deus pela vida e por Sua providência sempre fiel. À minha família por ter superado comigo todos os obstáculos que apareceram em nosso caminho ao longo dos últimos dois anos e por todo suporte para que eu me dedicasse à pesquisa. Aos técnicos do laboratório Adriano, Roseli e Josiane, por todos os cafezinhos que tomamos juntos nesses dois anos, por todo auxílio e ensinamentos. Aos colegas de laboratório: Izabela, Juliana, Giovana, Eliane, Lucas, Laís, Malu e William pelo convívio e troca de experiências. Em especial, gostaria de agradecer à Laura e à Sarah pelo encorajamento em momentos de desânimo, auxílio no planejamento de experimentos e esclarecimento de dúvidas. À Natália Cordano pela amizade que construímos graças ao mestrado e pelo suporte em tantos momentos, seja de perto ou de longe. A todos que foram ombro amigo durante esses dois anos. Dedico essa dissertação de mestrado à minha avó, Maria Apparecida Rossi Barreto, que é um grande exemplo para mim e sempre me motivou a ser uma boa profissional. "Pode-se tirar tudo de um homem exceto uma coisa: a última das liberdades humanas – escolher a própria atitude em qualquer circunstância, escolher o próprio caminho." Viktor Frankl RESUMO Os microrganismos probióticos associados a produtos alimentícios já são amplamente utilizados para a manutenção da saúde e redução do risco de certas doenças. Nesse contexto, diversos estudos mostram que cepas probióticas específicas têm grande potencial de manutenção e melhora da saúde oral, apresentando atividade anticariogênica, redução da halitose e prevenção de infecções oportunistas, como a candidose, frequentemente presente em indivíduos imunocomprometidos. Essas condições patogênicas apresentadas estão relacionadas à formação e estabelecimento de biofilmes na cavidade oral. As infecções provenientes de biofilmes são duradouras, altamente resistentes à farmacoterapia e de difícil eliminação. Dessa forma, o objetivo do presente estudo foi obter filmes orodispersíveis (ODF) contendo cepa probiótica Enterococcus faecium CRL 183, avaliando sua capacidade de inibir a formação e/ou maturação de biofilme de Candida albicans in vitro. Essa cepa foi selecionada devido as suas características de cultivo, informações disponíveis sobre efeito probiótico, segurança e caráter inovador, pois não há na literatura informações sobre a atividade antifúngica dessa cepa na cavidade oral. A segurança da cepa probiótica foi avaliada através do teste susceptibilidade a antimicrobianos. Para testar a capacidade de inibição de biofilmes de C. albicans in vitro foram formados biofilmes polimicrobianos (C. albicans + E. faecium), em diferentes proporções de fungo: probiótico (1:1; 1:10; 1:100), a serem avaliados durante os períodos de formação (24 horas) e maturação (48 horas) do biofilme. A cepa probiótica foi incorporada em um filme orodispersível (ODF) para avaliar sua resistência durante o processamento e armazenamento e efeito inibitório contra os biofilmes de C. albicans. Os resultados evidenciaram que o microrganismo E. faecium CRL 183 foi capaz de se estabelecer em um biofilme polimicrobiano com C. albicans ATCC 90028 sem perder sua viabilidade celular, ao passo que antagonizou significativamente a formação (até 99,9% de redução em 24 horas) e maturação (até 99% de redução em 48 horas) do biofilme de C. albicans. Os filmes orodispersíveis com e sem probióticos (PF e CF, respectivamente) apresentaram aspecto e textura uniformes, pH neutro, baixa atividade de água e não apresentaram contaminação por bolores, leveduras ou coliformes totais durante o armazenamento. O probiótico resistiu à produção do ODF, apresentando viabilidade elevada (> 9 log10 UFC/g), até 90 dias de armazenamento à temperatura ambiente. A incorporação do probiótico na matriz polimérica do ODF não alterou significativamente as propriedades mecânicas, o tempo de desintegração in vitro, o intumescimento, a atividade de água e a mucoadesividade em relação a CF. Entretanto o probiótico aumentou o parâmetro de cor amarelo (+b*), o teor residual de umidade, a espessura e a permeabilidade ao vapor d’água dos filmes. O filme probiótico (PF) foi capaz de reduzir em 68,99% (0,52 log) a formação e em 91,24% (1,15 log) a maturação do biofilme de C. albicans em comparação aos biofilmes estimulados com o filme orodispersível controle (CF). Dessa forma, conclui-se que os filmes orodispersíveis são uma forma eficiente de veicular o probiótico E. faecium CRL 183, com potencial de prevenir o estabelecimento de infecções causadas por C. albicans na cavidade oral. Palavras-chave: Probióticos, atividade anti-Candida, Biofilmes, Filmes orodispersíveis, Candida albicans ATCC 90028, Enterococcus faecium CRL 183. ABSTRACT Probiotic microorganisms associated with food are already widely used to maintain health and reduce risk of certain diseases. In this context, several studies show that certain probiotic strains have great potential for maintenance and improvement of oral health, presenting anticariogenic activity, reduction of halitosis and prevention of opportunistic infections, such as candidiasis, often present in immunocompromised individuals. These pathogenic conditions are related to formation and establishment of biofilms in the oral cavity. Infections caused by biofilms are long-lasting, highly resistant to pharmacotherapy and difficult to eliminate. Thus, the objective of the present study is to obtain orodispersible films (ODF) containing Enterococcus faecium CRL 183, evaluating its capacity of inhibiting the formation and / or maturation of Candida albicans biofilms in vitro. This strain was selected due to its cultivation characteristics, available information on probiotic effect and safety and innovative character, as there is no information on the antifungal activity of this strain in the oral cavity. The safety of the probiotic strain was evaluated by antimicrobial susceptibility test. In order to test the ability to inhibit the formation of C. albicans biofilms in vitro, combined polymicrobial biofilms (C. albicans + E. faecium) with different proportions of opportunistic pathogens: probiotic were formed (1: 1, 1:10, 1: 100), during the formation (24 hours) and maturation (48 hours) periods of the biofilm. The probiotic strain was incorporated into an orodispersible film (ODF) to evaluate its resistance during processing and storage and inhibitory effect against C. albicans biofilms. The results showed that E. faecium CRL 183 microorganism was able to establish itself in C. albicans ATCC 90028 polymicrobial biofilm without losing its cellular viability. It significantly antagonized the formation (up to 99.9% reduction in 24 hours) and maturation (up to 99% reduction in 48 hours) of C. albicans biofilm. The orodispersible films with and without probiotics (PF and CF, respectively) presented uniform appearance and texture, neutral pH, low water activity and did not show contamination by molds, yeasts or total coliforms during storage. The probiotic resisted to the ODF production, presenting high viability (> 9 log10 CFU / g), up to 90 days of storage at room temperature. The incorporation of the probiotic in the polymer matrix of ODF did not significantly alter the mechanical properties, in vitro disintegration time, swelling, water activity and mucoadhesiveness in relation to CF. However, the probiotic increased the yellow color parameter (+ b *), the residual moisture content, the thickness and the water vapor permeability of the films. The probiotic film (PF) was able to reduce the formation of C. albicans biofilm in 68.99% (0.52 log) formation and in 91.24% (1.15 log) the biofilm maturation compared to the biofilms stimulated with orodispersible control film (CF). Thus, it is concluded that orodispersible films are an efficient way of transporting the probiotic E. faecium CRL 183, with potential to prevent the establishment of infections caused by C. albicans in the oral cavity. Keywords: Probiotics, anti-Candida activity, Biofilms, Orodispersible films, Candida albicans ATCC 90028, Enterococcus faecium CRL 183. SUMÁRIO INTRODUÇÃO ............................................................................................................ 5 Objetivo Geral .......................................................................................................... 15 Capítulo 1. Probiotic Enterococcus faecium CRL 183 inhibits Candida albicans in vitro. ..................................................................................................................... 16 Capítulo 2. Development of orodispersible films loaded with probiotic E. faecium CRL 183 for oral candidiasis prevention................................................. 39 CONSIDERAÇÕES FINAIS ...................................................................................... 68 REFERÊNCIAS BIBLIOGRÁFICAS ......................................................................... 69 5 INTRODUÇÃO Probióticos são definidos como microrganismos vivos que, quando administrados em quantidades adequadas, conferem benefícios à saúde do hospedeiro (1,2). Tais microrganismos devem ser preferencialmente de origem humana, dentre os quais destacam-se Bifidobacterium spp, as bactérias produtoras de ácido lático como os gêneros Lactobacillus spp. e, em menor escala, Enterococcus spp. (2–4). Também são representantes dessa classe leveduras como Saccharomyces spp. (3,4). Probióticos devem ainda apresentar resistência às condições adversas do trato gastrintestinal (especialmente se os efeitos esperados forem sistêmicos), modular positivamente os sistemas fisiológicos do paciente, ser inócuos e não devem transmitir genes de resistência antimicrobiana (2,3). A Agência Nacional de Vigilância Sanitária (ANVISA) recentemente revisou os requisitos para comprovação da segurança e dos benefícios à saúde dos probióticos para uso em alimentos (Resolução da Diretoria Colegiada - RDC Nº 241, de 26 de julho de 2018), estabelecendo que a identificação da linhagem do microrganismo deve ser inequívoca contemplando a nomenclatura binomial, identificação fenotípica e genotípica, origem e depósito em coleção de cultura internacionalmente conhecida (5). Além disso, a comprovação da segurança do probiótico deve ser demonstrada através de estudos científicos e documentos técnicos que assegurem o histórico de uso seguro, ausência de registros de eventos adversos, ausência de fatores de virulência e patogenicidade, ausência de resistência potencialmente transferível a antibióticos relevantes para a saúde humana; e susceptibilidade a, pelo menos, dois antibióticos (5). A resolução ainda determina que os benefícios à saúde devem ser comprovados por meio de estudos que envolvam um grupo representativo da população, considerando a quantidade mínima de cepa probiótica sugerida para obtenção desse benefício (5). Está bem estabelecido na literatura que probióticos têm um impacto positivo no funcionamento do trato gastrintestinal e auxiliam no tratamento ou redução de risco de diversas doenças como: diarreia decorrente do uso de antibióticos, síndromes metabólicas, vaginites, doenças respiratórias, hipercolesterolemia e 6 doenças inflamatórias intestinais (6,7). Em relação à saúde oral, estudos têm evidenciado que o consumo de certas cepas de microrganismos probióticos pode diminuir a halitose, a incidência de cáries em crianças e de doença periodontal, incluindo sangramento causado por gengivite, além de combater infecções orais por Candida spp. (8,9). A maioria das doenças da cavidade oral inicia-se com a formação dos biofilmes microbianos que revestem os dentes, popularmente conhecidos como placa dentária. Os microrganismos presentes nesses biofilmes metabolizam carboidratos da dieta e podem promover a desmineralização e destruição localizada de tecidos dentários, inicialmente na superfície do esmalte, mas podendo atingir a dentina no caso de cáries mais profundas e até comprometer a polpa dental (9,10). Para que doenças orais ocorram, a interação de três fatores é indispensável: o hospedeiro (dentes, saliva e sistema imune), a constituição da microbiota oral residente e a dieta (tipo e frequência) (10). Sabe-se que biofilmes são ecossistemas microbianos complexos caracterizados por células ligadas entre si e a um substrato, ou à superfícies bióticas e abióticas envoltas por uma matriz de polímero extracelular (Exopolissacarídeos - EPS) (11). O EPS confere aos microrganismos proteção contra o sistema imune e a agentes antimicrobianos, promove o sequestro de nutrientes do meio, facilita a dispersão para outras regiões não infectadas, entre outras vantagens (12–15). Na ausência de higiene oral um biofilme é capaz de estar completamente formado em um período de apenas quatro horas após as refeições e, mesmo com a prática da escovação dental diária e o uso do fio dental, não ocorre a eliminação completa desses biofilmes que, além de estarem envolvidos na gênese da cárie dentária, podem também atuar na periodontite e candidíase oral (8) Diferentes microrganismos estão envolvidos no desenvolvimento de doenças que afetam a saúde bucal, entre eles merecem destaque: Streptococcus mutans, Candida albicans e Lactobacillus spp. (16–20). Diversas cepas probióticas apresentam capacidade de se estabelecer em biofilmes e de reduzir a população de patógenos altamente prevalentes na cavidade oral como Actinomyces viscosus, Staphylococcus aureus, Candida albicans e Streptococcus mutans. (4,7). Apesar de algumas espécies de Lactobacillus spp. estarem presentes em lesões de cárie (17), o consumo de produtos contendo cepas 7 específicas, como Lactobacillus rhamnosus GG, foi relacionado à diminuição na incidência de cáries, bem como, redução na população de Streptococcus mutans em crianças, comparado a um grupo controle que não ingeriu o produto contendo essa cepa probiótica (21). Samot e Badet (2013) investigaram a ação probiótica de 66 espécies de lactobacilos, e observaram que todas essas espécies foram capazes de inibir o crescimento de S. mutans e A. viscosus, e a maioria desses lactobacilos foram capazes de antagonizar Fusobacterium nucleatum e Porphyromonas gingivalis, duas cepas presentes em doenças periodontais (22). Além do gênero Lactobacillus spp., as cepas probióticas Enterococcus faecium WB2000, Bifidobacterium adolescentes SPM1005 e Bifidobacterium BB12 foram efetivas na inibição da formação e crescimento de biofilmes (23). Os mecanismos pelos quais os microrganismos probióticos atuam na melhora ou manutenção da saúde oral incluem redução de microrganismos patogênicos formadores de biofilmes, em função da produção de substâncias com propriedade antimicrobiana (peróxido de hidrogênio e bacteriocinas), proteases, e por competição direta pelos nutrientes disponíveis e sítios de adesão na cavidade oral (3). Pujia e colaboradores (2017) sugeriram que os probióticos também afetam os patógenos por serem capazes de modular as ações locais e sistêmicas do sistema imune, neutralizar fatores de virulência e interferir nos sistemas de sinalização bacteriana (quórum sensing) (9). O fungo oportunista da pele e mucosa humana, Candida albicans, está presente na cavidade oral de mais de 75% da população (19). Altamente associado a infecções hospitalares provenientes de sondas e cateteres, é o quarto microrganismo mais isolado da corrente sanguínea nesses casos (24). Devido a mudanças ambientais, alterações na microbiota e competência do sistema imunológico do hospedeiro, a espécie C. albicans pode se tornar patogênica, especialmente em pacientes em quimioterapia, transplantados, soropositivos, idosos, pacientes com debilidades motoras, usuários de prótese totais ou parciais e recém-nascidos de baixo peso (19, 25–27). As principais doenças causadas por C. albicans em seres humanos são as infecções superficiais, como a candidíase oral (muito prevalente em imuno- comprometidos) ou vaginal, que acomete 75% das mulheres ao menos uma vez na vida (19). Os pacientes imunossuprimidos correm o risco dessas infecções 8 superficiais tornarem-se invasivas, podendo atingir a corrente sanguínea (candidemia) ou órgãos vitais como rins, fígado e coração (27). C. albicans é considerada a espécie mais prevalente e virulenta do gênero, pois entre os seus principais fatores de virulência, pode-se destacar o polimorfismo entre leveduras e hifas, que é um mecanismo de evasão do sistema imune inato por dificultar a fagocitose, bem como a formação de biofilmes e produção de toxinas e enzimas hidrolíticas (25, 28, 29). Como a maioria das infecções causadas por biofilmes, as infecções por C. albicans são duradouras, de difícil eliminação e com elevada resistência à farmacoterapia. Os biofilmes de C. albicans geralmente apresentam resistência aos principais antifúngicos existentes como: anfotericina B, fluconazol, voriconazol e nistatina (15). Tanto os fungos quanto os humanos são eucariontes e, no nível molecular, suas células são semelhantes. Isso torna mais difícil encontrar ou projetar drogas que atinjam fungos sem afetar as células humanas (30). Dessa forma, é urgente a busca por alternativas para o tratamento e prevenção de doenças, em especial fúngicas, causadas por biofilmes. Como uma dessas alternativas, a literatura demonstra que muitas cepas potencialmente ou comprovadamente probióticas apresentam atividade anti- Candida. Em uma revisão de literatura conduzida em 2016 demonstrou-se através de estudos in vivo e in vitro, que cepas probióticas como Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Saccharomyces boulardii, Streptococcus thermophilus, Streptococcus salivarius, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus plantarum, bem como vários isolados clínicos, teriam atividades inibitórias contra Candida spp. (4). Um estudo duplo-cego randomizado avaliou o efeito de uma mistura de probióticos - Lactobacillus rhamnosus HS111, Lactobacillus acidophillus HS101 e Bifidobacterium bifidum – na forma de cápsulas liofilizadas, no desenvolvimento de candidíase oral assintomática em pacientes com prótese total (dentadura). Após cinco semanas de tratamento, a taxa de detecção de Candida spp. foi de 92% no grupo placebo e 16,7% no grupo tratado com probióticos, evidenciando a sua capacidade de reduzir a colonização da cavidade oral por esse microrganismo (31). 9 Matsubara e colaboradores (2016) observaram que suspensões de L. rhamnosus, L. casei e L. acidophilus reduziram significativamente a formação de biofilmes de C. albicans SC5314 em 32, 59 e 56,3%, respectivamente. Quando biofilmes maduros (48 horas) foram estimulados com as mesmas suspensões probióticas, a redução dos biofilmes foi de 61,8% (L. rhamnosus), 54,7% (L. casei) e de 34,2% (L. acidophilus) (27). Ao estimular os biofilmes de C. albicans com o sobrenadante filtrado dessas culturas de lactobacilos, a redução foi menos efetiva na formação, e não houve redução na maturação dos biofilmes. Esse resultado demonstra que a ação inibitória dessas cepas sobre C. albicans não é mediada unicamente pela produção de metabólitos (27). Mailänder-Sánchez e colaboradores (2017) observaram que a cepa probiótica Lactobacillus rhamnosus GG impediu a adesão, invasão, formação de hifas (prevenindo danos epiteliais), depleção de glicose e da síntese de ergosterol de C. albicans (32). Vilela e colaboradores (2015) relataram redução de até 57,5% da contagem de C. albicans em biofilmes estimulados com culturas de L. acidophilus ATCC 4356 e redução de 45,10% em biofilmes estimulados com sobrenadante filtrados dessa cultura probiótica (33). Ambos os estímulos (suspensão de células probióticas ou filtrado) foram eficientes na supressão da formação de hifas. Nesse estudo, Galleria mellonella foi utilizada como modelo de candidíase experimental e o tratamento e a profilaxia com células de L. acidophilus ou com seus metabólitos aumentou consideravelmente a sobrevivência desse inseto (33). Ribeiro e colaboradores (2017) observaram efeito semelhante na interação de C. albicans com células de L. rhamnosus: diminuição de 52,2 % na quantificação de biofilme em UFC/ mL e redução de 48% na atividade metabólica. O sobrenadante filtrado de L. rhamnosus diminuiu o crescimento de C. albicans em 39,4% e atividade metabólica em 61% (34). L. rhamnosus também promoveu a regulação negativa de diversos genes associados a fatores de virulência de C. albicans como ALS3 e HWP1 (relacionados à adesão e filamentação) e de BCR1 e CPH1 (genes reguladores do processo de transcrição celular) (34). L. rhamnosus foi capaz de proteger G. mellonella de candidíase experimental (34). 10 Sabe-se também que não apenas os microrganismos probióticos podem competir com C. albicans influenciando sua viabilidade e patogenicidade. Há na literatura diversos estudos que avaliam a interação entre esse fungo e bactérias patogênicas como Staphylococcus aureus, Streptococcus mutans, Streptococcus gordonii, Actinomyces viscosus, espécies de Fusobacterium spp., Enterococcus spp., Escherichia spp. e até mesmo Pseudomonas aeroginosa em casos de fibrose cística (29, 35). Dentre esses microrganismos, a relação entre os gêneros Enterococcus spp. e Candida spp. mostrou-se particularmente interessante, pois a literatura mostra que o fungo favorece a bactéria, porém essa relação não é mutuamente benéfica. Dois estudos conduzidos por Mason e colaboradores (2012) demonstraram que C. albicans SC5314 promoveu aumento e persistência da colonização gástrica e entérica de Enterococcus spp. ao passo que antagonizou outras espécies de bactérias láticas como, por exemplo, Lactobacillus spp., durante a recuperação da microbiota após tratamento com antibiótico cefoperazona (36,37). Apesar do mecanismo envolvido não ter sido elucidado, os autores observaram efeito promotor semelhante em relação a populações de Bacteroidetes e sugerem que C. albicans poderia auxiliar a sua adesão e fixação ao ceco; que glucanos da levedura poderiam ser fonte potencial de alimento para essas bactérias, ou ainda, C. albicans poderia suprimir outras bactérias que antagonizam Bacteroidetes (37). Por outro lado, Enterococcus spp. diminui a patogenicidade de C. albicans através da ação de proteases, peptídeos e bacteriocinas. Pesquisadores identificaram a ação de duas proteases (peptídeos de ativação da biossíntese de gelatinase - GBAP) ativadoras do sistema regulador da virulência de Enterococcus spp., que ao serem secretadas por essa bactéria, impediram o polimorfismo de C. albicans (38). O polimorfismo é um dos principais fatores de virulência associados a esse fungo e a formação de hifas está intrinsecamente ligada à formação de biofilmes e à invasão das células hospedeiras (19,25,38). Cruz e colaboradores (2013) demonstraram ainda que Caenorhabditis elegans, modelo de estudo de infecções polimicrobianas, apresentou uma mortalidade ocasionada por C. albicans menor quando era co-inoculado por 11 Enterococcus spp. Sendo a taxa de sobrevivência do nematódeo dose-dependente, quanto maior o número de colônias de Enterococcus spp. inoculadas, maior a proteção contra a patogenicidade de C. albicans (38). Este estudo determinou ainda que a patogenicidade de ambos os microrganismos era atenuada durante a infecção polimicrobiana em C. elegans. O mesmo efeito foi observado ao tratar C. elegans, já contaminado por C. albicans, com o sobrenadante filtrado e esterilizado de um inóculo de Enterococcus faecalis, constatando-se redução considerável da formação de hifas e, consequentemente, da patogênese do fungo (38). Shekh e Roy (2012) caracterizaram e purificaram parcialmente uma proteína anti-candida (ACP) produzida por Enterococcus faecalis que apresentou atividade de amplo espectro contra oito cepas de C. albicans multirresistentes (30). Em um estudo de 2017 foi descrita a ação da bacteriocina EntV, codificada por um gene que está presente em todas as cepas de E. faecalis sequenciadas até o momento, o gene Ef1097 (39). Essa enterocina sozinha, bem como seu análogo sintético, foi suficiente para impedir o polimorfismo de C. albicans SC5314 e a formação de biofilmes em diferentes substratos e condições experimentais. Biofilmes de C. albicans pré-formados (24 horas/ ∼30 μm de espessura) foram tratados com EntV e sofreram grande perturbação, com redução de sua espessura para ∼15 μm. Os biofilmes que não foram tratados com EntV aumentaram sua espessura para > 50 μm (39). O peptídeo também protegeu macrófagos aumentando sua atividade antifúngica, reduziu a invasão epitelial, a inflamação e a carga fúngica em um modelo murino de candidíase orofaríngea (39). Bachtiar e colaboradores (2016) observaram que E. faecalis cps2 (um isolado clínico não encapsulado) e E. faecalis ATCC 29212, bem como os metabólitos produzidos por essas cepas, não inibiram a formação de biofilmes de C. albicans ATCC 10231 e promoveram a expressão de genes associados à formação de biofilme. No entanto, E. faecalis cps2 e E. faecalis ATCC 29212 reduziram significativamente (>50%) a maturação desses biofilmes (40). Esses resultados foram confirmados pela regulação negativa promovida pelas cepas de E. faecalis e seus metabólitos sobre genes de adesão ALS1 e ALS3 e sobre EFB1, gene utilizado para medir quantitativamente os efeitos prejudiciais de agentes antifúngicos contra biofilmes de candida maduros (40). 12 É importante salientar que em nenhum dos estudos citados (36-40) que avaliaram o potencial anti-Candida de Enterococcus spp., a cepa bacteriana utilizada foi descrita como sendo inócua, muito menos tendo alegação probiótica. Sabe-se, também que existe muita preocupação em relação a Enterococcus spp. devido à sua alta incidência em infeções nosocomiais, e alta capacidade de tornar-se resistente a antibióticos e transferir genes de resistência, em especial à vancomicina, para outros patógenos (41). Provavelmente por essa razão, nesses estudos foram explorados majoritariamente a ação de metabólitos, peptídeos e bacteriocinas ao passo que a interação célula – célula não foi considerada viável. Como visto anteriormente, a ação probiótica também pode ser mediada pela secreção de metabólitos, porém a competição célula-célula por sítios de adesão e nutrientes pode ser um potencializador do efeito anti-Candida (9,27,31,33,34). Dessa forma, seria interessante avaliar se o efeito dos metabólitos produzidos Enterococcus spp. seria também potencializado por essa competição direta, uma vez que algumas cepas desse gênero são consideradas seguras e probióticas (2, 41). A cepa probiótica Enterococcus faecium CRL 183 foi isolada do queijo Tafi, tradicional no Vale do Tafí, noroeste da Argentina e tem documentada a sua nomenclatura binomial, identificação genotípica, histórico de uso seguro, ausência de registros de eventos adversos, ausência de fatores de virulência, patogenicidade e resistência antimicrobiana (42). Vários benefícios sistêmicos promovidos por essa cepa probiótica estão documentados, como modulação do sistema imunológico e da microbiota intestinal, redução dos sintomas relacionados à colite ulcerativa (43), regulação do perfil lipídico e inibição do desenvolvimento da lesão aterosclerótica (44-45) sendo uma alternativa ou adjuvante para terapia medicamentosa. E. faecium CRL183 também preveniu o adenocarcinoma de mama (46), câncer de cólon quimicamente induzido (47) e ganho de peso corporal (48) em diferentes modelos murinos. É importante ressaltar que, em alguns desses estudos, o E. faecium CRL183 foi utilizado como cultura inicial de um produto à base de soja fermentado, combinado ou não com outras cepas probióticas, avaliando seus efeitos sistêmicos (43,45,47,48). Objetivando ampliar a utilização da referida cepa probiótica, Witzler e colaboradores (2017) demonstraram que a cepa probiótica E. faecium CRL 183 13 liofilizada após microencapsulação pelo método de coacervação complexa, manteve-se viável à temperatura ambiente por um período de 280 dias e foi capaz de inibir o crescimento de S. mutans, constatado pela formação de um halo de inibição 1,7 ± 0,3 mm de diâmetro (43). Esse mesmo estudo evidenciou que E. faecium CRL183 foi resistente à saliva humana. Sendo capaz de se liberar da matriz alimentícia, sobreviver às enzimas salivares e se multiplicar (aumento de até 3,64 log10 UFC/mL) na saliva após 24h, indicando uma possível capacidade de colonização da cavidade oral por esse probiótico (49). A cepa probiótica encapsulada e liofilizada foi posteriormente incorporada em uma pastilha alimentícia diet com potencial funcional. Entretanto, a viabilidade da cepa durante 28 dias de armazenamento à temperatura ambiente foi reduzida a valores considerados baixos para produtos probióticos (1,86±0,07 UFC/g) (49). Outros estudos veicularam cepas probióticas como L. reuteri ou L. brevis em gomas de mascar e pastilhas de manitol obtendo efeitos benéficos como redução significativa de placa, população de S. mutans e de sangramentos gengivais (50, 51). Porém esses estudos não avaliaram aspectos tecnológicos da formulação como a sobrevivência do probiótico ao longo do tempo de armazenamento. Tais resultados apontam para a necessidade do aprimoramento do processo de obtenção de formulações probióticas orais, que permitam a liberação controlada de doses conhecidas de organismos viáveis, resistam ao processo de fabricação, mantenham a estabilidade durante o armazenamento a longo prazo, de preferência sem necessidade de refrigeração, e cujos custos de produção sejam economicamente favoráveis (52). Uma opção de veículo para microrganismos probióticos são os filmes orodispersíveis (ODF), que foram desenvolvidos na década de 1970, para populações com dificuldades de deglutição, ou disfagia, tornando-se uma alternativa a comprimidos, cápsulas e xaropes (53). Porém, a primeira forma comercializada deste produto surgiu apenas em 2001, como refrescante bucal “Listerine” da Pfizer (53). Esta forma farmacêutica pode ser definida como uma fina película de fácil dissolução e elevada superfície de contato, que permite a liberação de todo componente ativo na cavidade oral. Dentre as vantagens atribuídas ao ODF pode-se citar: facilidade de manuseio e administração, não há necessidade de ingestão de água, evita o metabolismo de primeira 14 passagem e maior adesão do paciente ao tratamento (54). Tanto a nomenclatura oficial, quanto a popular tem ampla variação. O FDA utiliza o termo “filmes solúveis”, enquanto que a agência europeia de medicamentos (EMA) adotou “filmes orodispersíveis”. Popularmente, esses filmes são conhecidos como “strip oral”, “filmes orais”, “tira oral”, “filme solúvel oral”, “dissofilme”, “filme solúvel bucal”, “filme mucoadesivo” e “filme transmucoso”, porém, apesar de serem usados de forma intercambiável, podem designar diferentes produtos (53). A permanência do filme solúvel na cavidade oral, durante o tempo de desintegração, pode promover uma liberação mais eficiente de princípios ativos se comparado a outros produtos como géis orais que são facilmente retirados pelo fluxo salivar (55). Os filmes orodispersíveis são compostos por macromoléculas, naturais ou sintéticas, formadoras de matriz polimérica, plastificantes - que aumentam as propriedades filmogênicas dos polímeros conferindo maior flexibilidade e menor fragilidade – e o ingrediente farmacêutico ativo (API) ou composto bioativo de interesse (53,54). A composição e proporção desses excipientes pode ser alterada de acordo com as características desejadas para o produto e a adição de edulcorantes, estimulante salivar, aromatizantes, flavorizantes, pigmentos, tensoativos e estabilizantes pode ser considerada (53). De acordo com as características dos filmes, estes constituem veículos de substâncias bioativas ideais para pacientes com condições que favoreçam a disfagia como idosos, pacientes com debilidades motoras, imunocomprometidos, portadores de Alzheimer ou Parkinson. Coincidentemente, nesta população se observa alta prevalência de candidose (19, 25-27,53-55). Considerando o exposto, o objetivo do presente estudo é avaliar se a cepa probiótica E. faecium CRL 183 é capaz de desestabilizar a formação e/ou maturação de biofilme de C. albicans ATCC 90028 in vitro. E se os filmes orodispersíveis seriam uma forma eficiente de veicular essa cepa na cavidade oral, promovendo a redução de risco de doenças orais ocasionadas por este fungo oportunista. 15 Objetivo Geral Desenvolvimento de filmes orodispersíveis contendo Enterococcus faecium CRL 183, capaz de inibir a formação e/ou maturação de biofilme de Candida albicans in vitro. Objetivos específicos - Avaliar se a cepa probiótica Enterococcus faecium CRL 183 é capaz de se estabelecer e sobreviver em biofilmes polimicrobianos com Candida albicans ATCC 90028. - Avaliar se essa cepa probiótica é capaz de desestabilizar a formação e/ou maturação de biofilmes de Candida albicans in vitro. -Incorporar o microrganismo probiótico em filmes orodispersíveis e avaliar a viabilidade da cepa, bem como a estabilidade físico-química do produto durante o período de armazenamento. - Avaliar se o filme orodispersível probiótico é capaz de desestabilizar a formação e/ou maturação de biofilmes de Candida albicans in vitro. 16 Capítulo 1. Probiotic Enterococcus faecium CRL 183 inhibits Candida albicans in vitro. (Artigo submetido para a revista Applied Microbiology and Biotechnology) 17 Title: Probiotic Enterococcus faecium CRL 183 inhibits Candida albicans in vitro. Authors: Virgínia Barreto Lordello1, (virginialordello@gmail.com |+5516992013891) Sarah Raquel de Annunzio1, Maria Pía Taranto2, Carla Raquel Fontana1, Daniela Cardoso Umbelino Cavallini1,* (d.cavallini@unesp.br | +551633014691) 1 Sao Paulo State University (UNESP), School of Pharmaceutical Sciences, Rodovia Araraquara Jaú, Km 01 – s/n, Campus Ville, 14800-903 Araraquara, Brazil. 2 Reference Center for Lactobacilli (CERELA-CONICET), San Miguel de Tucumán, Chacabuco 145 - T4000ILC, Tucumán, Argentina. Abstract: Candida albicans is the most prevalent fungal microorganism of human microbiota presenting a commensal or pathogenic phenotype depending on the host’s immune defense capacity. Similarity between fungal and host cells promotes several adverse effects during antifungal pharmacotherapy and antimicrobial resistance increase is a major concern. Therefore, the search for alternative treatments and prevention strategies is urgent. In this context, probiotic bacteria seem to be a viable alternative with their benefits documented in the literature, through in vitro, in vivo and even clinical studies. The probiotic strain Enterococcus faecium CRL 183 has a well-documented safety use and history of benefits to the immune system, and activity against pathogens. Thus, the aim of this study was to evaluate if this probiotic strain was able to prevent C. albicans biofilm colonization in vitro. In order to test the anti-Candida activity of the probiotic strain E. faecium CRL 183 against Candida albicans ATCC 90028, combined polymicrobial biofilms (C. albicans + E. faecium) with different proportions of funghi: probiotic were formed (1:1, 1:10, 1: 100), during the formation (24 hours) and maturation (48 hours) periods of the biofilm. The results showed that E. faecium was able to establish itself with C. albicans in polymicrobial biofilms without losing its cellular viability. The probiotic strain significantly antagonized (p<0,0001) C. albicans biofilm formation (up to 99.9% reduction in 24 hours) and maturation (up to 99.43% reduction in 48 hours). According to these results E. faecium CRL183 may be a promising resource to prevent the formation of those biofilms. Key words: Probiotics, Biofilms, Anti-Candida activity, Polymicrobial biofilms, Enterococcus faecium CRL 183, Candida albicans ATCC 90028 Acknowledgements: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. V.B.L. was funded by a CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) master scholarship. mailto:virginialordello@gmail.com mailto:d.cavallini@unesp.br 18 Introduction Candida albicans is the most prevalent fungal species of human microbiota that innocuously colonizes the human skin and mucosa, as well as the gastrointestinal and reproductive tract (Nobile and Johnson 2015). It is also one of the few fungi capable of causing diseases in humans, which is commonly characterized as superficial infections such as oral or vaginal candidiasis, which affects 75% of women at least once in their lifetime (Mayer et al. 2013; Nobile and Johnson 2015). In certain groups such as: transplant patients, under chemotherapy, seropositive, elderly, patients with motor weakness, users of prostheses, catheters or pacemakers, and low birth weight infants, there is a greater risk that superficial infections may become invasive reaching the bloodstream (candidemia) or vital organs such as kidneys, liver and the heart (Han et al. 2013; Mattei et al. 2013; Mayer et al. 2013; Matsubara et al. 2016b). This can occur when C. albicans overgrows because an imbalance in the host microbiota, due to changes in local pH and nutritional availability, or impairment of the host immune defenses (Nobile and Johnson 2015). Highly associated with nosocomial infections, it is the fourth most isolated microorganism in the bloodstream with mortality rates up to 40% (Andes et al. 2004; Nobile and Johnson 2015). Among its main virulence factors, we can highlight the polymorphism, production of toxins and hydrolytic enzymes as well as the formation of biofilms (Niewerth and Korting 2001; Han et al. 2013; Zago et al. 2015). Like most infections caused by biofilms, C. albicans infections are long-lasting, difficult to eliminate and with high resistance to pharmacotherapy such as: amphotericin B, fluconazole, voriconazole and nystatin (Fanning and Mitchell 2012). Furthermore, both fungi and humans are eukaryotes and, at the molecular level, their cells are similar. This makes it more difficult to find or design drugs that target fungi without affecting human cells (Shekh and Roy 2012). In this way, the search for alternatives for the treatment and prevention of diseases, especially the ones caused by fungal biofilms is urgent. Among the current alternatives, researchers have been investigating the action of probiotics, which are defined as “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Hill et al. 2014). Such microorganisms should preferably be of human origin, among 19 which stand out the genera Lactobacillus, Bifidobacterium and, to a lesser extent, Enterococcus. Streptococcus, and Saccharomyces (Matsubara et al. 2016a) It is well established that a great variety of probiotic strains were able to impair C. albicans biofilm formation and maturation (Vilela et al. 2015; Matsubara et al. 2016b; Ribeiro et al. 2017), inhibit the fungal polymorphism, adhesion and invasion of host tissues (Ribeiro et al. 2017; Mailänder-Sánchez et al. 2017). Probiotics were also able to downregulate several genes associated with virulence factors of C. albicans such as ALS3, HWP1, BCR1 and CPH1 (Ribeiro et al. 2017). In vitro, in vivo and even clinical studies have demonstrated the ability of probiotics to prevent and treat mucosal candidiasis (Ishikawa et al. 2015; Matsubara et al. 2016a). But not only the interaction between probiotic-fungus has been investigated, there are several studies in the literature which evaluate the effect caused on Candida spp., by pathogenic bacteria such as: Staphylococcus aureus, Streptococcus mutans, Streptococcus gordonii, Actinomyces viscosus, species of Fusobacterium, Enterococcus, Escherichia and Pseudomonas aeroginosa in cases of cystic fibrosis (Nobile and Johnson 2015; Zago et al. 2015). Among these microorganisms, the interaction between the genera Enterococcus spp. and Candida spp. has been shown to be particularly interesting. Both are commonly co-isolated from the same niches, either as commensal members of the microbiota, colonizing the gut, or in polymicrobial infections losing in prevalence in those cases only to S. aureus (Garsin and Lorenz 2013). The literature also shows that the fungus favors this bacterium (Mason et al. 2012a; Mason et al. 2012b), whereas Enterococcus spp. significantly attenuates the fungus virulence in polymicrobial infections through the secretion of proteases, bacteriocins and lactic acid.(Shekh and Roy 2012; Garsin and Lorenz 2013; Cruz et al. 2013; Bachtiar et al. 2016; Graham et al. 2017). These findings show that several Enterococcus spp. metabolites where able to considerably reduce polymorphism and biofilm total biomass (Cruz et al. 2013; Bachtiar et al. 2016; Graham et al. 2017), increase the survival of Caenorhabditis elegans infected with Candida albicans (Cruz et al. 2013) and reduce epithelial invasion, inflammation, and fungal load in a murine model of oropharyngeal candidiasis through the increase of macrophages antifungal activity (Graham et al. 2017). 20 Another anti-Candida protein (ACP) characterized by Shekh and Roy (2012) showed a broad-spectrum activity against eight strains of multidrug resistant C. albicans. It is important to highlight that in none of those studies, the bacterial strain used against Candida was described as being innocuous, much less having probiotic claim (Shekh and Roy 2012; Garsin and Lorenz 2013; Cruz et al. 2013; Bachtiar et al. 2016; Graham et al. 2017). There is a lot of concern about Enterococcus spp. due to its high ability to become resistant to antibiotics and to transfer resistance genes, especially to vancomycin, to other pathogens (Hanchi et al. 2018). Probably because of that, the anti-Candida activity of Enterococcus spp. metabolites was evaluated whereas the cell - cell interaction was not considered suitable. However, the direct competition for adhesion sites and nutrients may be a potentiator of the anti-Candida effect promoted by bacteria metabolites (Vilela et al. 2015; Ishikawa et al. 2015; Matsubara et al. 2016b; Pujia et al. 2017; Ribeiro et al. 2017). Witzler et al (2017) shown that the probiotic strain E. faecium CRL 183, isolated from an Argentinian traditional cheese, was able to inhibit S. mutans growth verified by the formation of an inhibition halo of 1.7 ± 0.3 mm diameter. This same study pointed out that E. faecium CRL183 was resistant to human saliva enduring the salivary enzymes and being able to grow (up to 3.64 log10 CFU / mL) in this environment after 24 hours, indicating a possible ability of oral cavity colonization by this probiotic strain (Witzler et al. 2017). There is no documentation of adverse effects caused by E. faecium CRL 183, this strain has known genotypic identification, absence of virulence factors, pathogenicity and antimicrobial resistance(Saavedra et al. 2003). Nevertheless, several beneficial systemic effects promote by this probiotic strain are documented, i.e. modulation of the immune system and intestinal microbiota, reduction of symptoms related to ulcerative colitis (Celiberto et al. 2017), regulation of lipid profile and inhibition of atherosclerotic lesion development (Cavallini et al. 2009; Cavallini et al. 2011), being an alternative or adjuvant for drug therapy. E. faecium CRL183 also has prevented breast adenocarcinoma (Kinouchi et al. 2012), chemically induced colon cancer (Sivieri et al. 2008) and body weight gain (Marchesin et al. 2018) in different murine models. It is important to highlight that in some of those studies E. faecium CRL183 was used as a starter culture of a fermented soy-based product, 21 combined or not with other probiotic strains (Cavallini et al. 2011; Kinouchi et al. 2012; Celiberto et al. 2017; Marchesin et al. 2018). Therefore, the aim of this study was to evaluate if E. faecium CRL 183 would be able to prevent C. albicans biofilm formation and maturation through direct competition, once the antifungal probiotic strain has never been tested. Material and methods Material The probiotic strain E. faecium CRL 183 was obtained from Reference Center for Lactobacilli – CERELA/CONICET (San Miguel de Tucumán, Argentina) and C. albicans ATCC 90028 were kindly provided by the Laboratory of Clinical Microbiology of the School of Pharmaceutical Sciences, UNESP (Araraquara, Brazil). The microorganisms were kept frozen at -80ºC in a cryogenic tube containing proper culture media with addition of 20% glycerol, up to the time of its use. Methods Strains and growth conditions Prior to their use, the strains were thawed and subcultured in specific culture media: E. faecium in Bile Esculin Agar (Acumedia, Michigan, EUA) and C. albicans in Sabouraud Dextrose Agar supplemented with chloramphenicol (0.05 g L-1) (SDA - Acumedia, Michigan, EUA) and incubated at 37°C for 48h. Assurance of probiotic strain safety As stated before, E. faecium CRL 183 has its safety consumption attested through many in vivo and clinical studies. It does not show any antibiotic resistance genes nor virulence factors (Saavedra et al. 2003). The E. faecium probiotic strain was submitted to the antimicrobial susceptibility test by disc diffusion performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2019). A standard inoculum (turbidity equivalent to a standard solution of McFarland 0.5) was seeded with sterile swab in 140 x 15 mm Petri dishes (Corning, New York, USA) containing Mueller Hinton Agar (Difco, Detroit, USA). Disks containing antimicrobials: Ciprofloxacin 5 mg; Chloramphenicol 30 mg; Erythromycin 15 mg; Nitrofurantoin 300 22 mg; Norfloxacin 10 mg; Tetracycline 30 mg; and Vancomycin 30 mg (SENSIFAR - Cefar Diagnostica, São Paulo, Brazil), were placed on the inoculated agar with the aid of sterile forceps. The plates were incubated at 35 ± 2ºC, with incubation time of 16-18 h for most antimicrobials, except for vancomycin, which requires 24 hours of incubation. After this period, the presence and diameter of the growth inhibition halos caused by the antimicrobial discs were assessed. The results were interpreted according to the criteria recommended by the CLSI (2019). Biofilm formation The biofilm assays were carried out following the methods described by Fontana et al. (2009) and Zago et al., (2016), with modifications. The microorganisms freshly cultivated as described in growth conditions were inoculated with a sterile loop in 2mL of enriched broth containing: 26 g/L of brain–heart infusion (BHI- Kasvi, Curitiba, Brazil), 10 g/L of yeast extract (YE- Acumedia, Michigan, USA), 20 g/L of trypticase soy broth (TSB - Acumedia, Michigan, USA) and supplemented with 20% sucrose (Synth, Diadema, Brazil) to obtain yeast and probiotic bacteria inoculum. The suspensions obtained were standardized by spectrophotometry reading on a Synergy H1M microplate reader (Biotek, Winooski, USA). The two standardized microbial suspensions were then diluted to obtain different inoculum of yeast (106 or 108 CFU/mL) and probiotic (107 or 108 CFU/mL). Monospecies biofilms assays were conducted on 96-well microplates with each well containing 75 μL of yeast or probiotic suspension plus 75 μL of enriched broth, i.e. 150 μl per well. Polymicrobial biofilm assay were also performed in 96-well microplates with each well containing the suspension of each microorganism in the following proportions: 75 μL C. albicans 108 CFU/mL+ 75 μL E. faecium 108 CFU/mL (1:1), 75 μL C. albicans 106 CFU/mL 75+ μL E. faecium 107 CFU/mL (1:10) and 75 μL C. albicans 106 CFU/mL 75+ μL E. faecium 108 CFU/mL (1:100). The microplates were incubated at 35 ± 2ºC for 24h to evaluate the interaction of microorganisms in the biofilm adhesion and formation and for 48h to evaluate it in the biofilm maturation. In the case of the 48 hours biofilms, they were split in two groups (48A and 48B) to evaluate if the metabolites produced by E. faecium within the first 24 hours play or not a role in the destabilization of C. albicans biofilms. In the group 48A after 23 the first 24 hours, the supernatant from each well was carefully replaced with 150 μL of fresh enriched broth and the plates returned to the incubator. Therefore, the metabolites produced were removed and new nutrients were added to the microenvironment. In the group 48B no intervention has been made during the 48 hours of incubation. Biofilm pH evaluation After the formation (24h) and maturation (48h) times of the biofilms, all the supernatant from the wells were aspirated carefully to avoid removal of the adhered biofilms and the pH of those supernatants were measured with pH-fix (Neumann- Neander, Düren, Germany). After this procedure the supernatant was discarded. Biofilm quantification of viable cells The adhered biofilms were scraped off the wells and resuspended vigorously in 150 μL of enriched broth with a sterile pipette tip for 30s. Serial decimal dilutions of those suspensions were made using as diluent the culture medium itself. Monospecies biofilms were quantified by plating the obtained dilutions in SDA supplemented with chloramphenicol (for C. albicans) and Bile Esculin Agar (for E. faecium). The polymicrobial biofilms were plated onto both culture media. The plates were incubated for 24h at 35 ± 2ºC and the number of fungal and probiotic cells present in the biofilms was determined by counting colony-forming units (CFUs). The experiments were performed in four replicates and repeated in three independent assays. Determination of the anti-Candida activity of E. faecium CRL 183. The decimal reduction (DR) of Candida albicans cell viability in the presence of probiotic strain was determined by equation 1: DR = Log10 (Cam) - Log10 (Cap) (1) Where Cam is the colony-forming units (CFU/ mL) values of C. albicans present in the monospecies biofilms (positive control) and Cap is the colony-forming units (CFU/ mL) values of C. albicans present in the polymicrobial biofilms. Then, the percentual reduction (PR) of fungal population in the polymicrobial biofilms after 24 or 48 hours were calculated with the equation 2: 24 PR = (1-10-DR) x 100 (2) Where DR is the decimal reduction obtained by equation 1. Scanning electron microscopy (SEM) This analysis was performed through FEG- SEM (JEOL JSM-7500F) to visualize the interactions between probiotic and yeasts within the biofilms. The experimental conditions were: polymicrobial biofilm at 1:100 pathogen: probiotic ratio and monospecies biofilms: C. albicans (initial inoculum of yeast: 106 CFU/mL) and E. faecium (initial inoculum of bacteria 108 CFU/mL). Biofilms were formed as described above in a sterile coverslip placed at the bottom of 6 -well microtiter plates. After the periods of incubation (24h or 48h), the biofilms supernatants were gently removed and the coverslip were washed with PBS. The biofilms were fixed with a 2.5% glutaraldehyde solution (Merck, Darmstadt, Germany) then dehydrated with an ethanol series (30%, 50%, 70%, 85% and 95% ethanol solution for 15 min each; two washes with 100% ethanol for 15 min). The samples were dried at 37ºC and kept in a vacuum desiccator until the analysis day. The coverslips containing adhered biofilms were then attached to the stub surfaces with double sided adhesive tape, coated with a layer of carbon and observed through FEG- SEM (JEOL JSM-7500F). The photomicrographs were taken at 1000x; 3000× and 5000x magnifications. Statistical analysis To verify the statistical significance, the results were submitted to One-way ANOVA followed by Tukey’s multiple comparisons test (parametric data). It was performed using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, California USA) with a minimum significance level of 5% (p ≤ 0.05). Results Assurance of probiotic strain safety. The antimicrobial susceptibility test results presented in table 1 were interpreted according to the standard table for interpretation of inhibition halos present in the diagnostic test package insert following CLSI recommendations (CLSI 2019). The 25 probiotic strain was sensitive to all classes of antimicrobials tested, including vancomycin. Table 1: E. faecium CRL 183 antimicrobial susceptibility test by disc diffusion Biofilm pH evaluation For C. albicans monospecies biofilms in formation, the supernatant pH was 7.0 at all concentrations tested. After 48 hours, with or without culture medium renewal, the pH was reduced to 5.0. There was no difference between the pH of E. faecium monospecies biofilms supernatants and the polymicrobial biofilms supernatants in any experimental conditions (initial concentration, incubation time, culture medium exchange), which remained in the order of 5.0. Quantification of viable cells and determination of the anti-Candida activity of E. faecium CRL 183 The interaction between fungus and probiotic was analyzed in polymicrobial biofilms. The survival of E. faecium in an environment co-colonized by C. albicans was evaluated by quantifying its colonies and comparing it with the results obtained in the E. faecium monospecies biofilm (control). It was observed that this probiotic is able to establish itself and survive in the microenvironment, competing for space and nutrients with C. albicans. There was significant statistical difference in relation to the control group only in the 1:1 ratio of fungus/ probiotic (Figure 1). Renewal of the culture medium in the 48 hours biofilms (48A) did not result in a statistically significant difference in relation to the non-renewal group (48B) in the ratios 1:10 and 1:100 (Figure 1B). In the ratio 1:1 the renewal of culture medium (48A) impaired E. Antimicrobials Inhibition halos (millimeters) Interpretation Ciprofloxacin 5 mg 30 Sensitive Chloramphenicol 30 mg 27 Sensitive Erythromycin 15 mg 22 Sensitive Nitrofurantoin 300 mg 30 Sensitive Norfloxacin 10 mg 25 Sensitive Tetracycline 30 mg 24 Sensitive Vancomycin 30 mg 19 Sensitive 26 faecium survival in the presence of C. albicans if compared with the group 48B. Presumably, metabolites produced within the first 24 hours helped E. faecium to survive in the microenvironment where there was a high concentration of C. albicans. Despite this positive effect that C. albicans exerts in relation to Enterococcus spp. the relation between these microorganisms cannot be considered as mutually beneficial. Especially in the context of the present study, since the presence of the fungus impaired the viability of E. faecium only in the 1:1 ratio (Figure 1), while the probiotic negatively affected the cellular viability of C. albicans in all experimental conditions (Figure 2). The reduction of C. albicans within the 24 hours biofilms range from 1.57 to 3.08 Log10 CFU/mL, these results indicate that, during the formation of biofilms, the fungal reduction depends on probiotic concentration. However, in terms of percentual reduction, which ranged from 97.12% to 99.92% for 24 hours biofilms, there was no statistical difference between 1:1; 1:10 and 1:100. In mature polymicrobial biofilms (48 hours) C. albicans reduction ranged from 2.00 to 2.30 Log10 CFU/mL (Figure 2) corresponding to PR of 99 to 99.43% for the group where was the removal of metabolites and renewal of nutrients (48A). Which was not significantly different from 48B group where it was observed reduction from 1.46 to 2.15 Log10 CFU/mL (Figure 2) or 96.54 to 99.30%. The 48 hours results indicate that, for mature biofilms, the fungal reduction was not dependent on the initial probiotic dose, since there was no significant statistical difference between the groups (1:1, 1:10 and 1: 100). Scanning electron microscopy (SEM) The SEM analysis showed in Figure 3 allowed to illustrate the results obtained by counting colony-forming units. In monospecies biofilms, or controls, it was possible to observe cellular aggregates, surrounded by EPS, scattered throughout the coverslips. A lower number of fungal cells (3A,3B,3C and 3D) was observed in comparison to bacteria (3E,3F,3G,3H), which corroborates the cell counting results obtained for monospecies biofilms. In polymicrobial biofilms (3I,3J,3K and 3L), it is possible to observe that there was a considerable reduction in the number of C. albicans cells compared to the control biofilms (3A,3B,3C and 3D) of this species. This does not occur with the number of E. faecium seen in the polymicrobial biofilm which is apparently equal to that found in the monospecies biofilm. Furthermore, the 27 images of the polymicrobial biofilms (3I,3J,3K and 3L) allow to evaluate that there is a physical interaction between the fungus and the probiotic cells. Fig. 1 - Cell viability of E. faecium in the presence of C. albicans. A. 24 hours – biofilm formation. B. 48 hours – biofilm maturation; 48A: group with metabolites withdrawal and nutrients renewal. 48B: group without interventions. CTRL - monospecies biofilms of E. faecium (control); 1: 1; 1:10; 1: 100 - polymicrobial biofilms in different ratios of C. albicans / E. faecium. Each column represents the mean of three independent experiments, each performed in quadruplicate (n = 12), the bars represent the standard deviation. The asterisks indicate when there was a statistically significant difference in relation to the control group (*p = 0.03; ***p = 0.0003). ANOVA, followed by Tukey's post-test. Fig. 2 - Cell viability of C. albicans in the presence of E. faecium. A. 24 hours – biofilm formation. B. 48 hours – biofilm maturation; 48A: group with metabolites withdrawal and nutrients renewal. 48B: group without interventions. CTRL - monospecies biofilms of C. albicans (control); 1:1; 1:10; 1:100 - polymicrobial biofilms in different ratios of C. albicans / E. faecium. Each column represents the mean of three independent experiments, each performed in quadruplicate (n = 12), the bars represent the standard deviation. The asterisks indicate when there was a statistically significant difference in relation to the control group (****p < 0.0001). ANOVA, followed by Tukey's post-test. 28 Fig. 3. Scanning electron micrographs (SEM) of biofilms recorded at 1000x (E, G); 3000x (A, C, F, H, I, K) or 5000x (B, D, J, L) magnifications. A-B: C. albicans monospecies biofilm at 24 hours. C-D: C. albicans monospecies biofilm at 48 hours. E-F: E. faecium monospecies biofilm at 24 hours. G-H: E. faecium monospecies biofilm at 48 hours. I-J: Polymicrobial biofilms at 24 hours. K-L: Polymicrobial biofilms at 48 hours. Discussion Assurance of probiotic strain safety Whilst adverse effects due to administration of probiotic bacteria are uncommon, it is mandatory to assure its susceptibility to antimicrobials. It is known that antibiotic resistance can be intrinsic or acquired as a result of a chromosomal mutation or by horizontal gene transfer (Celiberto et al. 2018). There is a special concern about the spread of Vancomycin-Resistant Enterococci (VRE) since some species of the genus may become a nosocomial pathogen and a reservoir for resistance genes, leading to clinical isolates resistant to all antibiotics (Hanchi et al. 2018). The strain E. faecium CRL 183 has documented absence of antimicrobial resistance genes (Saavedra et al. 2003) and was susceptible to all classes of antimicrobials tested and, therefore, it can be considered safe. Biofilm pH evaluation Factors such as nutrients availability, temperature and pH are determinants for the survival and behavior of microorganisms, especially in polymicrobial environments. It is a consensus that probiotic strains can produce lactic acid and A B E F D G H I J K L C 29 other organic acids. Which, in your turn, can cross the plasma membrane of yeast cells, increasing the activity of H+-ATPase. This mechanism promotes energy exhaustion of yeast cells inhibiting their growth and promoting cell death (Jørgensen et al. 2017). Furthermore, neutral to alkaline is the optimal extracellular pH for Candida spp. because it aids hyphal morphogenesis, among other fungal virulence strategies (Davis 2003). E. faecium is a lactic acid bacteria (LAB) and the acid production could be one of the key mechanisms by which this probiotic impairs C. albicans viability (Hanchi et al. 2018). A study which evaluated the inhibition on several Candida species promoted by L. reuteri has shown that, the almost complete inhibition was only achieved in pH 3.6, which is a very acidic level. However, extensive acidification may be a concern regarding the application in oral cavity due to the role of acid in caries lesions (Jørgensen et al. 2017). In our study the biofilm supernatants pH remained within the range of healthy human saliva pH which is 5.3 to 7.8, depending on the stimulation state (Gittings et al. 2015). Quantification of viable cells and determination of the anti-Candida activity of E. faecium CRL 183 Similar to the results shown in Figure 1; Mason et al. (2012a, b) demonstrated that C. albicans SC5314 promoted gastric and enteric colonization of Enterococcus spp. while antagonizing other lactic bacteria such as Lactobacillus spp., during recovery of gut microbiota following treatment with the antibiotic cefoperazone. Although the mechanism involved was not elucidated, the authors suggest that it was similar to the one C. albicans exerts on bacteria from phylum Bacteroidetes. The fungus could aid its adhesion and fixation to the cecum; that yeast glucans could be a potential source of food for these bacteria, or else, C. albicans could suppress other bacteria that antagonize Enterococcus spp. (Mason et al. 2012a). The reduction of C. albicans viable cells during biofilm formation was affected by the fungal: probiotic ratio. Cruz et al. (2013) demonstrated that Caenorhabditis elegans, a study model of polymicrobial infections, presented less mortality caused by C. albicans when it was co-inoculated by Enterococcus spp. The survival rate of the nematode was dose-dependent: the higher the number of colonies of Enterococcus spp. inoculated, the greater the protection against C. albicans pathogenicity. This study further determined that the pathogenicity of both microorganisms was attenuated during polymicrobial infection in C. elegans (Cruz et 30 al. 2013). For biofilm maturation, the microorganism ratio did not influence in the result. Matsubara et al. (2016b) also did not observe a significant relationship between inoculated probiotic dose and biofilm destabilization. Among the Enterococcus spp. mechanism of action to downregulate C. albicans spp. pathogenicity the literature majorly suggests the action of proteases, peptides and bacteriocins secreted by the bacteria(Shekh and Roy 2012; Cruz et al. 2013; Bachtiar et al. 2016; Graham et al. 2017). For example, researchers identified the action of two proteases (gelatinase biosynthesis activating cluster peptide - GBAP) that activate the virulence regulating system of Enterococcus spp., which when secreted by this bacterium prevented C. albicans polymorphism and, consequently, fungal pathogenesis (Cruz et al. 2013). The same effect was observed when C. elegans, previously infected with C. albicans, was treated with the supernatant (filtered and sterilized) of an Enterococcus faecalis inoculum. Thus, Cruz et al. (2013) determined that in their study, the inhibitory activity was secreted instead of a direct cellular competition between the bacterium and the fungus. In a more recent research, the action of the EntV, an enterocin encoded by Ef1097 gene, which is present in all strains of E. faecalis sequenced to date was described (Graham et al. 2017). This enterocin alone, as well as its synthetic version, was able to prevent C. albicans polymorphism and biofilm formation on different substrates and experimental conditions. Preformed C. albicans biofilms (24 hours / ~ 30 μm thick) were treated with EntV and underwent a large perturbation with a reduction of their thickness to ~15 μm. Biofilms that were not treated with EntV increased their thickness to >50 μm. The peptide also protected macrophage by enhancing its antifungal activity, reduced epithelial invasion, inflammation, and fungal load in a murine model of oropharyngeal candidiasis (Graham et al. 2017). Bachtiar et al. (2016) observed that E. faecalis cps2 (a non-encapsulated clinical isolate) and E. faecalis ATCC 29212, as well as the metabolites produced by them, did not inhibit C. albicans biofilm formation. However, both strains and their secreted metabolites significantly reduced (>50%) the maturation of these biofilms. These results were confirmed by the downregulation promoted on ALS1 and ALS3 (adhesion related genes) and on EFB1, a gene used to quantify the harmful effects of antifungal agents against mature candida biofilms (Bachtiar et al. 2016). 31 In our study, it was not possible to determine if the anti-Candida activity of E. faecium CRL 183 was modulated by its metabolites as suggested in literature cited above. However, as can be seen in Figure 2B, the renewal of the culture medium (48A) did not significantly influence C. albicans survival compared to the group in which there was no intervention (48B). Therefore, the removal of E. faecium metabolites secreted during the first 24 hours and the new nutrient supply did not favor the cellular viability of C. albicans. In addition, the E. faecium population was approximately 11 log10 CFU/mL in the polymicrobial biofilms and C. albicans population was approximately 7 log10 CFU/mL, under all experimental conditions. It means that the probiotic growth was much greater than the fungal growth within polymicrobial biofilms. This may suggest that in our study model, the inhibitory action on C. albicans may not be only mediated by metabolites produced by E. faecium, but most likely due to direct cell competition for nutrients and space. This mechanism of direct competition and limitation of environmental nutrients can provoke a metabolic reprogramming on C. albicans, reducing its virulence. Mailänder-Sánchez et al. (2017) suggest that this is the mechanism by which the probiotic strain Lactobacillus rhamnosus GG prevented adhesion, invasion, formation of hyphae (preventing epithelial damage), glucose depletion and ergosterol synthesis of C. albicans. Another inhibitory mechanism may have been the culture medium acidification in polymicrobial biofilms promoted by E. faecium which is a lactic acid bacteria (LAB). This pH reduction promotes an energy exhaustion of yeast cells, inhibiting their growth and disfavor C. albicans filamentation (Davis 2003; Jørgensen et al. 2017). Hypha formation is one of the main virulence factors of C. albicans and is directly related to its pathogenicity and to biofilm stability (Mayer et al. 2013; Nobile and Johnson 2015). Vilela et al. (2015) suggest this same mechanism in the suppression of C. albicans polymorphism when it was stimulated with cultures of L. acidophilus ATCC 4356 or filtered supernatants from this probiotic culture. They also reported a reduction of up to 57.5% in CFU-counting of C. albicans biofilms stimulated with the probiotic cell culture and 45.10% reduction in biofilms stimulated with probiotic culture filtered supernatants. Galleria mellonella was used as an experimental candidiasis model and either treatment or prophylaxis with L. acidophilus cells, or their metabolites, greatly increased the survival of this insect (Vilela et al. 2015). 32 Ribeiro et al. (2017) observed a similar effect on the interaction of C. albicans with L. rhamnosus cells: a 52.2% reduction in biofilm quantification in CFU / mL and a 48% reduction in metabolic activity. The stimulation with only the filtered supernatant of L. rhamnosus decreased, in a lesser extent, C. albicans growth (39.4%) and metabolic activity (61%). Matsubara et al. (2016b) observed that L. rhamnosus, L. casei and L. acidophilus cell suspensions significantly reduced C. albicans SC5314 biofilm formation in 32, 59 and 56.3%, respectively. When 48 hours biofilms were stimulated with the same probiotic suspensions, the C. albicans reduction was 61.8% (L. rhamnosus), 54.7% (L. casei) and 34.2% (L. acidophilus). By stimulating C. albicans biofilms with the filtered supernatant of these lactobacillus cultures, the reduction was less effective in the formation, and there was no reduction in the biofilm maturation. This result demonstrates that the inhibitory action on C. albicans was also not mediated only by probiotic secreted metabolites(Matsubara et al. 2016b). Therefore, our results seem to be very promising once E. faecium CRL183 presented an ability to impair C. albicans biofilm formation in up to 99.92% and its biofilm maturation in up to 99.43%, which are much higher rates than those presented by other Enterococcus spp. (Shekh and Roy 2012; Cruz et al. 2013; Bachtiar et al. 2016; Graham et al. 2017) or probiotic strains (Vilela et al. 2015; Matsubara et al. 2016b; Jørgensen et al. 2017; Ribeiro et al. 2017). Scanning electron microscopy (SEM) The images obtained through SEM permitted to visually confirm the hypothesis that in the present study direct competition and limitation of environmental nutrients, play an important role. It is very interesting to observe that C. albicans not only appears in a lesser amount but also with its morphology very damaged in 48 hours polymicrobial biofilms (3K and 3L) in comparison with C. albicans control biofilms at the same period (3C and 3D), which could be caused by the lactic acid crossing yeasts membrane (Jørgensen et al. 2017). It was expected to see hyphal formation in C. albicans biofilms in our SEM images, which did not occur. It may be explained by the lack of molecular factors favoring C. albicans polymorphism in the culture medium used in the experimental protocol, since it is known that the culture medium plays a fundamental role in the growth, morphology and biofilm formation of 33 C. albicans (Weerasekera et al.; Davis 2003). Another reason could be the several washes with ethanol removed hyphae and pseudohyphae, since these structures develop in the outermost layers of biofilms, while yeasts initially inoculated, are found in the basal layers adhered to the biotic or abiotic substrate (Uppuluri et al. 2010). Yet, in the context of prevention, our results remain promising as yeasts are the infecting forms which initiates biofilms and are responsible for spread to no infected sites (Uppuluri et al. 2010; Lohse et al. 2017). Both the counting colony-forming units technique and SEM analysis showed that E. faecium CRL 183 promotes a great reduction in C. albicans viability. In conclusion, according to the methodologies applied in this in vitro study, E. faecium CRL 183 was able to significantly inhibit the formation and maturation of fungal biofilms in all evaluated pathogen / probiotic relationships. 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Virulence 6:29–39 . doi: 10.4161/21505594.2014.981486 Weerasekera MM, Wijesinghe GK, Jayarathna TA, Gunasekara CP, Fernando N, Kottegoda N, Samaranayake LP Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development. doi: 10.1590/0074-02760160294 Witzler JJP, Pinto RA, Font de Valdez G, de Castro AD, Cavallini DCU (2017) Development of a potential probiotic lozenge containing Enterococcus faecium CRL 183. LWT - Food Sci Technol 77:193–199 . doi: 10.1016/j.lwt.2016.11.011 Zago CE, Silva S, Sanitá PV, Barbugli PA, Dias CMI, Lordello VB, Vergani CE (2015) Dynamics of biofilm formation and the Interaction between Candida albicans and methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA). PLoS One 10:1–15 . doi: 10.1371/journal.pone.0123206 39 Capítulo 2. Development of orodispersible films loaded with probiotic E. faecium CRL 183 for oral candidiasis prevention. Artigo formatado de acordo com as normas da revista Carbohydrate Polymers (Elsevier). 40 Development of orodispersible films loaded with probiotic E. faecium CRL 183 for oral candidiasis prevention. Virgínia Barreto Lordello1, (virginialordello@gmail.com |+5516992013891) Andréia Bagliotti Meneguin1, Sarah Raquel de Annunzio1, Maria Pía Taranto2, Marlus Chorilli1, Carla Raquel Fontana1 Daniela Cardoso Umbelino Cavallini 1,* (d.cavallini@unesp.br | +551633014691) 1 Sao Paulo State University (UNESP), School of Pharmaceutical Sciences, Rodovia Araraquara Jaú, Km 01 – s/n, Campus Ville, 14800-903 Araraquara, Brazil. 2 Reference Center for Lactobacilli (CERELA-CONICET), San Miguel de Tucumán, Chacabuco 145 - T4000ILC, Tucumán, Argentina. Abstract: The aim of this study was to develop and characterize an orodispersible film (ODF) capable of deliver the probiotic Enterococcus faecium CRL 183 in the oral cavity evaluating its in vitro antifungal activity. The ODF was composed by cheap and natural polymers and physical-chemically characterized; the probiotic resistance and viability during processing and storage was evaluated as well as the in vitro anti- Candida activity. The obtained films were thin, resistant and flexible, with neutral pH, low Aw values and microbiologically safe. Furthermore, they presented good barrier properties. The probiotic resisted the obtaining process of ODF, demonstrating high viability (> 9 log10 CFU / g), up to 90 days of storage at room temperature. The Probiotic Film (PF) promoted 68.9% of reduction in fungal biofilm formation (24h) and 91.24% in its maturation (48h) compared to the group stimulated with the control film (CF). Those promising results were confirmed through SEM images. Keywords: Probiotics, anti-Candida activity, Biofilms, Orodispersible films, Candida albicans ATCC 90028, Enterococcus faecium CRL 183. mailto:virginialordello@gmail.com mailto:d.cavallini@unesp.br 41 1. Introduction It is well established that probiotics, which are defined as living microorganisms that, when administered in suitable amounts, confer benefits to the host health, are able to improve dysbiotic microbiota, regulate intestinal transit, neutralize carcinogens, promote vitamin synthesis and bile salt metabolism, reinforce gut barrier and the immune system and directly antagonize and compete with pathogens (Hill et al., 2014). In this context, several studies have shown that certain probiotic strains have great potential for maintenance and improvement of oral health, presenting anticariogenic activity, reduction of halitosis and prevention of opportunistic infections, such as candidiasis, often present in immunocompromised individuals (Ishikawa et al., 2015; Jørgensen, Kragelund, Jensen, Keller, & Twetman, 2017; Mailänder-Sánchez et al., 2017; Matsubara, Bandara, Mayer, & Samaranayake, 2016; Pujia et al., 2017). These pathogenic conditions are related to formation and establishment of biofilms in the oral cavity. Infections caused by biofilms, especially fungal biofilms, are long-lasting and difficult to eliminate. Therefore, high doses of antifungals are often required, which promote serious adverse effects due to the similarity between host and fungal cells, and leads to pharmacotherapy resistance (Fanning & Mitchell, 2012; Pujia et al., 2017; Shekh & Roy, 2012). Thus, alternatives for fungal infections must be searched and prevention seems to be the best strategy. Our research group has demonstrated that the probiotic strain Enterococcus faecium CRL 183 was able to inhibit up to 99.9% in vitro formation of Candida albicans biofilms and 99% biofilm maturation, without losing its own viability. This study also has shown that probiotic initial inoculum, previously considered low (107 CFU / mL), was as capable of impairing the fungal biofilms as higher initial inoculums (data not-published). Witzler et al. (2017) has shown that this same probiotic strain was able to inhibit the growth of Streptococcus mutans ATCC 25175, another important pathogen of oral cavity. Furthermore, E. faecium CRL 183 resisted to salivary enzymes, being able to significantly grow in this environment. The probiotic strain was incorporated in a lozenge in an attempt to develop an alternative way to deliver it into the oral cavity, 42 but the probiotic viability was quickly reduced in that vehicle (Witzler, Pinto, Font de Valdez, de Castro, & Cavallini, 2017). One of the greatest challenges concerning probiotic products is to develop non-dairy alternatives focusing on local, instead of systemic actions, such as: capsules, gels or incorporation in food. These novel products should grant probiotic viability during processing and storage, should not alter product flavor or consistency and, on the oral cavity concern, should stay in the mouth long enough to release the probiotic (Heinemann, Carvalho, & Favaro-Trindade, 2013; Saha, Tomaro- Duchesneau, Daoud, Tabrizian, & Prakash, 2013; Witzler et al., 2017) An option that meets all these requirements is the ODF, also known as soluble films, oral disintegrating films, oral thin films or mucoadhesive films – the nomenclature changes depending on their characteristics and functionalities (Lee, Kim, Kim, Choi, & Jeong, 2017). This pharmaceutical form can be defined as a thin film of easy dissolution and high contact surface, which allows the release of all active component into the oral cavity. Among the advantages attributed to those films are: ease of obtention and handling, storage at room temperature, and easy administration, since it does not need water intake, facilitating patient compliance to treatment. Lastly, as the oral mucosa is highly vascularized, ODF can also avoid the first-pass metabolism (Dixit & Puthli, 2009). Another advantage is that the permanence of the ODF in the oral cavity, during the disintegration time, can promote a more efficient release of active components compared to other products such as oral gels that are easily removed by the salivary flow (Borges, Silva, Cervi-Bitencourt, Vanin, & Carvalho, 2016). Researchers have shown that this dosage form may be the best strategy to immobilize probiotics and deliver them into specific areas such as the oral cavity. Probiotic ODF presented high microbial viability after processing allowing a proper shelf-life, suitable disintegration time allowing the local probiotic release, and good appearance and mechanical properties (de Barros, Scherer, Charalampopoulos, Khutoryanskiy, & Edwards, 2014; Heinemann et al., 2013; Saha et al., 2013). Those films are usually composed of macromolecules, natural or synthetic, forming polymer matrix, plasticizers - which increase the filmogenic properties of the polymers giving greater flexibility and less fragility - and the active pharmaceutical ingredient (API) or bioactive compound of interest. The composition and proportion of 43 such excipients can be altered according to the characteristics desired for the product and the addition of sweeteners, salivary stimulants, flavorings, pigments, surfactants and stabilizers can be considered (Lee et al., 2017). The chosen polymers should have a good shelf life, should not be toxic to the probiotic, guaranteeing its viability and metabolic activity, as well as a quick and efficient release. In addition, they should not cause damage to the patient (Heinemann, Vanin, De Carvalho, Trindade, & Fávaro-Trindade, 2017; Saha et al., 2013). Carboxymethylcellulose (CMC) and hydroxypropyl methylcellulose (HPMC) appears in several studies as a good choice for films that carry probiotics (De Barros et al., 2014; Heinemann et al., 2013; Saha et al., 2013). However, Heinemann et al (2013) demonstrated that films composed of CMC and gelatin blend were more efficient at protecting probiotic cells during the drying process of the films. And that films composed of CMC, gelatin and starch allowed the survival of probiotics in a high concentration (> 9 CFU / g) for a longer period (Heinemann et al., 2013). Borges et al. (2016) also suggests that the gelatin, nor only confers advantageous properties to the product as viscoelasticity, transparency and excellent solubility at room temperature, but it also can be an important source of peptides promoting a healthier nutritional status (Borges et al., 2016). According to the characteristics of the films, these constitute carriers of bioactive substances ideal for patients with conditions that favor dysphagia such as: the elderly, patients with motor debilities and immunocompromised. Coincidentally, in this population there is a high prevalence of candidiasis (Borges et al., 2016; Dixit & Puthli, 2009; Lee et al., 2017; Nobile & Johnson, 2015). Considering the above, this study aimed to develope and characterize a probiotic orodispersible film with the addition of E. faecium CRL 183, evaluating its antifungal potential against C. albicans ATCC 90028, in vitro. 2. Material and methods 2.1. Material The probiotic strain E. faecium CRL 183 was obtained from Reference Center for Lactobacilli – CERELA/CONICET (San Miguel de Tucumán, Argentina) and C. albicans ATCC 90028 were kindly provided by the Laboratory of Clinical Microbiology of the School of Pharmaceutical Sciences, UNESP (Araraquara, São Paulo, 44 Brazil).The microorganisms were kept frozen at -80ºC in a cryogenic tube containing proper culture media with addition of 20% glycerol, up to the time of its use. Carboxymethylcellulose (CMC) was donated by Biovital (São Carlos, São Paulo, Brazil), gelatine (260 bloom / 30 mesh) was provided by Gelita (Cotia, São Paulo, Brazil), Sorbitol (Neosorb P60W) was supplied by Labonathus (São Paulo, São Paulo, Brazil), alcohol-free mint flavoring was provided by Firmenich, (Cotia, São Paulo, Brazil) and potato starch was purchased from Yoki (Pouso Alegre, Minas Gerais, Brazil). 2.2. Methods 2.2.1. Strains and growth conditions Prior to their use, the strains were thawed and subcultured in specific culture media: C. albicans in Sabouraud Dextrose Agar supplemented with chloramphenicol (0.05 g L-1) (SDA - Acumedia, Michigan, EUA) and E. faecium in Bile Esculin Agar or M17 agar (Acumedia, Michigan, EUA) and incubated at 37°C for 48h. C. albicans colonies, freshly cultivated as described above, were inoculated with a sterile loop in enriched broth (26 g/L of BHI- Kasvi, Curitiba, Brazil; 10 g/L of YE - Acumedia, Michigan, USA; 20 g/L of TSB - Acumedia, Michigan, USA; 20% of sucrose Synth, Diadema, Brazil). This yeast suspension was standardized (106 CFU/mL) by spectrophotometry reading on a Synergy H1M microplate reader (Biotek, Winooski, USA). To prepare the probiotic inoculum, E. faecium freshly cultivated colonies were inoculated in 5 mL of M17 Broth (Himedia, Mumbai, India) followed by incubation at 35 ± 2 ° C for 14-16 hours. After this period, the tubes containing the inoculum were pelleted by centrifuging at 4000 rpm (80-2B Centribio-Equipar, Curitiba, PR, Brazil) for 20 min, washed twice with 0.9% (w/v) NaCl solution (Synth, Diadema, SP, Brazil) and resuspended in lower volume of 0.9% (w/v) NaCl. The bacterial suspension obtained was standardized at ~1010 CFU/mL. 2.2.2. Orodispersible films preparation Two formulations of ODFs - Probiotic Film (PF) and Control Film (CF) - were prepared by solvent casting as described by Heinemann et al. (2013), with modifications. The procedure is outlined in Figure 1. 45 Probiotic Film (PF) Three dispersions of macromolecules (polymers) and plasticizer were prepared using a magnetic stirrer (Fisatom, São Paulo, Brazil): G - 2.00 g gelatin + 0.4 g sorbitol in 100 mL distilled water; PS - 2.00 g potato starch + 0.4 g sorbitol in 100 ml distilled water; CMC- 1.00 g carboxymethylcellulose + 0.2 g sorbitol in 100 ml distilled water. These dispersions were mixed in the proportion 1CMC: 2PS: 2G (v / v) to obtain the film-forming dispersion (FD), to which 0.50% (w/w) mint powder flavoring was added. The film-forming dispersion was sterilized at 121 ° C / 15 minutes in a vertical autoclave (Phoenix, Araraquara, Brazil) and then cooled to 45 °C. After that, all the steps were conducted within the laminar flow under sterile conditions. To the sterile film-forming dispersion 10% v / v. of E. faecium CRL 183 inoculum freshly cultured as described