Universidade Estadual de Campinas Faculdade de Odontologia de Piracicaba
JOÃO GABRIEL SILVA SOUZA
INTERAÇÃO ENTRE MICRORGANISMOS EM BIOFILMES ORAIS E POTENCIAL DE TRATAMENTO DE SUPERFÍCIE NO TITÂNIO PARA REDUÇÃO DA
PROLIFERAÇÃO MICROBIANA
MICROORGANISMS INTERACTION IN ORAL BIOFILMS AND EFFECT OF SURFACE TREATMENT ON TITANIUM TO REDUCE MICROBIAL
ACCUMULATION
PIRACICABA 2019
JOÃO GABRIEL SILVA SOUZA
INTERAÇÃO ENTRE MICRORGANISMOS EM BIOFILMES ORAIS E POTENCIAL DE TRATAMENTO DE SUPERFÍCIE NO TITÂNIO PARA REDUÇÃO DA
PROLIFERAÇÃO MICROBIANA
MICROORGANISMS INTERACTION IN ORAL BIOFILMS AND EFFECT OF SURFACE TREATMENT ON TITANIUM TO REDUCE MICROBIAL
ACCUMULATION
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor em Clínica Odontológica, na Área de Prótese Dental.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Clinical Dentistry in Dental Prosthesis area.
Orientador: Prof. Dr. Valentim Adelino Ricardo Barão
Este exemplar corresponde à versão final da tese defendida pelo aluno João Gabriel Silva Souza e orientada pelo Prof. Dr. Valentim Adelino Ricardo Barão.
PIRACICABA 2019
Ficha catalográfica Universidade Estadual de Campinas Biblioteca da Faculdade de Odontologia de Piracicaba
Marilene Girello - CRB 8/6159 Souza, João Gabriel Silva,
So89i SouInteração entre microorganismos em biofilmes orais e potencial de tratamento de superfície no titânio para redução da proliferação microbiana / João Gabriel Silva Souza. – Piracicaba, SP : [s.n.], 2019.
SouOrientador: Valentim Adelino Ricardo Barão.
SouTese (doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
Sou1. Biofilme. 2. Titânio. 3. Bactérias. 4. Fungos. I. Barão, Valentim Adelino Ricardo, 1983-. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Microorganisms interaction in oral biofilms and effect of surface treatment on titanium to reduce microbial accumulation
Palavras-chave em inglês: Biofilms
Titanium Bacteria Fungi
Área de concentração: Prótese Dental Titulação: Doutor em Clínica Odontológica Banca examinadora:
Valentim Adelino Ricardo Barão [Orientador] Jaime Aparecido Cury
Magda Feres
Livia Maria Andaló Tenuta Marlise Inês Klein Furlan Data de defesa: 23-08-2019
Programa de Pós-Graduação: Clínica Odontológica
Identificação e informações acadêmicas do(a) aluno(a) - ORCID do autor: https://orcid.org/0000-0001-5944-6953 - Currículo Lattes do autor: http://lattes.cnpq.br/8428014988839178
UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Odontologia de Piracicaba
A Comissão Julgadora dos trabalhos de Defesa de Tese de Doutorado, em sessão pública realizada em 23 de Agosto de 2019, considerou o candidato JOÃO GABRIEL SILVA SOUZA aprovado.
PROF. DR. VALENTIM ADELINO RICARDO BARÃO
PROFª. DRª. MAGDA FERES
PROFª. DRª. MARLISE INÊZ KLEIN
PROFª. DRª. LIVIA MARIA ANDALÓ TENUTA
PROF. DR. JAIME APARECIDO CURY
A Ata da defesa, assinada pelos membros da Comissão Examinadora, consta no SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da Unidade.
AGRADECIMENTOS
Agradeço a Faculdade de Odontologia de Piracicaba (FOP) da Universidade Estadual de Campinas (UNICAMP) por me proporcionar a oportunidade para condução deste projeto e formação adquirida; ao Programa de Pós-graduação em Clínica Odontológica, em especial o Departamento de Prótese e Periodontia, no qual está inserida a área de Prótese Total, pela estrutura, oportunidade e contribuição na minha formação profissional e realização desta pesquisa. Agradeço imensamente a área de Prótese Dental e Laboratório de Bioquímica Oral da FOP-Unicamp pela estrutura e oportunidades de crescimento profissional.
Agradeço a Fundação de Amparo à Pesquisa do Estado de São Paulo pela concessão da bolsa de doutorado durante o curso e apoio financeiro. O presente trabalho foi realizado com apoio da Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), processo nº 2015/23118-2).
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) - Código de Financiamento 001.
Ao meu orientador, o Prof. Valentim Barão, meu muito obrigado por todos ensinamentos, valores e importante contribuição na minha formação profissional. Agradeço imensamente pela orientação na condução desta tese, assim como demais estudos, e pelo incentivo na carreira científica, me permitindo pensar, executar e desenvolver as minhas habilidades e ideias. Quero expressar aqui a minha admiração pelo profissional que ele é, seja como professor ou como pesquisador, e por ser um exemplo para mim de profissional focado em sua carreira acadêmica, sou grato e honrado por ter tido a oportunidade de ter sido orientado por alguém tão dedicado e competente.
A University of Connecticut Health Center, agradeço pela estrutura, e oportunidade de execução de estágio no exterior para realização desta tese. Meu muito obrigado a professora Anna Dongari-Bagtzoglou, o qual tanto admiro e que me orientou durante o estágio no exterior e na execução deste trabalho. Obrigado pelos ensinamentos, oportunidades de crescimento cientifico e profissional, e por
compartilhar comigo seus conhecimentos. Agradeço imensamente a Martinna Bertolini, não apenas pela amizade e apoio, mas pela dedicação e ensinamentos transmitidos a mim durante o meu estágio e na execução deste trabalho. A lab manager Angela Thompson, meu muito obrigado por toda paciência e apoio para execução deste trabalho. Aos amigos da UConn pelas contribuições e todo apoio: Andre, Jessica e Morena.
Agradeço aos demais professores que tiveram contribuição ímpar na minha formação profissional na FOP-Unicamp: a professora Livia Tenuta, o qual tanto admiro, por ser um exemplo de profissional na carreira acadêmica e que representa a figura de uma mentora na minha formação; ao professor Jaime Cury, por ser um exemplo de pesquisador de excelência e por compartilhar com seus alunos valores e conhecimentos relacionados a pesquisa cientifica; meu muito obrigado também a professora Altair Antoninha Del Bel Cury pela importante contribuição na minha formação profissional; e ao professor Antônio Pedro, por todos os ensinamentos e contribuições, assim como por todas as dúvidas sanadas. Agradeço a professora Magda Feres da Universidade de Guarulhos pela importante parceria científica e ensinamentos, assim como pela contribuição na minha formação profissional. Agradeço também a professora Elidiane Rangel da UNESP-Sorocaba por toda dedicação e contribuição para execução desta tese.
Meu muito obrigado aos amigos e colegas da Prótese Total por todo apoio, contribuições e companhia durante o doutorado, e que fizeram essa experiência ainda melhor: Bruna, Jairo, Raphael, Carolini, Guilherme, Heloisa, Thais. Agradeço também aos amigos da Cariologia: Juliana, Aline, Mateus, Debora, Luziana, Luiz, Alfredo. Em especial, quero agradecer todo o apoio e amizade aos colegas de pós-graduação que viraram irmãos para a vida: Carol, Aline, Vinicius, Barbara e Mayara, sem vocês este processo não teria sido o mesmo, muito obrigado por tudo! Muito obrigado para aqueles que de alguma forma me incentivaram e me apoiaram nesta jornada: Andrea, Matheus, Sara, Nestor, Bruno, Raquel.
Por fim, agradeço em especial a minha família. Aos meus pais (Ivani e Djalma) meu muito obrigado por todo apoio dado, educação e valores passados a mim, que sem dúvidas iram refletir na minha postura profissional, não teria chegado
ate aqui sem o apoio de você, muito obrigado! Estendo estes agradecimentos também aos meus irmãos (Tamirys e Andrey) por todo apoio durante este processo. Aos meus sobrinhos (Arthur, Caio e Bernardo) que apesar de ainda não entenderem todo esse processo e sentirem falta do tio que mora em SP, são fundamentais na minha vida.
No mais, um muito obrigado a todos que contribuíram de alguma forma na condução deste projeto e na minha formação como Doutor.
RESUMO
A formação de biofilmes orais na superfície de implantes dentários a base de titânio (Ti) é o principal fator etiológico de infecções orais que levam a falha do tratamento reabilitador. Entre os colonizadores iniciais na cavidade bucal, destaca-se os Estreptococos do grupo mitis (composto principalmente por S. mitis, S gordonii, S sanguinis e S. oralis) que interagem e formam biofilme com Candida albicans, principal fungo oportunista da cavidade bucal. No entanto, a interação entre estas espécies bacterianas com C. albicans na superfície do Ti e seu potencial efeito no dano tecidual não tem sido explorado. (1) Desta forma, o primeiro estudo avaliou por modelo in vitro a habilidade de estreptococos e C. albicans formar biofilmes mono-espécie (72 h), assim como interagir na superfície do Ti. Além disso, avaliou-se o potencial patogênico dessa interação na destruição da mucosal oral por meio de modelo 3D tecidual. Identificou-se que, a presença de C. albicans favorece o acúmulo de biofilme no Ti (p<0,05), promovendo o crescimento de estreptococos do grupo mitis, principalmente de S. oralis. Um efeito sinérgico para esta interação foi observado, já que biofilmes multi-espécie resultaram em uma elevada expressão de genes relacionadas a virulência do fungo (p<0,05). Em adição, esta interação levou há um maior dano tecidual. (2) A interação de Candida com estreptococos pode ser mediada por glucanos sintetizados por enzimas glucosiltransferase (gtf). S. oralis, que interage com Candida em biofilmes e que possui gtf (gtfR), ainda não teve o papel dessa enzima caracterizado nesta interação. Portanto, no segundo estudo utilizou-se cepa referência de S. oralis e cepa mutante com deleção do gene da gtfR na interação com C. albicans em biofilmes formados em superfície plástica, Ti e modelo 3D de mucosa oral. Os resultados identificados demostram que a gtfR e seus glucanos produzidos aumentam a biomassa de biofilmes devido a produção de matriz (p<0,05). Na presença de Candida, cepa referência contendo a gtfR apresentou maior crescimento bacteriano, comparado a cepa mutante, em biofilmes formados em todas as superfícies testadas (p<0,05). No entanto, essa interação foi modulada pela superfície, já que Candida foi capaz de aumentar a expressão de gtfR pelo S. oralis em biofilmes crescendo no Ti (p<0,05). (3) Considerando o fato de que o Ti também é substrato para adesão microbiana por patógenos orais, assim como o papel de biofilmes no desenvolvimento das doenças peri-implantares, o terceiro estudo propôs o desenvolvimento de um tratamento de superfície
superhidrofóbico para o Ti a partir de tecnologia de plasma de carga incandescente de baixa pressão. O tratamento proposto apresentou biocompatibilidade celular (p<0,05), aumentou a resistência a corrosão do Ti (p<0,05) e reduziu a adesão bacteriana e de C. albicans e, consequenmente, promoveu menor formação de biofilme multi-espécie (p<0,05). Conclui-se que, Estreptococos do grupo mitis interage com C. albicans em biofilmes formados na superfície do Ti, promovendo o crescimento bacteriano e seu potencial patogênico. Esta interação entre S. oralis e Candida é mediada por polímeros extracelulares e modulada pela superfície onde os biofilmes são formados. Para reduzir a adesão microbiana e formação de biofilmes no Ti, a superfície superhidrofóbica desenvolvida é uma estratégia eficaz e promissora.
ABSTRACT
Biofilm formation on dental implant surface made of titanium (Ti) is the main etiologic factor to trigger oral infection that lead to rehabilitation treatment failure. Among initial colonizers on oral cavity Streptococcus from mitis group (S. mitis, S. gordonii, S. sanguinis and S. oralis) interacts and form biofilms with C. albicans, the main opportunistic fungus on oral environment. However, the interaction among these organisms on Ti surface and its effect on tissue destruction has not been evaluated. (1) Then, the first study evaluated by in vitro model the ability of Streptococcus and C. albicans to form single-specie biofilm (72h) and to Interact on Ti surface. Moreover, the pathogenic potential of these interaction on tissue destruction was evaluated using a 3D-tissue model. The results showed that C. albicans presence favored biofilm formation on Ti (p<0.05), promoting bacteria growth, mainly for S. oralis. Synergistic effect was found for this interaction, since bacteria up-regulated genes related to Candida virulence expression. Additionally, this interaction led to a higher tissue destruction. (2) The interaction between Candida and streptococcus is also mediate by glucan polymers synthesized by glucosyltranferase (gtf) enzyme. S. oralis which Interact with C. albicans in biofilm state and has a single gene of gtf (gtfR), has not been evaluated in relation the role of gtfR on cross-kingdom interaction with Candida. Therefore, second study used S. oralis wild type (WT) strain and mutant strain lacking gtfR gene to evaluate the interaction with C. albicans on biofilms formed on plastic, surface, Ti and 3D-tissue. The results showed that gtfR and glucans synthesized increased S. oralis biofilm biomass due the effect on biofilm matrix. On Candida presence, mixed biofilms with WT showed higher bacteria count, compared to mutant strain on biofilms formed on all surfaces. However, this interaction was modulated by surface where biofilm is growing, since Candida up-regulated gtfR expression by S. oralis on Ti. (3) Considering that Ti surface is also substrate for microbial adhesion by oral pathogens, as well the role of biofilm on peri-implant disease, the third study aimed to develop a superhydrophobic surface coating on Ti using low pressure glow discharge plasma. Surface coating showed biocompatibility, promoted corrosion resistance and reduced bacteria and Candida adhesion and, consequently, multi-specie biofilm formation on Ti. In conclusion, Streptococcus from mitis group interacts with C. albicans on biofilms formed on Ti surface, promoting bacteria growth and its pathogenic potential. The interaction
between S. oralis and Candida is mediated by extracellular polymers and modulated by surface where the biofilms is growing. To reduce microbial adhesion and biofilm formation on Ti surface the superhydrophobic coating developed is a promising strategy.
SUMÁRIO
1 INTRODUÇÃO 13
2 ARTIGOS 16
2.1 Artigo: CROSS-KINGDOM INTERACTION BETWEEN CANDIDA ALBICANS AND STREPTOCOCCUS FROM MITIS GROUP ENHANCE BIOFILM FORMATION ON TITANIUM SURFACE AND TISSUE DAMAGED
16
2.2 Artigo: ROLE OF GLUCOSYLTRANSFERASE R IN BIOFILM INTERACTIONS BETWEEN STREPTOCOCCUS ORALIS AND CANDIDA ALBICANS
48
2.3 Artigo: TARGETING BIOFILM SUPERHYDROPHOBIC COATING TO REDUCE MICROBIAL ACCUMULATION ON TITANIUM SURFACE
89 3 DISCUSSÃO 118 4 CONCLUSÃO 121 REFERÊNCIAS ANEXOS
Anexo 1 – Comprovante de submissão 125
Anexo 2 - Relatório de verificação de originalidade e prevenção de plágio 126
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1 INTRODUÇÃO
O titânio (Ti) têm sido o principal biomaterial de escolha para fabricação de implantes dentários, devido sua excelente biocompatibilidade aos tecidos bucais e adequadas propriedades físico-químicas (Albrektsson et al., 2004; Lambert et al., 2009), favorecendo a longevidade clínica do tratamento. No entanto, assim como as superfícies bucais, os implantes dentários também são substratos para adesão microbiana e consequente acúmulo de biofilme. Biofilmes orais são estruturas tri-dimensionais de comunidades microbianas envoltas por uma matriz extracelular polimérica (Bowen et al., 2018). O crescimento microbiano na forma de biofilmes favorece a interação entre espécies e seu metabolismo, assim como a resistência contra antimicrobianos e resposta imune do hospedeiro (Bowen et al., 2018). De fato, biofilmes polimicrobianos são responsáveis por desencadear infecções orais (Arciola et al., 2018), sendo considerado um fator de “estresse” físico-químico para ativação da resposta inflamatória dos tecidos bucais (Marsh et al., 2011). Infecções microbianas na superfície de implantes dentários podem acometer a integridade dos tecidos moles, denominada mucosite, assim como afetar o osso de suporte, causando peri-implantite (Pontoriero et al., 1994; Salvi et al., 2017; Schincaglia et al, 2017). Dessa forma, o entendimento do processo de formação de biofilmes, assim como a interação entre microrganismos que modulam a virulência do mesmo, podem subsidiar a criação de estratégias que visem o controle da doença peri-implantar.
Após inseridos na boca os implantes dentários são imediatamente expostos a formação de pelicular saliva pela adsorção de proteínas (Pantaroto et al., 2019), o que favorece a adesão microbiana inicial (Rabe et al., 2011). Entre os colonizadores iniciais na cavidade bucal, destacam-se os Estreptococos do grupo mitis (composto principalmente por S. sanguinis, S. oralis, S. gordonii e S. mitis), que representam 60-90% da colonização inicial das superfícies dentais, sendo capazes também de colonizar a mucosa bucal (Rickard et al., 2003; Diaz et al., 2006; 2012). Essas espécies também são altamente capazes de colonizar e formar biofilmes na superfície do Ti, principalmente sob exposição de açúcares da dieta (Souza et al., 2018), sendo identificadas em biofilmes nas fases iniciais e avançadas da doença peri-implantar (Kumar et al., 2012). Além disso, Estreptococos do grupo mitis têm sido reconhecidos por interagir e formar biofilmes com Candida albicans, principal fungo oportunista presente na cavidade oral (Xu et al., 2014; Bertolini et al., 2015). De fato, estudos prévios têm demonstrado o efeito sinérgico da interação de
Estreptococos do grupo mitis e C. albicans em superfícies abióticas e bióticas (superfície plástica e mucosa oral), favorecendo a virulência de biofilmes e levando a uma exacerbada resposta inflamatória (Xu et al., 2014). No entanto, apesar da C. albicans colonizar a superfície do Ti e ter sua contagem elevada em sítios comprometidos por doença peri-implantar (Kumar et al., 2012), sua interação sinérgica com espécies de Estreptococos do grupo mitis na superfície do Ti e seu consequente efeito na resposta inflamatória da mucosa bucal não têm sido explorados. Tal interação foi experimentalmente avaliada no Ti considerando apenas a formação inicial de biofilme (Monte-longo-jauregui et al., 2018) ou na presença de outros microrganismos, como o P. gingivalis e S. Mutans (Cavalcanti et al., 2016).
A interação entre C. albicans e espécies de Estreptococos pode ser física mediada por proteínas de superfície presentes em ambos os microrganismos (Xu et al., 2014), assim como por polímeros extracelulares (glucanos) sintetizados por exoenzimas glucosiltransferase (gtf) (Koo et al., 2018) das bactérias. Gtfs utilizam a sacarose como substrato para sintetizar α-glucanos, importante componente da matriz do biofilme (Bowen et al., 2018). A utilização da molécula de glicose proveniente da sacarose pela gtf permite a síntese de glucanos insolúveis, compostos por ligações α (1 → 3) em sua cadeia principal, e/ou glucanos solúveis, cuja cadeia principal é composta por ligações tipo α (1 → 6) (Aires et al., 2011; Bowen e Koo, 2011). Estes polímeros diferem quanto à sua estrutura e função, promovendo a adesão e agregação bacteriana (Bowen e Koo, 2011), assim como estruturação do biofilme (Xiao et al., 2012; Koo et al., 2013). Além disso, α-glucanos sintetizados pela gtf tem sido associados há um aumento na formação de biofilme multi-espécies com C. albicans, o que acarreta em um aumento na virulência e patogenicidade do biofilme (Koo et al., 2018). Em geral, Estreptococos do grupo mitis também são reconhecidos por possuir enzimas gtf (Vickerman et al., 1996; Fujiwara et al., 2000). S. oralis, o qual interage com C. albicans formando biofilme hiper-virulento (Bertolini et al., 2015; Xu et al., 2014; 2017), possui uma cópia gênica de gtf, chamada de gtfR (Fujiwara et al., 2000). No entanto, o papel da gtfR e α-glucanos sintetizados por ela na formação de biofilmes de S. oralis e na interação com C. albicans ainda não foi explorado.
Considerando o potencial patogênico de biofilmes orais para desencadear infecções bacterianas, destaca-se a necessidade da criação de estratégias que
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visem o controle do acúmulo microbiano. Para tal, tratamentos de superfícies tem sido propostos para implantes dentários no intuito de diminuir a formação de biofilmes (Ferrari & Spriano, 2016). Dentre os tratamentos para reduzir a adesão microbiana e consequente formação de biofilme, superfícies superhidrofóbicas são alternativas promissoras devido suas propriedades não-adesivas e apresentar resultados promissores na redução da adesão microbiana (Falde et al., 2016). No entanto, este efeito para superfícies de Ti têm sido demosntrado apenas na adesão ou formação inicial de biofilme utilizando microrganismos específicos (ex: Staphylococcus aureus e Escherichia coli) (Fadeva et al., 2011; Hwang et al., 2017; Chang et al., 2018), o qual não mimetiza a característica polimicrobiana de biofilmes orais formados na superfície de implantes. Dessa forma, filmes superhidrofóbicos depositados na superfície do Ti que sejam biocompatíveis e reduzam a formação de biofilmes polimicrobianos não tem sido explorado experimentalmente. Portanto, esta tese tem como objetivos avaliar: (1) a formação de biofilme e interações microbianas entre C. albicans e Estreptococos do grupo mitis na superfície do Ti, assim como o seu efeito no dano tecidual da mucosa oral; (2) o papel da gtfR e polímeros extracelulares produzidos na interação de S. oralis e C. albicans em biofilmes formados em superfícies bióticas e abióticas; (3) o pontecial de um tratamento de superfície superhidrofóbico biocompativel para redução do acúmulo polimicrobiano no Ti.
CAPÍTULO 1 – Artigo a ser submetido no Journal of Oral Microbiology
Cross-kingdom interactions between Candida albicans and mitis group Streptococci
enhance biofilm formation on titanium surfaces
Running title: Candida and Streptococcus Biofilms on Titanium
João Gabriel Silva Souza1, Martinna Bertolini2, Angela Thompson2, Valentim Adelino
Ricardo Barao1, Anna Dongari-Bagtzoglou2*
1 Department of Prosthodontics and Periodontology, Piracicaba Dental School, University of
Campinas (UNICAMP), Piracicaba, São Paulo, Brazil.
2 Department of Oral Health and Diagnostic Sciences, University of Connecticut School of
Dental Medicine, Farmington, CT, USA.
*Corresponding author
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ABSTRACT
Aim: Streptococci from the mitis group (represented mainly by Streptococcus mitis,
Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii) form robust,
biofilms with Candida albicans on mucosal surfaces. These microorganisms have been found in biofilms associated with peri-implant disease, but cross-kingdom interactions in biofilms forming on titanium surfaces (Ti) and their effect on oral mucosal inflammation has not been tested experimentally. Materials and methods: Single and mixed biofilms of C. albicans and each Streptococcus species were grown on Ti surfaces. Organotypic mucosal constructs were exposed to preformed Ti surface biofilms to test their effect on mucosal damage and inflammatory responses. Spent culture media from these organotypic mucosal constructs were used as growth supplements in biofilm media in order to evaluate their effect on
Candida biofilm growth. Results: C. albicans promoted bacterial biofilm biovolumes and
CFU counts in mixed biofilms with all mitis Streptococcus species on Ti surfaces (p<0.05). This relationship was mutualistic since all bacterial species up-regulated the efg1 hypha-associated gene in C. albicans. These interactions increased tissue damage when exposed to mixed biofilms, compared to Candida alone. Interestingly, mixed biofilms exposed to spent tissue culture media from tissues suppressed Candida growth on Ti surface. Conclusion: Cross-kingdom interactions between Streptococci from mitis group and C. albicans on Ti surface increased biofilm formation and fungal virulence attributes leading to enhanced oral mucosal destruction.
1 | Introduction
Biofilms are microbial communities enmeshed in a three-dimensional (3D) extracellular polysaccharide matrix (Costerton et al., 1995). Oral microorganisms adhere to dental surfaces or to the surface of biomaterials (Marsh et al., 2011). While biofilms forming on dental surfaces are responsible for dental caries or periodontal disease (Marsh et al., 2011), biofilms grown on implant surfaces made of titanium (Ti) can also be harmful to the peri-implant mucosa and anchoring bone (Lang et al., 1993). Polymicrobial biofilms formed on Ti surfaces are considered the main etiologic factor to trigger inflammatory disease processes known as peri-implant mucositis and peri-implantitis (Pontoriero et al., 1994; Salvi et al., 2017; Schincaglia et al, 2017). It has been proposed that biofilm accumulation, initially, by early colonizers with the ability to trigger mucosal inflammation may promote the colonization and growth of more virulent colonizers that increase the overall pathogenicity of the biofilm. Thus identifying commensal organisms that can trigger mucosal inflammation or initiate mixed microbial biofilm formation on Ti surfaces is important to understand the pathogenic processes in peri- implant mucositis and peri-implantitis.
Streptococci of the mitis group (represented mainly by Streptococcus mitis,
Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii) have been
recognized as main initial colonizers in biofilms formed on dental surfaces (Rickard et al., 2003; Diaz et al., 2006; Diaz et al., 2012a). Moreover, pyrosequencing analysis has revealed that members of this streptococcal group also dominate the oral mucosa of healthy individuals (Diaz et al., 2012b) and are present in biofilms in both early and late stages of peri-implantitis (Kumar et al., 2012). Importantly, mitis group streptococci have been termed “accessory pathogens” due to their ability to form multi-species biofilms and to enhance the community virulence (Whitmore and Lamont, 2011). Thus, mitis Streptococcus species have been shown to form robust, hypervirulent biofilms with Candida albicans (Diaz et al., 2012b; Xu et al., 2014b), an oral fungal opportunistic pathogen that forms biofilms on implanted
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materials and can cause disseminated infection (Kojic, 2004; Andes et al., 2004; Bertolini et al., 2019)
C. albicans has been isolated from implant biofilms associated with peri-implant
disease (Leonhardt et al., 1999; Schwarz et al., 2015), possibly due its ability to form robust mixed biofilms with Streptococcus species. In fact, co-infection with C. albicans and oral streptococci increase the biofilm growth and pathogenic synergy (Xu et al., 2014a; Bertolini et al., 2015; Xu et al., 2016; Xu et al., 2017) during the course of opportunistic infections (Xu et al., 2014b). These organisms interact mainly through cell wall surface proteins or glycoproteins on both organisms (Jenkinson et al., 1990; Koo et al., 2018).
Cross–kingdom interactions between C. albicans and mitis group species on Ti have been described in early biofilms or in biofilms inoculated with other microorganisms (i.e.
Porphyromonas gingivalis) (Cavalcanti et al., 2016; Monte-longo-jauregui et al., 2018).
However, peri-implant disease is a chronic disease (Lang et al., 1993; Renvert et al., 2018) and other species can modulate the initial interactions and biofilm virulence (Cavalcanti et al., 2016). To the best of our knowledge, no previous studies have studied the interaction between C. albicans and mitis group Streptococcus species in biofilms forming on Ti surfaces and their effect on mucosal inflammation. Moreover, a previous study showed that Tumor Necrosis Factor (TNF), a cytokine released during inflammation, decreases metabolic activity of C. albicans (Rocha et al., 2017). However, it is unlikely that biofilms are exposed to a single cytokine in the oral environment in vivo, and the cumulative effect of mixed mucosal cell mediators released during the inflammatory response on Candida growth has never been evaluated.
Therefore, in this study we tested whether: (1) Streptococcus species from the mitis group (S. mitis, S. oralis, S. sanguinis and S. gordonii) form biofilms with C. albicans on Ti surfaces; (2) mixed biofilms lead to oral mucosal inflammation and destruction; (3) the mucosal response to mixed biofilms modulates Candida growth on Ti. The overarching hypothesis of this work is that cross-kingdom interactions enhance biofilm formation on Ti
surfaces exacerbating the inflammatory response of oral mucosa and this response has a reciprocal effect on fungal biofilm growth.
2 | Materials and Methods
2.1 Strains and growth conditions
Bacterial strains used in the study were S. oralis 34 (kindly provided by P.E. Kolenbrander), S. gordonii CH1 (kindly provided by J.M. Tanzer), S. mitis ATCC 49456 and
S. sanguinis SK36 (ATCC BAA-1455). Bacteria were reactivated prior each experiment by
overnight growth in brain-heart infusion (BHI) medium (Becton, Dickinson and Company, Sparks, MD, USA) under static conditions at 37°C, in a 5% CO2 incubator. Fungal organisms
consisted of C. albicans strain SC5314, a laboratory strain originally isolated from a patient with bloodstream infection (Irwin and Fonzi, 1993, Kindly provided by Dr. A. Mitchell). C.
albicans was grown in yeast extract–peptone–dextrose (YPD), containing 5 g L-1 yeast
extract (Merck, Darmstadt, Germany), 10 g L-1 peptone (Becton, Dickinson and Company,
Sparks, MD, USA) and 20 g L-1 of dextrose (Merck, Darmstadt, Germany), under agitation
(70 rpm), aerobically, at 30°C.
2.2 Titanium disks
Commercially pure titanium disks (Grade 2 - American Society for Testing and Materials, grade 2 - surface area 223 mm2) were used as biofilm growth substratum. Samples were
polished through standardized metallography methods with sequential sandpaper (#320, #400, #600) to standardize the surface roughness (average surface roughness - Ra = 0.19 µm ± 0.13; average maximum height of the profile - Rz = 0.62 µm ± 0.07). Then, the disks were sterilized in an autoclave at 121°C prior each experiment.
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2.3 Biofilm growth on titanium surfaces
Monospecies and mixed species biofilms of C. albicans and each Streptococcus species were allowed to develop for 72 h in RPMI 1640 medium supplemented with 10% FBS and 10% BHI as this media formulation allows robust mixed species biofilm growth (Xu et al., 2017). Media were changed every 24 h. Prior to microbial inoculation, Ti disks were coated with FBS for 30 minutes at 37°C to increase initial organism adhesion.
For biofilm growth, overnight stationary-phase cultures of Streptococcus strains were inoculated in fresh BHI broth, allowed to reach exponential growth, and adjusted to OD600=1,
representing a final suspension of 107 cells/mL. Overnight cultures of C. albicans were prepared in YPD broth and cell concentrations were adjusted by counting in a Neubauer chamber. The final inoculum in each biofilm consisted of 105 cells of C. albicans and 107 cells
of Streptococcus strain. Biofilms were incubated under static conditions at 37°C in a 5% CO2
incubator.
In a second set of experiments, we tested the hypothesis that streptococci create a favorable environment for subsequent C. albicans biofilm growth. For this, monospecies biofilms of each Streptococcus from mitis group were allowed to develop for 48 h. Then, C.
albicans was inoculated subsequently on preformed Streptococcus biofilms for up to 24 h. C. albicans monospecies biofilms were used as control.
2.4 Biofilm effect on organotypic mucosa
The effect of titanium surface biofilms on oral mucosal inflammation was tested using an
in vitro organotypic mucosal construct, described in detail previously (Dongari-Bagtzoglou
and Kashleva, 2006). Briefly, the model consisted of human immortalized oral keratinocytes (SCC15 cells, ATCC) seeded (5 × 105 cells per well in a 6-well transwell plate) over a
collagen type I matrix, embedded with fibroblasts (3T3 cell line, ATCC) (Dongari-Bagtzoglou and Kashleva, 2006). The model results in a non-keratinizing stratified squamous epithelium which takes approximately 2–3 weeks to complete. After tissue maturation, culture media (infection medium - Dulbecco’s modified Eagle’s medium, supplemented with L-glutamine, hydrocortisone, Insulin-Transferrin-Ethanolamine-Selenium (ITES), O-phosphorylethanol- amine, adenine and triiodothyronine) (Dongari-Bagtzoglou and Kashleva, 2006; Bertolini et al., 2015) with no antibiotics were used for 24 h. Biofilms grown for 72h on Ti disks which were pre-fixed onto a sterile plastic holder, were transferred to each well containing a mucosal construct and suspended 0.5-1 mm above the apical epithelial surface. Biofilms submerged in infection media were exposed to the mucosal surfaces for additional 16 h at 37°C in a 5% CO2 incubator. Following this step culture media were collected from the basal
aspect of the organotypic mucosa and tissues were processed as described below.
2.5 Biofilm analyses by confocal laser scanning microscopy
Confocal laser scanning microscopy was used for biovolume and 3D structure analyses of biofilms. Biofilms were fixed with 4% paraformaldehyde for 2 h at 4ºC. C. albicans was visualized after staining for 2h at room temperature using a FITC-labeled anti-Candida polyclonal antibody (Meridian Life Science, ME, USA). For biofilms containing streptococci, this step was followed by Fluorescence In Situ Hybridization (FISH) with the pan-eubacterial probe EUB338 labeled with Alexa 633 (Amann et al., 1990; Dongari-Bagtzoglou et al., 2009). Biofilms were visualized using a Zeiss LSM 880 confocal scanning laser microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA) with an argon laser (458-nm, 488-nm and 514-nm), using air Plan-Apochromat x20/0.8. Stacks of z-plane images from at least 3 different fields of view per sample were acquired and then reconstructed into 3-D images using the IMARIS software (Bitplane, Inc., Saint Paul, MN, USA). Surface reconstructions using the surpass mode were used to calculate the biovolumes (in µm3) of biofilms.
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Representative images of a minimum of three experimental replicates were used in all analyses.
2.6 Histologic staining of mucosal biofilms
To visualize mucosal biofilms, tissues were fixed in 4% paraformaldehyde for 2 h, followed by a series of ethanol and xylene dehydrations before paraffin embedding. Hematoxylin & eosin-stained sections were used to observe tissue architecture. To visualize biofilms, tissue sections were also stained by immunofluorescence for C. albicans and FISH for
Streptococcus strains as described above, and counter-stained with Hoechst 33258
(Invitrogen, Carlsbad, CA, USA), a nucleic acid stain to visualize the epithelial layers (Diaz et al., 2012c). Images were obtained using a Zeiss Axio Imager M1 microscope and an ECPlan-Neofluar 920 NA 0.5 air objective and further analyzed using the AXIOVISION LE64 program.
2.7 Tissue damage by lactase dehydrogenase assay
Lactate dehydrogenase (LDH) release into the basal culture media was monitored as an indicator of tissue/cell damage. The CytoTox-ONE Homogeneous Membrane Integrity Assay kit (Pro- mega, Madison, WI) was used to assay LDH activity using an Opsys MRTM Microplate Reader (Dynex Technologies Inc., Chantilly, VA) and REVELATION QUICKLINK software (Thermo Labsystems, Chantilly, VA) according to the manufacturer’s protocol. The LDH data were expressed as optical density units (490 nm).
2.8 Cytokines released by oral mucosal constructs and the effect of mucosal spent
Cytokines in culture media collected from organotypic mucosal constructs were analyzed using the Luminex/MAGPIX system (RCYTOMSG-80K; Millipore). Levels of six cytokines (IL-10, IL-17A, IL-1β, IL-6, IL-8 and TNFα) released in crevicular fluid during peri-implant disease in humans (Duarte et al., 2016) were simultaneously quantified in each sample.
To test the effect of products released by the oral mucosa inflammation on biofilm growth of Ti, Candida alone and mixed biofilms were formed on Ti for 24h in RPMI + 10% BHI + 10% and supplemented with increasing concentrations (0, 5, 10 and 20% v/v) of culture media collected from organotypic mucosa.
2.9 Gene expression analyses
For Candida gene expression analysis RNA was extracted from monospecies (C.
albicans) and mixed-species biofilms formed on Ti for 6 h and 24 h (C. albicans with each Streptococcus strain), followed by cDNA synthesis and qPCR, according to a previous
protocol (Xu et al., 2016; 2017). RNA was purified using the RNeasy Mini Kit which includes a DNAse treatment step (QIAgen, Hilden, Germany). A second DNase treatment was conducted using the TURBO DNA-free® Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quality and quantity of RNA was measured by NanoDrop®. cDNA was synthesized using SuperScript III CellDirect cDNA Synthesis kits® (Invitrogen, Carlsbad, CA, USA). RT-qPCR was performed using Bio-Rad CFX96 cycler, using IQTM SYBR® Green Supermix kit (BIO-RAD, Hercules, CA, USA) with a final reaction volume of 20 µl (Xu et al., 2017). EFB1 gene was used as housekeeping control. Hyphae-associated genes ALS3, HWP1 and EFG1 were analyzed using primer sequences as previously described (Xu et al., 2017). Data were calculated by the ∆∆Cq method and gene expression in co-species biofilms was expressed as fold relative to single Candida biofilms.
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For microorganism viable count (effect of mucosal tissue media on biofilm growth assay), Ti disks were vortexed in 2 mL PBS for 10 seconds, followed by sonication at 7 W for 30 seconds. 100 µL of the sonicated suspension was serially diluted in PBS and each dilution were plated on BHI agar supplemented with Nystatin (250 U/ml) for Streptococcus and on Sabouraud Dextrose Agar (SDA) supplemented with chloramphenicol (1 mg/mL) for
C. albicans quantification. Streptococcus plates were incubated at 37°C in an atmosphere of
5% CO2 and Candida plates at 30°C in aerobic conditions for 2 days. Colony-forming units
(CFUs) were counted by stereomicroscopy, and the results were expressed as CFUs per biofilm.
C. albicans and Streptococcus biomass in biofilms growing on Ti was additionally
determined by qPCR. After cell lysis (lysozyme), the DNA was extracted using QIAGEN DNA Stool Mini Kit according to the company’s handbook. Primers for the 18S rRNA gene of C.
albicans were used (Xu et al., 2017). Quality and quantity of DNA was measured by
NanoDrop®. qPCR for Candida was performed with the IQTM SYBR Green Supermix kit (BIO-RAD) (xu et al., 2017). For Streptococcus quantification we used 16S rRNA gene universal primers and a TaqMan probe, as described previously (Nadkarni et al., 2002). qPCR was performed using Bio-Rad CFX96 cycler using previous protocol (Nadkarni et al., 2002).
2.11 S. gordonii and C. albicans interaction
Since S. gordonii and C. albicans interaction on plastic surface is also mediate by glucan polymers synthesized by glucosyltransferase enzyme (gtfG) from bacteria (Vickerman et al., 1996; Ricker et al., 2014) using sucrose as substrate, we tested how these organisms interact on Ti surface and on biotic surface (fibroblast layer). S. gordonii strain AMS12 which a ca. 1.7-kbp internal fragment of the gtfG structural gene was replaced with a lacZ/erm determinant, that produces a truncated gtfG protein with no Gtf activity or glucan activity was
used (Ricker et al., 2014). Candida alone and mixed biofilms were formed for 24 h on Ti surface and fibroblasts monolayer (3T3) (107 cells) under 1% sucrose exposure (substrate
for glucan polymers synthesis) and conditions mentioned above. Biofilms were analyzed by CFU and confocal microscopic (biovolume). Alexa Fluor 647-labeled dextran conjugate (1 µM; absorbance/fluorescence emission maxima, 647/668 nm) was added during biofilm formation to stain the α-glucan polymers in extracellular matrix. EUB338 probe labeled with Alexa 405 was used to stain bacteria.
2.11 Statistics
The Graph-Pad Prism® software (Graphpad, La Jolla, CA, USA) and SPSS 20.0 was used for statistical analyses and a significance level of 5% was adopted. ANOVA-one way followed by Tukey post-hoc test and Bonferroni t-test was applied to analyze the data. When necessary data were transformed in Log10 prior to statistical analysis. Experiments were made at least in duplicate using two samples per experiment.
3 | Results
3.1 Characteristics of single and mixed biofilm growth on Ti surfaces
First we tested the ability of C. albicans and mitis streptococci to form mono-species biofilms on Ti surfaces after 72 hours of growth. All organisms were able to form low density biofilms, with Candida hyphae and bacterial clusters sparsely distributed on on Ti surfaces (Fig. 1A). A higher biovolume was noted for S. oralis biofilms, compared to other bacterial strains (p<0.05) (Fig. 1B). This was consistent with a slightly higher 16S rDNA gene copy number for this species compared to the other three species as assayed by qPCR (Fig. 1C).
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Figure 1 – Single biofilms growing for 72 hours on titanium surface. (A) X–Y isosurfaces (top
panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. Bacteria (red) was visualized after fluorescence in situ hybridization with a EUB probe labeled with Alexa 633. Candida albicans (green) was visualized after staining with an FITC-conjugated anti-Candida antibody. Scale bars, 50 µm (X–Y isosurfaces) and 70 µm (three-dimensional reconstructions). (B) Average total biovolumes (in µm3) for 72-h biofilms. Biovolumes were measured in 2 different confocal
laser scanning microscopy image stacks from two independent experiments. (C) Average microrganism count in logarithmic scale by qPCR. ANOVA-one way, different letters represent statistical differences (p<0.05). The error bars indicate standard deviations.
We next inoculated each streptococcal species with C. albicans on Ti surfaces and analyzed mixed biofilms after 72 hours of growth. Confocal images of mixed biofilms suggested direct physical proximity between C. albicans cells and Streptococcus sanguinis in
72h biofilms with bacterial clusters interspersed among hyphal organisms (Fig. 2A). In contrast mixed biofilms with S. oralis, S. gordonii and S. mitis were composed of two almost distinct layers, with bacteria forming a basal layer in direct contact with Ti and fungi forming a biofilm mat over the bacterial biofilm surface. This is consistent with the mixed mucosal biofilm structure of S. oralis with C. albicans when tissues are submerged in growth media (Bertolini et al., 2015). As expected, mixed biofilms with C. albicans contributed to a higher total biovolume compared to single specie biofilms (Fig. 2B). Interestingly, co-inoculation with
C. albicans improved streptococcal growth on titanium surfaces for all species by
approximately two-fold, consistent with our previous observations with S. oralis on mucosal surfaces (Xu et al., 2014b; 2016) (Fig. 2C). In contrast, Candida biovolumes and corresponding biomass seems be negatively affected by all streptococci (Fig. 2D and E). Although the fungal biomass was reduced we noticed that C. albicans still formed rather long hyphae when growing with streptococci. In fact, in early biofilms (6h), all Streptococcus strains up-regulated expression of the hypha-associated gene efg1 in Candida (Fig. 2F). Moreover, in mixed biofilms with S. oralis and/or S. gordonii certain efg1-regulated genes were upregulated (HWP1 and als3 genes) (Fig. 2F). However, we found no significant influence of Streptococcus strains on the genes expression in 24 h biofilms (not shown).
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Figure 2 – Seventy-two hour biofilms of C. albicans with Streptococcus species on titanium surface (A) X–Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. Bacteria
(red) was visualized after fluorescence in situ hybridization with a EUB probe labeled with Alexa 633. Candida albicans (green) was visualized after staining with an FITC-conjugated anti-Candida antibody. Scale bars, 50 µm (X–Y isosurfaces) and 70 µm (three-dimensional reconstructions). (B) Average total biovolumes (in µm3) for each type of biofilm. Biovolumes
were measured in 2 different confocal laser scanning microscopy image stacks from two independent experiments. (C) Bacteria count (qPCR) expressed as fold of mixed over single biofilms (D) Average Candida biovolumes (in µm3). (E) Candida count by qPCR. (F) Relative expression of Candida genes levels assessed by RT-qPCR. Results represent mean fold change gene expression in mixed biofilms over Candida alone, in independent experiments. *p<0.05, using the Bonferroni t-test. The error bars indicate standard deviations.
Prompted by the bilayer structure of mixed biofilms with certain mitis species we tested whether preformed Streptococcus biofilms (48h) on Ti surfaces could increase Candida growth (24h). C. albicans formed a biofilm on all preformed Streptococcus biofilms (Fig. 3A), consistent with an increase in total biovolume, compared to single biofilms (Fig. 3B). However, there seems to be a negative effect on Candida growth by bacteria on preformed
Streptococcus biofilms, in terms of fungal count (Fig. 3D) and biovolume (Fig. 3E).
Remarkably, in this setting Candida was still able to increase Streptococcal biomass count for all strains (Fig. S1A), which was significantly higher only for S. oralis, compared to single biofilms (Fig. 3C). In summary, these data suggest that on Ti surface C. albicans promotes an increase in streptococcal cell numbers in late biofilms growth which improved biofilm biomass. Moreover, although Streptococcus did not affect Candida growth as mixed biofilms or creating a favorable environment, bacteria enhanced Candida virulence-associated genes in early stages on biofilm formation.
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Figure 3 – Forty-eight hours preformed Streptococcus biofilms + twenty-four hours Candida
growth on titanium surface. (A) X–Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. Bacteria (red) was visualized after fluorescence in situ hybridization with a EUB probe labeled with Alexa 633. Candida albicans (green) was visualized after staining with an FITC-conjugated anti-Candida antibody. (B) Average of total biovolume of mixed and single biofilms. (C) Bacteria count by qPCR. (D) Candida count by qPCR. (E) Average bacteria,
Candida and total biovolume of biofilms. *p<0.05, using the Bonferroni t-test. The error bars
3.2 Mucosal tissue damage triggered by Ti surface biofilms
Mixed oral mucosal biofilms formed by C. albicans and S. oralis stimulate an exaggerated inflammatory response (Xu et al., 2014b). Thus, we tested whether mixed biofilms growing on Ti affect oral mucosal inflammation and tissue damage. Although biofilms were placed at a 0.5-1 mm distance from the mucosal surface, during the co-incubation process fungal cells dispersed from Ti and formed a biofilm directly on the mucosal surface, but no bacteria was visualized on mucosal surface (Fig. 4A). Images suggest a higher biofilm layer for C. albicans + S. gordonii biofilm (Fig. 4A). In fact, mixed biofilm increased tissue damaged for all Streptococcus strains, compared to Candida alone biofilm (Fig. 4B). However, although IL-8 released from tissue was notably high for all groups, mixed biofilms did not increase cytokines releasing from tissue after 16h of infection, compared to Candida alone (Fig. 4C). These data suggest that cross-kingdom interaction between C. albicans and
Streptococcus from mitis group increase biofilm formation on Ti and affects negatively oral
mucosal inflammation, increasing tissue damaged but no difference in immune response for cytokines releasing.
Since previous study showed that TNFα inhibited Candida growth biofilm (Rocha et al., 2017), we tested how products released during mucosal inflammation and damage (media collected after tissue infection mentioned above) could affect biofilm growth on Ti surface as
Candida alone or mixed biofilms. Increasing concentrations (0, 5, 10 and 20%) of culture
media collected from organotypic mucosa tissue culture wells after infection inhibited
Candida growth during 24 h biofilm growth on Ti surface as single biofilm (Fig. 4D and Fig.
S2A) or mixed biofilm with S. gordonii (Fig. 4D). However, it did not affect Streptococcus growth (Fig. S2B). Considering the high concentration of IL-8 released after mucosal inflammation, we tested whether this cytokine could be responsible to affect Candida growth biofilm in a dose-response manner, but no effect was identified (Fig. S2C).
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Figure 4 – Seventy-two hours biofilms of C. albicans alone or in combination with
Streptococcus growing titanium surface and kept above on organotypic mucosal surfaces for
16h. (A) Tissue sections of mucosal with Bacteria (red) stained by EUB probe labeled with Alexa 633. Candida albicans (green) was visualized after staining with an FITC-conjugated anti-Candida antibody, and mucosal cell nuclei counterstained with the nucleic acid stain Hoechst 33258 (blue, top panel). Corresponding haematoxylin & eosin-stained tissue sections are shown in the bottom panels. (B) LDH assay expressed by OD. (C) Luminex assay showing cytokines released on media collected from tissue after infection. (D) Twenty-four hours Candida alone and mixed biofilms growing on titanium and supplemented with
increasing concentrations of media collected from tissue after 16 h of infection. Medium uninfected which was exposed to titanium surface for 16h without preformed biofilm was used as control. ANOVA-one way and Tukey pos-hoc test (p<0.05). The error bars indicate standard deviation.
3.3 α-glucan mediate the interaction between C. albicans and S. gordonii on Ti surface
Since previous study showed that glucan extracellular polymers synthesized by gtfG using sucrose as substrate is able to mediate the interaction between C. albicans and S.
gordonii on plastic surface (Ricker et al., 2014), we further explored the mechanism of this
interaction on Ti surface. Therefore, we tested the role of gtfG (glucosyltransferase from S
gordonii) and polymers synthesized on this cross-kingdom interaction. As expected, growth
of wild type strain with C. albicans under sucrose exposure led to the development of α-glucan matrix (Fig. 5A), increasing biofilm biovolume, compared to mutant strain (Fig. 5B). Surprisingly, α-glucan presence affected Candida growth on mixed biofilms on Ti surface increasing fungal count (Fig. 5C). To extend these findings we next investigated the influence of α-glucan in biofilms growing on the fibroblast cellular monolayer (biotic surface) as a substratum. Both gtfG mutant and WT strains formed a mixed biofilm with C. albicans on the fibroblast, demonstrating that the bacterial growth promoting effect of C. albicans does not require α-glucan synthesis (Fig. 5D). Similarly to abiotic Ti surface, WT strain improved biofilm biovolume and Candida count on mixed biofilms (Fig. 5E and F).
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Figure 5 – Twenty-four biofilms of C. albicans (Ca) alone or with wild type S. gordonii (CH1)
and gtfG mutant strains. Biofilms were grown on titanium and fibroblast monolayer surfaces and media were supplemented with 1% sucrose. (A) X–Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. Candida albicans (green) was visualized after staining with
an FITC-conjugated anti-Candida antibody. S. gordonii (blue) was visualized after fluorescence in situ hybridization with a Streptococcus-specific probe conjugated to Alexa 405. Alexa Fluor 647-labeled dextran conjugate probe (red) was used to stain biofilm matrix (α-glucans). Scale bars, 50 µm (X–Y isosurfaces) and 70 µm (three-dimensional reconstructions). (B) Average total biovolumes (in µm3) for each type of biofilm on titanium.
(C) Candida CFU counts in biofilms on titanium. (D) X–Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms formed on fibroblast monolayer using strains mentioned above. (E) Average total biovolumes (in µm3) for each type of biofilm on monolayer. (F)
Candida CFU counts in biofilms on monolayer. *p<0.05, using the Bonferroni t-test. The
error bars indicate standard deviations.
DISCUSSION
The interaction of Streptococcus from mitis group with other microorganisms confers a mutual advantage in biofilm formation, enhancing its virulence (Xu et al., 2014a). Co-infection of these streptococci with C. albicans affect mixed biofilm growth, increasing the ability to trigger infection and modulating host-response (Xu et al., 2014b; Bertolini et al., 2015; Xu et al., 2017; Koo et al., 2018). This mutualistic relationship has been showed on biofilms state growing on biotic (oral mucosa) and abiotic (plastic or glass) surfaces, which promotes fungal virulence, severity of biofilm lesions and exacerbated inflammatory response (Diaz et al., 2012b; Xu et al., 2014b; Bertolini et al., 2015). Our study is the first one to provide experimental evidence that C. albicans and Streptococcus from mitis group species interacts in a synergistic manner in biofilms on Ti surface by the positive effect of fungal promoting streptococcal growth and enhanced Candida virulence-associated genes expression by bacteria presence. Moreover, this improved growth on late mixed biofilms is an important factor responsible to increase tissue damage. Interestingly, products released during
37
inflammatory process suppressed Candida growth biofilms on Ti surface, as single biofilm or with S. gordonii.
The interaction of C. albicans with oral Streptococcus species has been mainly explored in relation to the development of oropharyngeal candidiasis (Venkatesh et al., 2007; Nobbs et al., 2010; Xu et al., 2014b) or dental caries (Bowen et al., 2018; Koo et al., 2018). Growing in mixed biofilms these organisms changes the gene expression patterns and modulate host-response promoting the capacity of each other to form robust biofilms (Xu et al., 2017). Peri-implant diseases are polymicrobial opportunistic infections which endogenous microorganism species become pathogens under ecological shifts (Charalampakis and Belibasakis, 2015; Sanz-Martin et al., 2017). In fact, Streptococcus from mitis group does not have a high ability to colonize oral mucosa (Xu et al., 2014a); however, C. abicans promotes bacteria growth enhancing biofilm virulence (Xu et al., 2017). We found similar mechanism for Ti surface, since mixed biofilms showed higher bacteria count than single species, and it affected fungal virulence and tissue damaged.
Although bacteria level was improved by Candida presence, mixed biofilms did not favor Candida growth on Ti surface. It can be explained by the initial adhesion of oral bacteria on surfaces that directly reduce the opportunities of Candida to colonize (Wade, 2013). Additionally, some oral streptococci can produce small molecules with antibiotic-like activity that can inhibit the growth of other species and even affect yeast-to-hypha transition of C. albicans, affecting negatively fungal virulence (Lo et al., 1997; Vilchez et al., 2010), which explain that Streptococcus preformed biofilms on Ti did not favor Candida growth. However, on early biofilm all Streptococcus up-regulated Candida EFG1 gene, central regulatory gene of tissue invasion by hyphae, mucosal inflammation and expression of microbial co-aggregation-promoting adhesins (Bertolini et al., 2015; Xu et al., 2017). Therefore, this mutualistic relationship on biofilms growing on Ti surface suggests a high potential pathogenic of these organisms on implant sites.
Cross-kingdom interaction increased tissue damage but did not affect cytokines released during inflammatory process. Since the biofilm accumulation is the main etiologic factor for oral mucosal inflammation, as we expected the higher biofilm formation of mixed biofilm increased tissue damaged. Although some cytokines have been associated during peri-implantitis process (Duarte et al., 2016), the role of these components during immune-response still no clear, and some cytokines evaluated are mainly secreted by T-cells (Abusleme and Moutsopoulos, 2017), showing low concentrations in epithelial cells. In fact, some evidence showed that level of some cytokines, such as TNF-α, was not affected during biofilm accumulation on Ti (Schierano et al., 2008). Moreover, mixed biofilms are more virulent than Candida alone which could start inflammatory process earlier and reduced levels of cytokines identified on late stages of inflammation after complete tissue damaged. In this sense, tissue destruction in early stages by mixed biofilms may occur before cytokines releasing.
An increase in the production of cytokines is expected during cellular defense mechanisms in humans which can be stimulated by microbes (Calderone and Sturtevant, 1994; Castro et al., 1996). Previous study showed reduced metabolic activity and hyphae filamentation by C. albicans growing on plastic due exposure to increasing concentrations of TNF-α (Rocha et al., 2017), but the mechanism still unknown. Growing on cellular layer,
Candida growth was not affected by TNF-α, IL-1β, IFN-γ, and IL-4, but the addition of
granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in decreased fungal growth (Baltch et al., 2001). These data suggest that the effect of inflammatory products on fungal growth is associated with the activities of specific cytokines. A mix of products are released during inflammatory process and it suppressed Candida growth on Ti surface, as single biofilm and with S. gordonii. Therefore, the mechanism of this effect needs to be elucidated. Hematoxylin & eosin-stained and confocal images of tissues showed a higher biofilm layer for mixed biofilms with S. gordonni that suggest a more aggressive biofilm which can modulates inflammatory process and, consequently, suppressing Candida growth.
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Since images suggest a higher biofilm formation on mucosa surface for mixed biofilms with S. gordonii and previous study showed that this interaction on plastic surface is mediated by extracellular polymers synthesized by gtfG from bacteria (Ricker et al., 2014), we explored it for Ti and cellular surface. Although the interaction between these organism has been related to cell-wall proteins from both, such as SspA on bacteria and ALS3 on
Candida (Vickerman et al., 2007; Silverman et al., 2010; Xu et al., 2014a), α-glucan polymers
is also responsible to mediate the binding ability among these organisms (Ricker et al., 2014). On both surfaces, Ti and fibroblast layer, we found that α-glucan synthesized by gtfG promoted biofilm growth, due the effect on biofilm matrix, and Candida count on mixed biofilms, compared to S. gordonii mutant strain lacking gtfG gene. Therefore, glucan polymers contribute for biofilm matrix formation and fungal growth, promoting this cross-kingdom interaction.
In conclusion, for the first time we showed that Streptococcus from mitis group and C.
albicans interacts on biofilms growing Ti surface promoting bacteria growth and increasing
fungal virulence-associated gene expression. Cross-kingdom interaction between these organisms increased tissue damage; and products released during inflammatory process suppressed Candida growth on Ti as single biofilm or with S. gordonii, which interacts with
Candida mediated by extracellular polymers. These results contribute to a mechanistic
understanding of the biofilm growth and associated inflammatory processes that occur in implant sites, ultimately leading to the design of novel strategies to reduce the biofilm formation and to prevent peri-implant mucositis.
REFERENCES
1. Abusleme L, Moutsopoulos NM. IL-17: overview and role in oral immunity and microbiome. Oral Dis. 2017;23(7):854-865.
2. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, et al. Combination of 16S ribosomal RNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Env Microbiol. 1990;56: 1919–1925.
3. Andes D, Nett J, Oschel P, Albrecht R, Marchillo K, Pitula A. Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect Immun. 2004 Oct;72(10):6023-31.
4. Baltch AL, Smith RP, Franke MA, Ritz WJ, Michelsen PB, Bopp LH. Effects of cytokines and fluconazole on the activity of human monocytes against Candida albicans. Antimicrob Agents Chemother. 2001;45(1):96-104.
5. Bertolini M, Ranjan A, Thompson A, Diaz PI, Sobue T, Maas K, Dongari-Bagtzoglou A. Candida albicans induces mucosal bacterial dysbiosis that promotes invasive infection. PLoS Pathog. 2019;15(4):e1007717.
6. Bertolini MM, Xu H, Sobue T, Nobile CJ, Del Bel Cury AA, Dongari-Bagtzoglou A. Candida-streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes. Mol Oral Microbiol. 2015;30(4):307-22.
7. Bowen WH, Burne RA, Wu H, Koo H. Oral Biofilms: Pathogens, Matrix, and Polymicrobial Interactions in Microenvironments. Trends Microbiol. 2018 Mar;26(3):229-242.
8. Calderone R., Sturtevant J. (1994) Macrophage interactions with Candida. Immunol. Ser. 60:505–515.
9. Castro M., Bjoraker J. A., Rohrbach M. S., Limper A. H. (1996) Candida albicans induces the release of inflammatory mediators from human peripheral blood monocytes. Inflammation 20:107–122.
41
10. Cavalcanti YW, Wilson M, Lewis M, Del-Bel-Cury AA, da Silva WJ, Williams DW. Modulation of Candida albicans virulence by bacterial biofilms on titanium surfaces. Biofouling. 2016;32(2):123-34.
11. Charalampakis G, Belibasakis GN. Microbiome of peri-implant infections: lessons from conventional, molecular and metagenomic analyses. Virulence. 2015;6(3):183-7. 12. Charalampakis G, Belibasakis GN. Microbiome of peri-implant infections: lessons from conventional, molecular and metagenomic analyses. Virulence. 2015;6(3):183-7. 13. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial
biofilms. Annu Rev Microbiol. 1995;49:711-45.
14. Diaz PI, Chalmers NI, Rickard AH, Kong C, Milburn CL, Palmer RJ Jr, Kolenbrander PE. Molecular characterization of subject-specific oral microflora during initial colonization of enamel. Appl Environ Microbiol. 2006;72(4):2837-48.
15. Diaz PI, Dupuy AK, Abusleme L, Reese B, Obergfell C, Choquette L, et al. Using high throughput sequencing to explore the biodiversity in oral bacterial communities. Mol Oral Microbiol. 2012b;27(3):182-201.
16. Diaz PI, Xie Z, Sobue T, Thompson A, Biyikoglu B, Ricker A, Ikonomou L, Dongari-Bagtzoglou A. Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model. Infect Immun. 2012c;80(2):620-32. 17. Dongari-Bagtzoglou A, Kashleva H, Dwivedi P, Diaz P, Vasilakos J. Characterization
of mucosal Candida albicans biofilms. PLoS One. 2009; 4:e7967.
18. Dongari-Bagtzoglou A, Kashleva H. Development of a highly reproducible three-dimensional organotypic model of the oral mucosa. Nat Protoc. 2006; 1:2012-2018. 19. Douglas CM, D'Ippolito JA, Shei GJ, Meinz M, Onishi J, Marrinan JA, Li W, Abruzzo
GK, Flattery A, Bartizal K, Mitchell A, Kurtz MB. Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother. 1997;41(11):2471-9.
20. Duarte PM, Serrão CR, Miranda TS, Zanatta LC, Bastos MF, Faveri M, Figueiredo LC, Feres M. Could cytokine levels in the peri-implant crevicular fluid be used to
