Evaluation of microbial interactions and the role of Streptococcus oralis and Actinomyces oris adhesins in the formation and structure of mixed biofilms with Candida albicans

Orientador: Altair Antoninha Del Bel CuryTese (doutorado) Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A avaliação das interações microbianas é de suma importância para a compreensão do desenvolvimento de comunidades e seu controle. Na forma estruturada de biofilmes, bactérias estão aderidas às supefícies recobertas com película de saliva e continuam interagindo com as demais espécies no fenômeno de coadesão e coagregação. Essas interações ocorrem via moléculas denominadas adesinas, presentes na superfície dos micro-organismos. Dois importantes grupos de adesinas são o receptor de polissacarídeos RPS (do inglês Receptor of Polysaccharide) presente em S. oralis (So) e as fímbrias na superfície de Actinomyces oris (Ao), bactérias colonizadoras iniciais das superfícies orais. Ambas possuem papel essencial na adesão bacteriana à película de saliva, na interação com demais micro-organismos e na formação de biofilmes. O relacionamento desses micro-organismos e a base molecular das interações com o fungo C. albicans é motivo de estudo. Os objetivos deste estudo foram avaliar as interações microbianas entre S. oralis (So), A. oris (Ao) com C. albicans (Ca) e o papel de RPS e fímbrias tipo I e II nas comunidades mistas. Para isto, foram organizadas combinações duo e tri-espécies entre os micro-organismos em cepas silvestres: I (Ao + Ca), II (So + Ca) e III (So + Ao + Ca) e com cepas mutantes que não expressam RPS e fímbrias I/II: IV (Ao ?Fim + Ca), V (So ?RPS + Ca), VI (Ao ?Fim + So + Ca) e VII (Ao ?Fim + So ?RPS + Ca). Também foram desenvolvidos biofilmes mono-espécie de cada micro-organismo. Os micro-organismos cresceram em suspensão (planctonicamente) por 3 h em meio YPTG (YNB, peptona e triptona em tampão fosfato suplementado com glicose) e as interações presentes foram avaliadas por meio de microscopia de fluorescência. Os biofilmes foram desenvolvidos sobre discos de resina de polimetilmetacrilato (PMMA) e sobre lâminas de vidro, recobertas com película de saliva (2 h) e cresceram em mesmo meio até 24 h. A viabilidade celular foi determinada pela biomassa em biofilmes de 7 h e por Unidades Formadoras de Colônias (UFC/mL) em biofilmes de 1,5 e 24 h. O desenvolvimento e distribuição espacial dos micro-organismos foram avaliados pelas microscopias óptica, confocal a laser e de fluorescência. So e Ao interagem entre si na forma silvestre e diferentes formações de biofilmes foram observadas na presença de uma ou duas bactérias mutantes. Todas as bactérias interagem planctonicamente com Ca nas combinações e não competem por sítios de ligação na superfície do fungo. A presença de Ca em biofilmes duo-espécies influencia o aumento da comunidade bacteriana e a formação de biofilmes em cepa mutante de Ao, bem como as bactérias estimulam o crescimento de hifas. Há uma nova distribuição e predominância de células nos biofilmes tri-espécies contendo cepas mutantes. Com base nestes resultados, conclui-se que as bactérias colonizadoras iniciais So e Ao interagem entre si e com C. albicans na formas planctônicas e de biofilmes, porém as interações interespécies são independentes das adesinas RPS e fímbrias I/IIAbstract: The evaluation of microbial interactions is extremely important for the comprehension of communities¿ development and their control. As biofilm form, bacteria are adhered to salivary-pellicle covered-surfaces and still interacting with other species for coadhesion and coaggregation. These interactions occur due to adhesins molecules present onto microorganisms surfaces. Two important adhesins are the Receptor of Polysaccharide (RPS) in S. oralis (So) and fimbriae onto Actinomyces oris (Ao) surface, both bacteria are first colonizers of oral surfaces. These adhesins have essential role in bacterial adhesion onto the pellicle, in the interaction with other microorganisms and on biofilm formation. Their relationship and the molecular base of interactions with the fungus C. albicans are still under investigation. The aims of this study were to evaluate the interactions between S. oralis (So), A. oris (Ao) with C. albicans (Ca) and the role of RPS and Fimbriae I/II planktonically and as biofilms. Combinations of dual and triadic species in wild types and mutants strains lacking RPS and Fimbriae types I and II were designed for that: I (Ao + Ca), II (So + Ca), III (So + Ao + Ca), IV (Ao ?Fim + Ca), V (So ?RPS + Ca), VI (Ao ?Fim + So + Ca) and VII (Ao ?Fim + So ?RPS + Ca). The mono-species biofilms were developed as well. The microorganisms grown planktonically for 3 hours in YPTG medium (YNB, peptone, triptone in phosphate buffer supplemented with glucose) and the interactions evaluated by fluorescence microscopy. The biofilms were developed onto resin discs and glass cover slips covered with salivary pellicle (2 hours) in the same medium until 24 hours. The viability was determined by biomass in 7 hours biofilms and Colony-forming units (CFU/mL) in 1.5 and 24 hours biofilms. The temporal development and microbial distribution were evaluated by confocal, optical and fluorescence microscopies. So and Ao interacted as wild types and particular biofilm formation was observed in the presence of one or two mutant strains. All bacteria interacted planktonically with Ca in all combinations and do not compete for binding sites onto fungal surface. The presence of Ca in biofilms influenced the bacterial community improvement, Ao mutant strain to biofilm formation and bacteria stimulated hyphal growth. There is a new distribution and cell prevalence in three-species biofilms containing mutant bacteria. Based on these results, it can be concluded that first colonizers bacteria So and Ao interact with Ca planktonically or within biofilms, although the interspecies interactions are RPS and fimbriae independentDoutoradoProtese DentalDoutora em Clínica Odontológica2013/15884-108808/13-9FAPESPCAPESBE

Influence Of Substratum Position And Acquired Pellicle On Candida Albicans Biofilm.

The purpose of this study was to evaluate the influence of the substratum position and the saliva acquired pellicle (AP) on Candida albicans biofilm development. Poly(methylmethacrylate) (PMMA) disks were fabricated and randomly allocated to experimental groups: HNP (disks placed in a horizontal position and uncoated by pellicle), VNP (disks placed in a vertical position and uncoated by pellicle), HCP (disks placed in a horizontal position and coated by pellicle), and VCP (disks placed in a vertical position and coated by pellicle). Disks were placed in a 24-well plate and a suspension of 107 cells/mL of Candida albicans was added to each well for biofilm development. The plates were aerobically incubated at 35°C. The biofilms were evaluated at 1.5 (adhesion time point), 24, 48, 72, and 96 hours. The number of viable cells was quantified in terms of the colony-forming units per milliliter (CFU/mL). Metabolic activity was measured by the XTT assay. The biofilm structure was analyzed by scanning electron microscopy. The data were analyzed by three-way ANOVA followed by Tukey's test, with significance set at 5%. The vertical groups showed less biofilm formation and lower metabolic activity than the horizontal groups (p0.05). It can be concluded that the substratum position influenced biofilm development.27369-7

Influence of titanium nitride by cold-plasma on formation and development of multiespecies biofilms

Orientador: Altair Antoninha Del Bel CuryDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Tratamentos que alteram propriedades de superfície de titânio são realizados em implantes odontológicos visando uma melhor sinalização celular e a neoformação óssea. Porém, em determinadas situações clínicas, as superfícies de titânio expostas ao meio oral recobertas por película adquirida (PA) podem se tornar substratos para o desenvolvimento de biofilmes associados às doenças inflamatórias, como a peri-implantite. Diante desta observação, os objetivos deste estudo foram (i) caracterizar a superfície de titânio nitretada por plasma a frio quanto às propriedades de rugosidade, topografia, composição química e energia livre de superfície (ELS); (ii) determinar o perfil protéico da PA adsorvida às superfícies (iii) avaliar a influência do tratamento de superfície na formação e desenvolvimento de biofilmes multiespécies. Para o estudo, discos de titânio grau IV receberam polimento e acabamento e foram divididos aleatoriamente no grupo controle (Ti) e experimental TiN (nitretado por plasma a frio). As superfícies foram caracterizadas através da microscopia eletrônica de varredura (MEV) e a rugosidade e topografia determinadas pela Microscopia de Força Atômica (n = 4). A ELS foi avaliada pela técnica ácido-base e leitura em goniômetro (n = 9) e a composição química da superfície foi determinada por Espectroscopia Fotoeletrônica de Raios-X (XPS) (n = 4). Em seguida, os discos foram imersos em saliva para formação de PA por 2 horas e novamente a ELS foi determinada (n = 6). O perfil protéico da PA foi determinado por espectrometria de massas LC-MS/MS (n = 18). Um biofilme composto por um fungo e seis bactérias (Candida albicans, Veillonella dispar, Streptococcus mutans, Streptococcus oralis, Fusubacterium nucleatum e Actinomyces naeslundii), foi desenvolvido durante 64,5 horas sobre os discos com película. Após este período, os micro-organismos e o biofilme total foram quantificados em células viáveis (n = 12). A topografia e a organização dos biofilmes foram analisadas por MEV e pela microscopia confocal a laser. Os dados de células viáveis e perfil protéico foram avaliados estatisticamente pelo Teste t de Student e os dados de ELS pela análise de variância (ANOVA) de dois fatores seguida pelo teste de Tukey com nível de significância de 5 %. Os resultados demonstraram não haver diferença entre as propriedades de rugosidade e topografia entre os grupos e um maior pico de nitrogênio foi detectado na composição química da superfície nitretada. O tratamento não alterou a ELS, que aumentou apenas na presença de PA (p < 0.001). Diferentes proteínas se adsorveram a superfície nitretada. A quantidade de células viáveis do biofilme formado nas duas superfícies foi semelhante (p = 0.416), confirmado pelas microscopias, porém Fusobacterium nucleatum e Streptococcus oralis foram quantificados em maior número nos biofilmes do grupo TiN. Conclui-se que a nitretação por plasma a frio não alterou as propriedades de superfície de titânio e não influenciou a quantidade de biofilme formado. Porém, a superfície nitretada aumentou as contagens de Fusobacterium nucleatum e Streptococcus oralis e selecionou diferentes proteínas na PAAbstract: Surface treatments that alter titanium properties titanium are performed on dental implants in order to improve the cell signalization and bone formation. Although, in certain clinical situations, the surfaces exposed to oral cavity covered by acquired-pellicle (AP) may become substrates for development of biofilms associated with inflammatory diseases as periimplantitis. Given this observation, the aims of this study were (i) to characterize the surface properties of titanium nitride by cold plasma as roughness, topography, chemical composition and surface free energy (SFE), (ii) determine the protein profile of AP adsorbed to the surfaces (iii) evaluate the influence of surface treatment on formation and development of multispecies biofilms. For the study, titanium discs grade IV received polish and finish and were randomly allocated to control (Ti) and experimental TiN (nitride by cold-plasma) groups. The surfaces were characterized by scanning electron microscopy (SEM) and roughness and topography determined by Atomic Force Microscopy (n = 4). The SFE was evaluated using the acid-base technique and read in goniometer (n = 9). The surface chemical composition was determined by x-ray photo-electron spectroscopy (XPS) (n = 4). Then, the discs were immersed in saliva for AP formation per 2 hours and again the SFE was determined (n = 6). The AP protein profile was determined by mass spectrometry LC-MS/MS (n = 18). A biofilm composed by five bacteria and one fugal (Candida albicans, Veillonella dispar, Streptococcus mutans, Streptococcus oralis, Actinomyces naeslundii and Fusobacterium nucleatum) was conducted for 64.5 hours on coated-discs. After this period, the viable cells of biofilms were determined (n = 12). The biofilms topography and organization were analyzed by SEM and by confocal laser microscopy. The data of viable cells and protein profile were evaluated statistically by Student's t test and SFE data by two-way ANOVA followed by Tukey's test with a significance level of 5%. The results showed no difference between the properties of roughness and topography in groups. A high peak of nitrogen was detected in the chemical composition of the nitride surface. The treatment did not alter the SFE, that increased in the presence of AP (p <0.001). Different protein adsorbed to nitride surface. The amount of viable cells in the biofilm formed on both surfaces was similar (p = 0.416), confirmed by microscopies. The number of viable cells of Streptococcus oralis and Fusobacterium nucleatum were higher in TiN. It was concluded that the cold plasma nitriding did not alter the titanium surface properties and did not affect the amount of biofilm. However, it increased the counts of Fusobacterium nucleatum and Streptococcus oralis and selected different proteins in APMestradoProtese DentalMestra em Clínica Odontológic

Influence of substratum position and acquired pellicle on <italic>Candida albicans</italic> biofilm

<p>The purpose of this study was to evaluate the influence of the substratum position and the saliva acquired pellicle (AP) on <italic>Candida albicans</italic> biofilm development. Poly(methylmethacrylate) (PMMA) disks were fabricated and randomly allocated to experimental groups: HNP (disks placed in a horizontal position and uncoated by pellicle), VNP (disks placed in a vertical position and uncoated by pellicle), HCP (disks placed in a horizontal position and coated by pellicle), and VCP (disks placed in a vertical position and coated by pellicle). Disks were placed in a 24-well plate and a suspension of 10⁷cells/mL of <italic>Candida albicans</italic> was added to each well for biofilm development. The plates were aerobically incubated at 35&#176;C. The biofilms were evaluated at 1.5 (adhesion time point), 24, 48, 72, and 96 hours. The number of viable cells was quantified in terms of the colony-forming units per milliliter (CFU/mL). Metabolic activity was measured by the XTT assay. The biofilm structure was analyzed by scanning electron microscopy. The data were analyzed by three-way ANOVA followed by Tukey's test, with significance set at 5%. The vertical groups showed less biofilm formation and lower metabolic activity than the horizontal groups (<italic>p</italic>< 0.05). Significant differences in cell viability and metabolic activity were observed between the adhesion and other time points (<italic>p</italic>< 0.05), but these variables were not affected by the presence of the pellicle (<italic>p</italic> > 0.05). It can be concluded that the substratum position influenced biofilm development.</p

Semiautomatic device for in vitro/ experimental bone perforation in dental implant research

The present study presents a semiautomatic device developed to perform in vitro experiments using surgical drills for assisting dental implant research. It was built to perform tests independent of human direct contact, and contains an adjustable toolholder for engaging different types of implant contra angle hand pieces, in which different drills can be adapted. The researcher is able to make a range of adjustments on the machine, such as controlling the drilling force and depth. Materials and methods: The device was tested on samples of both synthetic and natural bone with type I density, and a sequence of drills selected to perform the perforations. Drilling time and perforation force exerted during drilling were evaluated, as both parameters are required to be standardized. Results: It was observed that the drilling performed using the device was uniform using both types of bone, although the drilling time for the synthetic bone was higher. All perforations were exactly on the spot previously determined, and without variations in drill angulations. The perforation force was higher for the lance pilot drill for both bone types, and the natural bone required a higher axial force than the synthetic bone. Conclusion: Thus, we consider this device trustable to perform standardized analysis and provide accurate results. It can be used for tests performed in universities and companies that develop dental implant materials and products47699199