Avaliação de biofilmes multiespécies formados em diferentes substratos e expostos a concentrações salivares de agentes antimicrobianos

Orientador: Altair Antoninha Del Bel CuryTese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A colonização de diferentes substratos presentes na cavidade oral por micro-organismos e o desenvolvimento de biofilme são fatores etiológicos da maioria das doenças orais. Além dos dentes, materiais como titânio e polimetilmetacrilato são comumente encontrados neste ambiente e o papel que estes substratos desempenham na prevalência de populações bacteriana e fúngica em biofilmes orais são pouco compreendidas. Além disso, o comportamento da população microbiana de biofilmes orais multiespécies na presença de antimicrobianos liberados na saliva permanece desconhecido. Assim, o objetivo deste estudo foi (i) avaliar o efeito de diferentes substratos na prevalência de micro-organismos em biofilmes orais multiespécies e (ii) o efeito de antimicrobianos liberados na saliva na população microbiana de biofilmes multiespécies. Para o primeiro estudo, discos de hidroxiapatita, titânio e polimetilmetacrilato (PMMA) foram utilizados como substrato para o desenvolvimento do biofilme mimetizando esmalte dental, implantes dentários e base de prótese, respectivamente. O modelo de biofilme multiespécies foi composto por cinco bactérias (Streptococcus oralis, Streptococcus mutans, Actinomyces naeslundii, Veillonella dispar e Fusobacterium nucleatum) e um fungo (Candida albicans). Biofilmes maduros (64,5 h de desenvolvimento) foram removidos por ondas ultrassônicas, plaqueados em meio ágar e as contagens de UFC de cada micro-organismo foram calculadas. A microscopia eletrônica de varredura foi utilizada para visualizar a superfície dos materiais. Os dados foram analisados por ANOVA um critério. Para o segundo estudo o mesmo modelo de biofilme multiespécies foi utilizado. Dois antibióticos, azitromicina e metronidazol, e um antifúngico, fluconazol, foram avaliados. Biofilmes maduros (64,5 h de desenvolvimento) foram expostos a azitromicina, metronidazol ou fluconazol em concentrações encontrada na saliva de 2,12 ug/mL, 15,15 ug/mL e 2,56 ug/mL, respectivamente, por 24h. Após este período, o biofilme foi removido por ondas ultrassônicas, plaqueados em meio ágar e as contagens de UFC de cada micro-organismo foram calculadas. Microscópio eletrônico de varredura e microscópio a laser de varredura confocal com células coradas por hibridização in situ por fluorescência (FISH) foram utilizados para avaliar a estrutura do biofilme. Os dados foram analisados por teste t para amostras independentes e testes não paramétricos de Mann-Whitney. O primeiro estudo não mostrou diferença na população para cada micro-organismo no biofilme entre os entre três materiais avaliados (p>0,05). No segundo estudo, todos os antimicrobianos avaliados foram capazes de alterar a população microbiana (p0.05). In the second study, all antimicrobials evaluated were able to change microbial population (p<0.05), however none of the antimicrobials was able to completely eliminate a specific microorganism from the biofilm. Azithromycin reduced A. naeslundii and V. dispar population while increased C. albicans (p<0.05). Metronidazole reduced all the microorganisms evaluated, with a great reduction for V. dispar and F. nucleatum (p<0.001). Fluconazole reduced C. albicans and F. nucleatum population and increased S. oralis and V. dispar counts (p<0.05). It can be concluded that the substrata were not able to interfere with the formation of multispecies biofilms and antimicrobials in concentrations similar to those released in the saliva changed microbial population, however they were not able to eliminate microorganismsDoutoradoProtese DentalDoutor em Clínica Odontológic

Dental caries prevalence, prospects, and challenges for latin america and caribbean countries: a summary and final recommendations from a regional consensus

Dental caries can be effectively managed and prevented from developing into cavitated lesions while preserving tooth structure at all levels. However, the strong correlation between caries and socioeconomic factors may compromise the efficacy of preventive strategies. The high prevalence of persistent inequalities in dental caries in Latin American and Caribbean countries (LACC) is a matter of concern. The estimates of the burden of disease in some countries in this region are outdated or absent. This paper aims to summarize and present the final recommendations of a regional Consensus for Dental Caries Prevalence, Prospects, and Challenges for LACC. This consensus is based on four articles that were written by a team of Latin American experts, reviewed by dental associations, and presented and discussed in two consensus events. The following domains were explored: epidemiology, risk factors, prevention strategies, and management of dental caries with a focus on restorative procedures. Dental caries can manifest throughout the lifespan of an individual, making it a matter of concern for infants, children, adults, and older people alike. The prevalence rates of untreated caries in deciduous and permanent teeth are high in many parts of the world, including LACCs. Previous evidence suggests that the prevalence of dental caries in 12-year-olds is moderate to high in most Latin American countries. Moreover, the prevalence of treatment needs and dental caries in the adult and elderly population can also be regarded as high in this region. The risk/protective factors (e.g., sugar consumption, exposure to fluoride, and oral hygiene) probably operate similarly in all LACCs, although variations in the interplay of these factors in some countries and within the same country cannot be ruled out. Although salt and water fluoridation programs are implemented in many countries, there is a need for implementation of a surveillance policy. There is also room for improvement with regard to the introduction of minimal intervention techniques in practice and public health programs. Dental caries is a marker of social disadvantage, and oral health promotion programs and interventions aimed at reducing the burden of dental caries in LACCs must consider the complexity of the socioeconomic dynamics in this region. There is an urgent need to promote engagement of stakeholders, policymakers, medical personnel, universities, dental associations, community members, and industries to develop regional plans that enhance the oral health agenda for LACCs. A list of recommendations has been presented to underpin strategies aimed at reducing the prevalence and severity of dental caries and improving the quality of life of the impacted LACC population in the near future

Biofilm extracellular polysaccharides degradation during starvation and enamel demineralization.

This study was conducted to evaluate if extracellular polysaccharides (EPS) are used by Streptococcus mutans (Sm) biofilm during night starvation, contributing to enamel demineralization increasing occurred during daily sugar exposure. Sm biofilms were formed during 5 days on bovine enamel slabs of known surface hardness (SH). The biofilms were exposed to sucrose 10% or glucose + fructose 10.5% (carbohydrates that differ on EPS formation), 8x/day but were maintained in starvation during the night. Biofilm samples were harvested during two moments, on the end of the 4th day and in the morning of the 5th day, conditions of sugar abundance and starvation, respectively. The slabs were also collected to evaluate the percentage of surface hardness loss (%SHL). The biofilms were analyzed for EPS soluble and insoluble and intracellular polysaccharides (IPS), viable bacteria (CFU), biofilm architecture and biomass. pH, calcium and acid concentration were determined in the culture medium. The data were analyzed by two-way ANOVA followed by Tukey's test or Student's t-test. The effect of the factor carbohydrate treatment for polysaccharide analysis was significant (p 0.05). Larger amounts of soluble and insoluble EPS and IPS were formed in the sucrose group when compared to glucose + fructose group (p < 0.05), but they were not metabolized during starvation time (S-EPS, p = 0.93; I-EPS, p = 0.11; and IPS = 0.96). Greater enamel %SHL was also found for the sucrose group (p < 0.05) but the demineralization did not increase during starvation (p = 0.09). In conclusion, the findings suggest that EPS metabolization by S. mutans during night starvation do not contribute to increase enamel demineralization occurred during the daily abundance of sugar

Biofilm extracellular polysaccharides degradation during starvation and enamel demineralization - Fig 2

<p><b>Tridimensional reconstruction of CLSM images of <i>S</i>. <i>mutans</i> biofilms formed under exposure to glucose + fructose (A and B) or sucrose (C and D) 8x/daily</b>. Images A and C show biofilms visualized at abundance moment, while images B and D at starvation moment. In green, <i>S</i>. <i>mutans</i> cells stained with SYTO 9. In red, extracellular polysaccharides labeled with Alexa Fluor 647—dextran conjugate. Oil immersion objective of 40x (numeric aperture 1.25).</p

Amounts (μg) per enamel slab of extracellular polysaccharides, soluble (S-EPS) and insoluble (I-EPS), and intracellular polysaccharides (IPS) according to the treatments (glucose + fructose or sucrose) and the harvest moment (abundance or starvation).

<p>Distinct capital letters indicate significant statistically differences among groups for each polysaccharide type (p < 0.05) (Mean ± SD; n = 12).</p

pH values of the culture medium according to the biofilm treatments (glucose + fructose or sucrose) and biofilm development time (h) as an indicator of biofilm acidogenicity.

<p>Time points at 80 h and 96 h refer to the harvest moment at abundance and starvation of carbohydrates, respectively. Asterisks indicate statistically significant difference between treatments at the time point evaluated (p < 0.05). (Means ± SD; n = 12).</p

Concentration of organic compounds (mM) in the culture medium according to the biofilm treatments (glucose + fructose or sucrose) and the harvest moment (abundance or starvation).

<p>Distinct capital letters indicate significant statistically differences among groups for each type organic compound (p < 0.05). (Mean ± SD; n = 6).</p

Mean ± SD (n = 12) of biofilm dry weight (mg), CFU counts (Log₁₀), CFU counts /mg biofilm dry weight (CFU Log₁₀/mg), %SHL, cumulative calcium released from enamel (μg) and biomass (μm³/μm²) according to the treatments (glucose + fructose or sucrose) and the harvest moment (abundance or starvation).

<p>Mean ± SD (n = 12) of biofilm dry weight (mg), CFU counts (Log₁₀), CFU counts /mg biofilm dry weight (CFU Log₁₀/mg), %SHL, cumulative calcium released from enamel (μg) and biomass (μm³/μm²) according to the treatments (glucose + fructose or sucrose) and the harvest moment (abundance or starvation).</p