Estudo in vitro e in situ da ação antimicrobiana no laser diodo associado a fotossensibilizador sobre microrganismos bucais

Orientador: Reginaldo Bruno GonçalvesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A redução de microrganismos patogênicos da superfície dental é um dos fatores principais envolvidos na prevenção e controle da doença cárie. Neste estudo avaliou-se o efeito antimicrobiano da terapia fotodinâmica sobre microrganismos bucais in vitro e in situ utilizando um laser diodo de 660 nm (meio ativo de arseneto-gálio-alumínio da DMC Equipamentos Ltda, emissão contínua, potência de entrada na sonda de 21 mW, sonda de 0,8 cm de diâmetro com perda de 24%, potência de saída na sonda de 16 mW e densidade de energia de 28,8 J/cm2) associado ao corante azul de toluidina O na concentração de 100 'mu'/mL (Sigma CI 52040). Suspensões bacterianas foram submetidas aos tratamentos com corante, com laser e com corante e laser ao mesmo tempo. A total inibição de crescimento foi encontrada in vitro para Streptococcus mutans, Streptococcus sobrinus, Streptococcus. cricetus, Laclobacillus acidophilus e Lactobacillus casei. Reduções estatisticamente significativas foram encontradas para Streptococcus sanguinis e a ausência de efeito antimicrobiano foi observado para Cândida albicans. A aplicação da terapia fotodinâmica sobre pool de saliva humana in vitro demonstrou inibição total de crescimento para estreptococos grupo mutans e inibição parcial de crescimento para estreptococos totais. Os resultados da sensibilização dos microrganismos crescidos em biofilme dental in situ demonstraram total inibição de crescimento para estreptococos grupo mutans, estreptococos totais e lactobacilos com reduções nas contagens respectivamente de 2,4 x 10 'POT. 5¿, 4,8 X 10 'POT. 6¿ ,e 9,7 x 10 'POT. 5¿ nos grupos não tratados para zero nos grupos submetidos à terapia fotodinâmica. A candida apresentou inibição parcial de crescimento estatisticamente significativa in situ. Assim, a terapia fotodinâmica é efetiva para a redução de bactérias cariogênicas in vitro e in situ nos parâmetros testadosAbstract: Elimination of pathogenic microorganisms from dental surface has been one of the most important factors related to dental caries control and prevention. In order to test the antimicrobial effect of photodynamic therapy on the susceptibility of oral microorganisms, the diode laser at 660 nm (a gallium-aluminium-arsenide laser from DMC Equipments Ltda, continuous mode, power input of 21 mW, sound diameter of 0,8 cm with loss of 24%, power output of 16 mW and energy density of 28,8 J/cm2) in association with toluidine blue O at a concentration of 100 'mu¿g/mL (Sigma CI 52040) was evaluated in vitro and in situ. Suspensions of microorganisms were exposed to treatment with the dye, the laser and to dye and the laser together. Streptococcus mutans, Streptococcus sobrinus, Streptococcus cricetus, Lactobacillus acidophilus and Lactobacillus casei showed total growth inhibition in vitro. A statistically significant reduction in microorganism was observed for Streptococcus sanguis, and no effect on the viability of Candida albicans was found after therapy. Total growth inhibition to mutans streptococci and partial growth inhibition to total streptococci were found when human saliva was exposed to the photodynamic therapy. Following, an in situ experiment was conducted in order to verify the effect of this therapy on dental biofilms. Microorganisms grown in biofilm presented total growth inhibition of mutans streptococci, total streptococci, and lactobacilli with the viable count, being respectively reduced from 2,4 x 10 'POT. 5¿, 4,8 X 10 'POT. 6¿, e 9,7 x 10 'POT. 5¿ (control groups) to zero (groups exposed to both laser and dye). A partial growth inhibition statistically significant in microorganism was observed for candida in situ. In conclusion, it was observed that the photodynamic therapy can eftectively reduce in vitro and in situ cariogenic bacteria on tested parametersMestradoCariologiaMestre em Odontologi

Occurrence and antimicrobial susceptibility of enteric rods and pseudomonads isolated from the dental prostheses biofilm

ABSTRACT Aspiration of oral bacteria leads to cardiac and respiratory infectious diseases and dentures can act as a reservoir for pathogenic microorganisms. Objective: To determine the occurrence and the in vitro antimicrobial susceptibility of enteric rods and pseudomonads from the denture biofilm of 52 subjects at the Center for Dental Specialties of Sobral/ Ceara, Brazil. Material and Methods: Denture biofilm was collected and samples plated on MacConkey agar. The isolated bacterial colonies were identified using the BBL Crystal enteric/non-fermenter system. Antibiotic bacterial susceptibility was assessed by the disc diffusion method of amoxicillin, amoxicillin/clavulanic acid, doxycycline, tetracycline, tobramycin, imipenem, cefotaxime, and ciprofloxacin. The Minimum Inhibitory Concentration (MIC) of cefotaxime, tobramycin, doxycycline, imipenem, and ciprofloxacin was determined for 40 species by E-test. Results: 34 subjects (65.4%) harbored enteric rods in their prostheses. Klebsiella pneumoniae (26.5%), Escherichia coli (23.5%), and Enterobacter aerogenes (23.5%) were the most prevalent species. All organisms were susceptible to ciprofloxacin and most species were resistant to amoxicillin or amoxicillin/clavulanic acid, demonstrating variable sensitivity patterns to other antimicrobials. However, the MIC showed the emergence of strains with reduced sensitivity to ciprofloxacin (MIC90≥3 μg/ mL) and cefotaxime (MIC90≥2 μg/mL). Conclusion: The findings show high prevalence of nosocomial diseases-related bacterial species and low susceptibility to antimicrobial drugs. Therefore, these results imply caution against the indiscriminate use of broad spectrum antibiotics in dental practice

Antimicrobial effect of photodynamic therapy on oral biofilms

Orientadores : Reginaldo Bruno Gonçalves, Jaime Aparecido CuryTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A terapia fotodinâmica (TFD) é um tratamento antibacteriano alternativo para as doenças causadas por biofilmes como a cárie dental. Esta tese, apresentada na forma de 3 artigos, teve por objetivos: (1) revisar a literatura enfocando o efeito da TFD em bactérias cariogênicas, (2) validar um modelo de formação de biofilme de mono-espécies de estreptococos e avaliar o efeito da associação do azul de orto-toluidina (AOT) e um diodo emissor de luz (LED) vermelho sobre biofilmes formados pela associação dessas bactérias e, (3) avaliar o efeito da TFD utilizando um laser de HeNe ou uma luz LED vermelha sobre biofilmes de S. mutans crescidos em fermentador de espessura e fluxo contínuos (FEFC). No artigo 2, biofilmes de Streptococcus mutans, Streptococcus sobrinus e Streptococcus sanguinis foram formados in vitro sobre blocos de esmalte dental bovino. Nos biofilmes avaliou-se a contagem microbiológica e a concentração de polissacarídeo extracelular insolúvel, sendo que no esmalte foi determinada a perda de mineral. O potencial antimicrobiano da associação AOT a 100 mg/ml e um LED com densidade de energia de 85,7 J/cm2 sobre estes biofilmes foi avaliado através de contagem das bactérias viáveis. No artigo 3, foi testado o efeito antimicrobiano da TFD utilizando densidades de energia de um laser de HeNe ou uma luz LED vermelha variando entre 49 e 294 Jcm-2, sobre biofilmes de S. mutans crescidos em um FEFC. O efeito da TFD foi avaliado através da análise da viabilidade bacteriana e microscopia confocal a laser. A revisão da literatura demonstrou a relevância e a escassez de estudos utilizando a TFD sobre biofilmes cariogênicos. Os resultados do artigo 2 demonstraram que o modelo de formação de biofilme in vitro testado foi efetivo para simular situações de alto desafio cariogênico promovendo a desmineralização do esmalte, sendo que a aplicação da TFD foi eficaz para reduzir significantemente a viabilidade bacteriana nos biofilmes (p < 0,05). No terceiro estudo, reduções significativas (p < 0,05) da viabilidade S. mutans foram observadas após a TFD tanto com utilização do laser de HeNe como pela luz LED, porém este efeito se limitou as camadas externas dos biofilmes. No conjunto, os resultados desses estudos indicam que tanto o laser HeNe quanto a luz LED na presença de AOT apresentam efeito antimicrobiano sobre biofilmes de estreptococosAbstract: Photodynamic therapy (PDT) is an alternative antibacterial therapeutic to biofilm-related diseases such as dental caries. This thesis, comprised by 3 manuscripts was designed (1) to review the literature about the use of PDT on cariogenic bacteria, (2) to validate a streptococci biofilm model and to evaluate the effect of toluidine blue O (TBO) and a red Light Emission Diode (LED) association on viability of biofilmes formed with these bacteria and, (3) to evaluate the effect of PDT using either a HeNe laser or a red LED light on the viability of S. mutans biofilms grown in a constant depth film fermentor (CDFF). In the second manuscript, Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguinis biofilms were grown on bovine enamel blocks. In the biofilms the microbiological counting and water-insoluble polysaccharide concentrations were analyzed; while mineral loss analysis were performed on enamel blocks. Also, the antimicrobial potential of TBO at 100 mg/ml associated with a LED at 85.7 Jcm-2 on single-species biofilms were evaluated by microbiological counting. In the manuscript 3, the antimicrobial effect of PDT on S. mutans biofilmes growth on CDFF were verified either using a HeNe laser or a red LED with energy density ranging from 49 to 294 Jcm-2. The effect of TFD was verified by analyzing bacterial viability and confocal scanner laser microscopy. The review of literature demonstrated the importance and shortage of studies about PDT on cariogenic biofilms. The results of manuscript 2 showed that the tested biofilm was effective to simulate high cariogenic challenger promoting enamel demineralization. Also, significant reductions (p = 0.05) in bacteria viability were founded after PDT. At the third study, significant reductions in S. mutans biofilms counting was observed after PDT using either a HeNe laser or a LED light the both light (p = 0.05), however, the effect was restricted to external biofilm layers. In conclusion, the results of these studies showed that either a HeNe laser or a LED light in the presence of TBO showed antimicrobial effect on streptococci biofilmsDoutoradoCariologiaDoutor em Odontologi

Occurrence and antimicrobial susceptibility of enteric rods and pseudomonads isolated from the dental prostheses biofilm

Aspiration of oral bacteria leads to cardiac and respiratory infectious diseases and dentures can act as a reservoir for pathogenic microorganisms. Objective: To determine the occurrence and the in vitro antimicrobial susceptibility of enteric rods and pseudomonads from the denture biofilm of 52 subjects at the Center for Dental Specialties of Sobral/ Ceara, Brazil. Material and Methods: Denture biofilm was collected and samples plated on MacConkey agar. The isolated bacterial colonies were identified using the BBL Crystal enteric/non-fermenter system. Antibiotic bacterial susceptibility was assessed by the disc diffusion method of amoxicillin, amoxicillin/clavulanic acid, doxycycline, tetracycline, tobramycin, imipenem, cefotaxime, and ciprofloxacin. The Minimum Inhibitory Concentration (MIC) of cefotaxime, tobramycin, doxycycline, imipenem, and ciprofloxacin was determined for 40 species by E-test. Results: 34 subjects (65.4%) harbored enteric rods in their prostheses. Klebsiella pneumoniae (26.5%), Escherichia coli (23.5%), and Enterobacter aerogenes (23.5%) were the most prevalent species. All organisms were susceptible to ciprofloxacin and most species were resistant to amoxicillin or amoxicillin/clavulanic acid, demonstrating variable sensitivity patterns to other antimicrobials. However, the MIC showed the emergence of strains with reduced sensitivity to ciprofloxacin (MIC90≥3 μg/ mL) and cefotaxime (MIC90≥2 μg/mL). Conclusion: The findings show high prevalence of nosocomial diseases-related bacterial species and low susceptibility to antimicrobial drugs. Therefore, these results imply caution against the indiscriminate use of broad spectrum antibiotics in dental practice

Occurrence and antimicrobial susceptibility of enteric rods and pseudomonads isolated from the dental prostheses biofilm

ABSTRACT Aspiration of oral bacteria leads to cardiac and respiratory infectious diseases and dentures can act as a reservoir for pathogenic microorganisms. Objective: To determine the occurrence and the in vitro antimicrobial susceptibility of enteric rods and pseudomonads from the denture biofilm of 52 subjects at the Center for Dental Specialties of Sobral/ Ceara, Brazil. Material and Methods: Denture biofilm was collected and samples plated on MacConkey agar. The isolated bacterial colonies were identified using the BBL Crystal enteric/non-fermenter system. Antibiotic bacterial susceptibility was assessed by the disc diffusion method of amoxicillin, amoxicillin/clavulanic acid, doxycycline, tetracycline, tobramycin, imipenem, cefotaxime, and ciprofloxacin. The Minimum Inhibitory Concentration (MIC) of cefotaxime, tobramycin, doxycycline, imipenem, and ciprofloxacin was determined for 40 species by E-test. Results: 34 subjects (65.4%) harbored enteric rods in their prostheses. Klebsiella pneumoniae (26.5%), Escherichia coli (23.5%), and Enterobacter aerogenes (23.5%) were the most prevalent species. All organisms were susceptible to ciprofloxacin and most species were resistant to amoxicillin or amoxicillin/clavulanic acid, demonstrating variable sensitivity patterns to other antimicrobials. However, the MIC showed the emergence of strains with reduced sensitivity to ciprofloxacin (MIC90≥3 μg/ mL) and cefotaxime (MIC90≥2 μg/mL). Conclusion: The findings show high prevalence of nosocomial diseases-related bacterial species and low susceptibility to antimicrobial drugs. Therefore, these results imply caution against the indiscriminate use of broad spectrum antibiotics in dental practice