Efeito do óleo essencial de folha de canela verdadeira (Cinnamomum verum) contra biofilmes in vitro de Candida e seu efeito citotóxico

Orientador: José Francisco HoflingDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O óleo essencial extraído das folhas de Cinnamomum verum tem sido usado a tempos como um promissor agente antibacteriano e antifúngico. O objetivo desta pesquisa foi avaliar a atividade antifúngica do óleo essencial das folhas de C. verum contra cepas padrão de C. albicans (ATCC MYA-2876), C. tropicalis (ATCC 750) and C. dubliniensis (ATCC MYA-646). O efeito do óleo essencial C. verum na fase de vapor foi avaliado pela técnica da microatmosfera. A microdiluição em caldo CL27 M27-A3 foi empregada na determinação da CIM e MFC. O tempo necessário para agir sobre a Candida planctônica, o efeito na adesão inicial e a formação do tubo germinativo foram avaliados usando protocolos padrão. O efeito do óleo essencial de C. verum na formação do biofilme foi quantificado usando o ensaio de viabilidade XTT, visualizado por Microscopia Eletrônica de Varredura (MEV) e a progressão do biofilme na presença do óleo essencial foi visualizada no microscópio Time Lapse. O efeito do óleo essencial de C. verum nos biofilmes formados foi quantificado por teste de viabilidade XTT e SEM, respectivamente. Alterações intracelulares pós-exposição foram visualizadas usando Microscopia Eletrônica de Transmissão (MET). As avaliações toxicológicas foram levadas a efeito com a linha de células de queratinócitos humanos (HaCaT) e larvas de Galleria mellonella. Antifúngico comercial e digluconato de clorexidina foi utilizado como controle positivo. Todos os testes foram feitos em triplicatas. Múltiplas médias de mais de três conjuntos de dados foram comparadas usando ANOVA unidirecional e ANOVA bidirecional. O nível de significância adotado foi de 5% (p 2,0 mg/ml e 0,34 mg/ml para C. albicans, C. tropicalis e C. dubliniensis, respectivamente. 0,5 e 1,0 mg/ml inibe significativamente a formação de tubos germinativos de C. albicans e C. dubliniensis. A concentração mínima inibidora de biofilme (MBIC50) em formação e biofilmes estabelecidos foi 2.0 mg/ml and 0.34 mg/ml by C. albicans, C. tropicalis and C. dubliniensis respectively. 0.5 And 1.0 mg/ml significantly inhibit the germ tube formation of C. albicans and C. dubliniensis. Minimum Biofilm Inhibitory Concentration (MBIC50) for forming and established biofilms were <0.2 mg/ml for all test strains. C. verum leaf oil caused retardation in biofilm development. SEM images exhibited cell wall damages, cellular shrinkages and decreased hyphal formation of Candida. TEM indicated intracellular vacuolation, cell wall damages and cytoplasmic granulation. Cinnamon leaf oil caused no inhibition of HaCaT cell at maximum concentration tested (1000 mg/ml). Essential oil of Cinnamon leaf didn¿t exhibit a lethal effect at any concentration tested on G. mellonella in vivo assay. C. verum leaf oil is a potential alternative anti-Candida agent with minimal toxicityMestradoMicrobiologia e ImunologiaMestre em Biologia Buco-Dental132718/2018-9CNP

Ayurvedic, herbal extracts suppress Candidal biofilms in vitro

Plant derivatives have been used for centuries to treat various human afflictions including microbial infections. A vast majority of these infections are initiated and perpetuated by community dwelling, surface-attached organisms living in micro-econiches known as biofilms. We investigated the biofilm suppressant effect of phytomedicinal preparations used widely in traditional Ayurvedic medicine, Triphala, a mixture of Terminalia bellirica, Terminalia chebula and Emblica officinalis, and Mimusops elengi bark extract. Inhibitory effect of extracts were first investigated against the planktonic C. albicans and C. tropicalis using the well diffusion. Minimum biofilm inhibitory concentration for in-vitro biofilms was determined by MTT assay. The biofilm suppressant effect was determined by measuring biofilm viability at different time intervals, post-exposure to the two herbal extracts, and using MTT. Scanning electron microscopy was performed to assess the post-exposure biofilm architecture. Triphala inhibited both species of the planktonic yeasts, and only the biofilm phase C. tropicalis and mixed species, and not C. albicans. M. elengi had no inhibitory effect on either the planktonic or the biofilms of either Candida species. Ultrastructural microscopy revealed increased cell density of C. albicans biofilm, but not that of C. tropicalis which was significantly reduced in size after Triphala exposure. Triphala, but not M. elengi, extracts exhibit selective and differential biofilm inhibitory activity against Candida. C. albicans biofilms are more resistant to the anti-biofilm activity of Triphala

The genotypes and virulence attributes of C. albicans isolates from oral leukoplakia

There is a debate as to whether some types of oral leucoplakias (OL) are caused by Candida species, and whether they contribute to the malignant transformation, associated with a minority of such lesions. As no detailed population analysis of yeast isolates from OL is available, we evaluated the virulence attributes, and genotypes of 35 C. albicans from OL, and compared their genotypes with 18 oral isolates from healthy individuals. The virulence traits evaluated were esterase, phospholipase, proteinase, haemolysin and coagulase production, and phenotypic switching activity, and yeast adherence and biofilm formation. DNA from OL and control yeasts were evaluated for A, B or C genotype status. Phospholipase, proteinase, and coagulase activity and biofilm formation was observed in 80%, 66%, 97 % and 77 % of the isolates, respectively. Phenotypic switching was detected in 8.6%, while heamolytic, and esterase activity and adherence were noted in all isolates. The genotype A was predominant amongst both the OL and control groups. Due to the small sample size of our study a larger investigation to define the role of candidal virulent attributes in the pathogenicity of OL is warranted, and the current data should serve as a basis until then

Effect of natural curcuminoids‐intercalated layered double hydroxide nanohybrid against Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis: A bactericidal, antibiofilm, and mechanistic study

Abstract The study aimed to determine the antibacterial/antibiofilm effect and mechanism of interaction of curcuminoids‐intercalated Mg/Al layered double hydroxide (curcuminoids‐LDH) against three different bacteria. Antimicrobial effect of curcuminoids‐LDH nanohybrid was investigated against P. aeruginosa, S. aureus, and E. faecalis (for both standard strains and clinical isolates), using agar well diffusion method. Minimum inhibitory concentrations (MIC) of planktonic bacteria were determined using the broth microdilution method. MIC of biofilms (MBIC50) and killing time for 48 hr matured biofilms were determined by MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. Scanning electron microscopy (SEM) was used to determine pre‐ and postexposure architecture of biofilms. The mechanism of the antibiofilm activity of curcuminoids‐LDH was determined using UV‐visible spectroscopy. All tested bacteria had given a zone of inhibition in the presence of curcuminoids‐LDH. The MIC values were 0.200 g/ml for P. aeruginosa, 0.025 g/ml for S. aureus, and 0.100 g/ml for E. faecalis. The 48 hr matured biofilms were reduced by curcuminoids‐LDH with an MBIC50 of 0.100 g/ml. The minimum time to achieve MBIC50 was 3 hr, and the reduction was constant until 48 hr. SEM images showed a significant reduction of biofilm cell density and exopolymer matrics for all biofilms in the presence of curcuminoids‐LDH. UV‐visible studies revealed the antibiofilm activity of curcuminoids‐LDH as due to the auto‐oxidation of curcuminoids. The oxidation products are more limited in both product concentration per unit time and the variety of products, compared to pure curcuminoids, resulting in sharper UV‐visible peaks than in the case of the latter. Curcuminoids‐LDH has a potential antibacterial activity against P. aeruginosa, S. aureus, and E. faecalis. An antibiofilm activity has been achieved within 3 hr of the treatment. Curcuminoids released from the LDH showed the antibacterial activity due to oxidation products interfering with bacterial cell functions, and also encapsulation in the LDH causes curcuminoids to exhibit the activity in a persistent manner compared to pure curcuminoids

Addition of hydrogen peroxide to methylene blue conjugated to β-cyclodextrin in photodynamic antimicrobial chemotherapy in S. mutans biofilm

This study evaluated the effect of hydrogen peroxide addition on β-cyclodextrin-conjugated methylene blue in antimicrobial photodynamic therapy(a-PDT) in S. mutans biofilm model using laser or light emitting diode (LED) (λ = 660 nm). A preliminary assay was performed to evaluate the cytotoxicity of hydrogen peroxide in oral fibroblasts by the colorimetric method (MTT). Afterwards, groups were divided into (n = 3, in triplicate): C (negative control), CX – chlorhexidine 0.2% (positive control), P (methylene blue/β-cyclodextrin), H (Hydrogen Peroxide at 40 μM), PH, L (Laser), LP, LH (Laser+Hydrogen Peroxide), LPH, LED, LEDP, LEDH, and LEDPH. The biofilm was formed in 24 h with BHI + 1% sucrose (w/v). Light irradiations were conducted with laser, 9 J, 323 J/cm2, 113 s or with LED, 8.1 J, 8.1 J/cm2 for 90 s. Microbial reduction was evaluated by counting the viable microorganisms of the biofilm after the respective treatments, in a selective culture medium, and laser confocal microscopy evaluation. LP, LH, LPH, LEDP, LEDH, and LEDPH groups statistically reduced the counts of S.mutans compared with the C group and the log reductions were of 1.87, 1.94, 2.19, 0.91, 0.92, and 1.33, respectively; the addition of hydrogen peroxide did not potentiate the microbial reductions (LPH and LEDPH) compared with the LP and LEDP groups. The association of hydrogen peroxide with the conjugated β-cyclodextrin nanoparticle as photosensitizer did not result in an enhanced effect of a-PDT; hydrogen peroxide behaved as a photosensitizer, since it reduced the number of S. mutans when associated with laser light28226233CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP132211/2017-30012017/03263-