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Master's Dissertation
DOI
10.11606/D.60.2015.tde-02102015-161413
Document
Author
Full name
Amir Horiquini Barbosa
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2015
Supervisor
Committee
Candido, Regina Celia (President)
Pedroso, Reginaldo dos Santos
Watanabe, Evandro
Title in Portuguese
Avaliação de fatores de virulência e sensibilidade a antissépticos bucais de espécies de Candida isoladas da saliva de pacientes com próteses bucais
Keywords in Portuguese
ALS3
antissépticos bucais
biofilme
Candida
genes ALS2
proteinase
SAP1
SAP3.
Abstract in Portuguese
As espécies de Candida são patógenos oportunistas e as infecções nas cavidades bucais causadas por este gênero são comuns em pacientes com fatores predisponentes, entre eles imussuprimidos, diabéticos e usuários de próteses dentais. No Brasil o índice de endentados é elevado sendo necessária a utilização de próteses bucais, que são locais favoráveis à colonização de microrganismos e o desenvolvimento de biofilme. A utilização de antissépticos como medida complementar na higiene oral é cada vez mais difundida. O objetivo deste trabalho foi detectar em amostras de C. albicans e C. não-albicans, isoladas da saliva de portadores de próteses bucais, os fatores de virulência, proteinase e biofilme e genes relacionados à proteinase (SAP1, SAP3) e à adesão (ALS1 e ALS3) e avaliar a atividade antifúngica in vitro de três formulações comerciais à base de clorexidina 0,12%, cetilpiridínio 0,07% e 0,075% frente aos isolados de Candida e biofilmes. Foram coletadas amostras de saliva de 70 pacientes usuários de próteses bucais. As leveduras foram isoladas em meio de CHROMagar® Candida e identificadas pela metodologia clássica e a confirmação de C. albicans por biologia molecular (PCR). Leveduras do gênero Candida foram isoladas de 32 (45,7%) amostras. Destas foram identificadas: C. albicans (29), C. krusei (5), C. glabrata (4), C. tropicalis (3) e C. parapsilosis (2), totalizando 43 leveduras entre monoculturas e culturas mistas. Os genes pesquisados foram detectados somente em amostras de C. albicans, os genes ALS2, ALS3 e SAP3 foram identificados em 86% dos isolados e o SAP1 em 93%. A atividade de proteinase entre os dos isolados de C. albicans foi de 72,4% e entre das espécies de C. não-albicans 35,7%. A de formação de biofilme sobre placas de 96 poços, e após coloração com XTT, demonstrou que a maioria dos isolados de Candida e todas as culturas mistas desenvolveram biofilmes em 24 horas. A atividade de três antissépticos frente as leveduras foi de <=1,25%, equivalente a uma diluição de 1:80 do antisséptico. Concluímos os três antissépticos também foram eficazes frente aos biofilmes de monoculturas e culturas mistas, as cepas de C. albicans continua sendo a espécie prevalente na cavidade bucal, os genes SAP1, SAP3, ALS2 e ALS3 estão presentes na maioria dos isolados de C. albicans, nestas amostras a maioria foram proteinase e biofilme positivos. A atividade de proteinase foi superior nas espécies de C. albicans em comparação com C. não-albicans. Os biofilmes das espécies de Candida quando formados em culturas mistas se desenvolvem mais em comparação com a formação de biofilme em monocultura. As três formulações de antissépticos foram eficazes contra todos isolados de leveduras em forma planctônica e na maioria dos biofilmes.
Title in English
Evaluation of virulence factors and susceptibility to mouthwashes of Candida species isolated from saliva of patients with oral prostheses
Keywords in English
ALS2
ALS3
biofilm
Candida
genes SAP1
mouthwashes
proteinase
SAP3
Abstract in English
Candida species are opportunistic pathogens and infections in the oral cavity caused by these yeasts are common in patients with predisposing factors, such as diabetic or immunocompromised individuals with dental prosthesis. In Brazil, the number of toothless patients is high and the use of dental prosthesis is common, which represents a niche for colonization and formation of microbial biofilms in the oral cavity. The use of mouthwashes to aid in the oral hygiene has increased. The objective of this research was to isolate C. albicans and C. non-albicans from saliva of dental prosthesis users and to evaluate the isolates for the presence of virulence factors, proteinase production, biofilm formation and to search for genes encoding for proteinases (SAP1, SAP3) and adhesion factors (ALS1 and ALS3). Another objective was to evaluate in vitro the antifungal activity of three commercial mouthwashes containing 0.12% chlorhexidine and 0.07% or 0.075% cetylpyridinium, against planktonic Candida sp. cultures and in biofilms. Saliva samples of 70 patients with dental prosthesis were collected, the yeasts were isolated on CHOMagar® Candida and identified by classical biochemical tests combined with molecular test (PCR) for confirmation of C. albicans. Yeasts of Candida genus were isolated from 32 samples (45.7%) and were identified as C. albicans (29), C. krusei (5), C. glabrata (4), C. tropicalis (3) and C. parapsilosis (2), with a total of 43 yeasts, including mono and mixed cultures. The genes studied were detected only in C. albicans: ALS2, ALS3 and SAP3 genes were detected in 86% of the isolates and SAP1 in 93%. The proteinase activity among the isolates of C. albicans was 72.4% and among the species of C. non-albicans it was 35.7%. Assay done with cultivation in 96-wells microplates and staining with XTT, showed the majority of Candida isolates and all the mixed cultures were able to form biofilms within 24h. The activity of the three mouthwashes towards the yeasts was < 1.25%, equivalent to a dilution of 1:80 of the formulation. The three mouthwashes tested were also active against mono or multispecies biofilms. In conclusion, it was shown C. albicans is the most prevalent species. The genes SAP1, SAP3, ALS2 and ALS3 are present in the majority of C. albicans isolates, which were also positive for proteinase and biofilm formation. There was more proteinase activity in C. albicans in comparison with C. non-albicans. The biofilms of Candida species grew better in mixed cultures than in monoculture. The three commercial mouthwashes formulations were effective against all the yeast isolates in planktonic growth and they also inhibited the majority of yeasts in biofilm. Future work is needed to completely elucidate the role of Candida species in biofilms of oral cavity. It is also interesting to propose studies to standardize sensitivity tests for mono and multi-species biofilms.
 
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Release Date
2017-10-04
Publishing Date
2015-11-03
 
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