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Previous issue date: 2015-02-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Funda??o de Amparo ? Pesquisa do Estado do Rio Grande do Sul - FAPERGS / The species of the ?C. parapsilosis complex? are common etiological agents of nosocomial candidemia, and C. parapsilosis sensu stricto is one of non-albicans Candida species with the highest incidences in clinical infections, primarily related to implanted medical devices. In this study, we identified 29 isolates comprising the ?C. parapsilosis complex? to species level, all previously obtained from the hospital environment and medical tools. The isolates were also characterized for in vitro activity of aspartyl proteinase and phospholipase, as well as for their ability to form biofilms. The isolates identified as C. parapsilosis sensu stricto were also evaluated for their capability to interact with Staphylococcus aureus and Acinetobacter sp. in two-species polymicrobial biofilms. These biofilms were then analyzed for their tolerance to treatments with increasing concentrations of antimicrobials drugs and sanitizers. The results indicated that 75.9% of the isolates were identified as C. parapsilosis sensu stricto, 24.1% as Candida metapsilosis and none as Candida orthopsilosis. Regarding C. parapsilosis sensu stricto, all isolates were very strong producers of aspartyl proteinase, and 13.6% of phospholipase. Regarding C. metapsilosis, 71.4% and 14.3% of the isolates showed very strong aspartyl proteinase and phospholipase activity, respectively. Moreover, all isolates were able to form biofilm on polystyrene surface. Among the C. parapsilosis sensu stricto isolates, 9.1%, 45.4% and 45.4% were strong, moderate and weak biofilm producers, respectively. Regarding C. metapsilosis, 71.4% were moderate and 28.6% were weak biofilm producers.We also demonstrated that C. parapsilosis sensu stricto was capable of associating synergistically with both bacteria tested, since in polymicrobial biofilms a significantly higher number of bacterial cells adhered, compared to their own monomicrobial condition. However, bacterial cells did not interfere on the efficiency of yeast biofilm formation. When testing sanitizers, we showed that 70% ethyl alcohol and 1% sodium hypochlorite were effective to significantly decrease the number of viable cells, although they have not been able to eliminate the microbial populations of the biofilms. Our data also showed that the cells from all monomicrobial biofilms exhibited elevated tolerance to their respective antimicrobial drugs, at higher doses than their pre-determined minimal inhibitory concentration values. Nevertheless, biofilms of 6, 12 and 24 h of C. parapsilosis sensu stricto presented some decrease in cell viability to the treatment with ketoconazole, whereas 48 h biofilms were resistant to this antifungal. In polymicrobial biofilms, the bacterial cells did not affect the tolerance of yeast isolates to this antimicrobial. Conversely, S. aureus and Acinetobacter sp. cells were even more tolerant to the treatment with vancomycin and polymyxin B, respectively, when they were in polymicrobial biofilms, which was significantly different from their respective monomicrobial condition.The results from this work revealed the ability of ?C. parapsilosis complex? isolates from nosocomial environment to express important virulence factors, as well as to interact synergistically with bacterial species of clinical importance. This data is clinically relevant, since such phenotypic features may represent an important risk in terms of nosocomial infection to hospitalized patients. / As esp?cies do ?complexo Candida parapsilosis? mostram-se como frequentes agentes etiol?gicos de candidemias nosocomiais, sendo a C. parapsilosis sensu stricto uma das esp?cies n?o-albicans de Candida de maior incid?ncia em infec??es cl?nicas, estando relacionada principalmente com dispositivos m?dicos implantados. Neste estudo, identificamos, em n?vel de esp?cie, 29 isolados pertencentes ao ?complexo Candida parapsilosis?, todos previamente obtidos a partir do ambiente hospitalar e de dispositivos m?dicos. Os isolados tamb?m foram caracterizados quanto ? atividade in vitro de aspartil proteinase e fosfolipase, e quanto ? habilidade de formar biofilmes. Os isolados identificados como C. parapsilosis sensu stricto foram avaliados quanto ? capacidade de interagir com Staphylococcus aureus e Acinetobacter sp. no desenvolvimento de biofilmes polimicrobianos de duas esp?cies. Estes biofilmes foram ent?o analisados quanto ? sua capacidade de tolerar o tratamento com concentra??es crescentes de f?rmacos antimicrobianos e sanitizantes. Como resultados, 75,9% dos isolados foram identificados como C. parapsilosis sensu stricto, 24,1% como Candida metapsilosis e nenhum como Candida orthopsilosis. Em rela??o ? C. parapsilosis sensu stricto, todos os isolados mostraram-se como produtores muito fortes de aspartil proteinase e 13,6% de fosfolipase. Em rela??o ? C. metapsilosis, 71,4% e 14,3% dos isolados apresentaram atividade muito forte de aspartil proteinase e fosfolipase, respectivamente.Al?m disso, todos os isolados foram capazes de formar biofilme em superf?cie de poliestireno. Dentre os isolados de C. parapsilosis sensu stricto, 9,1%, 45,4% e 45,4% mostraram-se como produtores fortes, moderados e fracos de biofilme, respectivamente. Em rela??o aos de C. metapsilosis, 71,4% foram moderados e 28,6% foram fracos formadores de biofilme. Demonstramos tamb?m que a C. parapsilosis sensu stricto foi capaz de se associar de forma sin?rgica com ambas as bact?rias testadas, pois nos biofilmes polimicrobianos um n?mero significativamente maior de c?lulas bacterianas se aderiram em compara??o ? sua condi??o monomicrobiana. Entretanto, as bact?rias n?o interferiram na efici?ncia de forma??o de biofilmes da levedura. O tratamento com sanitizantes mostrou que o ?lcool et?lico a 70% e o hipoclorito de s?dio a 1% foram eficazes em diminuir significativamente o n?mero de c?lulas vi?veis, mas n?o foram capazes, mesmo assim, de eliminar as popula??es microbianas dos biofilmes. Al?m disso, nossos dados mostraram que as c?lulas integrantes de todos os biofilmes monomicrobianos avaliados apresentaram elevada toler?ncia aos seus respectivos antimicrobianos, em doses superiores aos seus valores de concentra??o inibit?ria m?nima, previamente determinados.Mesmo assim, os biofilmes de C. parapsilosis sensu stricto de 6, 12 e 24 h mostraram alguma perda de viabilidade celular ao tratamento com cetoconazol, enquanto biofilmes de 48 h se mostraram resistentes a este antif?ngico. Nos biofilmes polimicrobianos, a presen?a das c?lulas bacterianas n?o influenciou a toler?ncia das leveduras ao antimicrobiano. Por outro lado, as c?lulas de S. aureus e Acinetobacter sp. foram ainda mais tolerantes ao tratamento com vancomicina e polimixina B, respectivamente, quando em biofilmes polimicrobianos, de forma significativa em compara??o ?s suas respectivas condi??es monomicrobianas. Os resultados deste trabalho revelaram a capacidade de isolados do ?complexo C. parapsilosis? do ambiente nosocomial de expressar importantes fatores de virul?ncia, bem como de interagir sinergicamente com bact?rias de import?ncia cl?nica. Em conjunto, estas informa??es mostram-se de grande relev?ncia cl?nica, em fun??o de tais caracter?sticas fenot?picas representarem um importante risco em termos de infec??o hospitalar a pacientes internados.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/6332 |
| Date | 25 February 2015 |
| Creators | Mattiello , Shaiana Paula |
| Contributors | Oliveira , Silvia Dias de |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, Brasil, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 600, 600, 600, 600, 600, 36528317262667714, -1634559385931244697, 2075167498588264571, -3614735573891122254 |
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