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Previous issue date: 2016-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Staphylococcus spp. plays an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant specie due to the production of virulence factors such as ?slime?, which is required for biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolates from bovine mastitis, to detect and quantify the expression of genes involved in its production and regulation, as well as to detect the phenogenotypic resistance to beta-lactam in order to evaluate the possible relation between biofilm production and antimicrobial resistance. The isolates were characterized by MALDI-TOF and phenogenotypic identification assays. Also they were submitted to the phenotypic tests to evaluate biofilm production and the susceptibility to beta-lactams. Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration in the Biofilm (MICB) were determined to three isolates presenting distinct biofilm production. Futherly, a PCR for the detection of ?slime? production genes (icaA and icaD), Bap protein (bap), beta-lactamase (blaZ) and protein altered penicillin-binding (mecA). Also, the Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Scanning Electron Microscopy (SEM) was performed to determine the most suitable time interval for biofilm analysis. Real-time PCR (qPCR) was performed at the chosen times to quantify the expression of icaA, icaD and hld genes in the three studied isolates. All 20 isolates were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II presented a prevalence of 70%. The SEM showed gradual changes in bacterial arrangement during the biofilm formation along the phases of the growth curve. The peak was reached at the stationary phase. Transcriptional analysis revealed increased expression of ica genes at 8 h of growth and of hld at 24 h. However, for the N-365 strain the ica expression was of low yield. For this study, the penicillin resistance was related to the production of beta-lactamase otherwise the high MBC detected for cefoxitin may be associated to biofilm protection, evidentiated by the fact that the isolates have MICB values higher than MICs tested for planktonic cells / Staphylococcus spp. tem papel importante na etiologia da mastite bovina. Staphylococcus aureus ? considerada a esp?cie mais relevante devido ? produ??o de fatores de virul?ncia, tais como ?slime?, o que favorece a forma??o do biofilme. O presente trabalho teve por objetivo detectar a express?o fenot?pica da forma??o de biofilme em 20 isolados de S. aureus oriundos de mastite bovina, detectar e quantificar a express?o dos genes envolvidos na sua produ??o e regula??o, al?m de detectar a resist?ncia fenogenot?pica aos beta-lact?micos para avalia??o da poss?vel rela??o da produ??o de biofilme com a resist?ncia antimicrobiana. Os isolados foram caracterizados atrav?s de testes fenogenot?picos e MALDI-TOF, submetidos ?s provas fenot?picas de detec??o da forma??o de biofilme e avalia??o da suscetibilidade aos beta-lact?micos. A Concentra??o Inibit?ria M?nima (CIM), Concentra??o Bactericida M?nima (CBM) e a Concentra??o Inibit?ria M?nima no Biofilme (CIMB) foram determinadas para tr?s isolados selecionados com base na varia??o da intensidade da produ??o de biofilme. Posteriormente, todos os isolados foram submetidos ? t?cnica de PCR para detec??o dos genes de produ??o de ?slime? (icaA e icaD), prote?na Bap (bap), beta-lactamase (blaZ) e prote?na ligante de penicilina alterada (mecA). Al?m de detec??o do sistema regulador Agr (agr RNAIII) e da tipifica??o do sistema Agr (agr I, agr II, agr III e agr IV). Foi realizada Microscopia Eletr?nica de Varredura (MEV) para determinar o intervalo de tempo mais adequado para a an?lise do biofilme. A PCR em tempo real (qPCR) foi realizada nos tempos selecionados para quantificar a express?o dos genes icaA, icaD e hld em tr?s isolados com produ??o variada de biofilme. Todos os isolados foram produtores de biofilme e a maioria apresentou os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr tipo II mostrou preval?ncia de 70%. A MEV mostrou mudan?as graduais no arranjo bacteriano durante a forma??o de biofilme ao longo das fases da curva de crescimento que atingiu seu pico de forma??o na fase estacion?ria. A an?lise transcricional evidenciou maior express?o dos genes ica no tempo de 8 h de crescimento e hld em 24 h. Contudo, a cepa N-365 mostrou baixa produ??o dos genes ica. Para este estudo, a resist?ncia ? penicilina foi relacionada com a produ??o de beta-lactamase, enquanto a elevada CBM detectada para cefoxitina pode estar associada ? prote??o que o biofilme oferece, epis?dio evidenciado pelo fato dos isolados apresentarem valores de CIMB superiores aos CIMs testados para as c?lulas planct?nicas.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/2065 |
| Date | 02 March 2016 |
| Creators | Marques, Viviane Figueira |
| Contributors | Souza, Miliane Moreira Soares de, Coelho, Shana de Mattos de Oliveira, Santos, Huarrisson Azevedo, Senna, Jos? Proc?pio Moreno, Merval, Marcia Giambiagi de, Pereira, Ingrid Annes |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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