Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino

Detalhes bibliográficos
Autor(a) principal: Marques, Viviane Figueira
Data de Publicação: 2016
Tipo de documento: Tese
Idioma: por
Título da fonte: Biblioteca Digital de Teses e Dissertações da UFRRJ
Texto Completo: https://rima.ufrrj.br/jspui/handle/20.500.14407/9725
Resumo: Staphylococcus spp. tem papel importante na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais relevante devido à produção de fatores de virulência, tais como “slime”, o que favorece a formação do biofilme. O presente trabalho teve por objetivo detectar a expressão fenotípica da formação de biofilme em 20 isolados de S. aureus oriundos de mastite bovina, detectar e quantificar a expressão dos genes envolvidos na sua produção e regulação, além de detectar a resistência fenogenotípica aos beta-lactâmicos para avaliação da possível relação da produção de biofilme com a resistência antimicrobiana. Os isolados foram caracterizados através de testes fenogenotípicos e MALDI-TOF, submetidos às provas fenotípicas de detecção da formação de biofilme e avaliação da suscetibilidade aos beta-lactâmicos. A Concentração Inibitória Mínima (CIM), Concentração Bactericida Mínima (CBM) e a Concentração Inibitória Mínima no Biofilme (CIMB) foram determinadas para três isolados selecionados com base na variação da intensidade da produção de biofilme. Posteriormente, todos os isolados foram submetidos à técnica de PCR para detecção dos genes de produção de “slime” (icaA e icaD), proteína Bap (bap), beta-lactamase (blaZ) e proteína ligante de penicilina alterada (mecA). Além de detecção do sistema regulador Agr (agr RNAIII) e da tipificação do sistema Agr (agr I, agr II, agr III e agr IV). Foi realizada Microscopia Eletrônica de Varredura (MEV) para determinar o intervalo de tempo mais adequado para a análise do biofilme. A PCR em tempo real (qPCR) foi realizada nos tempos selecionados para quantificar a expressão dos genes icaA, icaD e hld em três isolados com produção 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 prevalência de 70%. A MEV mostrou mudanças graduais no arranjo bacteriano durante a formação de biofilme ao longo das fases da curva de crescimento que atingiu seu pico de formação na fase estacionária. A análise transcricional evidenciou maior expressão dos genes ica no tempo de 8 h de crescimento e hld em 24 h. Contudo, a cepa N-365 mostrou baixa produção dos genes ica. Para este estudo, a resistência à penicilina foi relacionada com a produção de beta-lactamase, enquanto a elevada CBM detectada para cefoxitina pode estar associada à proteção que o biofilme oferece, episódio evidenciado pelo fato dos isolados apresentarem valores de CIMB superiores aos CIMs testados para as células planctônicas.
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spelling Marques, Viviane FigueiraSouza, Miliane Moreira Soares de1076198732http://lattes.cnpq.br/0865211214618618Coelho, Shana de Mattos de Oliveira5466821705http://lattes.cnpq.br/3212438357088121Santos, Huarrisson AzevedoSenna, José Procópio MorenoMerval, Marcia Giambiagi dePereira, Ingrid Annes5788549701http://lattes.cnpq.br/96390853749544862023-12-21T18:43:10Z2023-12-21T18:43:10Z2016-03-02MARQUES, Viviane Figueira. Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino. 2016. 73 f. Tese (Doutorado em Ciências Veterinárias) - Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, 2016.https://rima.ufrrj.br/jspui/handle/20.500.14407/9725Staphylococcus spp. tem papel importante na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais relevante devido à produção de fatores de virulência, tais como “slime”, o que favorece a formação do biofilme. O presente trabalho teve por objetivo detectar a expressão fenotípica da formação de biofilme em 20 isolados de S. aureus oriundos de mastite bovina, detectar e quantificar a expressão dos genes envolvidos na sua produção e regulação, além de detectar a resistência fenogenotípica aos beta-lactâmicos para avaliação da possível relação da produção de biofilme com a resistência antimicrobiana. Os isolados foram caracterizados através de testes fenogenotípicos e MALDI-TOF, submetidos às provas fenotípicas de detecção da formação de biofilme e avaliação da suscetibilidade aos beta-lactâmicos. A Concentração Inibitória Mínima (CIM), Concentração Bactericida Mínima (CBM) e a Concentração Inibitória Mínima no Biofilme (CIMB) foram determinadas para três isolados selecionados com base na variação da intensidade da produção de biofilme. Posteriormente, todos os isolados foram submetidos à técnica de PCR para detecção dos genes de produção de “slime” (icaA e icaD), proteína Bap (bap), beta-lactamase (blaZ) e proteína ligante de penicilina alterada (mecA). Além de detecção do sistema regulador Agr (agr RNAIII) e da tipificação do sistema Agr (agr I, agr II, agr III e agr IV). Foi realizada Microscopia Eletrônica de Varredura (MEV) para determinar o intervalo de tempo mais adequado para a análise do biofilme. A PCR em tempo real (qPCR) foi realizada nos tempos selecionados para quantificar a expressão dos genes icaA, icaD e hld em três isolados com produção 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 prevalência de 70%. A MEV mostrou mudanças graduais no arranjo bacteriano durante a formação de biofilme ao longo das fases da curva de crescimento que atingiu seu pico de formação na fase estacionária. A análise transcricional evidenciou maior expressão dos genes ica no tempo de 8 h de crescimento e hld em 24 h. Contudo, a cepa N-365 mostrou baixa produção dos genes ica. Para este estudo, a resistência à penicilina foi relacionada com a produção de beta-lactamase, enquanto a elevada CBM detectada para cefoxitina pode estar associada à proteção que o biofilme oferece, episódio evidenciado pelo fato dos isolados apresentarem valores de CIMB superiores aos CIMs testados para as células planctônicas.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorStaphylococcus 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 cellsapplication/pdfporUniversidade Federal Rural do Rio de JaneiroPrograma de Pós-Graduação em Ciências VeterináriasUFRRJBrasilInstituto de VeterináriaAgr typesantimicrobial resistanceS. aureusicaAicaDhldtipos de Agrresistência antimicrobianaMicrobiologiaExpressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovinoGene expression in biofilm formation and resistance to beta-lactam in Staphylococcus aureus isolates from bovine milk mastiticinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisALATOOM, A.A.; CUNNINGHAM, S.A.; IHDE, S.M.; MANDREKAR, J.; PATEL, R. 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dc.title.por.fl_str_mv Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
dc.title.alternative.eng.fl_str_mv Gene expression in biofilm formation and resistance to beta-lactam in Staphylococcus aureus isolates from bovine milk mastitic
title Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
spellingShingle Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
Marques, Viviane Figueira
Agr types
antimicrobial resistance
S. aureus
icaA
icaD
hld
tipos de Agr
resistência antimicrobiana
Microbiologia
title_short Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
title_full Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
title_fullStr Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
title_full_unstemmed Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
title_sort Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino
author Marques, Viviane Figueira
author_facet Marques, Viviane Figueira
author_role author
dc.contributor.author.fl_str_mv Marques, Viviane Figueira
dc.contributor.advisor1.fl_str_mv Souza, Miliane Moreira Soares de
dc.contributor.advisor1ID.fl_str_mv 1076198732
dc.contributor.advisor1Lattes.fl_str_mv http://lattes.cnpq.br/0865211214618618
dc.contributor.advisor-co1.fl_str_mv Coelho, Shana de Mattos de Oliveira
dc.contributor.advisor-co1ID.fl_str_mv 5466821705
dc.contributor.advisor-co1Lattes.fl_str_mv http://lattes.cnpq.br/3212438357088121
dc.contributor.referee1.fl_str_mv Santos, Huarrisson Azevedo
dc.contributor.referee2.fl_str_mv Senna, José Procópio Moreno
dc.contributor.referee3.fl_str_mv Merval, Marcia Giambiagi de
dc.contributor.referee4.fl_str_mv Pereira, Ingrid Annes
dc.contributor.authorID.fl_str_mv 5788549701
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/9639085374954486
contributor_str_mv Souza, Miliane Moreira Soares de
Coelho, Shana de Mattos de Oliveira
Santos, Huarrisson Azevedo
Senna, José Procópio Moreno
Merval, Marcia Giambiagi de
Pereira, Ingrid Annes
dc.subject.eng.fl_str_mv Agr types
antimicrobial resistance
topic Agr types
antimicrobial resistance
S. aureus
icaA
icaD
hld
tipos de Agr
resistência antimicrobiana
Microbiologia
dc.subject.por.fl_str_mv S. aureus
icaA
icaD
hld
tipos de Agr
resistência antimicrobiana
dc.subject.cnpq.fl_str_mv Microbiologia
description Staphylococcus spp. tem papel importante na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais relevante devido à produção de fatores de virulência, tais como “slime”, o que favorece a formação do biofilme. O presente trabalho teve por objetivo detectar a expressão fenotípica da formação de biofilme em 20 isolados de S. aureus oriundos de mastite bovina, detectar e quantificar a expressão dos genes envolvidos na sua produção e regulação, além de detectar a resistência fenogenotípica aos beta-lactâmicos para avaliação da possível relação da produção de biofilme com a resistência antimicrobiana. Os isolados foram caracterizados através de testes fenogenotípicos e MALDI-TOF, submetidos às provas fenotípicas de detecção da formação de biofilme e avaliação da suscetibilidade aos beta-lactâmicos. A Concentração Inibitória Mínima (CIM), Concentração Bactericida Mínima (CBM) e a Concentração Inibitória Mínima no Biofilme (CIMB) foram determinadas para três isolados selecionados com base na variação da intensidade da produção de biofilme. Posteriormente, todos os isolados foram submetidos à técnica de PCR para detecção dos genes de produção de “slime” (icaA e icaD), proteína Bap (bap), beta-lactamase (blaZ) e proteína ligante de penicilina alterada (mecA). Além de detecção do sistema regulador Agr (agr RNAIII) e da tipificação do sistema Agr (agr I, agr II, agr III e agr IV). Foi realizada Microscopia Eletrônica de Varredura (MEV) para determinar o intervalo de tempo mais adequado para a análise do biofilme. A PCR em tempo real (qPCR) foi realizada nos tempos selecionados para quantificar a expressão dos genes icaA, icaD e hld em três isolados com produção 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 prevalência de 70%. A MEV mostrou mudanças graduais no arranjo bacteriano durante a formação de biofilme ao longo das fases da curva de crescimento que atingiu seu pico de formação na fase estacionária. A análise transcricional evidenciou maior expressão dos genes ica no tempo de 8 h de crescimento e hld em 24 h. Contudo, a cepa N-365 mostrou baixa produção dos genes ica. Para este estudo, a resistência à penicilina foi relacionada com a produção de beta-lactamase, enquanto a elevada CBM detectada para cefoxitina pode estar associada à proteção que o biofilme oferece, episódio evidenciado pelo fato dos isolados apresentarem valores de CIMB superiores aos CIMs testados para as células planctônicas.
publishDate 2016
dc.date.issued.fl_str_mv 2016-03-02
dc.date.accessioned.fl_str_mv 2023-12-21T18:43:10Z
dc.date.available.fl_str_mv 2023-12-21T18:43:10Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
format doctoralThesis
status_str publishedVersion
dc.identifier.citation.fl_str_mv MARQUES, Viviane Figueira. Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino. 2016. 73 f. Tese (Doutorado em Ciências Veterinárias) - Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, 2016.
dc.identifier.uri.fl_str_mv https://rima.ufrrj.br/jspui/handle/20.500.14407/9725
identifier_str_mv MARQUES, Viviane Figueira. Expressão gênica na formação do biofilme e resistência aos beta-lactâmicos em isolados de Staphylococcus aureus provenientes de leite mastítico bovino. 2016. 73 f. Tese (Doutorado em Ciências Veterinárias) - Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, 2016.
url https://rima.ufrrj.br/jspui/handle/20.500.14407/9725
dc.language.iso.fl_str_mv por
language por
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