Caracterização fenotípica e molecular de amostras de Staphylococcus spp. isoladas de fluído de sonicação de implantes ortopédicos infectados
Data
2021-05
Autores
Santos, Ingrid Nayara Marcelino 
Orientadores
Pignatari, Antonio Carlos Campos
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
As infecções associadas a dispositivos ortopédicos (ODRIs) são causadas principalmente por Staphylococcus spp. formadores de biofilme, incluindo Staphylococcus aureus e Staphylococcus coagulase negativo (CoNS). Nosso objetivo foi caracterizar a capacidade de formação de biofilme, virulência e o perfil de sensibilidade em isolados de Staphylococcus spp recuperados de fluido de sonicação de ODRIs, por métodos fenotípicos e moleculares. Foram coletados e analisados dados clínicos e microbiológicos de 32 pacientes, com ODRIs, entre os quais os implantes foram retirados e submetidos a sonicação. Os micro-organismos foram identificados pela técnica de dessorção em vôo de espectrometria de massa por ionização a laser assistida por matriz (MALDI-TOF MS). O teste de susceptibilidade antimicrobiana foi realizado por disco difusão e a técnica de microdiluição em caldo foram utilizadas para avaliar a concentração inibitória mínima (CIM) para a vancomicina. Os resultados foram interpretados de acordo com o European Committee on Antimicrobial Susceptibility Testing (EUCAST) e Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) (2020). A detecção do gene, mecA e icaAB foi realizada por reação em cadeia da polimerase (PCR). Foi realizado o ensao qualitativo para avaliação da produção de biofilme em superfícies abióticas. No estudo realizada comparações de resultados utilizado o cálculo estatístico para o coeficiente de Kappa. O sequenciamento do genoma completo (SGC) foi feito usando a plataforma Illumina MiSeq. No geral, 32 Staphylococcus spp. foram isolados, S. aureus em 50% (16/32), S. epidermidis em 25% (8/32) e S. haemolyticus em 16% (6/32). Todos os isolados de S. aureus foram resistentes a meticilina e mecA positivos. Entre os SCoN a resistência foi observada foi de 56,2%. Na avaliação qualitativa da capacidade de formação de biofilme dos isolados em superfícies abióticas, 75% (N = 24/32) dos isolados foram fortes produtores de biofilme. A concordância entre detecção do gene icaAB e formação de biofilme pelo coeficiente de Kappa foi fraca. A análise de sequenciamento mostrou que entre 3 cepas de S. aureus, duas eram ST 5 e uma ST105. Os Sccmec encontrados foram tipo I, II e V. Os três isolados pertenciam ao CC5. Os genes de resistência e virulência de S. aureus foram detectados incluindo múltiplos genes codificadores de AMEs, mecA, blaZ, fnbA, sdrC, sdrD, sdrE, ebp. Entre os SCoN, notou-se uma grande variabilidade quando a presença de genes de que expressam aderência (MSCRAMMs), sendo os genes mais frequentemente identificados foram, atl, ebp e ebh A realização do SGC nestes isolados forneceu informações importantes e mais completas com relação a presença de múltiplos genes de resistência e de virulência, incluindo os genes associados a formação de biofilme. Este estudo oferece ferramentas para a melhor compreensão dos mecanismos envolvidos na patogênese destas infecções.
Infections associated with orthopedic devices (ODRIs) are mainly caused by biofilm-forming Staphylococcus spp, including Staphylococcus aureus and coagulase negative Staphylococcus (CoNS). Our objective was to characterize the capacity of biofilm formation, virulence and the sensitivity profile in Staphylococcus spp isolates recovered from ODRI sonication fluid, by phenotypic and molecular methods. Clinical and microbiological data were collected and analyzed from 32 patients with ODRIs, among which the implants were removed and subjected to sonication. The microorganisms were identified by the flight desorption technique of matrix-assisted laser ionization mass spectrometry (MALDI-TOF MS). The antimicrobial susceptibility test was performed by disk diffusion and the broth microdilution technique was used to assess the minimum inhibitory concentration (MIC) for vancomycin. The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) (2020). The detection of the gene, mecA and icaAB was performed by polymerase chain reaction (PCR). Qualitative testing was carried out to assess biofilm production on abiotic surfaces. In the study, results were compared using the statistical calculation for the Kappa coefficient. The complete genome sequencing (SGC) was done using the Illumina MiSeq platform. Overall, 32 Staphylococcus spp. were isolated, S. aureus in 50% (16/32), S. epidermidis in 25% (8/32) and S. haemolyticus in 16% (6/32). All S. aureus isolates were resistant to methicillin and mecA positive. Among the CoNS, resistance was observed to be 56.2%. In the qualitative evaluation of the biofilm formation capacity of the isolates on abiotic surfaces, 75% (N = 24/32) of the isolates were strong producers of biofilm. The agreement between detection of the icaAB gene and biofilm formation by the Kappa coefficient was weak. Sequencing analysis showed that among 3 strains of S. aureus, two were ST 5 and one ST105. The Sccmec found were type I, II and V. The three isolates belonged to CC5. The resistance and virulence genes of S. aureus were detected including multiple genes encoding AMEs, mecA, blaZ, fnbA, sdrC, sdrD, sdrE, ebp. Among the CoNS, great variability was noted when the presence of genes that express adherence (MSCRAMMs), the most frequently identified genes being, atl, ebp and ebh. the presence of multiple resistance and virulence genes, including genes associated with biofilm formation. This study offers tools for a better understanding of the mechanisms involved in the pathogenesis of these infections.
Infections associated with orthopedic devices (ODRIs) are mainly caused by biofilm-forming Staphylococcus spp, including Staphylococcus aureus and coagulase negative Staphylococcus (CoNS). Our objective was to characterize the capacity of biofilm formation, virulence and the sensitivity profile in Staphylococcus spp isolates recovered from ODRI sonication fluid, by phenotypic and molecular methods. Clinical and microbiological data were collected and analyzed from 32 patients with ODRIs, among which the implants were removed and subjected to sonication. The microorganisms were identified by the flight desorption technique of matrix-assisted laser ionization mass spectrometry (MALDI-TOF MS). The antimicrobial susceptibility test was performed by disk diffusion and the broth microdilution technique was used to assess the minimum inhibitory concentration (MIC) for vancomycin. The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) (2020). The detection of the gene, mecA and icaAB was performed by polymerase chain reaction (PCR). Qualitative testing was carried out to assess biofilm production on abiotic surfaces. In the study, results were compared using the statistical calculation for the Kappa coefficient. The complete genome sequencing (SGC) was done using the Illumina MiSeq platform. Overall, 32 Staphylococcus spp. were isolated, S. aureus in 50% (16/32), S. epidermidis in 25% (8/32) and S. haemolyticus in 16% (6/32). All S. aureus isolates were resistant to methicillin and mecA positive. Among the CoNS, resistance was observed to be 56.2%. In the qualitative evaluation of the biofilm formation capacity of the isolates on abiotic surfaces, 75% (N = 24/32) of the isolates were strong producers of biofilm. The agreement between detection of the icaAB gene and biofilm formation by the Kappa coefficient was weak. Sequencing analysis showed that among 3 strains of S. aureus, two were ST 5 and one ST105. The Sccmec found were type I, II and V. The three isolates belonged to CC5. The resistance and virulence genes of S. aureus were detected including multiple genes encoding AMEs, mecA, blaZ, fnbA, sdrC, sdrD, sdrE, ebp. Among the CoNS, great variability was noted when the presence of genes that express adherence (MSCRAMMs), the most frequently identified genes being, atl, ebp and ebh. the presence of multiple resistance and virulence genes, including genes associated with biofilm formation. This study offers tools for a better understanding of the mechanisms involved in the pathogenesis of these infections.