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Caracterização de plasmídeos de Staphylococcus epidermidis e correlação com a resistência a compostos antimicrobianos mediada por efluxo
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Staphylococcus epidermidis coloniza a pele e mucosas do Homem e de animais, sendo considerado um importante agente patogénico oportunista em clínica humana e veterinária. Embora já estejam descritos diversos determinantes plasmídicos de resistência a compostos antimicrobianos nesta bactéria, a caracterização de plasmídeos de coleções alargadas de S. epidermidis e o seu papel na disseminação destas resistências é ainda relativamente escassa. O objectivo desta Dissertação foi identificar e caracterizar perfis plasmídicos de isolados comensais de S. epidermidis, correlacionando-os com a presença de genes de resistência a compostos antimicrobianos, com ênfase naqueles que codificam para bombas de efluxo.
Para isso, estudou-se uma coleção de 112 S. epidermidis de colonização nasal em profissionais e estudantes de Medicina Veterinária. O DNA plasmídico foi isolado pelo método de lise alcalina e o perfil plasmídico determinado por digestão com a enzima de restrição EcoRI (RFLP). A detecção de genes plasmídicos que codificam para bombas de efluxo associadas a resistência a antibióticos (msrA, tetK, vga(A) e vga(C)), a biocidas (qacG, qacJ) e a metais pesados (cadA, cadD e arsB) foi realizada por PCR e a presença destes correlacionada com o perfil de resistência estabelecido em trabalhos anteriores.
Este estudo permitiu demonstrar a presença de plasmídeos nos 112 isolados analisados, sendo a presença de plasmídeos de grandes dimensões (> 23 kb) predominante. A maioria dos isolados (66%) tinha dois ou mais plasmídeos. A análise por RFLP evidenciou uma grande heterogeneidade dos perfis plasmídicos. A pesquisa por PCR permitiu detectar o gene msrA em 35 de 57 isolados resistentes a macrólidos (61,4 %), o gene tet(K) em 25 dos 26 isolados resistentes à tetraciclina (96,2%), o gene vga(A) em 12 dos 19 isolados com resistência constitutiva à clindamicina (63,2%) e em 3 dos 19 com resistência indutível à clindamicina (15,8%) e o gene vga(C) em 15 dos 19 isolados com resistência constitutiva à clindamicina (78,9%) e em 15 dos 19 isolados com resistência indutível a este antibiótico (78,9%), naquela que deverá ser das primeira descrições deste gene em isolados de origem humana. Para os metais pesados, nomeadamente para o cádmio, apenas se detectou o gene cadA em 3 dos 112 isolados (2,7%), não se tendo identificado a presença do gene cadD. Por fim, nenhum isolado apresentou os genes qacJ e qacG, associados à susceptibilidade reduzida a biocidas e corantes.
Este trabalho demonstra a elevada frequência de plasmídeos e de determinantes que conferem resistência por efluxo a antibióticos em estirpes de S. epidermidis de colonização nasal em humanos em contacto próximo com animais. O alargamento desta análise a outros genes de resistência, permitirá explorar a importância destes e dos plasmídeos que os transportam na disseminação de estirpes resistentes, com especial destaque para a ligação entre as vertentes humana e veterinária.
Staphylococcus epidermidis colonizes the skin and mucous membranes of both Man and animals and it is considered to be an important opportunistic pathogen in human and animal medicine. Although several plasmid encoded determinants of antimicrobial resistance are known for this bacterium, the characterization of plasmids carried by large collections of S. epidermidis is scarce. The aim of this Dissertation was to identify and characterize plasmid profiles in S. epidermidis, correlating them with the presence of antimicrobial resistance genes, with emphasis on those coding for efflux pumps. In this work we studied a collection of 112 S. epidermidis of nasal colonization in professionals and students of Veterinary Medicine. Plasmid DNA was isolated by the alkaline lysis method and the plasmid profile determined by restriction with enzyme EcoRI (RFLP). Detection of plasmid genes encoding efflux pumps associated with resistance to antibiotics (msrA, tetK, vga(A) and vga(C)), biocides (qacG, qacJ) and heavy metal (cadA, cadD and arsB) was performed by PCR. The presence of these determinants was correlated with the resistance phenotypes previously established. This study demonstrated the presence of plasmids in the 112 isolates analyzed, with large plasmids (> 23 kb) predominating. The majority of the isolates (66%) carried two or more plasmids. RFLP analysis showed a great heterogeneity of the plasmid profiles. PCR screening detected the msrA gene in 35 out of 57 macrolide resistant isolates (61,4 %), the tet(K) in 25 of the 26 tetracycline resistant isolates (96.2%), the vga(A) in 12 of the 19 isolates with constitutive resistance to clindamycin (63,2%) and in 3 of 19 with inducible resistance to clindamycin (15.8%), whereas the vga(C) gene was identified in 15 of the 19 isolates with constitutive resistance (78.9%) and in 15 of the 19 isolates with inducible resistance to clindamycin (78.9%), in one of the first reports of vga(C) in humans. For heavy metals, particularly cadmium, the cadA gene was detected in 3 of 112 isolates (2.7%), while the cadD gene was not detected. Finally, no isolates showed the qacJ or qacG genes, associated with reduced susceptibility to biocides and dyes. This work demonstrates the high frequency of plasmids and determinants conferring efflux mediated resistance to antibiotics in S. epidermidis strains of nasal colonization in humans in close contact with animals. The extension of this analysis to other plasmid encoded resistance genes will allow exploring their importance in the dissemination of resistant strains, with particular emphasis on the link between the human and veterinary perspectives.
Staphylococcus epidermidis colonizes the skin and mucous membranes of both Man and animals and it is considered to be an important opportunistic pathogen in human and animal medicine. Although several plasmid encoded determinants of antimicrobial resistance are known for this bacterium, the characterization of plasmids carried by large collections of S. epidermidis is scarce. The aim of this Dissertation was to identify and characterize plasmid profiles in S. epidermidis, correlating them with the presence of antimicrobial resistance genes, with emphasis on those coding for efflux pumps. In this work we studied a collection of 112 S. epidermidis of nasal colonization in professionals and students of Veterinary Medicine. Plasmid DNA was isolated by the alkaline lysis method and the plasmid profile determined by restriction with enzyme EcoRI (RFLP). Detection of plasmid genes encoding efflux pumps associated with resistance to antibiotics (msrA, tetK, vga(A) and vga(C)), biocides (qacG, qacJ) and heavy metal (cadA, cadD and arsB) was performed by PCR. The presence of these determinants was correlated with the resistance phenotypes previously established. This study demonstrated the presence of plasmids in the 112 isolates analyzed, with large plasmids (> 23 kb) predominating. The majority of the isolates (66%) carried two or more plasmids. RFLP analysis showed a great heterogeneity of the plasmid profiles. PCR screening detected the msrA gene in 35 out of 57 macrolide resistant isolates (61,4 %), the tet(K) in 25 of the 26 tetracycline resistant isolates (96.2%), the vga(A) in 12 of the 19 isolates with constitutive resistance to clindamycin (63,2%) and in 3 of 19 with inducible resistance to clindamycin (15.8%), whereas the vga(C) gene was identified in 15 of the 19 isolates with constitutive resistance (78.9%) and in 15 of the 19 isolates with inducible resistance to clindamycin (78.9%), in one of the first reports of vga(C) in humans. For heavy metals, particularly cadmium, the cadA gene was detected in 3 of 112 isolates (2.7%), while the cadD gene was not detected. Finally, no isolates showed the qacJ or qacG genes, associated with reduced susceptibility to biocides and dyes. This work demonstrates the high frequency of plasmids and determinants conferring efflux mediated resistance to antibiotics in S. epidermidis strains of nasal colonization in humans in close contact with animals. The extension of this analysis to other plasmid encoded resistance genes will allow exploring their importance in the dissemination of resistant strains, with particular emphasis on the link between the human and veterinary perspectives.
Descrição
Palavras-chave
Ciências biomédicas Biologia molecular Plasmídeos de Staphylococcus epidermidis Resistência Efluxo Bacterioterapia
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical