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Estudo do efluxo na resistĂȘncia a tetraciclinas em Staphylococcus pseudintermedius
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A bactĂ©ria Staphylococcus pseudintermedius, agente comensal da pele e tecidos moles dos animais de companhia, Ă© tambĂ©m um agente patogĂ©nico oportunista, responsĂĄvel pela maioria das infeçÔes da pele e tecidos moles (SSTIs) nestes animais, em particular no cĂŁo, e que apresenta frequentemente resistĂȘncia aos principais antibiĂłticos utilizados na terapĂȘutica.
Este trabalho teve como ponto de partida um estudo anterior com 155 isolados de S. pseudintermedius associados a SSTIs em animais de companhia na regiĂŁo de Lisboa. Para o presente estudo, foram selecionadas as 28 estirpes dessa coleção que apresentaram resistĂȘncia Ă s tetraciclinas (o segundo fenĂłtipo de resistĂȘncia mais frequente), associado Ă presença dos genes tet(K) e tet(L), que codificam para bombas de efluxo. Neste estudo pretendeu-se avaliar a contribuição do efluxo para a resistĂȘncia Ă s tetraciclinas em S. pseudintermedius atravĂ©s de vĂĄrias abordagens, analisando em particular a relação entre o fenĂłtipo de suscetibilidade Ă tetraciclina, doxiciclina e minociclina com presença dos determinantes tet, perfil plasmĂdico e a atividade de efluxo de tetraciclinas.
Das 28 estirpes estudadas, vinte e uma (21/28, 75,0%) possuem um plasmĂdeo. Os plasmĂdeos detetados foram agrupados em quatro perfis plasmĂdicos, de acordo com o seu tamanho e perfil de restrição. A larga maioria das estirpes apresenta plasmĂdeos pequenos (< 5 kb) e apenas duas estirpes apresentaram plasmĂdeos de tamanho intermĂ©dio (â 15kb, 2/21, 9,5%).
O gene tet(K) foi detetado nos quatro perfis plasmĂdicos das 21 estirpes, 16 das quais pertencem Ă linhagem clonal ST71, prevalente na coleção em estudo e a mais disseminada na Europa. O gene tet(L) foi identificado em apenas uma estirpe da linhagem clonal ST25, para a qual nĂŁo foi detetado nenhum plasmĂdeo.
A transferĂȘncia por eletrotransformação do plasmĂdeo pV143, de â 4 kb e representativo do perfil P1, para Staphylococcus aureus, permitiu confirmar a presença do gene tet(K) neste plasmĂdeo e a sua associação Ă resistĂȘncia Ă tetraciclina e doxiciclina. A caracterização mais detalhada do transformante, S. aureus RN4220:pV143, permitiu identificar atividade de efluxo por ensaios de suscetibilidade Ă s tetraciclinas com diferentes inibidores de efluxo, incluindo o verapamil. Este mesmo transformante foi utilizado para otimizar um novo protocolo experimental de avaliação do efluxo de tetraciclinas em estafilococos, baseado numa anĂĄlise fluoromĂ©trica. Testaram-se diferentes condiçÔes experimentais, nomeadamente substratos de efluxo, temperatura do ensaio, pH e presença de iĂŁo Mg2+ no meio. Os resultados obtidos confirmaram atividade de efluxo de tetraciclina no transformante, associada ao determinante da bomba Tet(K). A presença do gene tet(M), determinante cromossĂłmico de proteção ribossĂłmica, foi tambĂ©m associada Ă resistĂȘncia Ă tetraciclina e doxiciclina, e nalgumas estirpes tambĂ©m Ă minociclina.
Este estudo demonstra assim, que diferentes mecanismos contribuem para a resistĂȘncia Ă s tetraciclinas em S. pseudintermedius, entre os quais o efluxo, este associado Ă presença do gene tet(K), maioritariamente plasmĂdico, podendo comprometer a terapĂȘutica de SSTIs, uma das causas mais frequentes de prescrição de antibiĂłticos em Medicina VeterinĂĄria.
The bacterium Staphylococcus pseudintermedius, a commensal of the skin and soft tissues of companion animals, is also an opportunistic pathogen, responsible for the majority of skin and soft tissue infections (SSTIs) in these animals, particularly in dogs, with increasing resistance to the main antibiotics used in therapy. The starting point for this work was a previous study with 155 isolates of S. pseudintermedius associated with SSTIs in companion animals in the Lisbon region. For the present study, we selected the 28 strains from this collection that showed resistance to tetracyclines (the second most common resistance phenotype), associated with the presence of the tet(K) and tet(L) genes, which encode efflux pumps. In this study, we aimed to evaluate the contribution of efflux to tetracycline resistance in S. pseudintermedius through several approaches, focusing on the relation between the susceptibility phenotype to tetracycline, doxycycline and minocycline and the presence of tet determinants, plasmid profile and tetracycline efflux activity. Twenty-one out of the 28 strains studied (21/28, 75.0%) carried a plasmid. The plasmids detected were grouped into four profiles, according to their size and restriction profile. The vast majority of strains have small plasmids (< 5 kb) and only two strains carried plasmids of intermediate size (â 15 kb, 2/21, 9.5%). The tet(K) gene was detected in the four plasmid profiles of the 21 strains, 16 of which belong to the ST71 clonal lineage, prevalent in the study collection and the most widespread in Europe. The tet(L) gene was identified in a single strain of the ST25 clonal lineage, for which no plasmid was detected. The transfer of plasmid pV143, â 4 kb and representative of the P1 profile, to Staphylococcus aureus by electrotransformation, confirmed the presence of the tet(K) gene in this plasmid and its association with resistance to tetracycline and doxycycline. Further characterization of the transformant, S. aureus RN4220:pV143, identified efflux activity by susceptibility assays with different efflux inhibitors, including verapamil. This same transformant was used to optimize a new experimental protocol to evaluate the efflux of tetracyclines in staphylococci, based on a fluorometric analysis. Different experimental conditions were tested, including efflux substrates, temperature, pH and presence of Mg2+ ion in the medium. The results obtained confirmed tetracycline efflux activity, associated with the Tet(K) pump determinant, in the transformant. The presence of the tet(M) gene, a chromosomal determinant of ribosomal protection, was also associated with resistance to tetracycline and doxycycline and to minocycline in a few strains. This study demonstrates that different mechanisms contribute to resistance to tetracyclines in S. pseudintermedius, including efflux, which is associated with the presence of the tet(K) gene, mostly plasmid, that may compromise the treatment of SSTIs, one of the most common causes for prescription of antimicrobials in Veterinary Medicine.
The bacterium Staphylococcus pseudintermedius, a commensal of the skin and soft tissues of companion animals, is also an opportunistic pathogen, responsible for the majority of skin and soft tissue infections (SSTIs) in these animals, particularly in dogs, with increasing resistance to the main antibiotics used in therapy. The starting point for this work was a previous study with 155 isolates of S. pseudintermedius associated with SSTIs in companion animals in the Lisbon region. For the present study, we selected the 28 strains from this collection that showed resistance to tetracyclines (the second most common resistance phenotype), associated with the presence of the tet(K) and tet(L) genes, which encode efflux pumps. In this study, we aimed to evaluate the contribution of efflux to tetracycline resistance in S. pseudintermedius through several approaches, focusing on the relation between the susceptibility phenotype to tetracycline, doxycycline and minocycline and the presence of tet determinants, plasmid profile and tetracycline efflux activity. Twenty-one out of the 28 strains studied (21/28, 75.0%) carried a plasmid. The plasmids detected were grouped into four profiles, according to their size and restriction profile. The vast majority of strains have small plasmids (< 5 kb) and only two strains carried plasmids of intermediate size (â 15 kb, 2/21, 9.5%). The tet(K) gene was detected in the four plasmid profiles of the 21 strains, 16 of which belong to the ST71 clonal lineage, prevalent in the study collection and the most widespread in Europe. The tet(L) gene was identified in a single strain of the ST25 clonal lineage, for which no plasmid was detected. The transfer of plasmid pV143, â 4 kb and representative of the P1 profile, to Staphylococcus aureus by electrotransformation, confirmed the presence of the tet(K) gene in this plasmid and its association with resistance to tetracycline and doxycycline. Further characterization of the transformant, S. aureus RN4220:pV143, identified efflux activity by susceptibility assays with different efflux inhibitors, including verapamil. This same transformant was used to optimize a new experimental protocol to evaluate the efflux of tetracyclines in staphylococci, based on a fluorometric analysis. Different experimental conditions were tested, including efflux substrates, temperature, pH and presence of Mg2+ ion in the medium. The results obtained confirmed tetracycline efflux activity, associated with the Tet(K) pump determinant, in the transformant. The presence of the tet(M) gene, a chromosomal determinant of ribosomal protection, was also associated with resistance to tetracycline and doxycycline and to minocycline in a few strains. This study demonstrates that different mechanisms contribute to resistance to tetracyclines in S. pseudintermedius, including efflux, which is associated with the presence of the tet(K) gene, mostly plasmid, that may compromise the treatment of SSTIs, one of the most common causes for prescription of antimicrobials in Veterinary Medicine.
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Microbiologia