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Estudo do potencial de virulência de Staphylococcus pseudintermedius associado a infeções de pele em animais de companhia
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Resumo(s)
A espécie Staphylococcus pseudintermedius é um agente patogénico oportunista,
responsável pela maioria das infeções de pele e tecidos moles (SSTIs) em animais de
companhia, nomeadamente em cães. A emergência e disseminação de estirpes resistentes
à meticilina (MRSP) e multirresistentes (MDR) é uma crescente ameaça à saúde pública.
A virulência em S. pseudintermedius é afetada por múltiplos fatores, incluindo a presença
da leucocidina LukI e a produção de biofilme, ambos sob regulação do sistema acessório
regulador agr. Os objetivos desta Dissertação foram: (i) caracterizar molecularmente o
sistema agr; (ii) pesquisar a presença do determinante da leucocidina LukI; (iii) avaliar a
capacidade de formação de biofilme e; (iv) avaliar o potencial de virulência de estirpes
de interesse no modelo de infeção de Galleria mellonella.
Para tal, foi estudada uma coleção de 155 S. pseudintermedius associados a SSTIs
em cães, gatos e um coelho. A caracterização molecular do sistema agr foi feita através
de PCR e sequenciação. A capacidade de formação de biofilme foi avaliada pelo método
de adesão em placa com coloração por violeta de cristal. Foram pesquisados por PCR
genes de interesse, nomeadamente o gene lukF que codifica para LukI e os genes icaADB
associados à produção de biofilme. Estas características foram relacionadas com
características previamente estabelecidas por meio de análise estatística, utilizando o teste
do Qui-Quadrado. Finalmente, foram selecionadas duas estirpes produtoras de biofilme e
pertencentes a linhagens clonais de interesse, para avaliar o seu potencial de virulência
no modelo de infeção animal de G. mellonella, comparando-as com a estirpe tipo S.
pseudintermedius DSM21284T.
Os resultados obtidos revelaram agrIII como o tipo de agr mais frequente nesta
coleção (45,8%, 71/155), seguindo-se agrIV (27,1%, 42/155), agrII (17,4%, 27/155) e
agrI (9,7%, 15/155). A produção de biofilme foi detetada num terço da coleção (33,6%,
52/155), sendo influenciada pela concentração de NaCl para 26,5% (41/155) das estirpes.
Salienta-se ainda que a produção de biofilme mostrou ser uma característica frequente em
estirpes da linhagem ST71-agrIII. Os genes lukF e icaADB foram detetados em toda a
coleção. A análise estatística destes dados revelou uma relação estatisticamente
significativa entre o tipo de agr e a formação de biofilme, estando agrIII e agrI associados
à produção e não produção de biofilme, respetivamente. Os ensaios de infeção em G.
mellonella e a análise das curvas de sobrevivência de Kaplan-Meier sugerem um maior
potencial de virulência da estirpe produtora de biofilme pertencente à linhagem ST118-
agrII.
Este estudo demonstra que a produção de biofilme é uma característica frequente
em linhagens clonais de S. pseudintermedius resistentes aos antibióticos e associadas a
SSTIs. É ainda sugerido um maior potencial de virulência de estirpes da linhagem clonal
ST118. Estes resultados reforçam a necessidade de uma abordagem Uma Só Saúde no
estudo de infeções causadas por esta bactéria de modo a otimizar o seu controlo e
terapêutica.
The species Staphylococcus pseudintermedius is an opportunistic pathogen, responsible for most skin and soft tissue infections (SSTIs) in companion animals, particularly dogs. The emergence and spread of methicillin-resistant (MRSP) and multidrug-resistant (MDR) strains is a growing threat to public health. Virulence in S. pseudintermedius is affected by multiple factors, including the presence of leucocidin LukI and biofilm production, both under regulation of the agr accessory gene regulator. The aims of this dissertation were: (i) to molecularly characterize the agr system; (ii) to search for the presence of the leucocidin determinant LukI; (iii) assess the ability to form biofilms and; (iv) evaluate the virulence potential of interest strains in the Galleria mellonella infection model. To this end, a collection of 155 S. pseudintermedius associated with SSTIs in dogs, cats and one rabbit was studied. Molecular characterization of the agr system was performed using PCR and sequencing. The ability to form biofilm was evaluated by the plate adhesion method with crystal violet staining. Genes of interest were searched by PCR, namely the lukF gene that encodes for LukI and the icaADB genes associated with biofilm production. These traits were related to previously established characteristics through statistical analysis, using the Chi-square test. Finally, two biofilm producing strains belonging to clonal lineages of interest were selected to assess their virulence potential in the G. mellonella animal infection model, comparing them with the S. pseudintermedius DSM21284T type strain. The results obtained revealed agrIII as the most frequent type of agr in this collection (45.8%, 71/155), followed by agrIV (27.1%, 42/155), agrII (17.4%, 27/155) and agrI (9.7%, 15/155). Biofilm production was detected in one third of the collection (33.6%, 52/155) and was influenced by the NaCl concentration for 26.5% (41/155) of the strains. It should also be noted that the production of biofilm proved to be a frequent trait in strains of the ST71-agrIII lineage. The lukF and icaADB genes were detected in all the collection. Statistical analysis of these data revealed a statistically significant relationship between agr type and biofilm formation, with agrIII and agrI being associated with biofilm production and non-production, respectively. Infection assays in G. mellonella and the analysis of Kaplan-Meier survival curves suggest a greater virulence potential of the biofilm-producing strain belonging to the ST118-agrII lineage. This study demonstrates that biofilm production is a frequent trait in clonal lineages of S. pseudintermedius resistant to antibiotics and associated with SSTIs. It is also suggested a greater virulence potential of ST118 clonal lineage strains. These results reinforce the need for a One Health approach in the study of infections caused by this bacterium to optimize its control and treatment.
The species Staphylococcus pseudintermedius is an opportunistic pathogen, responsible for most skin and soft tissue infections (SSTIs) in companion animals, particularly dogs. The emergence and spread of methicillin-resistant (MRSP) and multidrug-resistant (MDR) strains is a growing threat to public health. Virulence in S. pseudintermedius is affected by multiple factors, including the presence of leucocidin LukI and biofilm production, both under regulation of the agr accessory gene regulator. The aims of this dissertation were: (i) to molecularly characterize the agr system; (ii) to search for the presence of the leucocidin determinant LukI; (iii) assess the ability to form biofilms and; (iv) evaluate the virulence potential of interest strains in the Galleria mellonella infection model. To this end, a collection of 155 S. pseudintermedius associated with SSTIs in dogs, cats and one rabbit was studied. Molecular characterization of the agr system was performed using PCR and sequencing. The ability to form biofilm was evaluated by the plate adhesion method with crystal violet staining. Genes of interest were searched by PCR, namely the lukF gene that encodes for LukI and the icaADB genes associated with biofilm production. These traits were related to previously established characteristics through statistical analysis, using the Chi-square test. Finally, two biofilm producing strains belonging to clonal lineages of interest were selected to assess their virulence potential in the G. mellonella animal infection model, comparing them with the S. pseudintermedius DSM21284T type strain. The results obtained revealed agrIII as the most frequent type of agr in this collection (45.8%, 71/155), followed by agrIV (27.1%, 42/155), agrII (17.4%, 27/155) and agrI (9.7%, 15/155). Biofilm production was detected in one third of the collection (33.6%, 52/155) and was influenced by the NaCl concentration for 26.5% (41/155) of the strains. It should also be noted that the production of biofilm proved to be a frequent trait in strains of the ST71-agrIII lineage. The lukF and icaADB genes were detected in all the collection. Statistical analysis of these data revealed a statistically significant relationship between agr type and biofilm formation, with agrIII and agrI being associated with biofilm production and non-production, respectively. Infection assays in G. mellonella and the analysis of Kaplan-Meier survival curves suggest a greater virulence potential of the biofilm-producing strain belonging to the ST118-agrII lineage. This study demonstrates that biofilm production is a frequent trait in clonal lineages of S. pseudintermedius resistant to antibiotics and associated with SSTIs. It is also suggested a greater virulence potential of ST118 clonal lineage strains. These results reinforce the need for a One Health approach in the study of infections caused by this bacterium to optimize its control and treatment.
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Palavras-chave
Staphylococcus pseudintermedius Ciências biomédicas Biologia molecular