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Pesquisa de genes envolvidos no fenótipo de tolerância à oxacilina em populações clínicas de Staphylococcus aureus
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A tolerância a antibióticos é um termo utilizado para descrever a capacidade de uma
população bacteriana sobreviver a exposições com antibióticos bactericidas, mesmo em
concentrações que de outra forma seriam letais, sem ocorrer alteração da concentração
mínima inibitória (MIC). Comparativamente à resistência bacteriana, a compreensão
sobre a tolerância é escassa não existindo testes laboratoriais utilizados rotineiramente na
prática clínica capazes de identificar este fenómeno; consequentemente, estirpes
tolerantes tendem a ser classificadas incorretamente resultando em falhas terapêuticas e
em infeções bacterianas recorrentes. Adicionalmente, tem vindo a ser proposto que um
background de tolerância pode promover o desenvolvimento de resistência aos
antibióticos, reforçando a necessidade de compreender e estudar este fenómeno. A
presente dissertação de mestrado, tem como objetivo aprofundar os conhecimentos
associados aos mecanismos de tolerância para uma das classes de antibióticos mais
amplamente utilizada na prática clínica, a classe dos antibióticos β-lactâmicos, no
patógeno humano Staphylococcus aureus, através da identificação, por sequenciação total
do genoma (WGS), de genes que possam estar envolvidos no fenótipo de tolerância.
Para tal, usámos uma coleção bacteriana composta por trinta estirpes clínicas de S. aureus
sensíveis à meticilina (MSSA), recuperadas em 2017 de infeções sanguíneas, com o
intuito de obter estirpes tolerantes. A tolerância foi promovida pela exposição
independente destas estirpes a dois químicos (mupirocina e carbonil cianeto m-clorofenil hidrazona ou CCCP), que, segundo a literatura, são indutores do fenótipo de tolerância.
Esta técnica permitiu a identificação de duas estirpes tolerantes. A análise genómica
revelou que o multilocus sequence type dos derivados tolerantes era diferente das
correspondentes estirpes parentais; por conseguinte, as análises bioinformáticas
realizadas neste trabalho incidiram sobre três estirpes tolerantes, previamente isoladas
antes da realização desta tese de mestrado, através de um novo método de deteção de
células tolerantes denominado de “Replica Plating Tolerance Isolation System”
(REPTIS). A análise genómica realizada entre as estirpes tolerantes e as respetivas
estirpes parentais, aponta para o envolvimento nestas estirpes de quatro mecanismos no
processo de aquisição de tolerância: a via biossintética dos ácidos gordos; o sistema
regulador VraTSR; modificações ribossomais; e, a ligação de aminoácidos aos RNAs de
transferência (tRNA) correspondentes. Por fim, os resultados apresentados nesta
dissertação, além de proporem um novo mecanismo de tolerância em S. aureus
envolvendo o gene vraT, corroboram propostas de obtenção de tolerância descritas na
literatura para Escherichia coli, o que sugere que acontecimentos de natureza semelhante
possam ocorrer em S. aureus.
Abstract Antibiotic tolerance is a term used to describe the ability of a bacterial population to survive exposure to bactericidal antibiotics, even at concentrations that would otherwise be lethal, without changing its minimum inhibitory concentration (MIC). Compared to bacterial resistance, information about tolerance is scarce and there are no laboratory tests routinely used in clinical practice capable of identifying this phenomenon; consequently, tolerant strains tend to be incorrectly classified causing therapeutic failures and recurrent bacterial infections. Additionally, it has been proposed that a tolerance background can promote the development of resistance to antibiotics, reinforcing the need to understand and study this phenomenon. This master's thesis aims to deepen the knowledge associated with tolerance mechanisms for one of the most widely used classes of antibiotics in clinical practice, the β-lactam antibiotics class, in the human pathogen Staphylococcus aureus, through the identification, by whole genome sequencing (WGS), of genes that may be involved in the tolerance phenotype. To this end, we used a bacterial collection consisting of thirty clinical methicillin susceptible S. aureus (MSSA) strains, recovered in 2017 from blood infections, in order to obtain tolerant strains. Tolerance was promoted by independently expose the strains to two chemical products (mupirocin and carbonyl cyanide m-chlorophenylhydrazone or CCCP), which, according to the literature, are tolerance phenotype inducers. This technique allowed the identification of two tolerant strains. The genome analysis revealed that the multilocus sequence type of the tolerant derivatives did not match with the parental strains; therefore, the bioinformatic analyzes performed in this work focused on three tolerant strains, previously isolated before the start of this master's thesis, through a new method of detection of tolerant cells called "Replica Plating Tolerance Isolation System" (REPTIS). The genetic analysis performed between the tolerant strains and their respective parental strains, hint to the involvement in these strains of four mechanisms in the process of tolerance acquisition: the biosynthetic pathway of fatty acids, the VraTSR regulatory system, ribosomal modifications and the binding of amino acids to their corresponding transfer RNAs (tRNA). Finally, the results presented in this dissertation, in addition to proposing a new mechanism of tolerance in S. aureus involving the vraT gene, corroborate proposals for granting tolerance described in the literature for Escherichia coli, which suggests that events of a similar nature may occur in S. aureus.
Abstract Antibiotic tolerance is a term used to describe the ability of a bacterial population to survive exposure to bactericidal antibiotics, even at concentrations that would otherwise be lethal, without changing its minimum inhibitory concentration (MIC). Compared to bacterial resistance, information about tolerance is scarce and there are no laboratory tests routinely used in clinical practice capable of identifying this phenomenon; consequently, tolerant strains tend to be incorrectly classified causing therapeutic failures and recurrent bacterial infections. Additionally, it has been proposed that a tolerance background can promote the development of resistance to antibiotics, reinforcing the need to understand and study this phenomenon. This master's thesis aims to deepen the knowledge associated with tolerance mechanisms for one of the most widely used classes of antibiotics in clinical practice, the β-lactam antibiotics class, in the human pathogen Staphylococcus aureus, through the identification, by whole genome sequencing (WGS), of genes that may be involved in the tolerance phenotype. To this end, we used a bacterial collection consisting of thirty clinical methicillin susceptible S. aureus (MSSA) strains, recovered in 2017 from blood infections, in order to obtain tolerant strains. Tolerance was promoted by independently expose the strains to two chemical products (mupirocin and carbonyl cyanide m-chlorophenylhydrazone or CCCP), which, according to the literature, are tolerance phenotype inducers. This technique allowed the identification of two tolerant strains. The genome analysis revealed that the multilocus sequence type of the tolerant derivatives did not match with the parental strains; therefore, the bioinformatic analyzes performed in this work focused on three tolerant strains, previously isolated before the start of this master's thesis, through a new method of detection of tolerant cells called "Replica Plating Tolerance Isolation System" (REPTIS). The genetic analysis performed between the tolerant strains and their respective parental strains, hint to the involvement in these strains of four mechanisms in the process of tolerance acquisition: the biosynthetic pathway of fatty acids, the VraTSR regulatory system, ribosomal modifications and the binding of amino acids to their corresponding transfer RNAs (tRNA). Finally, the results presented in this dissertation, in addition to proposing a new mechanism of tolerance in S. aureus involving the vraT gene, corroborate proposals for granting tolerance described in the literature for Escherichia coli, which suggests that events of a similar nature may occur in S. aureus.
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Palavras-chave
Microbiologia médica Staphylococcus aureus Tolerância Via biossintética dos ácidos gordos VraT Tradução de proteínas