Staphylococcus aureus is a Gram positive potentially pathogenic bacterium which has a propensity to gather resistance determinants. Mupirocin is a novel topical antibiotic active against many Gram positive species, including staphylococci and effective in the treatment and prevention of staphylococcal infections. Mupirocin acts by competitively inhibiting the charging of isoleucyl tRNA-synthetase (IRS) with isoleucine. Resistance has been observed in Staphylococcus aureus and coagulase negative staphylococci. Intermediate-level resistance (MIC >8μg ml^-1 >512μg ml^-1) is thought to be due to spontaneous mutations in the native IRS. High-level resistance (>512μg ml^-1) is conferred by a second IRS protein, encoded by mupA which has a much lower affinity for mupirocin than isoleucine. The mupirocin resistance gene (mupA) is usually found on a 4.05kb EcoRI fragment of plasmids of otherwise varied EcoRI restriction pattern which are easily transferred between strains by filter mating. Prior to the onset of these studies, mupirocin resistance had not been found linked to another resistance determinant. Initial investigations intended to identify mechanisms of gene flux resident on mupA plasmids revealed a family of related mupA plasmids, the p3356 family which includes three plasmid types: p3356, p3356D and p3358. p3356 contains a single copy mupA flanked by direct repeats of the staphylococcal insertion sequence IS257. p3356D is identical to p3356 except for the duplication of a "mupA-IS257" cassette in tandem repeat. p3358 is related to p3356D by the insertion of a pT181-like plasmid (tetracycline resistant) accompanied by the duplication of an IS257 in direct repeat to flank the inserted pT181; thus p3358 is the first documented example of linked resistance between mupirocin and another resistance determinant, namely tetracycline. IS257 has been implicated as the recombinogenic site in the gene duplication event involved in the evolution of p3356D from p3356. IS257 co-integrative transposition has been demonstrated to allow the co-integration of pOX7 with p3356 to generate a p3358-type plasmid in which pT181 is replaced by pOX7. Therefore, it is concluded that IS257 transposition and recombination is a mechanism by which staphylococcal replicons can evolve to form multiply resistant replicons.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Rhodococcus fascians is a Gram-positive phytopathogenic bacterium which induces the development of leafy galls, local amplifications of multiple buds, on most infected plants. This process is linked to the production of phytohormones along with the presence of essential virulence-associated genes like the plasmid loci att and fas and the chromosomal gene vicA. However, the presence of these genes is not sufficient to ensure the infection phenotype development, indicating that other genes play a role in R. fascians pathogenicity. In this work, we studied the metabolic modifications occurring when the bacterium interacts with its host using a proteomic approach. A comparison between virulent and avirulent strains showed variations in the expression of catalases. In the virulent strain, besides the transitory induction of the att locus expression, the bacterium changes its metabolism from the Krebs cycle to the glyoxylate shunt, a process which is frequently observed in bacteria confronted to a hostile environment. The expression of the shunt-specific enzyme isocitrate lyase increased, while expression of fumarate hydratase and pyruvate dehydrogenase decreased. Hence, we focused on the link between the glyoxylate shunt and virulence. A screening of a R. fascians mutant library based on the capacity of bacteria to use acetate as the sole carbon source, a metabolic pathway depending on the glyoxylate shunt, resulted in the identification of a new gene essential for R. fascians pathogenicity. This gene encodes a glycosyl transferase, an enzyme known to be involved in the bacterial cell wall biosynthesis but possibly also implicated in cytokinin secretion. A mutant in this gene harboured an altered colony phenotype and could not induce malformations on infected plants. Accordingly, our results were integrated in the leafy gall pathology model recently presented by Stes et al. (2011). Finally, the several questions that are raised by this work, allowed us to suggest further research perspectives in order to unveil a little more of the R. fascians mysterious ways to interact with the plant./Rhodococcus fascians est une bactérie Gram-positive phytopathogène qui induit le développement de galles feuillées, des amplifications locales de multiples bourgeons, sur la plupart des plantes infectées. Ce processus est lié à la production de phytohormones ainsi qu’à la présence de gènes essentiels associés à la virulence tels que les loci plasmidiques att et fas et le gène chromosomique vicA. Cependant, la présence de ces gènes ne suffit pas à garantir le développement du phénotype d’infection, indiquant que d’autres gènes jouent un rôle dans la pathogénicité de R. fascians. Dans ce travail, nous avons étudié les modifications métaboliques qui se produisent lorsque la bactérie interagit avec son hôte par une approche protéomique. Une comparaison entre les souches virulente et avirulente a mis en évidence des variations d’expression au niveau des catalases. Dans la souche virulente, outre l’induction transitoire de l’expression du locus att, la bactérie change son métabolisme pour passer du cycle de Krebs au shunt du glyoxylate, un processus fréquemment observé chez les bactéries confrontées à un environnement hostile. L’expression de l’isocitrate lyase, enzyme spécifique au shunt, augmente, tandis que celle de la fumarate hydratase et de la pyruvate déhydrogénase diminue. Nous nous sommes donc intéressés au lien entre le shunt du glyoxylate et la virulence. Le screening d’une banque de mutants de R. fascians basé sur la capacité de la bactérie à utiliser l’acétate comme seule source de carbone, une voie métabolique dépendant du shunt du glyoxylate, a permis d’identifier un nouveau gène essentiel pour la pathogénicité de R. fascians. Ce gène code pour une glycosyl transferase, une enzyme impliquée dans la biosynthèse de la paroi bactérienne mais également dans la sécrétion des cytokinines. Un mutant dans ce gène présente un phénotype de colonie altéré et ne peut induire de malformations chez les plantes infectées. Finalement, nos résultats et les pistes d’interprétations que nous avons émisent nous permettent de compléter le modèle de l’interaction R. fascians-plante proposé récemment par Stes et al. (2011). Des perspectives de recherches visant une meilleure compréhension de ce pathosystème sont proposées. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished