The impact of glycopeptide- and methicillin- resistance on "Staphylococcus aureus" and its virulence in localized and systemic infections

Abstract

Adaptation of bacteria to their environment is a necessity to keep their fitness and survival under limiting conditions. In chapter I of this work we demonstrated that adaptation of glycopeptide-resistant S. aureus (GISA) happens at the cost of resistance loss. An in vitro generated step selected teicoplanin-resistant mutant was phenotypically and genotypically characterized and compared to its glycopeptide-susceptible parent S. aureus strain SA113, prior to and after challenging the host in a foreign body infection model. Slower growth, increased thickness of the bacterial cell wall, increased N-acetylglucosamine incorporation, decreased hemolysis, and downregulation of some virulence-associated genes were determined upon in vitro step selection. In vivo host pressure together with the absence of antibiotics were shown to impair this GISA phenotype and to result in the selection of fitter variants at the cost of resistance loss. In a further study we compared the virulence and pathogenicity of isolates of two epidemic ST8 and ST5 health-care associated methicillin-resistant S. aureus (MRSA) clones with MSSA isolates of variable STs that are prevalent in French clinical settings and with MSSA of the same ST in a murine sepsis model. Mouse mortality, induced by the different groups of MRSA ST8 and ST5 and MSSA ST8, ST5 and var. ST was related to phenotypic and genotypic features. MRSA isolates of both clones ST8 and ST5 caused higher mortality than MSSA expressing var. ST. However, higher mortality was not related to the presence of SCCmec, since only ST5 but not ST8 isolates showed a higher virulence in the presence of this resistance determinant. Despite the same genetic background individual clonal MRSA isolates had heterogeneous virulence properties. No specific virulence factor determined in vitro, was found related to mortality in mice. In conclusion, in a bacteraemic model, lethality varied with the sequence type, and was modulated by SCCmec. Within the same ST, clonality was not associated with a homogenous outcome in the murine sepsis model. Whole genome sequencing of clonal lethal and non-lethal isolates will allow identifying virulence factors relevant to the outcome in this model. In the third part of this thesis we investigated phenotypic properties of the clonal MRSA ST8 isolates that had a variable outcome in the murine sepsis model and measured innate and adaptive immune responses of 7 lethal and 3 non-lethal isolates. Clonality of the 10 MRSA ST8 isolates was certified by MLVA. In vitro assays, simulating steps that occur during the course of infection were performed in order to detect host response discriminating differences between lethal and non-lethal isolates. While chemotaxis, bactericidal activity and apoptosis of PMN were similar for lethal and non-lethal isolates, a preliminary study yielded a different specific immune response induced by a lethal compared to a non-lethal isolate. The role of the adaptive immunity in the outcome of an infection with lethal and non-lethal isolates will be investigated in further.