Control of intracellular Listeria monocytogenes growth by antibiotics and granulocyte-macrophage colony stimulating factor

Listeria monocytogenes is a facultative intracellular bacterium that causes life-threatening infections in human. The treatment of listeriosis with conventional antibiotics therapy remains a challenge (mortality still up to 30%), and the intracellular forms have proved to be difficult to eradicate in the absence of a fully effective immune response and seem to be responsible for the recurrence of the infection. Our work was therefore aimed at the study of the influence of two different treatment regimens against THP-1 intracellular infection by Listeria monocytogenes, namely antimicrobials and an immunomodulator (the granulocyte-macrophage colony stimulating factor). We first considered the activity of several classes of antibiotics against extracellular and intracellular L. monocytogenes during 5h. The bacteria were sensible to all compounds and as expected beta-lactams and azithromycin were bacteriostatic when gentamicin and the fluoroquinolones were bactericidal against bacteria in broth. However, despite the fact that they are considered as the first choice therapy for listeriosis, gentamicin had no activity and ampicillin was only slightly bactericidal against intracellular forms. Conversely moxifloxacin was highly bactericidal in a concentration-dependent fashion. Moxifloxacin could therefore be considered as an alternative to the common antimicrobial treatment of listeriosis and clinical trials seems therefore interesting. We then compared the intracytosolic pharmacodynamic properties of antibiotics with the extracellular ones at concentrations achievable in humans after conventional administration. We showed that these properties could be quite different. Indeed, (i) gentamicin is completely inactive against intracytosolic pathogen when eradicating all bacteria in broth in less than 5h; (ii) the great accumulation of azithromycin in macrophages does not allow them to become bactericidal intracellularly, confirming their intrinsically bacteriostatic character; (iii) beta-lactams are slowly bactericidal at high concentration after 24h, which is consistent with the fact that they are considered as time-dependent antibiotics, but more surprisingly are much more active intracellularly than extracellularly despite their lack of cellular accumulation and the fact that their MBCs are higher than the extracellular concentration used, suggesting some sort of cooperation between the beta-lactams and the macrophages; and (iv) although fluoroquinolones show the same pattern of activity against extracellular and intracellular forms and that we showed that they are concentration-dependent antibiotics, the cellular accumulation does not allow them to become more active, in fact moxifloxacin showed exactly the same activity in both cases and ciprofloxacin was twice less active intracellularly. Finally we studied the activity of the granulocyte-macrophage colony stimulating factor used as immunomodulator on the intracellular growth of L. monocytogenes. We showed that GM-CSF has a concentration-dependent activity within a range of concentrations achievable in human that inhibit up to 40% of the intracellular growth of the bacteria. GM-CSF action seems to be mediated by autocrine activation following TNF-alpha secretion and the action of free radicals following induction of iNOS. Both processes are necessary but not sufficient, suggesting an additive or a synergistic activity. Indeed, when suppressing one of the phenomenons the activity of GM-CSF is completely abolished.Thèse de doctorat en sciences pharmaceutiques (FARM 3) -- UCL, 200