Pharmacokinetics and pharmacodynamics of antibiotics in model of persistent infections

Bruxelles Woluwe

Bacterial persistent or recurrent infections are associated with two specific lifestyles, namely intracellular survival and biofilms. We are studying antibiotic activity against these two forms of infections in relationship with antibiotic pharmacokinetics (factors determining antibiotic access to the target). 

  1. Cellular pharmacokinetics
    We study the cellular accumulation (including the mechanisms of entry) and the subcellular localization of novel molecules in preclinical and clinical development, as a basis for further studies examining their intracellular activities in specific compartments. We try to decipher the mechanisms for their penetration and distribution within the cells. Over the last years, we have focused our interest on new antibiotic classes, like lipoglycopeptides, ketolides, new fluoroquinolonesand new oxazolidinones now present on the market. We are now examining innovative antibiotic classes acting on still unexploited targets in order to define their capacity to accumulate within the cells and then to define their interest for the treatment of intracellular infections.
  2. Cellular pharmacodynamics
    In parallel, we study the activity of antibiotics against intracellular bacteria sojourning in different subcellular compartments, mainly Listeria mono-cytogenes(cytosol), Staphylococcusaureus(phagolysosomes), andPseudomonasaeruginosa.We have also extended this model to other bacterial species of medical interest. We developed an in vitro pharmacodynamic approach to comparethe efficacy and the potency of the drugs. In brief, we showed thatantibiotics are in general less effective but equipotent against intracellular than against extracellular bacteria, irrespective of their accumulation level. The data generated with these models have been incorporated inthe dossier having led to the registration of the last antibiotics brought on the market.            We are now trying to elucidate the mechanisms by which intracellular bacteria become tolerant to antibiotics. We specially focus on trying to identify genes involved in intracellular persistence. To this effect, we ran a transcriptomic analysis of intracellular S. aureus surviving antibiotic exposure within permissive eukaryotic cells. We found that these survivors were persisters, i.e. phenotypic variants exhibiting transient non-growing state and antibiotic tolerance. This phenotype was stable but reversible upon antibiotic removal, unveiling a reservoir for relapsing infection.These persisters harboreda major transcriptomic reprogramming but remain metabolically active. Regulation mechanisms were not solely dependent on stringentresponse but includeda network of responses displaying multiples entries, comprising the activation of cell wall stress stimulon, SOS and heat shock responses. These changes led to multidrug tolerance after exposure to a single antibiotic.We have also characterized the capacity to survive inside these cells of clinical isolates collected from persistent infections.

Overview of intracellular persistence regulation of S. aureus. In vacuolar nutrient-rich compartments, persisters are metabolically active cells shielding cell wall, DNA and translation products. Under environmental factors from the host cell, including a carbon source shift and antibiotic pressure, persisters promote a network of stress or adaptive responses displaying multiple entries. Stringent response does not show signs of activity for prolonged periods but rather contributes partly to initiate the switch to a persister phenotype through (i) post-translational modifications, contributing to an almost immediate blockade of bacterial division, and (ii) transcriptional regulation, silencing energy-consuming processes. Regulation circuits also include the cell wall stress stimulon, the SOS response, and the heat shock response. These active responses, together with a decrease in oxidative phosphorylation and in translation levels, lead to multidrug tolerance upon exposure to a single antibiotic. This stable phenotype allows bacteria to maximize the chances of long-term survival. Finally, depending on the level of stress, this state could either revert to replicative forms, or promote the evolution to resistant forms, through increased probability of mutations and horizontal gene transfer. Peyrusson et al. 2020, Nature Communication.