Accumulation of a bactericidal antibiotic by tolerant bacteria and insights into bacterial persistence

Aminoglycoside (AG) is a family of antibiotic which target bacterial ribosome. Few examples of this family are neomycin, gentamicin and streptomycin. When these antibiotics bind to ribosomes, they cause miscoding or inhibit protein synthesis which consequently leads to cell death. Although discovery of these antibiotics was more than half a century ago, there are many facts about AGs’ action mechanism which remain unknown. AG accumulation in the bacterial cells happens in three steps. First step is cell membrane attachment. This step is driven by an electrostatic interaction with the cationic AGs. Second step is an energy dependent phase I (EDPI). In EDPI, the antibiotic enters into the cytoplasm and reaches ribosomes, causing miscoding and production of misfolded proteins. EDPI depends on cellular energy level, however to date the mechanism by which AGs pass through membranes and enter cytoplasm is unknown. The third step is energy dependent phase II (EDPII) in which the antibiotic enters into the cytoplasm in larger amount due to damages in the membrane that resulted from EDPI. The aim of this PhD was to create new tools to study the interaction of AGs with bacteria and apply the methodology to study fast growing bacteria as well as persister cells. We have made fluorescently-tagged AGs with preserved bactericidal properties. We used these conjugates to track down the interaction of AG at single cell level by fluorescence microscopy. We combined fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis to measure AGs accumulation in the cells at different time points to capture the kinetics of antibiotic penetration. This study showed that there are two accumulations patterns for the drug in cells: in the first step there is a peripheral accumulation, which corresponds to specific binging to cell membrane. Next there is a cytoplasmic accumulation in which the antibiotic in entering into the cytoplasm. According to microscopy time laps study, low levels of cytoplasmic accumulation is tolerated by cells and did not cause cell death. Using FACS analysis, we used an inhibitor of EDPI and EDPII and proved that with this technique we can distinguish different steps of AGs accumulation. During protocol adjustment steps we found that AGs can enter into the cytoplasm as a result of mechanosensation and activation of mechanosensitive (MS) channels. These channels have already been shown to have affinity to AG and here this is a first time that we observed that mechanical manipulation of cells lead to opening of MS channel causing massive cytoplasmic accumulation. This unpredictable result may lead us to a better understanding of the mechanism of AG entrance into the cytoplasm. After studying AG accumulation in fast growing cells, we studied AG tolerance for non-growing cells, which are called persisters. Persisters are antibiotic tolerant sub-population among susceptible bacterial cell population. Persisters are non-growing, dormant cells which tolerate high concentrations of antibiotic. In the absence of antibiotic, they exit this dormant state and grow into an antibiotic susceptible population. By fluorescence microscopy we showed that persister cells have peripheral accumulation of AG. Thanks to our methodology, we have a powerful tool by which we can determine the patterns of AG accumulation. Prior to this study, it was only possible to know the levels of accumulation and not the corresponding patterns. We applied the method to investigate AG accumulation in two mutants of E. coli, which are less tolerant to AG and defined their pattern of accumulation. Finally, we developed a coated microfluidic system, which is adapted to our antibiotics for studying in real time drug accumulation by persister cells.