טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentJammal Joanna
SubjectSensitization of Gram-Negative Bacteria to Rifampin
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Amram Mor
Full Thesis textFull thesis text - English Version


Abstract

The development of new tools to fight antibiotic-resistant pathogenic bacteria is of essential need. Due to their structural simplicity, pharmacokinetic stability and relative low production cost, oligomers of acylated lysines (OAKs) represent a potentially useful approach for designing new antibacterial agents. The OAK C12(ω7)KKc12K has demonstrated synergistic action with some antibiotics against Gram-negative bacteria (GNB). The OAK showed the ability to cause partial membrane depolarization at sub-minimal inhibitory concentrations (MIC), hence depriving bacteria of the proton motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics such as erythromycin. Preliminary in vivo results showed that combined treatment of the OAK and erythromycin was effective, unlike the combined treatment with rifampin. In attempt to improve activity of OAK-rifampin combined treatment in vivo, we extend these studies and exploit accumulated knowledge for further developing the OAK approach, while focusing on short antibacterial sequences. Thus, design principles that emerged from previous studies regarding the relative importance of charge and hydrophobicity were used in the current work, to inspire the design of new derivatives towards improving the antibacterial activity of the reference OAK (C12(ω7)KKc12K) in vitro and in vivo.

In vitro efficacy assays demonstrated that OAK and rifampin were individually incapable to affect bacterial growth efficiently as the MICs were generally ≥16 µg/ml, whereas combinations of these drugs revealed to be mutually synergistic since OAKs MIC were reduced by >100 folds in presence of sub-MIC rifampin and inversely, rifampin MIC was reduced by >5,000 folds in presence of sub-MIC OAKs. Mechanistic data suggest that chemo-sensitization is likely to result from OAK-mediated outer membrane (OM) permeabilization, such as proposed for polymyxins, considered gold standard references owing to their high affinity binding to lipopolysaccharides. Efficacy was demonstrated in vivo as well, using the peritonitis-sepsis model. The individual drugs led to 80-90% death of infected mice when the combination therapy achieved 60% survival. Collectively, these findings suggest a potentially useful combination therapy approach for expanding the sensitivity spectrum of GNB to include impermeable antibiotics.