טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLiran Livne
SubjectOAK and Antibiotic Combination Effects on Antibacterial
Activity
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Mor Amram
Full Thesis textFull thesis text - English Version


Abstract

Antibiotic resistance represents a worldwide health problem where treatment failure of an ever increasing number of pathogens is associated with severe outcomes such as increased mortality and morbidity. Host defense peptides (HDPs) represent a class of promising agents in fighting the growing bacterial resistance to antibiotics and might provide a viable alternative for the treatment of raging multidrug-resistant infectious diseases. In addition to their antimicrobial action some HDPs show evidence of synergistic activity in the presence of conventional antibiotics, although until now the molecular basis for their combined mode of action was not examined experimentally. However, HDPs exhibit crucial drawbacks that severely hamper their potential uses in medicine, including high toxicity and poor bioavailability due to non-specific modes of action and susceptibility to proteolytic degradation, as well as high production costs.


The oligo-acyl-lysyl (OAK) sequence C12K-7α8 is a chemical mimic of host defense peptides, designed to address disadvantageous characteristics of conventional peptides, with documented in-vitro and in-vivo antibacterial properties. Here, to probe new means for overcoming antibiotic resistance phenomena, we investigated the role of this membrane-active peptide upon combination with various classes of antibiotics, as assessed against E. coli strains that are typical of multi-drug resistant (MDR) Gram-negative bacteria. In-vitro susceptibility assays revealed combinations with sub-MIC OAK concentrations that acted synergistically with several antibiotics (e.g., erythromycin, clarithromycin, tetracycline, rifampicin and ciprofloxacin) whose MIC was lowered by up to orders of magnitude. Our attempts to shed light into the molecular basis for this synergism using relevant mutant strains and biochemical assays, provided evidence in support of the view that bacterial sensitization to antibiotics was derived mainly from the OAK’s capacity to overcome the efflux-enhanced mechanism for resistance, by promoting backdoor entry of otherwise excluded antibiotics In addition, to facilitate simultaneous delivery of the pooled drugs to the infection site, we developed a novel OAK-based cochleate system with demonstrable stability in whole blood and subsequent slow drug release. To assess the potential therapeutic use of such cochleates, we performed preliminary experiments that imitate systemic treatment of neutropenic mice infected with lethal inoculums of MDR E. coli and treated by a single intravenous dose. Unlike individual treatments with free erythromycin or cochleated OAK, their co-encapsulation has increased mice survival in a dose-dependent manner, indicating that drug combination in OAK-based cochleates can decrease drug toxicity and increase systemic therapeutic efficacy. Collectively, the data suggest a potentially useful approach for fighting efflux-enhanced drug resistance.