COMBINATION STRATEGIES AGAINST PERSISTENCE AND RESISTANCE IN Acinetobacter baumannii

Abstract

The damaging effects of antimicrobial resistance have globally manifested themselves as a continuing challenge to human health, costing hundreds and thousands of lives. The emergence of multidrug resistant (MDR) Gram negative bacterial pathogens, particularly Metallo-β- lactamase harbouring Acinetobacter baumannii poses a major threat to the existing antibiotic arsenal. The increasing success of A. baumannii as a clinically relevant pathogen lies in its ability to acquire genes which enable it to survive harsh environmental conditions in the healthcare settings and facilitates its adherence onto abiotic and biotic surfaces forming complex biofilms. Global outbreaks of A. baumannii in hospitals and military bases concomitant with the emergence of highly resistant and virulent isolates, places this pathogen in the category of six most problematic MDR pathogens, by Infectious Diseases Society of America (IDSA). The introduction of new antibiotics in clinics made it clear that antibiotic treatment often fails to completely eradicate a bacterial subpopulation, termed as ‗Persisters‘. Bacterial persisters represent non inheritable drug tolerant populations that are linked to recalcitrance of infections in health care settings. The rise of antibiotic resistance and depletion of new antibiotics in drug discovery pipeline has made the task of persister eradication more daunting. The work performed in this thesis has investigated the potential of antibiotic combinations to combat the problem of resistance and persistence against MDR pathogen A. baumannii. A dual fluorescence system was constructed to detect persister cells of A. baumannii. This system was used for screening of a potential antibiotic candidate that displayed killing of polymyxin B induced persisters. Among the selected antibiotics, treatment of rifampicin led to complete eradication of polymyxin B induced persisters of A. baumannii. Furthermore, biochemical and cell based assays were performed to decipher the mechanism of persister formation upon polymyxin B treatment and their subsequent eradication in the presence of rifampicin. This finding was further validated in a murine wound persister infection model, demonstrating polymyxin B and rifampicin combination to be an effective therapeutic option against A. baumannii persisters. Metallo-β-lactamases (MBL) are zinc containing enzymes that display broad substrate spectrum and can inactivate all bicyclic β-lactam antibiotics. It was identified that a Food and Drug Administration (FDA) approved drug disulfiram, in vitro potentiated meropenem against New Delhi Metallo-β-lactamase (NDM) and Imipenemases (IMP) carrying Gram negative pathogens. In combination with meropenem, disulfiram improves the pharmacodynamic properties against MBL expressing A. baumannii. It was demonstrated that disulfiram inhibits NDM -1 and IMP-7 by chelation of active site zinc atom at a concentration far below the Lethal Dose 50 (LD50) value for Peripheral Blood Mononuclear Cells (PBMCs). This study demonstrated that disulfiram in combination with meropenem results in significant reduction in bacterial burden in pneumonia and systemic mice infection models in contrast to mice administered with antibiotic alone. Results obtained showed the therapeutic potential of disulfiram in overcoming therapeutic failure of MBL positive Gram-negative pathogens. Quorum sensing is a process by which bacterial cells communicate amongst themselves and fine tune the expression of different virulence genes accordingly. One novel strategy to treat A. baumannii could be the use of antibiotics in combination with anti-quorum sensing agents that may attenuate the expression of virulence factors in the pathogen, allowing conventional antibiotics to act synergistically and leading to its eradication. Towards this end, I have also performed antibiotic interaction screen to decipher a novel synergistic interaction between streptomycin and rifampicin which exhibited excellent bactericidal activity against MDR A. baumannii clinical isolates. The combination displayed effective penetration and disruption of preformed A. baumannii biofilms, thus causing a considerable reduction in the viable cell count. In combination, streptomycin and rifampicin downregulate the expression of csuA/BABCDE genes of the type I pilus system, which plays an essential role in A. baumannii biofilm formation on biotic and abiotic surfaces. Studies on the murine wound infection model further established this combination to be a promising treatment option for topical administration. Overall, this study reports novel antibiotic combinations as chemotherapy and endeavours to minimize or overcome the problem of antibiotic resistance and persistence in healthcare settings. This work highlights the mechanism of pathogen eradication by these novel combinations and based on the in vivo efficacy results, hold promise for use in clinics in the fight against A. baumannii.