Antibiotic combinations to tackle Gram-negative bacterial pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu Campus, New Zealand

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Antimicrobial resistance, especially in Gram-negative bacterial pathogens, is one of the most serious threats with which humans have been confronted. Coordinated efforts from industrial, academic and government sectors have been called and executed to introduce novel antibiotics into the market to meet clinical demand. Despite that, the rate of successful antibiotic development appears to be lagging behind the emergence of antibiotic resistant pathogens in the arms race between humans and super-bugs. In this dire context, alternative approaches are required to tackle Gram-negative bacterial infections. This thesis reports synergistic interaction between a secondary bile salt, sodium deoxycholate (DOC), and 5-nitrofuran pro-drugs, an old class of synthetic antibiotics, in inhibiting/killing Gram-negative enterobacteria, such as Escherichia coli, Salmonella enterica and Citrobacter gillenii. Using a genetic approach, the underlying mechanism of the synergy between the two drugs was found to involve 5-nitrofuran-mediated inhibition of TolC-associated efflux pumps that otherwise exclude DOC from bacterial cells. This synergistic combination provides a promising tool to combat infections caused by enterobacterial pathogens. The mechanism of action of individual drugs, DOC and 5-nitrofurans, was also investigated using whole-genome sequence analyses of selected resistant mutants, followed by genetic and biochemical studies. A novel nitrofuran-activating enzyme, AhpF, was identified in E. coli that reduces 5-nitrofuran prodrugs in a manner different from that of an established 5-nitrofuran activation enzyme NfsB. This discovery opens new avenues to counteract nitrofuran-resistant clinical isolates by screening for molecules that upregulate AhpF expression or catalytic activity, or designing nitrofuran analogues activated at high efficiency by the AhpF enzyme. Also, this thesis identified mutations that cause a low-level resistance to DOC in efflux-pump-deficient genetic background. These all resulted in growth-slowing phenotype, the majority of which were involved in cAMP signaling. Singe mutations conferring high-level DOC-resistance were not identified in the mutant screen, supporting the use of DOC/nitrofuran combinations.