Synergistic triple combination antibiotic therapy for Gram-negative bacterial infections : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu, New Zealand

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The continuing emergence of multidrug-resistant bacteria and the slowing down of the discovery and development of novel antibiotics have made antimicrobial resistance an ominous threat to human health. As reflected in the World Health Organization’s priority pathogens list, this problem is notably more severe in multidrug-resistant Gram-negative bacteria. This situation needs to be rectified through alternative approaches, such as the revival of 'old' antibiotics and the development of combination therapies. This thesis focuses on the combination of the 'old' antibiotics, nitrofurans and vancomycin (VAN) with the secondary bile salt sodium deoxycholate (DOC). The synergistic interaction of these antibacterials was demonstrated in the in vitro growth inhibition and killing of Gram-negative bacteria, including the clinically relevant pathogens such as carbapenemase-producing Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. The synergy increased the efficacy and reduced the doses of each of the components compared to monotherapy use, with the advantage of mitigating nitrofuran mutagenicity. Using a transcriptomics approach, underlying mechanisms of the individual and combined action of nitrofurans, VAN, and DOC in E. coli were elucidated. The nitrofuran antibiotic, furazolidone (FZ), and DOC elicited highly similar gene perturbations indicative of iron starvation response, decreased respiration and metabolism, and translational stress. VAN, on the other hand, induced extracytoplasmic stress response in agreement with its known role in peptidoglycan synthesis inhibition. Through genetic and biochemical approaches, Fur (ferric uptake regulator) protein inactivation was confirmed to be important in the synergy of FZ and DOC and to contribute to the synergy of the triple combination. Similarly, the SOS response to DNA damage was shown to be essential for the synergy between FZ and VAN and to also contribute to the synergy of the triple combination. Taken together, the findings of this thesis strongly suggest the presence of multiple interaction points, that leads to the triple synergy, and support the proposed mechanism of synergy where the combined effects lead to the amplification of damaging effects and suppression of resistance mechanisms. Overall, this thesis shows the synergistic triple combination of nitrofurans, DOC, and VAN as a promising therapy for Gram-negative infections. Furthermore, this work significantly increases the understanding of drug interaction mechanisms that lead to synergy, which is hoped to help advance this combination further into the development pipeline. Transcriptomics analyses and the follow-up experiments provide key fundamental insights into the physiological impact that these three antimicrobials have on enterobacterium E. coli and highlight the advantage of combined targets in bacterial killing. These findings, in turn, will help design novel antibiotics, mono- or combined therapies, against multidrug-resistant bacteria.
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Appendix B Supplementary files 1-5 are available from Figshare, and files 6-13 from NCBI Sequence Read Archive under BioProject Accession PRJNA642878
Antibiotics, Gram-negative bacterial infections, Treatment, Drug interactions, Drug resistance in microorganisms