New Insights on Polymyxin Resistance in Acinetobacter baumannii

Abstract

The global health landscape is increasingly threatened by the rapid emergence of multidrug- resistant (MDR) Gram-negative ‘superbugs’, including Acinetobacter baumannii. Polymyxins, namely polymyxin B and colistin, stand as critical last-line therapeutic options. However, resistance to polymyxins continues to rise worldwide. Polymyxin resistance in A. baumannii primarily involves modifications to lipid A in the outer membrane mediated by the PmrAB two-component system. Additionally, the development of polymyxin-dependence during exposure to these antibiotics also contributes to this resistance phenotype. The overall objective of this thesis is to generate new mechanistic insights into polymyxin resistance in MDR A. baumannii. In Chapter 2, we developed an effective genome manipulation method for MDR A. baumannii, facilitating an in-frame pmrB deletion to explore the role of PmrAB in polymyxin resistance. We also reconstructed new genetic tools, crucial for phenotypic screening post homologous recombination and complementation/overexpression experiments in MDR A. baumannii. Overall, our method and genetic tools are critical for molecular research on the functions of antibiotic resistance genes in MDR A. baumannii strains. Chapter 3 investigated the role of PmrAB in the polymyxin resistance of AB5075. Deletion of pmrB resulted in increased tolerance to higher concentrations of polymyxin B (16 mg/L and 32 mg/L), without any observed modifications in lipid A. Further, a missense mutation in the gene ABUW_1353 was identified in the polymyxin-resistant AB06-32 strain. This gene is homologous to the response regulator of the QseBC system, known to be associated with polymyxin resistance in other bacterial species, but not previously reported in A. baumannii. Chapter 4 explored the effect of pmrB deletion on the A. baumannii transcriptome in the absence and presence of 4 mg/L polymyxin B. RNA sequencing showed 467 differentially expressed genes (DEGs) in AB06 (ΔpmrB) relative to AB5075. COG enrichment analysis indicated the regulatory role of PmrAB extends to various pathways, including energy production, virulence, stress responses, cell membrane structure and TCSs. Additionally, we examined the initial (1 h) and later (4 h) responses to polymyxin B, identifying a reduced number of DEGs in AB06 compared to AB5075 at both time points. Notably, the paa operon was downregulated in AB5075 post-polymyxin B exposure but upregulated in AB06 (ΔpmrB) before and unaffected afterwards. Our results are the first to implicate the PAA pathway in responding to polymyxin exposure and its potential connection to the PmrAB system in A. baumannii. In Chapter 5, we employed the mutagenesis method developed in Chapter 2 to achieve an in- frame deletion of the astA gene in the polymyxin-dependent AB5075D strain. Our results demonstrated that deletion of astA improved growth, increased polymyxin resistance, and reduced dependency. Our findings highlight the link between ast operon and arginine metabolism in A. baumannii polymyxin resistance. To the best of our knowledge, this is the first study to make a gene knockout in a polymyxin-dependent A. baumannii strain to investigate the mechanism of polymyxin dependence. In conclusion, this thesis developed a new molecular approach to investigate mechanisms underlying polymyxin resistance in A. baumannii. These mechanistic findings provide new insights into polymyxin resistance in A. baumannii and will aid in optimising the clinical use of this critical class of last-line antibiotics against MDR A. baumannii.