Saudi Cultural Missions Theses & Dissertations
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Item Restricted New Insights on Polymyxin Resistance in Acinetobacter baumannii(Monash University, 2024-11) Alsaadi, Yasser; Li, JianThe 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.13 0Item Restricted Isolation and characterization of bacteriophage in Providencia rettgeri.(University of Manchester, 2024) Albalawi, Hind Faleh; Hargreaves, KatP. rettgeri is a gram-negative bacterium and a member of the Enterobacteriaceae family. It. an opportunistic pathogen, which can cause many infections but primarily urinary tract infections and is commonly associated with hospital-acquired infections. This bacterium is part of the natural microbiome found in the human gut. Also, is a ubiquitous bacterium that can be found in water sources, soil, and animals. The emergence of resistance in antibiotics in particular carbapenem is a significant concern. Also, it plays a role in the dissemination of this resistance across the world. Therefore, the exploration of alternative therapies is required. One of the most promising alternative therapies is bacteriophages. Hence, this study aims to isolate and characterize bacteriophages that target Providencia rettgeri. 12 environmental samples were collected from the outskirts of Manchester to isolate phages. In addition, bioinformatic tools analysis was used to predict and highlight the role of prophage in the bacteria genome. However, there were some challenges such as Phage induction and failure of phage propagation therefore, understanding and exploiting the P. rettgeri defence mechanism against phages is critical for further research.16 0Item Restricted The transcriptional landscape and small RNAs in Acinetobacter baumannii AB5075 during antibiotic and environmental stress(University of Dublin, 2024-06-28) Bakheet, Ali Ahmad; Kröger, CarstenMulti-drug resistant Acinetobacter baumannii is considered a major challenge because it causes a variety of serious infections. Studying these bacteria at the level of their genetic response could facilitate finding a way to treat them. Tracking gene expression throughout the growth phases or when affected by any environmental stresses can deepen our understanding of how genes work and how cells function. Here, we studied global gene expression of A. baumannii AB5075 using RNA-seq under the influence of 12 different stresses divided into three groups. The first group includes five growth stages in rich medium, the second group includes four environmental shocks (osmotic, iron limitation and two antibiotic shocks), and the third group contains temperatures shift experiments (between 22°C and 37°C). An important finding is the dramatic up-regulation of the phenyl-acetic acid (paa) pathway including paaA, paaB, paaC, paaJ, paaK and paaZ in late exponential and early stationary phase in rich medium which influences virulence, antibiotic resistance, and biofilm formation. In the environmental shock conditions, we reveal that the transcriptional responses were similar between osmotic shock and iron limiting conditions, and the effect of antibiotics (exposure to chloramphenicol and tetracycline), respectively, indicating that similar pathways are involved in these stress responses. These included genes encoding siderophore Acinetobactin encoding genes that were up-regulated in both osmotic shock and iron limiting conditions. The two antibiotic shocks showed gene expression changes to a much larger than osmotic shock and iron limiting conditions suggesting that protein synthesis stress require a much larger transcriptomic response to cope with the stress compared to the osmotic and iron limitation stress. A response to the antibiotic stress was observed to be a down-regulation of the NADH-quinone oxidoreductase complex, a central protein complex in the respiratory chain, indicating the antibiotics could influence the proton gradient across the membrane or that further growth and ATP synthesis should be immediately stopped. When growing at different temperatures, there were large changes to the transcriptome in all temperature comparisons. The virulence-associated otsAB operon and paa genes were strongly up-regulated upon temperature shift. Type IV pilus encoding genes were down-regulated genes when cells were experiencing a temperature upshift (TS) which could mean that DNA uptake is at least transiently down-regulated when A. baumannii enters a warm-blooded host. In contrast, a temperature downshift induced expression of the Type 6 Secretion System and Csu pili. We also provide a first expression landscape of sRNAs in the suite of 12 conditions. We observed dynamic expression of many small RNAs across the 12 conditions. The sRNAs sRNA99 and sRNA100 very quickly dropped in abundance when the cells experienced antibiotic shocks which was independently validated by Northern blotting for sRNA99 and sRNA100. Deletion of sRNA99 and sRNA100, resulted in large expression changes in MEP in the mutants compared to the WT, which will require further analysis. The sRNA44 was investigated mechanistically in more detail in this study because previous data in A. baumannii AB5075 suggested that sRNA44 was ligated to the mRNA of the biofilm-associated bap gene. We show that sRNA44 translationally represses bap translation in E. coli and that overexpression of sRNA44 reduces bap mRNA levels, likely induced by a base-pairing mechanism. Deletion of sRNA44 and bap showed that bap is required for full biofilm formation after 24h, while deletion of sRNA44 increased biofilm formation suggesting that sRNA44 is a regulator of A. baumannii biofilm formation.46 0