Membrane remodeling occurring in Gram-negative bacteria is a fundamental process involved in many aspects of bacterial physiology. Bacteria have evolved a variety of membrane modifications, e.g., outer membrane vesicles (OMVs), nanotubular membrane structures, lipopolysaccharide alteration, allowing them to better cope with a constantly changing, often hostile, environment. The overall goal of this dissertation was to investigate the influence of the macromolecular modification of the cell wall of Gram-negative bacteria on the development of antibiotic resistance and formation of outer membrane vesicles. One section of this work examines the phenomenon of the bacterial OMVs with respect to the mechanism underlying their formation and their contribution to antibiotic resistance. This research showed that all of the investigated opportunistic pathogens including Acinetobacter baumannii, Citrobacter freundii, Enterobacter sp., Escherichia coli and Serratia marcescens were able to continuously release vesicles into the surrounding milieu during in vitro growth. I validated that OMVs constitute an ubiquitous secretion system that may play a pivotal role in the transmission of enzymatically active compounds (e.g., active ß-lactamases), antibiotic resistance genes (e.g., KPC-2), and overall bacterial survival. However, the general mechanism underlying OMV formation still needs to be understood. Here, I demonstrated that the hemolysin F gene (hlyF), a putative virulence factor associated with highly virulent strains of avian pathogenic E. coli and neonatal meningitis E. coli, is involved in OMV formation. Overexpression of hlyF increased OMV production in E. coli and the presence of the truncated version of this gene led to a hypovesiculation phenotype. Therefore, hlyF appears to be part of a natural biological switch, regulating the vesiculation process in Gram-negative bacteria. Furthermore, I demonstrated that some clinical isolates of C. freundii may release OMVs acting as vehicles for transferring antibiotic resistance genes over longer distances. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) visualized the shedding of DNA-containing OMVs. Exposure to OMVs derived from the carbapenemase gene KPC-2-containing donor cells resulted in gene transfer to E. coli. The second section of this work, focused on the modification of lipopolysaccharide (LPS), mediated by the plasmid-borne mcr-1 gene, leading to colistin resistance in Enterobacteriaceae. I established a methodology for purification of a full-length MCR-1 and showed an in vitro activity of this enzyme for catalyzing phosphoethanolamine (pEtN) hydrolysis from a lipid substrate. Lastly, I discovered that an optimized level of Ca2+ is required for the functionality of the mcr-1-mediated resistance. With this, I was able to develop a novel calcium-enhanced medium for the improved determination of Colistin resistance and the detection of mcr-1-producing Enterobacteriaceae. The medium devised here has been patented and a related patent application examining conditions associated with MCR-1 activity is ongoing.
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