Caracterização de vesículas extracelulares de bactérias gram-negativas e seu papel na disseminação da resistência antimicrobiana

Abstract

Antimicrobial resistance is currently one of the greatest public health challenges worldwide. The issue is particularly critical in hospital settings, where the extensive use of these drugs favors the selection of resistant microorganisms. In this context, multidrug-resistant Gram-negative bacilli (GNB) represent a major concern, as they resist last-resort antimicrobials and possess remarkable genomic plasticity, enabling them to readily acquire and transfer resistance genes. Considering this last point, one mechanism of horizontal gene transfer (HGT) has been suggested as a potential disseminator: extracellular vesicles (EVs), although little is known about their actual contribution to the problem. Thus, this thesis conducted an in-depth study of EVs produced by GNB resistant to last-resort antimicrobials, isolated from diverse sources, and established a methodology for their isolation from solid medium cultures. A collection of multidrug-resistant, genetically diverse, and genome-sequenced strains was assembled, comprising six bacterial genera (Acinetobacter, Aeromonas, Enterobacter, Escherichia, Klebsiella, and Pseudomonas), with two representatives per genus. From this collection, the EVs produced were isolated and characterized, with analyses of size distribution, protein and phospholipid content, resistance gene packaging, and transformation capacity. We confirmed that the proposed method was highly efficient and reproducible, yielding between 1010 and 1012 nanoparticles/mL depending on the producing strain, with co-isolation of filaments in the genera Aeromonas, Escherichia, Enterobacter, and Pseudomonas. The highest and lowest EV producers belonged to the genera Pseudomonas and Aeromonas, respectively. Size profiles were strain-specific, preventing extrapolation at the genus level. Mean diameters ranged from 120 nm to 250 nm, with larger averages in Acinetobacter and Klebsiella, and smaller ones in Pseudomonas, Escherichia, and Enterobacter. Quantification of macromolecules revealed a positive correlation between protein content and EV quantity, whereas phospholipids showed a negative correlation with EV diameter. Regarding genetic material packaging, more than 70% (in Escherichia and Acinetobacter) was found to be intravesicular and protected from enzymatic degradation. Resistance genes were detected in plasmids of various sizes (8 Kb to 350 Kb), partially or fully packaged within EVs, carrying multiple last-resort resistance determinants (blaNDM-1, blaOXA-181, and blaKPC-2) in Acinetobacter, Escherichia, and Klebsiella. Transformation assays, however, did not reveal broad HGT potential, with only Klebsiella sp. KP13 showing results consistent with a transformation event. Although phenotypic resistance changes were confirmed, no intact plasmid was detected in the transformants, suggesting the transfer of only a plasmid fragment containing a resistance gene—a phenomenon unprecedented in the literature. Nevertheless, it was not possible to determine which gene was transferred, requiring further studies. In conclusion, this work provides a detailed characterization of EVs produced by a diverse collection of multidrug-resistant GNB and the methodology for their isolation, while simultaneously indicating that EV-mediated HGT may not be as widespread and significant as previously hypothesized.