http://hdl.handle.net/11422/31 Tue, 07 Apr 2026 07:23:40 GMT 2026-04-07T07:23:40Z http://hdl.handle.net/11422/28281 Title: Desvendando Staphylococcus nepalensis: avaliação da larva Tenebrio molitor como modelo de patogenicidade e caracterização de plasmídeos carreadores de resistência antimicrobiana Author(s)/Inventor(s): Oliveira, Ana Luisa Andrade Advisor: Marval, Marcia Giambiagi de Abstract: Staphylococcus nepalensis is a coagulase-negative Staphylococcus (CoNS) initially identified as part of goat’s normal microbiota. However, it is now known to be found in different locations and hosts, including some cases of opportunistic infections in humans. Similar to other CoNS, S. nepalensis may serve as a reservoir for antimicrobial resistance (AMR) genes, potentially impacting more pathogenic species like Staphylococcus aureus. The aim of this project is to characterize the S. nepalensis species and its plasmids: i) evaluating virulence and horizontal gene transfer in an in vivo model and ii) establishing an efficient protocol for plasmid extraction to sequence and characterize the plasmids obtained. First, we established Tenebrio molitor larvae as an in vivo model to evaluate the virulence of Staphylococcus spp. Our findings demonstrated that S. aureus, Staphylococcus saprophyticus and S. nepalensis killed T. molitor in a dose-dependent manner and stimulates the cellular immune response of larvae. Notably, when compared at the same concentration, only the S. nepalensis 136b strain was able to kill more than 50% of the larvae at doses lower than 105 CFU (Colony forming unit at 72h post-infection. We also utilized T. molitor larvae as a model for plasmid transfer. For this, larvae were inoculated with the donnor strain S. aureus RN7242 (106 CFU), which carries the gentamicin resistance plasmid pGO1, and the recipient strain S. aureus RN2677 105 CFU. We successfully obtained transconjugant colonies with a frequency of 1.6 × 10−5 per donor cell, demonstrating the suitability of T. molitor as a host for staphylococcal horizontal gene transfer. Next, we tested different plasmid extraction methods to ensure higher-quality samples for sequencing. To evaluate the efficiency of plasmid recovery, we compared two approaches using the S. nepalensis 136b strain: i) total DNA extraction and ii) plasmid-enriched DNA extraction. The plasmid-enriched extraction using Qiagen’s miniprep kit, with an additional lysozyme step, improved assembly results, allowing the recovery of four complete plasmids. Three mobilizable plasmids were identified carrying the resistance genes aadk, cat, and tetK, which confer resistance to streptomycin, chloramphenicol, and tetracycline, respectively. Comparative and phylogenetic analyses revealed high sequence similarity between these plasmids and mobile genetic elements found in various pathogenic and environmental bacteria, including S. aureus, S. epidermidis, Enterococcus sp., and Pseudomonas aeruginosa, indicating plasmid circulation across different genera. Additionally, a novel plasmid was identified, showing low similarity to any sequence deposited in databases, suggesting the existence of uncharacterized plasmid lineages in commensal Staphylococcus. This study highlights the importance of monitoring commensal species such as S. nepalensis, which can act as reservoirs of resistance genes, promoting their dissemination and posing a potential risk for opportunistic infections. Publisher: Universidade Federal do Rio de Janeiro Type: Tese Wed, 17 Dec 2025 00:00:00 GMT http://hdl.handle.net/11422/28281 2025-12-17T00:00:00Z http://hdl.handle.net/11422/28252 Title: Caracterização de vesículas extracelulares de bactérias gram-negativas e seu papel na disseminação da resistência antimicrobiana Author(s)/Inventor(s): Moreira, Roberto Guardatti Gambine Advisor: Picão, Renata Cristina 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. Publisher: Universidade Federal do Rio de Janeiro Type: Tese Tue, 18 Nov 2025 00:00:00 GMT http://hdl.handle.net/11422/28252 2025-11-18T00:00:00Z http://hdl.handle.net/11422/28227 Title: Atividade antivirulência do composto de coordenação CTP, [cobre(teofilina)2(1,10-fenantrolina)(H2O).5H2O], sobre espécies de Candida não-albicans: foco na inibição de aspártico peptidases secretadas Author(s)/Inventor(s): Barbosa, Pedro Fernandes Advisor: Santos, André Luis Souza dos Abstract: The rise of infections caused by Candida non-albicans species resistant to conventional antifungals, particularly in immunocompromised patients, highlights the urgent need for novel therapeutic strategies. Secreted aspartic peptidases (Saps) play key roles in fungal pathogenesis by facilitating adhesion, tissue damage, and immune evasion, making them promising drug targets. Coordination compounds involving transition metals and biologically active organic molecules, such as CTP [copper(theophylline)₂(1,10-phenanthroline)(H₂O).5H₂O], have shown potential against Candida spp. This study investigated the antivirulence activity of CTP against Saps from six Candida non-albicans species using in silico, in vitro, and in vivo approaches. Clinical isolates were cultured in YCB medium supplemented with bovine serum albumin (BSA) to induce Sap production. Significant BSA consumption was dose-dependently inhibited by CTP (IC₅₀: 40.5-70.7 µM) without fungicidal effects, suggesting direct interference with Sap activity. Agar-albumin plate assays demonstrated that pre-treatment of yeast cells with CTP (100-12.5 µM) significantly reduced Sap secretion, as evidenced by lower enzymatic activity compared to untreated cells. Flow cytometry experiments confirmed that CTP significantly decreased the expression of Sap homologs, reducing both the percentage of labeled cells and fluorescence intensity. Sap enzymatic activity was measured using a fluorogenic peptide substrate specific for aspartic peptidases in cell-free, Sap-rich supernatants. Results showed that Saps degraded the substrate with values ranging from 267.17 to 476.75 arbitrary fluorescence units (AFU). In the presence of CTP, proteolytic activity was dose-dependently inhibited (IC₅₀: 6.82-8.5 µM), similar to pepstatin A inhibition (IC₅₀: 4.62-6.85 µM). Molecular docking studies indicated that CTP effectively binds to the catalytic site of Candida Saps, forming an enzymeinhibitor complex with binding energy values ranging from -7.9 to -9.1 kcal/mol. In in vitro and in vivo assays, concentrated fungal supernatants exhibited high enzymatic activity (779.3-1041.7 AFU), which was completely inhibited by CTP and pepstatin A. CTP showed no cytotoxicity up to 20 µM (CC₅₀: 53.66 µM) and reversed the cytotoxic effects induced by C. auris, C. albicans, and C. parapsilosis supernatants. In Galleria mellonella models, fungal supernatants significantly increased larval mortality (30-70%), an effect eliminated by preheating or treatment with CTP and pepstatin A (10 µM), ensuring total larval survival. In infection assays, larval mortality (50-90%) was reduced by CTP, with greater efficacy at higher concentrations (2-5×MIC). These findings highlight CTP as a promising candidate for antivirulence therapies against Candida species resistant to conventional antifungals. Publisher: Universidade Federal do Rio de Janeiro Type: Dissertação Tue, 18 Mar 2025 00:00:00 GMT http://hdl.handle.net/11422/28227 2025-03-18T00:00:00Z http://hdl.handle.net/11422/28212 Title: Adesão, biofilme e invasão: investigando os mecanismos de virulência de Prevotella spp e Fusobacterium nucleatum Author(s)/Inventor(s): Marre, Andressa Temperine de Oliveira Advisor: Lobo, Leandro Araújo Abstract: The Prevotella genus and the species Fusobacterium nucleatum are strict anaerobic organisms associated with opportunistic infections in the vaginal, oral, and gastrointestinal cavities. During infection, virulence mechanisms such as adhesion to host tissues, invasion of cells and connective tissue, and evasion of the immune system are essential for bacterial establishment and host persistence. In the present study, we investigated the adhesion to human extracellular matrix proteins, biofilm formation, Matrigel invasion, and plasminogen activation of strains from the Prevotella species, including P. intermedia, P. melaninogenica, and P. nigrescens, as well as a strain of F. nucleatum. The bacterial adhesion capacity was quantified by the interaction of these bacteria with extracellular matrix proteins, including fibronectin, collagen type IV, collagen type I, laminin type 1, and Matrigel, previously immobilized on glass slides. P. intermedia and P. nigrescens demonstrated adhesion to fibronectin, type IV collagen, and Matrigel. F. nucleatum adhered to laminin, fibronectin, type IV collagen, and Matrigel. P. melaninogenica did not adhere to the substrates under the study conditions. To identify ligands in Prevotella and Fusobacterium, outer membrane protein extracts were purified from P. intermedia, P. nigrescens, and F. nucleatum and subjected to affinity chromatography using NHS-activated Sepharose columns containing immobilized laminin, fibronectin, and type IV and type I collagen. Eluted fractions containing potential ligands were sent for mass spectrometry analysis. In P. intermedia, six proteins were identified as potential laminin adhesins and 15 as potential type IV collagen adhesins. In P. nigrescens, five proteins were identified as potential laminin adhesins and three as potential type IV collagen adhesins. For F. nucleatum, 44 proteins were identified as potential fibronectin adhesins and 12 as potential type I collagen adhesins. Biofilm experiments were also conducted in the presence and absence of Matrigel. Biofilm formation was reduced in the presence of this substrate in P. intermedia and P. melaninogenica, while no significant difference was observed in the other species tested. To analyze the biofilm architecture, scanning electron microscopy was performed. It was observed that, in the presence of Matrigel, the biofilm surface of the analyzed species was altered. P. melaninogenica did not form biofilm on the glass surface used for SEM. A transwell invasion assay was performed for all Prevotella species. It was observed that only P. melaninogenica was capable of crossing the Matrigel layer. Matrigel degradation assays using SDS-PAGE showed that P. melaninogenica degrade matrix proteins and plasminogen. To understand the interaction between species and plasminogen, a plasminogen activation kinetic assay was conducted, in which only P. melaninogenica activated this molecule, likely utilizing this strategy to destroy tissues. Understanding the mechanisms involved in virulence may help develop new strategies to prevent periodontitis and biofilm formation in the gingival sulcus. Publisher: Universidade Federal do Rio de Janeiro Type: Tese Tue, 18 Feb 2025 00:00:00 GMT http://hdl.handle.net/11422/28212 2025-02-18T00:00:00Z