http://hdl.handle.net/11422/31 2026-07-30T05:57:45Z http://hdl.handle.net/11422/29839 Title: Salmonella sp. em águas superficiais de regiões agropecuárias do Estado do Rio de Janeiro: ocorrência, identidade, virulência e resistência a antimicrobianos Author(s)/Inventor(s): Moura, Vinicius de Carvalho Advisor: Bonelli, Raquel Regina Abstract: Salmonella sp. is a bacterial genus of the Enterobacteriaceae family, composed of two species: S. bongori and S. enterica, the latter being of greater clinical and economic relevance due to its subspecies S. enterica subsp. enterica (I), which is responsible for causing infections in warm-blooded animals such as humans, cattle, and poultry. This pathogen plays a central role in the context of Waterborne and Foodborne Diseases (WFDs), impacting human, animal, and environmental health. Its association with hosts such as cattle and broiler chickens makes agricultural production regions potential epicenters of dissemination, either through water sources or food products. In this context, the aim of this study was to investigate the occurrence of Salmonella in water bodies in agricultural regions of the State of Rio de Janeiro, focusing on area impacted by cattle farming, broiler chicken production, and horticulture, as well as to characterize aspects of the isolates, such as serovars, virulome, resistome, and plasmidome. A total of 313 water samples were collected from rivers, streams, and creeks within the hydrographic basins of Médio Paraíba do Sul (MPS; marked by cattle farming and horticulture) and Piabanha (Pb; with intense horticulture and broiler chicken farming). From 181 (58%) of these samples, Salmonella was successfully isolated. After isolation, screening by RAPD, sequencing of up to five isolates per water sample, and bioinformatics analyses, 308 isolates were selected as representatives of the collection (all those that were determined to be non-clonal within each evaluated water sample). Among them, 46 serovars were identified, with emphasis on Panama (44/308), Newport (43/308), Typhimurium (27/308), and Saphra (23/308), with the first three serovars present in both basins and the last one exclusive to MPS. A total of 146 virulence genes were detected, including operons such as spv, pef, rck, and yopJ/yopP, which were associated with plasmids of the incompatibility group IncF, commonly linked to invasive virulence genes. Additionally, resistance mechanisms to β-lactams, aminoglycosides, tetracyclines, phenicols, quinolones, sulfonamide-trimethoprim were identified in chromosomal genes (mutations in gyrA and ramR) and in contigs associated with plasmid markers such as Col440I (containing qnrB19), ColRNAI (aph(3'')-Ib, aph(6)-Id, sul2, and tet(A)), IncC/A2 (sul2 and tet(A)), IncFIC (floR and tet(A)), IncR, and IncX (blaTEM-1, blaTEM-135, and tet(A)), as well as genes blaCMY-2, aadA1, aadA2, tet(B), tet(C), qnrS1, and dfrA in plasmids without a defined Inc group. The fosA7 gene (fosfomycin) was found in the genome of all Saphra isolates. The results of this study provide unique insights into the phylogeny, virulence, resistance, and plasmidome of Salmonella in water bodies of agricultural regions in Rio de Janeiro, contributing to a better understanding of the dissemination and adaptation of this pathogen in this region. Publisher: Universidade Federal do Rio de Janeiro Type: Dissertação 2025-03-31T00:00:00Z http://hdl.handle.net/11422/29836 Title: Epidemiologia molecular e aspectos da formação de biofilme de Staphylococcus pseudintermedius: uma espécie de importância na medicina veterinária Author(s)/Inventor(s): Teixeira, Izabel Mello Advisor: Penna, Bruno de Araújo Abstract: Staphylococcus pseudintermedius is a component of the skin microbiota in healthy dogs. Despite this, it causes a variety of opportunistic infections such as pyoderma, otitis, and urinary tract infections. Some studies report S. pseudintermedius infections in humans, and contact between animals and their owners can facilitate zoonotic transmission. In recent years, a concerning factor has been the increasing antimicrobial resistance. Methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains often exhibit resistance to other antimicrobials, making them multidrug-resistant (MDR) strains. Previous studies have shown a significant clonal diversity among S. pseudintermedius, with over 2000 reported sequence types (STs). The most successful lineages reported are ST71, ST68, and ST45. S. pseudintermedius has the potential to harbor a wide variety of virulence factors, including protein A, cytotoxins, enterotoxins, toxins, and surface proteins, among others. Biofilm formation is another important virulence factor in Staphylococcus species, but it has been little reported in S. pseudintermedius. Therefore, this study aims to evaluate the composition of S. pseudintermedius biofilm and the presence of virulence and resistance factors in the species population. The samples used are from a collection of cultures from Gram-positive cocci Laboratory at UFF (Niterói). The samples were previously identified, and their resistance profile and biofilm formation capacity were studied. The biofilm composition was evaluated through a quantitative assay in microtiter plates. The presence of ica operon and Sps genes was investigated using conventional PCR. A total of 25 samples underwent whole genome sequencing, and their virulence and resistance gene profiles, circulating clone types, Agr and SCCmec types were evaluated. Most strains exhibited polysaccharide biofilm. The complete ica operon (ADBC) was found in 64.9% of strains. All samples presented SpsK, SpsA, and SpsL genes. Other tested genes showed variable distribution. Agr type III was the most prevalent among samples (56.0%). A total of 19 different resistance genes were identified, encoding resistance to a wide range of antimicrobials. Five of these 19 genes (26.3%) were identified in all samples. Six out of 16 sequenced MRSP samples belonged to the worldwide distributed ST71 clone; others belonged to unknown clones (ST2283, ST2285, ST2288, ST2124, ST2287). Most samples had SCCmec type IIIA (6/16 or 37.5%), and three had SCCmec type V (3/15 or 20.0%). Considering the One Health perspective, it is important to monitor animal-origin Staphylococcus species, as their zoonotic potential has been evaluated, and antimicrobial resistance is a growing problem. Little is known about the pathogenicity and carriage of virulence factors in S. pseudintermedius, as well as the characteristics of MDR strains in the region. Studies of this kind are increasingly necessary and relevant. Population dynamics and molecular epidemiology studies of infections caused by multidrug-resistant staphylococci in companion animals are important as they can provide critical data to guide public health programs. Publisher: Universidade Federal do Rio de Janeiro Type: Tese 2023-12-13T00:00:00Z 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 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 2025-11-18T00:00:00Z