Percorrer por autor "Pinto, Miguel"
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- An integrative assessment to determine the genotoxic hazard of estuarine sediments: combining cell and whole-organism responsesPublication . Costa, Pedro Manuel; Pinto, Miguel; Vicente, Ana M.; Gonçalves, Cátia; Rodrigo, Ana P.; Louro, Henriqueta; Costa, Maria Helena; Caeiro, Sandra; Silva, Maria JoãoThe application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors,e.g.,when dealing with estuarine sediments,that hampers the drawing of cause-effect relationships. Although the in vitro The application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors, e.g., when dealing with estuarine sediments, that hampers the drawing of cause-effect relationships. Although the in vitro Comet assay may circumvent confounding factors, its application in environmental risk assessment (ERA) still needs validation. As such, the present work aims at integrating genotoxicity and oxidative DNA damage induced by sediment-bound toxicants in HepG2 cells with oxidative stress-related effects observed in three species collected from an impacted estuary. Distinct patterns were observed in cells exposed to crude mixtures of sediment contaminants from the urban/industrial area comparatively to the ones from the rural/riverine area of the estuary, with respect to oxidative DNA damage and oxidative DNA damage. The extracts obtained with the most polar solvent and the crude extracts caused the most significant oxidative DNA damage in HepG2 cells, as measured by the formamidopyrimidine-DNA glycosylase (FPG)-modified Comet assay. This observation suggests that metals and unknown toxicants more hydrophilic than polycyclic aromatic hydrocarbons may be important causative agents, especially in samples from the rural part of the estuary, where oxidative DNA damage was the most significant. Clams, sole, and cuttlefish responded differentially to environmental agents triggering oxidative stress, albeit yielding results accordant with the oxidative DNA damage observed in HepG2 cells. Overall, the integration of in vivo biomarker responses and Comet assay data in HepG2 cells yielded a comparable pattern, indicating that the in vitro FPG-modified Comet assay may be an effective and complementary line-of-evidence in ERA even in particularly challenging, natural, scenarios such as estuarine environments.
- Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosisPublication . Štefánek, Matúš; Wenner, Sigurd; Borges, Vítor; Pinto, Miguel; Gomes, João Paulo; Rodrigues, João; Faria, Isabel; Pessanha, Maria Ana; Martins, Filomena; Sabino, Raquel; Veríssimo, Cristina; Nogueira, Isabel D.; Carvalho, Patrícia Almeida; Bujdáková, Helena; Jordao, LuisaBiofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism’s distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.
- Assessment of the Genotoxic Hazard of Estuarine Sediments Using an Integrative Approach With LacZ Plasmid‐Based Transgenic MicePublication . Pinto, Miguel; Sacadura, Joana; Costa, Pedro M.; Caeiro, Sandra; Louro, Henriqueta; Silva, Maria J.Under the influence of multiple anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as particularly sensitive ecosystems to contamination. The present study aimed at investigating the genotoxic potential of a contaminated sediment sample from an urban and industrial area of the Sado Estuary, by combining the analysis of multiple endpoints in the LacZ plasmid‐based transgenic mouse model exposed for 28 days to contaminated estuarine sediment extracts through drinking water. The DNA and chromosome damaging effects were monitored in peripheral blood at 7‐day intervals using the standard and enzyme‐modified Comet assay, as well as the micronucleus assays in peripheral blood cells. After euthanasia, DNA damage was analyzed in several mouse tissues, and LacZ mutant frequencies were determined in the liver. Livers were also surveyed for histopathological analysis. A time‐dependent increase in micronuclei frequency was seen at all tested doses, in spite of no induction of DNA damage in any organ or mutation induction in the liver of exposed mice. The liver from mice exposed to sediment extracts did not reveal major alterations besides evidence of inflammation. Overall, the integration of the endpoints analyzed in the mice is suggestive of potential chronic, rather than acute, adverse effects in vivo, and points to the need for further research in the resident human population in the area. This experimental design can be used to assess the genotoxicity of complex environmental mixtures, understand how they work, and reduce costs and resources while speeding up data collection and interpretation.
- Avaliação do potencial citotóxico e genotóxico de contaminantes de sedimentos do Estuário do Sado numa linha celular humanaPublication . Pinto, Miguel; Costa, Pedro Manuel; Louro, Henriqueta; Costa, Maria Helena; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoO presente estudo integra-se num projeto mais amplo que visa avaliar o risco ambiental – que inclui os riscos ecológicos e para a saúde humana - associado a este ambiente estuarino contaminado. Em particular, este estudo teve como objetivo caracterizar o potencial citotóxico e genotóxico de sedimentos colhidos em vários locais de pesca do Estuário do Sado numa linha celular humana, tendo em vista uma avaliação de eventuais efeitos nefastos para a saúde humana.
- Avaliação do risco ambiental e para a saúde pública de um ambiente estuarino contaminadoPublication . Pinto, Miguel; Louro, Henriqueta; Caeiro, Sandra; Costa, Pedro; Costa, Maria Helena; Lavinha, João; Silva, Maria JoãoO estuário do rio Sado é um ecossistema de elevado valor ecológico e económico. Apesar de ter vindo a ser afetado ao longo dos anos por várias fontes de poluição de origem urbana, industrial e agrícola, continua ainda a ser, para a população local, um local privilegiado para atividades piscícolas e agrícolas. Contudo, existe uma preocupação relacionada com a potencial bioacumulação de contaminantes nas partes edíveis de espécies estuarinas ou de produtos agrícolas locais que entrarão assim na cadeia alimentar humana, podendo representar um problema de saúde pública. O presente estudo integra-se num projeto mais amplo que visa avaliar os riscos ambientais e para a saúde humana, associados a esse ambiente estuarino contaminado. Em particular, este estudo teve como objetivo caracterizar o potencial citotóxico e genotóxico de sedimentos colhidos em vários locais de pesca do Estuário do Sado numa linha celular humana, tendo em vista uma avaliação de eventuais efeitos nefastos para a saúde humana. Observaram-se efeitos citotóxicos e genotóxicos diferentes em amostras de sedimentos derivadas da margem esquerda vs. direita do estuário que se podem correlacionar com diferenças nos níveis e tipos de contaminantes identificados nessas amostras. Estes refletem, provavelmente, as diversas pressões no ecossistema derivadas de uma área urbana e industrializada (margem esquerda) vs. uma área de intensa atividade agrícola (margem direita). A observação de que amostras de sedimentos estuarinos induzem efeitos citotóxicos e genotóxicos em células humanas, em consonância com os efeitos também detetados em espécies estuarinas, enfatiza a preocupação sobre o impacto do consumo de espécies contaminadas na saúde das populações locais e reforça a necessidade de serem tomadas medidas no sentido de reduzir e remediar a poluição desta área estuarina.
- Can Estuary Sediment Contaminants Interfere with the DNA Repair Capacity of HEPG2 Cells?Publication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro Manuel; Caeiro, Sandra; SIlva, Maria JoãoEstuarine sediments tend to act as reservoirs of pollutants, many of which are acknowledged genotoxicants and potential carcinogens for humans. In addition, many of these environmental contaminants, particularly metals, have the potential to interfere with DNA repair mechanisms. Taking an impacted estuary as a case study (the Sado, SW Portugal), previous studies showed that human hepatoma cells (HepG2) exposed to extracts of sediments collected from two areas (urban/industrial and riverine/agricultural), both contaminated by distinct mixtures of organic and inorganic toxicants, revealed differential cytotoxic and genotoxic effects, consistent with contamination indices. In this context, the present study aimed at determining whether contaminants present in sediment extracts are able to interfere with the DNA repair mechanisms of HepG2 cells. Organic and inorganic contaminants were extracted (methanol:dichloromethane) from sediment samples collected in different sites of the Sado Estuary, either heavily impacted by an urban/industrial environment (site P) or by agriculture (sites E and A); a sediment collected in a potentially “clean” site was also included (site C). The repair capacity of HepG2 cells towards ethyl methanesulfonate (EMS, 5 - 40 mM, 1h) induced DNA lesions was assessed after cells recovery in the presence of non-genotoxic concentrations of each sediment extract, during 24h and 48h, using the comet assay. Negative and solvent controls were included. Preliminary results show that at 24h and 48h after exposure, 60% and 85% of EMS-induced DNA strand breakage, respectively, was spontaneously repaired by HepG2 cells. However, exposure to extracts P and A after EMS challenge, partially inhibited DNA repair given that, relatively to the spontaneous DNA repair level, 20 and 30%, of DNA damage remained unrepaired, after 24h whereas 30 and 40% remained unrepaired after 48h. Extract E slightly interfered with HepG2 DNA repair capacity (10% unrepaired damage), independently of the period of exposure. Extract C and solvent control did not interfere with DNA damage recovery, 24 and 48h after EMS exposure. Our results suggest that extracts P and A, which display the highest concentrations of PAHs and metals, respectively, produce a clear interference with the DNA repair capacity of HepG2 cells while less or no contaminated extracts (E and C, respectively) resulted in a low or no influence on DNA repair mechanisms. These results, together with the previous observations that extracts P and A were genotoxic, raise more concern on the potential hazard of estuarine contaminants on the health of exposed populations.
- Carga treponémica em amostras biológicas correspondentes a diferentes fases clínicas de sífilisPublication . Pinto, Miguel; Antelo, Minia; Ferreira, Rita; Azevedo, Jacinta; Santo, Irene; Borrego, Maria José; Gomes, João Paulo[PT] Sífilis, a doença sexualmente transmitida causada por Treponema pallidum, apresenta diferentes manifestações clinicas. No entanto, os fatores que levam às diversas progressões da doença permanecem desconhecidos. Este estudo teve como objetivo a quantificação da carga treponémica, em diversas amostras biológicas, para contribuir para uma melhor compreensão da doença. Analisaram-se 309 amostras de DNA de distintos locais anatómicos associados a diferentes manifestações de sífilis. Uma quantificação absoluta por PCR em tempo-real foi utilizada para quantificar precisamente o número de células treponémicas e humanas, e calcular a carga treponémica em cada amostra. Os exsudados de lesões primárias apresentaram as cargas treponémicas mais elevadas contrastando com as amostras sanguíneas. Uma grande dispersão de concentrações bacterianas foi observada nas amostras sanguíneas dos casos de sífilis secundária. T. pallidum foi detetado em 37 amostras de indivíduos seronegativos e em 13 casos de sífilis tratada. Este estudo sugere um cenário onde a sífilis poderá ser caracterizada por: i) cargas elevadas e heterogéneas na sífilis primária, refletindo a duração do desenvolvimento e resolução da lesão; ii) uma potencial capacidade replicativa de T. pallidum na corrente sanguínea, sugerida pela elevada dispersão de concentrações na sífilis secundária; e iii) uma evasão bacteriana quer ao sistema imunitário do hospedeiro quer ao tratamento antibiótico.
- Catheter related bloodstream infection caused by E. cloacae and Candida parapsilosis: Are biofilms guilty?Publication . Štefánek, Matúš; Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel D.; Pinto, Miguel; Faria, Isabel; Pessanha, Maria Ana; Veríssimo, Cristina; Sabino, Raquel; Rodrigues, João; Matias, Rui; Carvalho, Patrícia Almeida; Gomes, João Paulo; Bujdáková, Helena; Jordao, LuisaBiofilm-associated infections is a public health concern in the context of healthcare associated infections (HAI) such as catheter related bloodstream infections (CRBSI). Here the dynamics of two top ten etiological agents of CRBSI, Enterobacter cloacae and Candida parapsilosis isolated from a CRBSI’s patient, were studied to get insights on the role played by biofilms on this HAI. Antimicrobial susceptibility of CVC and HC’s isolates was evaluated according to EUCAST guidelines. Single and/or mixed biofilms assembled on different materials in Mueller-Hinton broth with 2% glucose were assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification purposes and to assess microorganisms distribution within the biofilm (3D reconstruction) complemented with Focus Ion Beam (FIB)-SEM to assess biofilms assembled on the inner/outer CVC’s surfaces (tomograms). Whole-genome sequencing (WGS) was performed for all isolates. All isolates were antimicrobial resistant. Of note E.cloacae resistance to collistin and an additional resistance of the CVC compared to HC-isolate (ceftolozame-tazobactam) probably linked to a mutation in rpoB gene. Candida resistance to fluconazol might be explained by ERG11 gene mutation. Enterobacter and Candida assembled biofilms on glass, polystyrene and polyurethane being mixed biofilms denser when both microorganism were present from the beginning. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Using environmental isolates of the same species we showed that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), denser mixed biofilms richer in EPS were observed. A distinct phenotype was present on the patient’s CVC by SEM and FIB/SEM. WGS confirmed the genetic identity of the pair CVC/HC isolates, while corroborating the virulence potential and observed antimicrobial resistant character of the studied CRBSI-driving pathogens. The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Cephalosporin-resistant Neisseria gonorrhoeae isolated in Portugal, 2019Publication . Pinto, Miguel; Matias, Rui; Rodrigues, João Carlos; Duarte, Sílvia; Vieira, Luís; Gonçalves, Inês; Gonçalves, Maria João; Ramos, Maria Helena; Gomes, João Paulo; Borrego, Maria JoséWe report a multidrug resistant Neisseria gonorrhoeae exhibiting resistance to ceftriaxone and cefixime, isolated in Portugal in 2019. Whole-genome sequencing was performed for typing and identification of genetic determinants of antimicrobial resistance. Due to its antimicrobial susceptibility profile, awareness should be raised for the circulation of this strain.
- Characterization of cytotoxic and genotoxic effects of contaminated sediments from the Sado Estuary and potential human health riskPublication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro; Costa, Maria Helena; Caeiro, Sandra; Lavinha, João; Silva, Maria JoãoThe river Sado Estuary (W Portugal) is affected by various sources of pollution, such as heavy-industry, urbanism, mining, agriculture and maritime traffic. Mostly classified as a natural reserve, it also remains a privileged site for fishing activities performed by the local population, who not only consume but distribute their fishery. The present study is part of a broader project whose objective is to evaluate the environmental and human health risks associated with the estuarine benthic environment. This study aims to assess the cytotoxic and genotoxic potential of sediments from several local fishing areas of the Sado Estuary. Sediments were collected from four geochemically distinct and potentially contaminated sites of the Sado Estuary: sites C and P from the northern shore and sites E and A from the southern shore. A previously characterized sample (F) from the northern shore was added as a positive control. Total organic and inorganic contaminants were extracted with a mixture of methanol:dichloromethane (1:2) and recovered in DMSO. Cytotoxicity and genotoxicity were evaluated through the neutral red uptake assay and by the alkaline comet (coupled with DNA repair endonucleases) and the micronucleus assays, respectively, in the human HepG2 cell line. Cells were exposed for 48h to concentrations of each extract ranging from 0.1 to 20µl/ml of culture medium. A dose-related decrease in cell viability was observed for extracts F, P and E, indicating sediment contaminant-driven cytotoxicity, whereas no effect was observed for extracts C and A. No significant genotoxicity was observed for extract C, while extract F was clearly genotoxic, as expected. A significant increase in the level of DNA and chromosome damage was observed, by the comet and micronucleus assays, respectively, for sub cytotoxic concentrations of extracts P and E. The level of DNA damage was accentuated following treatment with the DNA repair endonuclease FPG, suggesting the existence of oxidative DNA damage. Extract A was genotoxic in the micronucleus assay and in the comet assay only after FPG treatment. Negative results from sample C leads us to consider it as potential clean reference for further studies. Moreover, sediment contamination analysis revealed high levels of metals in all samples except C, whereas only sample P exhibited high levels of known genotoxic polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and DDTs, similar to the previously described for sample F. The differential cytotoxicity and genotoxicity observed in samples from the northern (P) and southern areas (E and A) of the Sado Estuary probably reflects different pressures from a urban and heavy industrialized area versus an intense agricultural area, respectively. The observation that sediment samples have cytotoxic and genotoxic effects, together with the knowledge that contaminants can be accumulated in the edible parts of estuarine species or local agricultural products entering the human food chain, raise concern about a hazardous impact on the health of exposed populations that must be assessed. Work supported by the Foundation for Science and Technology (ref. PTDC/SAU-ESA/100107/2008).