Instituto Nacional de Saúde Doutor Ricardo Jorge
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Percorrer Instituto Nacional de Saúde Doutor Ricardo Jorge por Domínios Científicos e Tecnológicos (FOS) "Ciências Naturais"
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- Application of a real-time reverse transcription polymerase chain reaction for rapid detection of Escherichia coli in drinking water: an EU representative studyPublication . Gómez, Livia; Brandão, João; Navarro, Anna; Martines, Antonino; Sanseverino, Isabella; Porcel-Rodríguez, Elena; Marinov, Dimitar; Ableitner, Oksana; Allerberger, Franz; Atsma, Adrie; Briancesco, Rossella; Cenov, Arijana; Cíchová, Marianna; Coccia, Anna Maria; Libera, Simonetta Della; De Maeyer, Katrien; De Vries, Hendrik Jan; Elsinga, Goffe; Heijnen, Leo; Hokajärvi, Anna-Maria; Janská, Veronika; Hrabar, Jerko; Huse-Kutowska, Monika; Jozic, Slaven; Klatovsky, Stefanie; Kornschober, Christian; Kohls, Katharina; Krom, Adrie; Luden, Katrin; Mikula-Pratschke, Claudia; Pitkänen, Tarja; Poje, Mateja; Prokšová, Miloslava; Retelj, Matjaz; Rozwadowska, Beata; Rytkönen, Annastiina; Sarekoski, Anniina; Schlager, Sabine; Springer, Burkhard; Stojan, Iva; Stroobach, Eline; Šućić, Neven; Valério, Elisabete; Van Pelt, Gabi; Vranken, Jerom; Vrdoljak Tomaš, Ana; Vukić Lušić, Darija; Włos, Ewa; Wubbels, Gerhard; Ziombska, Maria; Zelenik, Katja; Ziuziakowski, Marcin; Lettieri, TeresaThe microbiological quality of water intended for human consumption in the EU is regulated by the recast Drinking Water Directive 2020/2184 (DWD), which sets strict parametric values for intestinal enterococci and Escherichia coli (E. coli), allowing no more than zero colony-forming units per 100 mL. Detection and enumeration of E. coli typically rely on culture-based reference methods or the most probable number approach, which require 1–2 days to produce results—potentially delaying timely action during contamination events. In contrast, molecular techniques can deliver results within hours. The DWD permits the use of alternative methods if they are as reliable as the reference method and developing and validating such methods requires multiple laboratory trials in line with ISO standard 16140-2. Following this, we conducted a representative EU study to validate a molecular method based on real-time reverse transcription polymerase chain reaction for rapid E. coli detection in drinking water. In a concerted action, the first of its kind for drinking water, nineteen laboratories across ten Member States participated. To ensure consistency, drinking water was artificially contaminated with E. coli. The alternative method showed slightly lower sensitivity than the reference method (91.1 % vs. 97.2 %) but delivered much faster results, making it a valuable screening tool. It can support rapid decision-making during contamination events, reducing the risk of waterborne outbreaks and protecting public health. For reliable routine performance, appropriate training in this alternative method is strongly recommended.
- Challenges of the Application of In Vitro Digestion for Nanomaterials Safety AssessmentPublication . Vital, Nádia; Gramacho, Ana Catarina; Silva, Mafalda; Cardoso, Maria; Alvito, Paula; Kranendonk, Michel; Silva, Maria Joao; Louro, HenriquetaConsidering the increase in the production and use of nanomaterials (NM) in food/feed and food contact materials, novel strategies for efficient and sustainable hazard characterization, especially in the early stages of NM development, have been proposed. Some of these strategies encompass the utilization of in vitro simulated digestion prior to cytotoxic and genotoxic assessment. This entails exposing NM to fluids that replicate the three successive phases of digestion: oral, gastric, and intestinal. Subsequently, the resulting digestion products are added to models of intestinal cells to conduct toxicological assays, analyzing multiple endpoints. Nonetheless, exposure of intestinal cells to the digested products may induce cytotoxicity effects, thereby posing a challenge to this strategy. The aim of this work was to describe the challenges encountered with the in vitro digestion INFOGEST 2.0 protocol when using the digestion product in toxicological studies of NM, and the adjustments implemented to enable its use in subsequent in vitro biological assays with intestinal cell models. The adaptation of the digestion fluids, in particular the reduction of the final bile concentration, resulted in a reduced toxic impact of digestion products.
- Ciguatera toxins, a potential health risk emerging in Europe: overview of progress and challengesPublication . Alvito, Paula; Gago-Martínez, Ana; AlvitoCiguatera Poisoning (CP) is considered an emerging risk of concern in several places worldwide. The risk of CP in Europe is currently being investigated, and important advantages have been achieved under two EuroCigua projects, which allowed the characterization of Caribbean Ciguatoxins as the main responsible for the contamination in the European Coastal areas investigated. Important challenges and limitations, particularly the ones related to the lack of reference materials and of relevant samples that enable to associate the contaminated fish consumption with human intoxication, are the most critical aspects hindering the progress from both analytical and toxicological perspectives. This review aims to provide an update on the most recent advances in CP in Europe on the source of the toxins involved in contamination, their toxicology and pharmacology, and their detection and characterization in order to contribute to envisioning the toxic mechanism of CP in humans.
- Exploring flagellar contributions to motility and virulence in Arcobacter butzleriPublication . Santos, Raquel; Mateus, Cristiana; Oleastro, Mónica; Ferreira, SusanaFlagella is a well-known bacterial structure crucial for motility, which also plays pivotal roles in pathogenesis. Arcobacter butzleri, an enteropathogen, possesses a distinctive polar flagellum whose functional aspects remain largely unexplored. Upon investigating the factors influencing A. butzleri motility, we uncovered that environmental conditions like temperature, oxygen levels, and nutrient availability play a significant role. Furthermore, compounds that are found in human gut, such as short-chain fatty acids, mucins and bile salts, have a role in modulating the motility, and in turn, the pathogenicity of A. butzleri. Further investigation demonstrated that A. butzleri ΔflaA mutant showed a reduction in motility with a close to null average velocity, as well as a reduction on biofilm formation. In addition, compared with the wild-type, the ΔflaA mutant showed a decreased ability to invade Caco-2 cells and to adhere to mucins. Taken together, our findings support the role of environmental conditions and gut host associated compounds influencing key physiological aspects of the gastrointestinal pathogen A. butzleri, such as motility, and support the role of the flagellum on bacterial virulence.
- Genomic Analysis of Antibiotic Resistance and Virulence Profiles in Escherichia coli Linked to Sternal Bursitis in Chickens: A One Health PerspectivePublication . Ribeiro, Jessica; Silva, Vanessa; Freitas, Catarina; Pinto, Pedro; Vieira-Pinto, Madalena; Batista, Rita; Nunes, Alexandra; Gomes, João Paulo; Pereira, José Eduardo; Igrejas, Gilberto; Barros, Lillian; Heleno, Sandrina A.; Reis, Filipa S.; Poeta, PatríciaAbstract: Sternal bursitis is an underexplored lesion in poultry, often overlooked in microbiological diagnostics. In this study, we characterized 36 Escherichia coli isolates recovered from sternal bursitis in broiler chickens, combining phenotypic antimicrobial susceptibility testing, PCRbased screening, and whole genome sequencing (WGS). The genetic analysis revealed a diverse population spanning 15 sequence types, including ST155, ST201, and ST58. Resistance to tetracycline and ciprofloxacin was common, and several isolates carried genes encoding β-lactamases, including blaTEM-1B. Chromosomal mutations associated with quinolone and fosfomycin resistance (e.g., gyrA p.S83L, glpT_E448K) were also identified. WGS revealed a high number of virulence-associated genes per isolate (58–96), notably those linked to adhesion (fim, ecp clusters), secretion systems (T6SS), and iron acquisition (ent, fep, fes), suggesting strong pathogenic potential. Many isolates harbored virulence markers typical of ExPEC/APEC, such as iss, ompT, and traT, even in the absence of multidrug resistance. Our findings suggest that E. coli from sternal bursitis may act as reservoirs of resistance and virulence traits relevant to animal and public health. This highlights the need for including such lesions in genomic surveillance programs and reinforces the importance of integrated One Health approaches.
- Hazard characterization of the mycotoxins enniatins and beauvericin to identify data gaps and improve risk assessment for human healthPublication . Behr, Anne-Cathrin; Fæste, Christiane Kruse; Azqueta, Amaya; Tavares, Ana M.; Spyropoulou, Anastasia; Solhaug, Anita; Olsen, Ann-Karin; Vettorazzi, Ariane; Mertens, Birgit; Zegura, Bojana; Streel, Camille; Ndiaye, Dieynaba; Spilioti, Eliana; Dubreil, Estelle; Buratti, Franca Maria; Crudo, Francesco; Eriksen, Gunnar Sundstøl; Snapkow, Igor; Teixeira, João Paulo; Rasinger, Josef D.; Sanders, Julie; Machera, Kyriaki; Ivanova, Lada; Gaté, Laurent; Le Hegarat, Ludovic; Novak, Matjaz; Smith, Nicola M.; Tait, Sabrina; Fraga, Sónia; Hager, Sonja; Marko, Doris; Braeuning, Albert; Louro, Henriqueta; Silva, Maria João; Dirven, Hubert; Dietrich, JessicaEnniatins (ENNs) and beauvericin (BEA) are cyclic hexadepsipeptide fungal metabolites which have demonstrated antibiotic, antimycotic, and insecticidal activities. The substantial toxic potentials of these mycotoxins are associated with their ionophoric molecular properties and relatively high lipophilicities. ENNs occur extensively in grain and grain-derived products and are considered a food safety issue by the European Food Safety Authority (EFSA). The tolerable daily intake and maximum levels for ENNs in humans and animals remain unestablished due to key toxicological and toxicokinetic data gaps, preventing full risk assessment. Aiming to find critical data gaps impeding hazard characterization and risk evaluation, this review presents a comprehensive summary of the existing information from in vitro and in vivo studies on toxicokinetic characteristics and cytotoxic, genotoxic, immunotoxic, endocrine, reproductive and developmental effects of the most prevalent ENN analogues (ENN A, A1, B, B1) and BEA. The missing information identified showed that additional studies on ENNs and BEA have to be performed before sufficient data for an in-depth hazard characterisation of these mycotoxins become available.
- HBM4EU chromates study: the Portuguese integrated and harmonized study on exposure to hexavalent chromium and related early effects.Publication . Viegas, Susana; Martins, Carla; Ribeiro, Edna; Ladeira, Carina; Pinhal, Hermínia; Nogueira, Ana; Santos, Sílvia; Tavares, Ana; Gomes, Bruno Costa; Afonso, Catarina Maia; Louro, Henriqueta; Silva, Maria JoaoIn the scope of the European Union (EU) human biomonitoring initiative, a multicentric study on different occupational settings from several European countries was performed, to provide information on occupational exposure to hexavalent chromium [Cr(VI)], a known lung carcinogen. Biomonitoring approaches were used to obtain exposure data to support the implementation of new risk management measures and policy actions at the national and European levels. This work describes the Portuguese contribution to the study, which aimed to assess workers' exposure to Cr, by using exposure biomarkers (urinary chromium [U-Cr]), and industrial hygiene samples (air and hand wipes) and to link exposure to potential long-term health effects by using effect biomarkers. Exposure determinants influencing exposure were explored from the contextual information and human biomonitoring data. The ultimate goal of the study was to appraise the risk management measures contributing to minimize exposure and protect workers' health. Several occupational settings and activities were considered, including plating, welding, and painting. A control group from the Portuguese general population was also included. Data on age, sex, and smoking habits from both groups were considered in the statistical analysis. Information on the risk management measures available for workers was collected and used to identify the ones that mainly contributed to reduce exposure. Environmental monitoring and human biomonitoring revealed that painters were the highest exposed group. The use of respiratory protection equipment showed an influence on total U-Cr levels for workers involved in painting activities. Concerning early health effects, the painters presented also a significantly higher level of DNA and chromosomal damage in peripheral blood cells, as compared to the control group, suggesting a plausible association between exposure to Cr(VI) and early genotoxic effects. The results showed that workers are exposed to Cr(VI) in those occupational settings. These findings point to the need to improve the prevention and risk management measures and the implementation and enforcement of new regulatory actions at the national level.
- Impact of non-pharmacological interventions on the first wave of COVID-19 in Portugal 2020Publication . Loyens, Dinis B.; Caetano, Constantino; Matias-Dias, CarlosIntroduction: The COVID-19 pandemic caused over 7 million global deaths. Without vaccines during the first wave, governments implemented nonpharmacological interventions (NPIs) such as lockdowns, school closures, and travel restrictions. This study quantifies the impact of NPIs on COVID-19 transmission in Portugal between 24th February and 1st May. Methods: A compartmental SEIR (Susceptible, Exposed, Infectious, Removed) model was employed to simulate the first COVID-19 wave in Portugal, using a Bayesian approach and symptom-onset incidence data. The effect of the lockdown, which began on March 22, 2020, on the effective reproductive number, R t was measured. A counterfactual scenario was created to ascertain the number of cases prevented by the NPIs during the first 15 days after the implementation of NPI. Results: The lockdown reduced overall transmission by 68·6 % (95%Credible Interval (95%CrI): 59·2 %; 77·5 %), almost immediately. This corresponds to a reduction in the effective reproductive number from 2·56 (95%CrI: 2·08; 3·40) to 0·80 (95%CrI: 0·76; 0·84). The counterfactual scenario estimated that the lockdown prevented 118052 (95%CrI: 99464; 145605) cases between 24th February and 6th April. Discussion: The lockdown significantly reduced COVID-19 transmission in Portugal, bringing Rt below 1, meaning each person infected fewer than one individual. While costly, lockdowns effectively control disease spread in the absence of vaccines. Conclusion: Our findings suggest NPIs curbed epidemic transmission, reducing Rt below 1 and easing hospital loads and deaths. This research will help inform future pandemic decision-making and infectious disease modeling worldwide.
- New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) projectPublication . Snapkow, Igor; Smith, Nicola M.; Arnesdotter, Emma; Beekmann, Karsten; Blanc, Etienne B.; Braeuning, Albert; Corsini, Emanuela; Sollner Dolenc, Marija; Duivenvoorde, Loes P.M.; Sundstøl Eriksen, Gunnar; Franko, Nina; Galbiati, Valentina; Gostner, Johanna M.; Grova, Nathalie; Gutleb, Arno C.; Hargitai, Rita; Janssen, Aafke W.F.; Krapf, Solveig A.; Lindeman, Birgitte; Lumniczky, Katalin; Maddalon, Ambra; Mollerup, Steen; Parráková, Lucia; Pierzchalski, Arkadiusz; Pieters, Raymond H.H.; Silva, Maria Joao; Solhaug, Anita; Staal, Yvonne C.M.; Straumfors, Anne; Szatmári, Tünde; Turner, Jonathan D.; Vandebriel, Rob J.; Zenclussen, Ana Claudia; Barouki, RobertAs a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).
- Optimizing MS Parameters for Data-Independent Acquisition (DIA) to Enhance Untargeted MetabolomicsPublication . Pinto, Frederico G.; Giddey, Alexander D.; Almarri, Rouda S. B.; Alkhnbashi, Omer S.; Garrett, Timothy J.; Uddin, Mohammed J.; Soares, Nelson C.Data-Independent Acquisition (DIA) has emerged as a powerful mass spectrometry (MS) strategy for comprehensive metabolomics. This study presents a novel short gradient (13 min) nanosensitive analytical method for human plasma analysis using DIA LC-MS/MS, focusing on in-depth optimization of MS parameters to maximize data quality and metabolite coverage. Key MS parameters, including scan speed, isolation window width, resolution, automatic gain control, and collision energy, were systematically tuned to balance the sensitivity and specificity while minimizing interferences. The optimized method enabled the detection of 2,907 features with 675 annotated compounds, leveraging recent progress in nano-LC-MS/MS for multiomics applications and showcasing the possibility of combining proteomics and metabolomics within a single chromatographic system. Ultimately, a comparison was performed between the data acquired through the DIA and DDA MS approaches in the context of untargeted metabolomics. This optimized analytical method yields more robust and reproducible results, thereby expanding the potential for meaningful discoveries across diverse biological fields.