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- Rsm-mediated post-translational control of the pseudomonas putida Type VI1 secretion systemPublication . Civantos, Cristina; Paredes, Carmen; Murillo-Torres, Marina; Botelho, João; Sánchez-Romero, María Antonia; Allsopp, Luke P.; Bernal, PatriciaThe Type VI secretion system (T6SS) is a bacterial nanoweapon that injects toxic effectors into prokaryotic and eukaryotic cells. It is widely found among gram-negative bacteria and provides a significant fitness advantage in interbacterial competition. Pseudomonas putida KT2440 possesses three T6SS clusters (K1-, K2- and K3-T6SS) that combat phytopathogens. This makes this strain a potent biocontrol agent that protects plants from pathogens and can be further enhanced by a better understanding of its T6SS regulation. Although the core components of T6SS are conserved, the elements controlling its regulation differ among bacterial species. T6SS activity is regulated by various factors acting at different levels, from transcription to post-translational modification, to ensure precise control of its activity. Here, we demonstrate the critical importance that the three Rsm proteins, RsmIEA, have in controlling the K1-T6SS structural components and related orphan elements at the post-transcriptional level in Pseudomonas putida. We identified multiple Rsm-binding sites responsible for directly repressing the translation of T6SS proteins (Hcp1 and Hcp5) and their associated effectors (Tke2 and Tke7). Derepression of K1-T6SS mRNA in the rsmIEA mutant led to enhanced translation and expression of the K1-T6SS components and effectors, and critically increased the number of cells in the population with assembled T6SS. This results in a greater capacity to secrete toxins and kill prey cells via the T6SS-dependent mechanism. Finally, we demonstrate the K1-T6SS ability to kill environmental pathogens, including Salmonella enterica and Erwinia amylovora.
- Metabarcoding of mediterranean monk seal (Monachus monachus) scats from the isolated population of Madeira reveals a diverse piscivorous dietPublication . Vijayakumar, Subramaniam; McIvor, Ashlie; Themudo, Goncalo Espregueira; Pires, Rosa; Thomsen, Philip Francis; Dinis, Ana; Canning-Clode, João; Carneiro, João; Campos, Paula F.The Mediterranean monk seal (Monachus monachus) is one of the world’s most endangered marine mammals, and information on its feeding ecology is needed to support conservation planning. Here, high-throughput DNA metabarcoding of scat samples from Madeira was used to characterise the diet of this geographically isolated population. Fourteen samples were analysed using universal fish primers targeting the mitochondrial 12S rRNA gene, enabling detection of both bony and cartilaginous fishes. Across samples, 38 prey taxa were identified, including records not previously reported in monk seal diet studies, such as Fistularia petimba, as well as cartilaginous fish such as Bathytoshia centroura, Dasyatis pastinaca, and Torpedo marmorata. Frequency of occurrence indicated a diet dominated by demersal and reef-associated taxa, with frequent detection of Sparidae, alongside evidence of opportunistic prey use. Overall, these results provide a high-resolution dietary profile for monk seals in Madeira and demonstrate the utility of scat metabarcoding for monitoring trophic ecology in small, threatened populations. This approach offers a scalable framework for assessing dietary shifts over time and informing adaptive management in human-impacted coastal ecosystems.
- Fire-resistant bio-based polyurethane foams designed with two by-products derived from sugarcane fermentation processPublication . Capeto, Ana Paula; Amorim, Manuela; Sousa, Sérgio; Costa, Joana R.; Uribe, Braian; Guimarães, Ana Sofia; Pintado, Manuela; Oliveira, Ana L. S.There is a great interest in replacing conventional fossil-based polymers and composites with inorganic or organic waste-based materials and filler-type additives, to promote environmental sustainability and circularity. The main objective of this study was the design of water-blown polyurethane rigid foams integrating two by-products derived from the Amyris fermentation process of production of β-farnesene. The distillation residue (FDR) was used as main polyol component in the neat formulation of the foams (PF) that it was supplemented (PFA) with 4.5% of sugarcane bagasse ash (SCBA) added as a filler with fire-retardant properties. The impact of both by-products on the foam density and morphology, and in the thermal, mechanical, and flame-retardant properties was assessed. SCBA presence led to a reduction in apparent density, cells size, and glass transition, delaying the thermal decomposition. The differences observed in the thermal conductivity and flammability test parameters suggest a visible impact of the ash incorporation, thus meeting the fire protection standard UL 94, class HB. Highlighting the need for further optimization, this work presents a practical example of the integration of different wastes derived from the same fermentation process in the formulation of sustainable PUR foams with reduced flammability.
- Green chitosan: thiourea dioxide cleaning gel for manganese stains on granite and glass substratesPublication . Campos, Bruno; Marco, Alexandra; Cadeco, Guihermina; Freire-Lista, David M.; Silvestre-Albero, Joaquin; Algarra, Manuel; Vieira, Eduarda; Pintado, Manuela; Moreira, PatríciaThe cleaning or removal of manganese stains on Cultural Heritage has not been much tested or successful so far. The aim of this article was to assess a new green cleaning gel for Mn-rich black-blue stains on different substrates. The black-blue stains were characterized at optical and chemical level through RGB data, colorimetry, optical microscope, FTIR, XRF and XPS. Mn-stained granite found on historical churches at Vila Real (North of Portugal) and glass jars of Leclanché cells, belonging to the ISEP’s Museum (Portugal) collection, were the ideal case studies to test the efficiency of Chitosan:Thiourea Dioxide (TD) cleaning gel. TD proved to be the best candidate to reduce insoluble manganese oxides, over Hydroxylamine Hydrochloride and Hydroxymethanesulfinic Acid. Cleaning assays performed on stained granite samples collected at a historical quarry and in situ application on stained granite churches showed the complete removal of the stains. Similar results were obtained on stained glass jars. This article presents an innovative, feasible and green Chitosan gel embedded with TD and phosphoric acid, applied to the cleaning of Mn-stained granite and glass substrates.
- Evaluation of the fungicide treatment with copper oxide and potassium phosphonate solutions for the sustainable management of P. pinaster trees infected with B. xylophilusPublication . López-Villamor, Adrián; Silva, Marta Nunes da; Vasconcelos, MartaFungicides induce changes in the plants promising to increase tolerance of Pinus pinaster against the pathogenic pinewood nematode (PWN). To test this hypothesis, P. pinaster plants were inoculated with the PWN, treated with copper oxide (CO) or potassium phosphonate (PP), and evaluated post-inoculation for: i) the extent of foliar symptoms; ii) nematode density inside stem tissues; iii) proxies for oxidative damage and antioxidant activity, iv) mineral concentration; and v) bacterial diversity. The mortality of infected plants reached 12.5% regardless of the treatment, but plants treated with fungicides, particularly with PP, had significantly lower PWN density (up to 0.61-fold). Plants treated with PP had substantially higher concentrations of anthocyanins at 14 dai than those treated with CO and non-Treated plants (by 1.47-fold), possibly contributing to the lower PWN colonization and degree of foliar symptoms observed. CO and PP led to increased lipid peroxidation at 28 dai (by 1.84- and 1.77-fold), and PP showed higher flavonoids concentration than CO (by 1.37- and 0.49-fold), corroborating its higher potential in increasing plant antioxidative response during infection. Fungicides also induced significant changes in micronutrient accumulation in plant tissues, resulting in a decrease in Zn and P concentrations in plants treated with either fungicide as compared to infected nontreated plants. Finally, CO treatment increased the diversity of the bacterial communities, while PP decreased microbial biodiversity. Altogether, results suggest that treatment with CO and PP increases tolerance against B. xylophilus by promoting the plant antioxidant system, changing the accumulation of essential minerals, and modulating plant-associated bacterial diversity.
- Skincare potential of a sustainable postbiotic extract produced through sugarcane straw fermentation by Saccharomyces cerevisiaePublication . Duarte, Marco; Carvalho, Maria João; Carvalho, Nelson Mota; Silva, João Azevedo; Mendes, Adélia; Ribeiro, Inês Pinto; Fernandes, João Carlos; Oliveira, Ana Lúcia; Oliveira, Carla; Pintado, Manuela; Amaro, Ana; Madureira, Ana RaquelPostbiotics, a new class of molecules derived from microorganism’s metabolism, are defined as a “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”. They can be produced by fermentation, using culture media with glucose as the carbon source, and lactic acid bacteria of the genus Lactobacillus, and/or yeast, mainly Saccharomyces cerevisiae as fermentative microorganisms. Postbiotics comprise different metabolites, and have important biological properties (antioxidant, anti-inflammatory, etc), which is why their use in cosmetics should be considered. During this work, the production of postbiotics was carried out by fermentation with sugarcane straw, as a source of carbon and other active compounds, and as a more sustainable and promising process to obtain more bioactive extracts at the end. For this, its saccharification process was carried out with cellulase at 55 ºC for 24 h. Fermentation was performed sequentially after saccharification at 30 ºC, for 72h, using S. cerevisiae. The cells-free extract was characterized regarding its composition, antioxidant activity, and skincare potential. Its use was safe at concentrations below ~ 20 mg.mL − 1 for keratinocytes and ~ 7.5 mg.mL − 1 for fibroblasts. It showed antioxidant activity, with ABTS IC50 of 1.88 mg.mL − 1 , and inhibited elastase and tyrosinase activities by 83.4% and 42.4%, respectively, at the maximum concentration tested (20 mg.mL − 1 ). In addition, it promoted the production of cytokeratin 14, and demonstrated anti-inflammatory activity at a concentration of 10 mg.mL − 1 . Finally, in the skin microbiota of human volunteers, the extract inhibited the Cutibacterium acnes bacterium and the Malassezia fungal genus. In short, postbiotics were successfully produced using straw as substrate, and as source of carbon and phenolic compounds. These postbiotics showed bioactive properties that potentiate their use in the development of cosmetic and skincare products, such as the treatment of acne or other skin diseases, due to their anti-inflammatory and inhibitory effect on the bacteria responsible for acne, as well as on potentially colonizing fungi.
- Evaluation of cow’s and goat’s fresh cheese preservation under hyperbaric storage at room temperature up to 60 days versus refrigeration regarding nutritional and physicochemical parametersPublication . Duarte, Ricardo Valente; Casal, Susana; Gomes, Ana Maria; Delgadillo, Ivonne; Saraiva, Jorge A.Two highly perishable dairy products (cow’s and goat’s fresh cheese (FC)), were stored under hyperbaric storage at room temperature (HS/RT, 50-100MPa) and compared with RF (4ºC), for 60 days. HS/RT (≥75MPa) lead to a more stable volatile organic and fatty acid profiles, reducing lipid oxidation rate, particularly for cow’s FC, resembling more to FC prior storage, contrarily to refrigerated cheeses. No variation was observed for total protein, while free amino acids increased over time after 60 days at 75/RT of 13- and 16-fold, and at 100/RT of 14- and 8-fold, respectively in cow’s and goat’s FC, which may have contributed to increased protein digestibility (5.2%) observed for goat’s FC after 60 days at 100/RT. These results indicate the overall increased preservation performance achieved by HS/RT for cow’s and goat’s FC when compared with RF, slowing down FC matrix degradation, possibly leading to considerable shelf-life extension.
- Dissemination of metallo-β-lactamase-producing Pseudomonas aeruginosa in Serbian hospital settings: expansion of ST235 and ST654 clonesPublication . Kabic, Jovana; Fortunato, Gianuario; Vaz-Moreira, Ivone; Kekic, Dusan; Jovicevic, Milos; Pesovic, Jovan; Ranin, Lazar; Opavski, Natasa; Manaia, Célia M.; Gajic, InaBackground: Pseudomonas aeruginosa is one of the most common nosocomial pathogens worldwide. Limited information on beta-lactamase-producing P. aeruginosa in Serbia is available. The aim of this nationwide study was to investigate the molecular characteristics of metallo-β-lactamase (MBL)- producing P. aeruginosa in Serbia, to characterize underlying resistance mechanisms, to analyze the genetic context of detected MBL genes and clonal relationship between isolates harboring genes encoding MBL. Methods: A total of 320 isolates of P. aeruginosa were identified out of 5334 bacterial isolates collected from 2018 to 2021. Carbapenem-resistant P. aeruginosa (CRPA) were screened for the presence of blaVIM, blaIMP , and blaNDM,genes whereas MBL-positive isolates were tested for the presence of the genes blaCTXM-2,blaPER,blaTEM,blaSHV,blaVEB , and blaGES . Multilocus sequence typing and phylogenomic analysis were performed for P. aeruginosa producing MBL. Results: P. aeruginosa isolates were recovered from the lower respiratory tract (n=120; 37.5%), wound specimens (n=108; 33.75%), and urine samples (n=71; 22.2%), mainly from patients admitted to the intensive care unit (n=178; 55.6%). CRPA isolates accounted for 43.1% (n=138) of the tested P. aeruginosa and 31 out of these were blaNDM-1 -positive (22.5%). Colistin was the most effective antibiotic, with a resistance rate of 0.3%. MLST analysis of all blaNDM-1 -positive isolates (n=31) revealed the occurrence of ST235 (n=25) and ST654 (n=6), mostly confined to Serbia. The distribution of betalactamase encoding genes in these isolates suggested clonal dissemination and possible recombination: ST235/blaNDM-1 , ST235/blaNDM-1 /blaPER-1 , ST654/blaNDM-1 , ST654/blaNDM-1 /blaPER-1 , and ST654/blaNDM1 /blaGES-5 . Conclusions: High-risk clones ST235 and ST654 identified for the first time in Serbia, are important vectors of acquired MBL and ESBL in Serbia and their associated multidrug resistance (MDR) phenotypes represent a cause for considerable concern.
- Microbial variation and shelf life of chicken breast fillets: a year-long study across two European producersPublication . Moen, Birgitte; Silva, Beatriz Nunes; Måge, Ingrid; Teixeira, Paula; Langsrud, Solveig; Berget, Ingunn; Jensen, Merete Rusås; Gaarder, Mari Ø.; Lian, Ina Aksnes; Fernandes, Dina; Bjørkøy, Solfrid; Ferreira, Diogo; Fagerlund, AnnetteDespite extensive research on poultry spoilage, variability across batches, seasons, and facilities remains poorly resolved. This limits the ability to determine whether microbiological data can be used to predict sensory shelf life at the batch level, and thereby support adaptive, data-driven shelf-life determination. This study examined how initial microbial status, growth dynamics, microbiota composition, and sensory changes varied in modified atmosphere packed (MAP) chicken breast fillets from two European producers (A, Norway; B, Portugal) sampled over one year. Fillets were analysed after production and during storage at 4 °C or under a 4 to 8 °C temperature shift. Initial total viable counts (TVC) were lower in fillets from Producer A than B, but both producers showed approximately 2-log variation between batches. Maximum growth rates were similar, but fillets from Producer B showed consistently shorter lag times, reaching 7 log10 CFU/g earlier. Lag time was the primary determinant of shelf-life variation, whereas initial aerobic TVC had limited predictive value. During late storage, microbiota communities converged toward producer-specific spoiler profiles that were stable across batches (A: Lactobacillales, Hafnia_Obesumbacterium; B: Brochothrix, Yersiniaceae). Sensory deterioration correlated with TVC and was accelerated but not qualitatively altered by elevated temperature. Overall, initial aerobic TVC was not a reliable predictor of batch-specific shelf life; instead, early measurements that enable estimation of batch-specific lag time (i.e., the fraction of bacteria able to grow in the product) may provide an actionable basis for adaptive shelf-life setting within producers.
- Are fermented foods safe for human health?: a narrative systematic analysis of literaturePublication . Assunção, Ricardo; Angelovski, Ljupco; Ciesarová, Zuzana; Russo, Pasquale; Salvetti, Elisa; Sarand, Inga; Silva, Beatriz Nunes; Torbica, Aleksandra; Zakrzewski, Arkadiusz; Kasikci, Müzeyyen Berkel; Guzel, Mustafa; Issa, Aline; Chassard, Christophe; Praćer, Smilja; Vergères, Guy; Laranjo, MartaBackground: Fermented foods (FF) are widely consumed across different dietary cultures and are increasingly recognised for their nutritional and functional properties. Although fermentation can improve food preservation and safety, there are still concerns about microbiological and chemical hazards. Given the growing consumption of FF, it is essential to conduct a systematic evaluation of their safety to support evidence-based risk-benefit assessments. Objectives: This review aimed to establish the safety of FF for human consumption and to identify the main microbiological and chemical hazards associated with these foods, including their occurrence, exposure and potential risks. Design: A systematic narrative review was conducted in accordance with a predefined protocol aligned with PRISMA principles. Relevant human studies were identified through searches of the PubMed, Scopus and Cochrane Library databases. The eligibility criteria were defined using a PECO framework and included all population groups and a broad range of FF across major food categories. Alcoholic beverages with an alcohol content above 1.25% were excluded. The outcomes included the occurrence of hazards, associated exposure and risk, and reported foodborne outbreaks. Results: Microbiological hazards were predominantly associated with pathogens such as Salmonella, pathogenic Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, and Clostridium botulinum, with dairy and fermented meat products most frequently implicated in outbreaks. However, these risks were largely linked to contaminated raw materials, inadequate processing conditions, or post-process contamination rather than fermentation itself. Chemical hazards included mycotoxins, heavy metals, polycyclic aromatic hydrocarbons, and acrylamide. Most available risk metrics indicated low concern under typical consumption scenarios, although certain products, such as specific cheeses and cereal-based foods, showed higher exposure levels. Importantly, several chemical hazards were associated with raw material contamination or thermal processing rather than the fermentation process. Conclusion: Overall, FF are generally safe when produced under appropriate hygienic and technological conditions. The identified risks are primarily driven by contamination upstream in the supply chain, processing practices, and environmental factors, rather than by fermentation itself. These findings emphasise the importance of targeted risk management strategies that focus on the quality of raw materials, process control and harmonised risk assessment approaches. This will support the safe integration of FF into modern diets.
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