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Microorganisms naturally colonize rock-based materials in outdoor environments, thereby contributing to their degradation. Fungi, especially in tropical environments with abundant water and favorable temperatures, play a significant role in biodegradation. However, many aspects of the microorganism-stone interaction, including fungal colonization dynamics and the impact of treatment applications, remain unclear. This study conducted a four-year in-situ evaluation of fungal community dynamics on limestone surfaces in the Temple of the Warriors at the Chichén Itzá archaeological site in Mexico, focusing on cleaning and treatment using nanoparticles (NPs). These NPs included zinc oxide (ZnO) and CaZn2(OH)6·2H2O (CZ)-based NPs synthesized via sol-gel (CZ-SG) and mechanochemical methods (CZ-MC), as well as CZ/Ca(OH)2-based products (CZ:Ca-SG). The microbial colonization cover was assessed using colorimetric measurements, and the surface was sampled for fungal community isolation and identification. The results demonstrated significant impacts of cleaning and nanomaterial applications on cultivable fungal communities (melanized filamentous, hyaline, and microcolonial fungi), altering composition, dynamics, and stone surface coloration. In particular, ZnO NPs caused 50 % decline in fungal species and individuals, whereas CZ:Ca-SG NPs displaced most species, indicating effective inhibition of the cultivable fungal community. Microcolonial fungi (MCF), known for their tolerance to withstand harsh environmental conditions, were the only fungal group found in the CZ:Ca-SG treatment. In contrast, CZ-SG and CZ-MC increased the abundance of melanized species, resulting in darkening and reduced color intensity. This study highlights the importance of microcolonial fungi that are tolerant to cleaning and coating procedures in the preservation of stone cultural heritage. These findings enhance our understanding of fungal colonization dynamics following treatment and provide valuable, European Union, CONACYT, Depto. de Mineralogía y Petrología, Fac. de Ciencias Geológicas, TRUE, pub

Nanoparticles derived from plant viruses play an important role in nanomedicine due to their biocompatibility, self-assembly and easily-modifiable surface. In this study, we developed a novel platform for increasing antibody sensing using viral nanoparticles derived from turnip mosaic virus (TuMV) functionalized with SARS-CoV-2 receptor binding domain (RBD) through three different methods: chemical conjugation, gene fusion and the SpyTag/SpyCatcher technology. Even though gene fusion turned out to be unsuccessful, the other two constructs were proven to significantly increase antibody sensing when tested with saliva of patients with different infection and vaccination status to SARS-CoV-2. Our findings show the high potential of TuMV nanoparticles in the fields of diagnostics and immunodetection, being especially attractive for the development of novel antibody sensing devices., Ministerio de Universidades (España), Comunidad de Madrid, Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The bioindicator potential of the microeukaryotic community in a full-scale A2O wastewater treatment process was assessed after deactivating the internal recirculation in one of the two parallel treatment lines of the plant. The deactivation of the internal recirculation in one of the lines (NIR-line), led to a strong decrease in nitrogen removal efficiency (53 % ammonia-uptake in the NIR-line versus 97 % in the unaltered line or IR-line), as well as to a lower organic matter removal (95 % BOD5 removal versus 97 % in the IR-line). The microeukaryote community structure changed greatly confirming trends observed in experimental studies and showed a decrease in abundance and richness in the NIR-line. However, this line showed a significant higher abundance of certain ciliate species, such as Epistylis camprubii and Vorticellides infusionum, which are common species in conventional activated sludge. Conversely, the IR-line showed useful indicators of internal recirculation, associated with much higher ammonium and BOD5 removal. These results show that identification to ciliate species level provides a more reliable bioindication in nutrient removal systems, supporting the importance of research on biological parameters, mainly protists, as simple and useful tools for plant operators to manage nutrient removal processes. These observations extend the current boundaries of bioindication., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Vultures have evolved adaptive mechanisms to prevent infections associated with their scavenging lifestyle. However, food-borne exposure to antimicrobial pharmaceuticals can promote opportunistic infections with adverse outcomes. Here, we used multivariate and network analyses to increase understanding of the behavior of the yeast communities causing oral mycosis outbreaks recently reported in wild nestling cinereous (Aegypius monachus), griffon (Gyps fulvus) and Egyptian (Neophron percnopterus) vultures (CV, GV and EV, respectively) exposed to antibiotics from livestock farming. Common and unique yeast signatures (of Candida, Debaromyces, Diutina, Meyerozyma, Naganishia, Pichia, Rhodotorula, Trichosporon and Yarrowia species) associated with oral mycoses were identified in the three vulture species. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) highlighted that oral lesions from CV and GV shared similar yeast signatures (of major causative pathogens of opportunistic mycoses, such as Candida albicans, Candida parapsilosis and Candida tropicalis), while EV had a distinct yeast signature (of uncommon pathogenic species, such as Candida dubliniensis, Candida zeylanoides, Pichia fermentans and Rhodotorula spp.). Synergistic interactions between yeast species from distinct fungal phyla were found in lesions from CV and GV, but not in EV. These formed co-occurrence subnetworks with partially or fully connected topology. This study reveals that the composition, assembly and co-occurrence patterns of the yeast communities causing oral mycoses differ between vulture species with distinct feeding habits and scavenging lifestyles. Yeast species widely pathogenic to humans and animals, and yeast co-occurrence relationships, are distinctive hallmarks of oral mycoses in CV and GV. These vulture species are more exposed to antibiotics from intensively medicated livestock carcasses provided in supplementary feeding stations and show higher incidence, Ministerio de Economía y Competitividad (MINECO), Fac. de Óptica y Optometría, TRUE, pub

A strain of the protozoan ciliate Tetrahymena thermophila adapted to increasing Pb(II) concentrations over two years has shown that one of the resistance mechanisms to this extreme metal stress is the lead biomineralization to chloropyromorphite, one of the most stable minerals in the earth's crust. Several techniques such as microanalysis coupled to transmission and scanning electron microscopy (X-Ray Energy Disperse Spectroscopy), fluorescence microscopy and X-ray power diffraction analysis have revealed the presence of chloropyromorphite as crystalline aggregates of nano-globular structure, together with the presence of other secondary lead minerals. This is the first time that the existence of this type of biomineralization in a ciliate protozoan is described. The Pb(II) bioremediation capacity of this strain has shown that it can remove >90 % of the toxic soluble lead from the medium. A quantitative proteomic analysis of this strain has revealed the main molecular-physiological elements involved in adaptation to Pb(II) stress: increased activity of proteolytic systems against lead proteotoxicity, occurrence of metallothioneins to immobilize Pb(II) ions, antioxidant enzymes to mitigate oxidative stress, and an intense vesicular trafficking presumably involved in the formation of vacuoles where pyromorphite accumulates and is subsequently excreted, together with an enhanced energy metabolism. As a conclusion, all these results have been compiled into an integrated model that could explain the eukaryotic cellular response to extreme lead stress., Ministerio de Ciencia y Competitividad, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Mitochondria play a wide diversity of roles in cell physiology and have a key functional implication in cell bioenergetics and biology of free radicals. As the main cellular source of oxygen radicals, mitochondria have been postulated as the mediators of the cellular decline associated with the biological aging. Recent evidences have shown that mitochondrial free radical production is a highly regulated mechanism contributing to the biological determination of longevity which is species-specific. This mitochondrial free radical generation rate induces a diversity of adaptive responses and derived molecular damage to cell components, highlighting mitochondrial DNA damage, with biological consequences that influence the rate of aging of a given animal species. In this review, we explore the idea that mitochondria play a fundamental role in the determination of animal longevity. Once the basic mechanisms are discerned, molecular approaches to counter aging may be designed and developed to prevent or reverse functional decline, and to modify longevity., Ministerio de Ciencia e Innovación (MICIN), Comunidad de Madrid, Generalitat de Catalunya, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Producción Científica, Bread is one of the most wasted products in distribution systems and in households. To consider reusing it, the presence of microorganisms must be taken into account. In this study, the microbial load of crumb and crust of four kinds of bread, sold in two different types of establishments, has been compared separately. Counts of Bacillus spp, sulphite reducing bacteria, coliforms, E. coli, Salmonella spp, lactic acid bacteria, Aerobic plate count, molds, and yeasts were determined. The microorganisms found were identified. The microbial load of discarded breads was similar to that of cereal flours found in other studies. In general, a greater presence of bacteria in breads from small stores than in those from supermarkets has been found. Some of the least sold breads in small bakeries, such as wholemeal or candeal, presented a higher microbial load. Although their quantity or type are not a concern for people's health, good practices must be implemented to reduce health risks in products made from discarded bread., LWT Volume 173, 2023, 114291, Junta de Castilla y León (project VA177P20)

Producción Científica, The present study investigated the physicochemical and microbiological changes occurring during the storage of simulated restaurant food waste (FW) and how such changes affected its biohydrogen and biogas production potential. FW was stored for 72 h in a closed atmosphere under two different scenarios: i) without and ii) with inoculation of a mixed microbial culture harboring lactic acid bacteria (LAB). Both storage scenarios resulted in similar biotic and abiotic changes in FW. Particularly, FW was pre-acidified and pre-hydrolyzed to some extent during the storage, resulting in a feedstock enriched in LAB (≈ 95 % total relative abundance) and lactate (10.5–12.3 g/L, 87.0–90.5 % selectivity). Biochemical hydrogen potential tests revealed that the use of stored FW resulted in similar or even higher hydrogen production efficiencies compared to that of non-stored FW, achieving up to 60 NmL H2/g VS added and a maximum volumetric hydrogen production rate of 9.7 NL H2/L-d. Metabolically, the conversion of lactate into hydrogen was crucial regardless of the use of non-stored or stored FW, albeit the presence of fermentable carbohydrates in the substrate was also essential either to produce lactate or to co-produce extra hydrogen. On the contrary, biochemical methane potential tests showed that the biogas production potential of FW was not affected by storage, yielding on average 400 NmL CH4/g VS added and revealing that lactate oxidation to methane precursors represented an important step in FW biomethanization., European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 894515, Ministerio de Ciencia e Innovación - AEI and European Union NextGenerationEU/PRTR (RYC2021-034559-I), Junta de Castilla y León and the European FEDER Programme (CLU 2017-09, CL-EI-2021-07 y UIC 315)

Aspergillus steynii is probably the most relevant species of section Circumdati producing ochratoxin A (OTA). This mycotoxin contaminates a wide number of commodities and it is highly toxic for humans and animals. Little is known on the biosynthetic genes and their regulation in Aspergillus species. In this work, we identified and analysed three contiguous genes in A. steynii using 5′-RACE and genome walking approaches which predicted a cytochrome P450 monooxygenase (p450ste), a non-ribosomal peptide synthetase (nrpsste) and a polyketide synthase (pksste). These three genes were contiguous within a 20742 bp long genomic DNA fragment. Their corresponding cDNA were sequenced and their expression was analysed in three A. steynii strains using real time RT-PCR specific assays in permissive conditions in in vitro cultures. OTA was also analysed in these cultures. Comparative analyses of predicted genomic, cDNA and amino acid sequences were performed with sequences of similar gene functions. All the results obtained in these analyseswere consistent and point out the involvement of these three genes in OTA biosynthesis by A. steynii and showed a co-ordinated expression pattern. This is the first time that a clustered organization OTA biosynthetic genes has been reported in Aspergillus genus. The results also suggested that this situation might be common in Aspergillus OTA-producing species and distinct to the one described for Penicillium species., Ministerio de Ciencia e Innovación (España), Universidad Complutense de Madrid y Banco de Santander, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Despite previously published methods, there is still a lack of rapid and affordable methods for genotyping the Meyerozyma guilliermondii yeast species. The development of microsatellite markers is a useful genotyping method in several yeast species. Using the Tandem Repeat Finder Software, a total of 19 microsatellite motifs (di-, tri-, and tetra- repetition) were found in silico in seven of the nine scaffolds published so far. Primer pairs were designed for all of them, although only fourwere used in thiswork. Allmicrosatellite amplifications showed size polymorphism, and the results were identical when repeated. The combination of three microsatellite markers (sc15F/R, sc32 F/R and sc72 F/R) produced a different pattern for each of the Type Culture Collection strains of M. guilliermondii used to optimize the method. The three primer pairs can be used in the same PCR reaction,which reduces costs, in tandem with the fluorescent labeling of only the forward primer in each primer pair.Microsatellite typing was applied on 40 more M. guilliermondii strains. The results showed that no pattern is repeated between the different environmental niches. Four M. guilliermondii strains were only amplified with primer pair sc32 F/R, and subsequently identified as Meyerozyma caribbica by Taq I-RFLP of the 5.8S ITS rDNA. Most out-group species gave negative results even for physiologically similarly species such as Debaryomyces hansenii. The microsatellite markers used in this work were stable over time, which enables their use as a traceability tool., Universidad Complutense de Madrid y Banco Santander, Ministerio de Economía y Competitividad (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Aspergillus steynii and Aspergillus westerdijkiae are the main ochratoxin A (OTA) producing species of Aspergillus section Circumdati. Due to its recent description, few data are available about the influence of cophysiological factors on their growth and OTA production profiles. In this work, the effect of temperature (20, 24 and 28ºC) and water activity (aw) (0.928, 0.964 and 0.995) on rowth, sporulation and OTA production by these fungi was examined in CYA and media prepared from paprika, green coffee, anise, grapes, maize and barley. Growth was positively affected by the highest temperature and aw values indicating that both species might be expected in warm climates or storage conditions. However, optimal growth conditions showed differences depending on the medium. OTA production was markedly affected by substrate and showed qualitative and quantitative differences. Both species, especially A. steynii, represent a great potential risk of OTA contamination due to their high production in a variety of conditions and substrates, in particular in barley and paprika-based media. Additionally, neither growth nor sporulation did result good indicators of OTA production by A. steynii or A. westerdijkiae;therefore, specific and highly-sensitive detection methods become essential tools for control strategies to reduce OTA risk by these species., Ministerio de Ciencia e Innovación (España), Universidad Complutense de Madrid y Banco de Santander, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Unlike previously reported methods that need a combination of several typing techniques, we have 22 developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage 23 species. Strains belonging to other species have also been included for comparison. We have demonstrated 24 that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in 25 this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z.bailii. 26 We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns 27 with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS 28 rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of 29 Zygosaccharomyces in a separate cluster., Ministerio de Educación y Ciencia (España), Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Aspergillus steynii and Aspergillus westerdijkiae are important ochratoxin A (OTA)-producing species frequently found in coffee. Although the processing of green coffee beans reduces markedly OTA contamination, levels exceeding the legal limitsmight remain in the final product. Environmental conditions are a crucial factor affecting growth and OTA production in fungal species; therefore, in thiswork,we analysed the effect of different levels of temperature (23, 28, 32, 37, 42 °C) andwater activity (aw) (0.89, 0.91, 0.93, 0.95, 0.97, 0.99) on growth and toxin production by A. steynii and A. westerdijkiae in green coffee-based medium. A. steynii was able to grow and produce OTA in a wider set of conditions than A. westerdijkiae. A new index (OTA risk index) has been described to integrate both fungal growth and OTA production and, according to it, A. steynii would pose a higher risk of OTA contamination in coffee than A. westerdijkiae at all the conditions tested. Neither A. steynii nor A. westerdijkiae were able to grow at the lowest value of aw (0.89) evaluated and OTA production was extremely low at 0.91. Therefore, the application of good practices during storage aiming to maintain low humidity levelsmight be essential to prevent OTA contamination in coffee at this stage. The optimal conditions of both species to grow and produce OTA were established at warm temperatures (28–32 °C) and high aw levels. Therefore, these species could be considered well-adapted in predicted climate change scenarios resulting in a potential high risk source of OTA contamination for this product., Ministerio de Ciencia e Innovación (MICINN), UCM-BSCH, Instituto Danone, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications., CEI Campus Moncloa, UCM-UPM (Madrid, Spain)-PICATA predoctoral fellowship, Ministerio de Educación, Cultura y Deporte (España)-FPU predoctoral fellowship, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgements We are grateful to Dr. Minna Zhang and Dr. Yinong Li from Northeast Normal University, Dr. Xincheng Li from Fudan University, and Dr. Shengen Liu from China Three Gorges University for the valuable feedback and suggestions for the data analysis in the earlier version. M.D-B. is supported by a Ramón y Cajal grant (RYC2018-025483-I), a “Ayuda P.P. 2020. Desarrollo Lineas Investigación Propias (UPO), a project from the Spanish Ministry of Science and Innovation (PID2020-115813RA-I00), and a project PAIDI 2020 from the Junta de Andalucía (P20_00879). H.Y.C. is supported by National Natural Science Foundation of China (32101335), China Postdoctoral Science Foundation (2021M690589), Innovation Project of Young Technological Talents in Changchun City (21QC07), and Fundamental Research Funds for the Central Universities (2412021QD014). J.P.V. is thankful to DST and SERB (Science and Engineering Research Board), India for financial support for plant-microbe interaction research. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government., The environmental factors controlling soil biodiversity along resource gradients remain poorly understood in wet tropical ecosystems. Aboveground biodiversity is expected to be driven by changes in nutrient availability in these ecosystems, however, much less is known about the importance of nutrient availability in driving soil biodiversity. Here, we combined a cross-continental soil survey across tropical regions with a three decades' field experiment adding nitrogen (N) and phosphorus (P) (100 kg N ha−1y−1 and 100 kg P ha−1y−1) to Hawai'ian tropical forests with contrasting substrate ages (300 and 4,100,000 years) to investigate the influence of nutrient availability to explain the biodiversity of soil bacteria, fungi, protists, invertebrates and key functional genes. We found that soil biodiversity was driven by soil acidification during long-term pedogenesis and across environmental gradients, rather than by nutrient limitations. In fact, our results showed that experimental N additions caused substantial acidification in soils from Hawai'i. These declines in pH were related to large decreases in soil biodiversity from tropical ecosystems in four continents. Moreover, the microbial activity did not change in response to long-term N and P additions. We concluded that environmental filtering drives the biodiversity of multiple soil organisms, and that the acidification effects associated with N additions can further create substantial undesired net negative effects on overall soil biodiversity in naturally tropical acid soils. This knowledge is integral for the understanding and management of soil biodiversity in tropical ecosystems globally., Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

CRUE-CSIC (Acuerdos Transformativos 2022), A comparative analysis of toxicities of both arsenic forms (arsenite and arsenate) in the model eukaryotic microorganism Tetrahymena thermophila (ciliate protozoa) has shown the presence of various detoxification mechanisms and cellular effects comparable to those of animal cells under arsenic stress. In the wild type strain SB1969 arsenate is almost 2.5 times more toxic than arsenite. According to the concentration addition model used in binary metallic mixtures their toxicities show an additive effect. Using fluorescent assays and flow cytometry, it has been detected that As(V) generates elevated levels of ROS/RNS compared to As(III). Both produce the same levels of superoxide anion, but As(V) also causes greater increases in hydrogen peroxide and peroxynitrite. The mitochondrial membrane potential is affected by both As(V) and As(III), and electron microscopy has also revealed that mitochondria are the main target of both arsenic ionic forms. Fusion/fission and swelling mitochondrial and mitophagy, together with macroautophagy, vacuolization and mucocyst extruction are mainly associated to As(V) toxicity, while As(III) induces an extensive lipid metabolism dysfunction (adipotropic effect). Quantitative RT-PCR analysis of some genes encoding antioxidant proteins or enzymes has shown that glutathione and thioredoxin metabolisms are involved in the response to arsenic stress. Likewise, the function of metallothioneins seems to be crucial in arsenic detoxification processes, after using both metallothionein knockout and knockdown strains and cells overexpressing metallothionein genes from this ciliate. The analysis of the differential toxicity of As(III) and As(V) shown in this study provides cytological and molecular tools to be used as biomarkers for each of the two arsenic ionic forms., Ministerio de Economía y Competitividad (España), Depto. de Biodiversidad, Ecología y Evolución, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

CRUE-CSIC (Acuerdos Transformativos 2022), In nature, bacteria exhibit a limited repertoire of behaviors in response to environmental changes. Synthetic biology has now opened up the possibility of programming cells or unicellular organisms in order to enable them to perform certain tasks, which would allow the programming of ’intelligent’ bacteria. Many of the theoretical ideas that Liberman proposed last century, for example his seminal idea that a cell is a computer, are now being put into practice with bacterial colonies in both wet and in silico experiments. These bacteria may one day be used to solve a wide range of problems whose solution requires their adaptation to external changes either within a bioreactor, organ or tissue of a patient or through the design of microbial-synthetic consortia oriented to their use in bioprocesses to produce medicines, biofuels or biomaterials. In this work, we show the possibility of programming synthetic bacteria with a previously trained perceptron neural network. First, we illustrate how a colony of bacteria endowed with a perceptron is able to solve an optimization problem in silico. Secondly, we study by means of in silico simulations how a perceptron can be applied to program behaviors in bacteria leading to social interactions and to the formation of complex communities that in the future would be useful in biotechnology. Finally, we go a step further, and study how the above perceptron designed to program bacterial behavior is implemented in a genetic circuit designed for this purpose. Once the genetic circuit was obtained, it was engineered into a plasmid., En la naturaleza, las bacterias muestran un repertorio limitado de comportamientos en respuesta a los cambios ambientales. La biología sintética ha abierto ahora la posibilidad de programar células u organismos unicelulares para que puedan realizar determinadas tareas, lo que permitiría programar "bacterias inteligentes". Estas bacterias podrán ser utilizadas algún día para resolver una amplia gama de problemas ya sea dentro de un biorreactor, el órgano o tejido de un paciente o mediante el diseño de consorcios microbianos-sintéticos orientados a su uso en bioprocesos para producir medicamentos, biocombustibles o biomateriales. En este trabajo, mostramos como un organismo "sin cerebro", las bacterias, pueden ser convertidos en seres inteligentes, introduciéndose una técnica con la que es posible dotar a las bacterias con un circuito neuronal perceptrón previamente entrenado. En primer lugar, mostramos cómo una colonia de bacterias dotada de un perceptrón es capaz de resolver un problema de optimización in silico. En segundo lugar, estudiamos también mediante experimentos de simulación, cómo una colonia de bacterías "inteligentes" forma colonias o comunidades complejas exhibiendo las bacterias interacciones sociales. Creemos que estas sociedades complejas formadas por bacterias inteligentes, al estar dotadas de un circuito genético que emula a un circuito neuronal, serán de utilidad práctica en el futuro para resolver problemas del mundo real. Por último, damos un paso más, y estudiamos cómo el perceptrón anterior - diseñado como un circuito genético con el que programar comportamientos bacterianos - puede ser insertado en un plásmido (https://doi.org/10.6084/m9.figshare.14399141.v4 Añadir un título al proyecto de Citavi utilizando este DOI) con el que sería posible transformar bacterias en organismos unicelulares inteligentes. Una de las aportaciones del trabajo es mostrar la equivalencia entre una red neuronal y un circuito genético, diseñándose el plásmido con CEL, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

CRUE-CSIC (Acuerdos transformativos 2022), Saccharomyces cerevisiae is a highly fermentative species able to complete the wine fermentation. However, the interaction with other non-Saccharomyces yeasts can determine the fermentation performance of S. cerevisiae. We have characterised three rare non-Saccharomyces yeasts (Cyberlindnera fabianii, Kazachstania unispora and Naganishia globosa), studying their impact on S. cerevisiae fitness and wine fermentation performance. Using a wide meta-taxonomic dataset of wine samples, analysed through ITS amplicon sequencing, we show that about a 65.07% of wine samples contains Naganishia spp., a 27.21% contains Kazachstania spp., and only a 4.41% contains Cyberlindnera spp; in all cases with average relative abundances lower than 1% of total fungal populations. Although the studied N. globosa strain showed a limited growth capacity in wine, both K. unispora and C. fabianii showed a similar growth phenotype to that of S. cerevisiae in different fermentation conditions, highlighting the outstanding growth rate values of K. unispora. In mixed fermentations with S. cerevisiae, the three yeast species affected co-culture growth parameters and wine chemical profile (volatile compounds, polysaccharides and proteins). K. unispora DN201 strain presents an outstanding capacity to compete with S. cerevisiae strains during the first stage of wine fermentation, causing stuck fermentations in both synthetic and natural grape musts., Ministerio de Ciencia e Innovación (MICINN) (10.13039/ 501100011033)., Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Research on the environmental impact of plastics, especially on the effect of microplastics (MPs), has become a priority issue in recent years, mainly in terrestrial ecosystems where there is a lack of studies. This work aims to assess the impact of two types of polyethylene MPs, white microbeads (W) and fluorescent blue microbeads (FB), and their interactions with two contaminants, ibuprofen (Ib) and simazine (Sz), on different organisms. A set of bioassays for Vibrio fischeri, Caenorhabditis elegans and Lactuca sativa was carried out, which helped to establish the ecotoxicological impact of those pollutants. C. elegans showed the least sensitivity, while V. fischeri and L. sativa showed a high toxicological response to MPs alone. We found that W and FB induced an inhibition of 27% and 5.79%, respectively, in V. fischeri, and the growth inhibition rates were near 70% in L. sativa for both MPs. MPs exhibited a potential role as contaminant vectors in V. fischeri since the inhibition caused by W-Ib or W-Sz complexes was near 39%. The W-Sz complex significantly reduced leaf development in L. sativa, and a reduction of 30% in seed germination was detected when the complex FB-Sz was tested. This study reveals the importance of designing a complete set of analyses with organisms from different trophic levels, considering the great variability in the effects of MPs and the high number of relevant factors., Ministerio de Ciencia, Innovación y Universidades (España), Sección Deptal. de Bioquímica y Biología Molecular (Veterinaria), Sección Deptal. de Farmacología y Toxicología (Veterinaria), Sección Deptal. de Fisiología (Veterinaria), Fac. de Veterinaria, TRUE, pub

We describe the first beetle mite (Oribatida) found in the lower Albian (Lower Cretaceous) amber-bearing site of Ariño, located in the Teruel Province (eastern Iberian Peninsula). It represents the first fossil record of the family Liacaridae (Acariformes: Oribatida: Gustavioidea). A new species, Liacarus (Procorynetes) shtanchaevae Arillo and Subías sp. nov., is described and compared with the living species of the subgenus Liacarus (Procorynetes). Notes on its biogeography and palaeobiology are provided. It corresponds to the first Cretaceous record of an extant oribatid subgenus and bears witness to the wide range of distribution that the ancient representatives of the subgenus may have had. Most of the oribatid species from Cretaceous ambers belong to living genera, which reflects the high degree of morphological stasis, or bradytely, over the evolutionary history of oribatid mites since the Early Cretaceous., Ministerio de Ciencia e Innovación (MICINN)/FEDER, Generalitat de Catalunya/FEDER, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

Nitrogen content of grape musts strongly impacts on fermentation performance and wine metabolite production. As nitrogen is a limiting nutrient in most grape musts, nitrogen supplementation is a common practice that ensures yeast growth during fermentation. However, preferred nitrogen sources -as ammonium- repress the genes related to alternative nitrogen sources consumption, usually involved in aromatic compounds production. Here, we describe the effect of high ammonium doses in Saccharomyces cerevisiae fermentation performance and wine properties, and how it is affected by yeast co-inoculation in mixed (S. cerevisiae + Torulaspora delbrueckii) fermentations. In addition, an RNA-seq analysis allowed us to study the S. cerevisiae transcriptional response to ammonium nutrition and yeast interaction, demonstrating that T. delbrueckii presence affects the global S. cerevisiae transcriptional response, reducing ammonium effects at both phenotypic -fermentation kinetics and metabolite production- and transcriptional levels, under experimental conditions., Ministerio de Economia, Industria y Competitividad. Centro para el Desarrollo Tecnológico Industrial (CDTI), Fundación para el Conocimiento madri+d/Universidad Rey Juan Carlos, Universidad Complutense de Madrid, Hessen State Ministry of Higher Education, Research and the Arts, Federation of European Microbiological Societies(FEMS), Agrovin S.A, Fundação para a Ciência e Tecnologia to Biosystems and Integrative Sciences Institute (BioISI), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Grapevine trunk diseases, caused by different fungal pathogens, are the most relevant group of diseases that affect vineyards, causing important economic losses. Quality control for sanitary purposes should be applied for different diseases in every stages of the plant development, from the nursery during propagation to the vineyard during grape production. Petri disease has been identified as one of the most dangerous for vine plants causing economic losses in cellars worldwide. Petri disease is caused by the infection of different fungal pathogens, mainly, Phaeomoniella chlamydospora, Phaeocremonium minimum and Cadophora luteo-olivacea. The molecular detection of plant pathogens is topical due to its facility, reproducibility and accuracy. Here we have described a PCR protocol based in a pair of new designed specific primers for the rapid detection of C. luteo-olivacea. The described procedure is intended for the direct inspection of plants suspected of being infected. Bovine serum albumin and MgCl2 were used as PCR adjuvants in order to cope with the presence of polymerase inhibitors from wood samples. Several assays have been settled out in order to assign the sensitivity and specificity of the developed technique. Under the conditions tested, the procedure has a low detection limit (10 pg) and it is specific for C. luteo-olivacea detection among other Cadophora species and fungal endophytes of Vitis vinifera., Ministerio de Economía y Competitividad (MINECO)/Centro para el Desarrollo Tecnólogico Industrial(CDTI), Pago de Carraovejas Estate Winery, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Brettanomyces bruxellensis is among the main spoilage yeasts in wine usually found in oak barrels. As routine, laboratories at wineries use selective-differential culture media to detect this yeast. Nevertheless, it is widely recognized that other microbial species can grow on these media, getting false positive signals. In this work, we have developed a conventional PCR method based on the vinylphenol reductase gene (VPR1) of B. bruxellensis applied to wines after a polyvinylpyrrolidone-based pre-treatment (7% w/v) and that allowed us to reach a low detection limit, up to 102 UFC/mL of wine. The procedure was conceived without time-consuming DNA extraction steps, simplifying the methodology and making it easy for applying in wineries., Ministerio de Economía y Competitividad (MINECO), Pago de Carraovejas SLU, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The aim of the present study was to investigate whether the late onset of diet-induced obesity (DIO) in middle-aged mice affected behavioral, immunological and oxidative stress parameters as well as life span of male and female mice. Also, it was analyzed whether the late DIO onset aggravated immunosenescence in old female mice. Late-adult male and female ICR/CD1 mice (28 weeks old) were fed either a high-fat diet or a standard diet during 14 weeks. After that, in these middle-aged (42 weeks old) diet-induced obese (DIO) and non-DIO controls, behavior as well as functions and redox state of peritoneal leukocytes were evaluated. These same parameters (excepting behavioral tests) were repeated when female mice were old (72 weeks old). The results showed lower exploratory activity and higher anxiety-like behavior in middle-aged male and female DIO than in controls. Moreover, these DIO animals from both sexes exhibited statistically significant impaired immune cell functions, such as chemotaxis of macrophages and lymphocytes, phagocytosis of macrophages, natural killer activity and lymphoproliferation in response to ConA and LPS, as well as an oxidative stress state in comparison with controls. Male DIO mice exhibited higher impairments in a variety of the evaluated parameters and a shorter life span than their female counterparts. In addition, female DIO mice, at old age, showed aggravated immunosenescence. In conclusion, the late DIO onset leads to impairments in behavior as well as in immune system functions of middle-aged male and female mice, males being significantly more affected than females., Universidad Complutense de Madrid, Instituto de Salud Carlos III (ISCIII), Fondo Europeo de Desarrollo Regional (FEDER), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The Aspergillus niger aggregate contains 15 morphologically indistinguishable species which presence is related to ochratoxin A (OTA) and fumonisin B2 (FB2) contamination of foodstuffs. The taxonomy of this group was recently reevaluated and there is a need of new studies regarding the risk that these species might pose to food security. 258 isolates of A. niger aggregate obtained from a variety of products from Spain were classified by molecular methods being A. tubingensis the most frequently occurring (67.5%) followed by A. welwitschiae (19.4%) and A. niger (11.7%). Their potential ability to produce mycotoxins was evaluated by PCR protocols which allow a rapid detection of OTA and FB2 biosynthetic genes in their genomes. OTA production is not widespread in A. niger aggregate since only 17% of A. niger and 6% of A. welwitschiae isolates presented the complete biosynthetic cluster whereas the lack of the cluster was confirmed in all A. tubingensis isolates. On the other hand, A. niger and A. welwitschiae seem to be important FB2 producers with 97% and 29% of the isolates, respectively, presenting the complete cluster. The genes involved in OTA and FB2 were overexpressed in producing isolates and their expression was related to mycotoxin synthesis., El grupo de especies del agregado de Aspergillus niger incluye importantes hongos contaminantes de alimentos que suponen un grave riesgo para la seguridad alimentaria debido a su capacidad de producir micotoxinas como la ocratoxina A o fumonisina B2, dos posibles carcinógenos humanos. En este trabajo, hemos estudiado en profundidad cual es la incidencia de estas especies en los productos alimentarios más frecuentemente contaminados por micotoxinas en España y su potencial capacidad para producir micotoxinas. La especie más frecuentemente detectada fue A. tubingensis con más de un 65% de los aislamientos en prácticamente todos los alimentos analizados. Sin embargo, ninguna de las cepas analizadas es potencial productora de ninguna de las toxinas ya que no presenta los genes de producción en su genoma. Por otro lado, A. niger y A. welwitschiae fueron aislados menos frecuentemente pero la mayoría sí son potenciales productores de toxinas y, por tanto, su presencia en alimentos debe ser tenida en cuenta para establecer medidas de control efectivas., Ministerio de Ciencia e Innovación (MICINN), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

IP13-01530, Objectives: Bacterial viability and enrichment of resistance resulting from three different amikacin administration schedules with respect to haemodialysis (HD) were assessed against three OXA-48-producing Klebsiella pneumoniae isolated during an outbreak in a Spanish hospital. Methods: A previously described two-compartment dynamic system was used. Three possible amikacin administration schedules were simulated: (i) haemodialysis immediately after amikacin infusion (pre-HD); (ii) infusion immediately after haemodialysis (post-HD); and (iii) infusion at 50% interdialytic period. Amikacin standard dose (SD) and double dose (DD) were simulated for each schedule. Values of Cmax/MIC, Cmax/MPC (mutant prevention concentration), AUC0-48h/MIC, AUC0-48h/MPC and %TMSW (percentage of time that the concentration was inside the mutant selection window) were determined with experimental data and were correlated with the area under the bacterial killing curve of the total population and the resistant subpopulation. Results: Both with SD and DD, the pre-HD schedule resulted in increases at 48h in bacterial counts of the total population at the expense of enrichment of pre-existing resistant subpopulations from 12h onwards for all strains. The estimated %TMSW that prevented enrichment of resistant mutants was <61.5%. The AUC0-48h/MPC (with values of ≈40 being associated with countering of increases in resistant subpopulations) was better than the %TMSW as a predictive parameter. Conclusion: This study showed that the longest times concentrations were above the MPC (i.e. highest AUC0-48h/MPC, lowest %TMSW), the lowest enrichment of resistant subpopulations. This implies use of the highest possible amikacin dose (limited by toxicity) and post-HD as the best administration schedule., European Commission, Ministerio de Ciencia y Tecnología (España), Instituto de Salud Carlos III (España), Depto. de Medicina, Fac. de Medicina, TRUE, pub

Nanoparticles are released in the environment causing a negative impact in several ecosystems such as microbial communities. To adapt to environmental changes some bacteria use a collective behaviour ruled by a cell-to-cell communication process called quorum sensing (QS). In this study, the impact of some of the most employed metal-based nanoparticles, such as zinc oxide nanoparticles (ZnONPs), titanium dioxide nanoparticles (TiO2NPs) and silver nanoparticles (AgNPs) on bacterial QS has been assessed by using two different strains of the model organism Chromobacterium violaceum and by employing different experimental conditions. TiO2NPs were tested with and without applying a previous step of UV-irradiation while the effect of AgNPs of two diameter sizes (40 and 60 nm) and two different coating agents (PVP and citrate) was evaluated. Results evidenced that all nanoparticles produced a significant effect on violacein production and therefore, in the QS system. ZnONPs mainly disrupted the QS steps related to signal perception and response whereas TiO2NPs and AgNPs affected the autoinducer biosynthesis. AgNPs with the smallest size and citrate as capping agent produced the most deleterious effect while the impact of TiO2NPs was not affected by UV irradiation. The present study provides new insights into the mechanisms by which these commonly employed metal-based nanoparticles disturb bacterial QS-based communication and clearly evidences the potential risk of releasing nanoparticles to the environment, especially for microbial communities which play a key role in many environmental and technological processes., Ministerio de Economía, Comercio y Empresa (España), Comunidad de Madrid, Fondo Europeo de Desarrollo Regional, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Fundación Mutua Madrileña (grant number 4131241)., In order to assess the role of the biotic and abiotic components of natural mineral water (NMW) in the spread of allochthonous bacteria in non-thermal spa facilities, we examined the effect of low mineralized NMWs on the growth of several commensal and pathogenic human strains. These NMWs were collected from two Spanish spring spas and had different microbiological characteristics. Microorganisms were exposed to untreated, filtered and autoclaved NMWs at the temperatures of 22 °C and 37 °C for 2 days, mimicking the early stage of starvation. Starvation stress was controlled by the effect identified after incubation in saline. Changes in culturability after exposure were used as a measure of the water’s antibacterial effect. The specific biotic and abiotic effect of NMWs on the suppression of bacterial growth was estimated after excluding the bacterial response to starvation stress, characteristic of this natural oligotrophic environment. The incubation temperature strongly modulated both the consequences of starvation and the impact of the natural biotic and abiotic components of NMWs on the growth of commensal and pathogenic bacteria. A temperature of 22 °C conferred cross-protection of microorganisms to starvation and NMW abiotic stress, whereas a temperature of 37 °C decreased the tolerance to both, and had a negative influence on the abundance and diversity of NMWs microflora. This temperature-dependent behaviour of the allochthonous and autochthonous bacteria explained the different culturability of microorganisms after exposure to untreated NMWs at 22 °C (≈1–2.4 log colony forming units per ml -CFU/ml- mean reduction) and at 37 °C (≈1.8–3.2 log CFU/ml mean reduction). Discarding the effect of starvation, we estimated that the antibacterial effect of NMWs at the temperature of 22 °C was mainly driven by the microecosystem of NMWs, which explained ≥95% of NMW response. In contrast, at optimal temperatures for the growth of commensal and pathogenic microorganisms, Fundación Mutua Madrileña, Depto. de Medicina, Depto. de Radiología, Rehabilitación y Fisioterapia, Fac. de Medicina, TRUE, pub

Cell-to-cell differences in bacterial gene expression can merely reflect the occurrence of noise. In certain cases, however, heterogeneous gene expression is a programmed event that results in bistable expression. If bistability is heritable, bacterial lineages are formed. When programmed bistability is reversible, the phenomenon is known as phase variation. In certain cases, bistability is controlled by genetic mechanisms (e. g., DNA rearrangement). In other cases, bistability has epigenetic origin. A robust epigenetic mechanism for the formation of bacterial lineages is the formation of heritable DNA methylation patterns. However, bistability can also arise upon propagation of gene expression patterns by feedback loops that are stable upon cell division. This review describes examples of bistability and phase variation in Salmonella enterica and discusses their adaptive value, sometimes in a speculative manner., Ministerio de Economía e Innovación (España), European Regional Fund, Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

Biological treatment of fermentation brines from table olive processing (FTOP) entails many difficulties due to their very high salinity and high COD concentration, which include some phenolic compounds. These extreme conditions limit the biodiversity of the microbial population. Experiments treating FTOP were performed in laboratory sequencing batch reactors (SBR) changing operating conditions during their operation, in order to study the effects on the SBR performance and on the protist population. The statistical study showed that the SBRs with high influent COD, pH and volatile solids and low influent phenol concentration, hydraulic retention time and temperature achieved the highest COD removal efficiencies. These operational conditions also provided the highest ciliate population and the lowest flagellate presence., Ministerio de Ciencia e Innovación (MICINN), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

In last years, non-Saccharomyces yeasts have emerged as innovative tools to improve wine quality, being able to modify the concentration of sensory-impact compounds. Among them, varietal thiols released by yeasts, play a key role in the distinctive aroma of certain white wines. In this context, Torulaspora delbrueckii is in the spotlight because of its positive contribution to several wine quality parameters. This work studies the physiological properties of an industrial T. delbrueckii strain, for the production of wines with increased thiol concentrations. IRC7 gene, previously described in S. cerevisiae, has been identified in T. delbrueckii, establishing the genetics basis of its thiol-releasing capability. Fermentations involving T. delbrueckii showed improvements on several parameters (such as glycerol content, ethanol index, and major volatile compounds composition), but especially on thiols release. These results confirm the potential of T. delbrueckii on wine improvement, describing new metabolic features regarding the release of cysteinylated aroma precursors., Centro para el Desarrollo Tecnológico Industrial (CDTI), Agrovin S.A, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The Next Generation Sequencing methodologies are considered the next step within DNA-based methods and their applicability in different fields is being evaluated. Here, we tested the usefulness of the Ion Torrent Personal Genome Machine (PGM) in food traceability analyzing candies as a model of high processed foods, and compared the results with those obtained by PCR-cloning-sequencing (PCR-CS). The majority of samples exhibited consistency between methodologies, yielding more information and species per product from the PGM platform than PCR-CS. Significantly higher AT-content in sequences of the same species was also obtained from PGM. This together with some taxonomical discrepancies between methodologies suggest that the PGM platform is still pre-mature for its use in food traceability of complex highly processed products. It could be a good option for analysis of less complex food, saving time and cost per sample., Ministerio de Universidades, Principado de Asturias, Fundación Mafre, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Saccharomyces cerevisiae is the most important yeast species for the production of wine and other beverages. In addition, nowadays, researchers and winemakers are aware of the influence of non-Saccharomyces in wine aroma complexity. Due to the high microbial diversity associated to several agro-food processes, such as winemaking, developing fast and accurate methods for microbial identification is demanded. In this context, MALDI-TOF MS mass fingerprint provides reliable tool for fast biotyping and classification of microorganisms. However, there is no versatile and standardized method for fungi currently available. In this study, an optimized sample preparation protocol was devised for the biotyping of yeasts of oenological origin. Taking into account that commercially available reference databases comprise almost exclusively clinical microorganisms, most of them bacteria, in the present study a database of yeasts isolated from vineyards and wineries was created, and its accuracy was tested using industrial and laboratory yeast strains. In addition, the implementation of a program for MALDI-TOF MS spectra analysis has been developed as an extensible open-source platform for MALDI data processing and analysis with statistical techniques that has arisen from our previous experience working with MALDI data. The software integrates two R packages for raw MALDI data preprocessing: Continuous Wavelet Transform (CWT)-based algorithm and MassSpecWavelet. One of the advantages of the CWT is that it can be directly applied to a raw spectrum, without prior baseline correction. Mass fingerprints of 109 S. cerevisiae strains and 107 non-Saccharomyces isolates were generated by MALDI-TOF MS upon optimized sample preparation and instrument settings and analyzed for strain, species, and genus-level differentiation. As a reference method, for S. cerevisiae differentiation at strain level, the analysis of the polymorphism in the inter-delta region was chosen. The data revealed that, Ministerio de Economía y Competitividad (MINECO), Agrovin S.A., Pago de Carraovejas, Depto. de Genética, Fisiología y Microbiología, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

Based on IGS-PCR RFLP polymorphism, we previously detected two Z. rouxiistrains (CECT 11923 and CECT 10425) that clustered with hybrid strains (NCYC 1682, NCYC 3060 and NCYC 3061). Given the recently recognized important industrial role of hybrids, their detection is very useful. Based on the IGS1 rDNA region alignment of hybrid strains and the Z. rouxii CECT 11923 and CECT 10425, in this work, we developed a pair of Zygosaccharomyces hybrid-specific primers, HibZF/HibZR. Positive amplicons were only obtained in the Zygosaccharomyces spp. hybrids included in this study and the CECT 11923 and CECT 10425 strains analyzed here. In the present study, we applied molecular tools to highlight the nature of these strains; they are quite different from each other as well as from Z. rouxii type strain. Based on the presence of two heterologous copies of nuclear-encoded genes (SOD2 and HIS3), the sequences of divergent 5.8S-ITS rDNA, D1/D2 26S rDNA copies and, the amplification with species-specific primer for Z. rouxii and Z. pseudorouxii, we hypothesize that the CECT 11923 strain might be a hybrid strain. Whereas, CECT 10425, the sequence analysis of 5.8S-ITS rDNA and D1/D2 26S rDNA copies presented 99–100% sequence identity with Zygosaccharomyces sp. NBRC 10669 (LN849119.1) and Z. sapae ABT 301T. Nevertheless, we discard that it could be a Z. sapae strain based on the results obtained in this study. Namely, the amplification with hybrid-specific primer designed in this study, the number of divergent copies of HIS3 (2), the fact that it only possesses one SOD2 gene and the amplification with species-specific primer for Z. pseudorouxii, therefore it could be a new species or a hybrid strain., Ministerio de Economía y Competitividad (MINECO), Universidad Complutense de Madrid/Santander-Hispano, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ailanthus altissima (Mill.) Swingle and Robinia pseudoacacia L. are two aggressive invasive trees in riparian areas in Central Spain. We aim to test whether soil properties, nutrient mineralization rates and soil bacterial communities of riparian forest dominated by the native Populus alba L. can be altered by the presence of A. altissima or R. pseudoacacia. In autumn 2011 and spring 2012 we conducted a field soil sampling in three sites where invasive and native trees were paired. In addition, in a 6-month greenhouse experiment (GHE), we grew A. altissima, R. pseudoacacia and P. alba from seeds in a soil collected from a native area. We quantified soil organic matter (OM), nitrogen (N), phosphorous (P), nitrate (NO3 eN), ammonium (NH4 þeN), pH, potential net ammonification and nitrification rates, phosphomonoesterase (PME) activity and the composition of soil bacterial community in soils from the field study and from the GHE. Both the field and the GHE results showed the capability of A. altissima to decrease soil total N and of R. pseudoacacia to increase soil mineral N. In the field, all invaded soils had greater NO3 eN than P. alba soils. R. pseudoacacia field soils had greater PME activity, total N and net ammonification rates while A. altissima soils had lower OM, NH4 þeN, net nitrification and total N mineralization rates than those of P. alba. Differences in the composition of soil bacterial communities were only found in the field, being more evident between A. altissima and P. alba than between R. pseudoacacia and P. alba field soils. Symbiotic N2 fixation could explain the capability of R. pseudoacacia to increase soil mineral N, while the potential of A. altissima to decrease total soil N may be attributed to changes in the balance between N input and losses from the soil. Although the GHE results indicated that the invasive trees can start changing soil conditions during early stages of establishment, more impacts found in the field study suggests that, Ministerio de Economía y Competitividad (MINECO), Comunidad de Madrid, Junta de Comunidades de Castilla-La Mancha, FCT and the European Social Fund, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

Recent studies have suggested that depression is accompanied by an increased intestinal permeability which would be related to the inflammatory pathophysiology of the disease. This study aimed to evaluate whether experimental depression presents with bacterial translocation that in turn can lead to the TLR-4 in the brain affecting the mitogen-activated protein kinases (MAPK) and antioxidant pathways. Male Wistar rats were exposed to chronic mild stress (CMS) and the intestinal integrity, presence of bacteria in tissues and plasma lipopolysaccharide levels were analyzed. We also studied the expression in the prefrontal cortex of activated forms of MAPK and some of their activation controllers and the effects of CMS on the antioxidant Nrf2 pathway. Our results indicate that after exposure to a CMS protocol there is increased intestinal permeability and bacterial translocation. CMS also increases the expression of the activated form of the MAPK p38 while decreasing the expression of the antioxidant transcription factor Nrf2. The actions of antibiotic administration to prevent bacterial translocation on elements of the MAPK and Nrf2 pathways indicate that the translocated bacteria are playing a role in these effects. In effect, our results propose a role of the translocated bacteria in the pathophysiology of depression through the p38 MAPK pathway which could aggravate the neuroinflammation and the oxidative/nitrosative damage present in this pathology. Moreover, our results reveal that the antioxidant factor Nrf2 and its activators may be involved in the consequences of the CMS on the brain, Instituto de Salud Carlos III, CIBERSAM, Fundación Mutua Madrileña, Depto. de Farmacología y Toxicología, Depto. de Medicina, Fac. de Medicina, TRUE, pub

Since ciliates rarely possess structures that easily fossilize, we are limited in our ability to use paleontological studies to reconstruct the early evolution of this large and ecologically important clade of protists. Tintinnids, a group of loricate (house-forming) planktonic ciliates, are the only group that has a significant fossil record. Putative tintinnid fossils from rocks older than Jurassic, however, possess few to no characters that can be found in extant ciliates; these fossils are best described as ‘incertae sedis eukaryotes’. Here, we review the Devonian fossil Nassacysta reticulata and propose that it is likewise another incertae sedis eukaryote due to the lack of any unambiguous ciliate characters. Future tintinnid fossil descriptions would be most helpful if: (i) neutral terminology is used in the descriptions but ciliate-specific terminology in the interpretations; (ii) the current ciliate classification is used, although fossil data may expand or modify classifications based on modern forms; (iii) close collaboration with specialists studying extant ciliates is done; and (iv) editors include an expert of extant ciliates in the review process., Deutsche Forschungsgemeinschaft, National Science Foundation, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

MAPK pathways are conserved and complex mechanisms of signaling in eukaryotic cells. These pathways mediate adaptation to different stress conditions by a core kinase cascade that perceives changes in the environment by different upstream elements and mediates adaptation through transcription factors. In the present work, the transmembrane protein Opy2 has been identified and functionally characterized in Candida albicans. This protein is required to trigger Cek1 phosphorylation by different stimuli such as the resumption of growth from stationary phase or the addition of the cell wall disturbing compounds zymolyase and tunicamycin. opy2 mutants display susceptibility to cell wall disturbing compounds like Congo red. However, it does not play a role in the adaptation to high osmolarity or oxidative stress, in close contrast with the situation for the homologous protein in Saccharomyces cerevisiae. The over-expression of Opy2 in a S. cerevisiae opy2ssk1 mutant partially complemented the osmosensitivity on solid medium by a Hog1-independent mechanism as well as the abnormal morphology observed in this mutant under high osmolarity. The electrophoretic pattern of CaOpy2 tagged version in S. cerevisiae suggested similar post-translational modification in both microorganisms. This protein is also involved in pathogenesis as revealed by the fact that opy2 mutants displayed a significantly reduced virulence in the Galleria mellonella model., Ministerio de Educación, Formación Profesional y Deportes (España), Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

Background: A common challenge of nail avulsion surgery is the associated bacterial contamination and infection that can manifest. The toe has a difficult anatomy to antiseptically prepare and properly maintain throughout the surgical procedure, lending to this widespread problem. Objective: We conducted a controlled, prospective randomized study to examine the antiseptic efficacy of 3 intraoperative irrigation methods during nail avulsion surgery. Methods: We compared intraoperative antiseptic irrigation using 0.9% saline solution (24 patients), 0.2% nitrofurazone (22 patients), and 0.1% polihexanide (25 patients). Swab samples were taken from each patient at 5 distinct stages throughout the surgical procedure, and bacterial culture analysis was performed (positive culture rate, total inocula count, reduction of bacterial load, and identification of specific micro-organisms). Results: All 3 intraoperative irrigation methods reduced the total bacterial load, but polihexanide was significantly more effective. Furthermore, no patient from the polihexanide group developed postoperative infection. The reduction in bacterial load was lost for all 3 methods after partial nail avulsion surgery, returning to similar values as the initial presurgical bacterial load. An intraoperative irrigation step after partial nail avulsion with saline, nitrofurazone, and polihexanide was effective in reducing the bacterial load by 95.2%, 96.6%, and 99.5%, respectively. Limitations: Our patients underwent phenol-based nail avulsion, resulting in no bacterial load after complete nail removal because of the intrinsic antiseptic nature of the phenol. Conclusions: Intraoperative irrigation with 0.1% polihexanide substantially reduced the bacterial load and subsequent infections, highlighting the importance of an irrigation step in nail avulsion surgery., Depto. de Medicina, Depto. de Enfermería, Fac. de Medicina, Fac. de Enfermería, Fisioterapia y Podología, TRUE, pub

Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis., Universidad Complutense de Madrid, Comunidad de Madrid, Agrovin, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

This study explored tigecycline exposure-bacterial responses in pharmacodynamic simulations (in vitro kinetic model) using different inocula. One meticillin-resistant vancomycin-heteroresistant Staphylococcus aureus, one Enterococcus faecium and one extended-spectrum beta-lactamase-producing Escherichia coli with equal tigecycline minimum inhibitory concentrations/minimum bactericidal concentrations (MICs/MBCs) (0.12/0.25 microg/mL) were used. A computerised pharmacodynamic bicompartmental model simulated three tigecycline twice-daily dosing regimens over 48h: 50mg (100mg loading dose); 100mg; and 150 mg. Areas under bacterial growth curves were calculated, and differences between the growth curve used as control and the killing curve of bacteria exposed to tigecycline (ABBC) were determined. With standard inocula [ca. 1 x 10(6)colony-forming units (CFU)/mL], linear increases in area under the concentration-time curve (AUC)/MIC (25.6 for 50mg, 53.76 for 100mg and 79.52 for 150 mg) produced linear increases in activity against Gram-positive organisms (mean ABBCs of 120.60, 143.20 and 195.80 log CFU x h/mL for S. aureus and of 95.75, 172.55 and 216.90 log CFUxh/mL for E. faecium, respectively), with the activity of the 150 mg regimen being significantly higher (P<0.01) than that of the other two regimens. ABBCs obtained with the 100mg regimen using standard inocula were similar to those obtained with the 150 mg regimen when using high inocula (ca. 1 x 10(7)CFU/mL). Against E. coli, the highest dosing regimen was required to obtain significant antibacterial activity compared with control (mean ABBCs of 145.75 log CFU x h/mL with standard inocula and 63.33 log CFU x h/mL with high inocula). An increase in tigecycline dosing appears to be an interesting therapeutic option to maximise antibacterial activity owing to its linear pharmacokinetics and pharmacodynamics, especially when severe infections with high bacterial load are suspected., Wyeth Farma S.A., Depto. de Medicina, Fac. de Medicina, TRUE, pub

Background: Orthopedic surgical procedures involving the foot and ankle are associated with high rates of infection. The optimal method of preparing the skin and nails for foot and ankle surgery remains unknown. Objective: This study was conducted to compare the efficacy of 4 different methods of skin and nail preparation of the foot using various antiseptic solutions. Methods: In this prospective, randomized study, 4 methods of skin and nail preparation were compared in terms of their efficacy in eliminating bacteria from the hallux nailfold and first web space of the normal foot in 28 healthy adult volunteers. Efficacy was determined by evaluating the difference in the total bacterial load before and after skin preparation. The foot-preparation solutions evaluated were 4% chlorhexidine gluconate, 70% isopropyl alcohol, and 7.5% to 10% povidone-iodine. Results: The addition of alcohol to povidone-iodine was found to increase the efficacy of the preparation method. The nailfold remained contaminated after any of the preoperative skin- and nail-preparation methods studied. Limitations: This study did not measure clinically relevant infections, and the results may not correlate with decreased rates of infection after surgery. Conclusion: Incorporation of alcohol and povidone-iodine into the preoperative skin- and nail-preparation process may help reduce the bacterial load. Every effort should be made to lower the risk of contamination from the nail., Depto. de Enfermería, Depto. de Medicina, Fac. de Enfermería, Fisioterapia y Podología, Fac. de Medicina, TRUE, pub

The in vitro activity of tinidazole against anaerobic periodontal pathogens (25 Prevotella buccae, 18 Prevotella denticola, 10 Prevotella intermedia, 6 Prevotella melaninogenica, 5 Prevotella oralis, 10 Fusobacterium nucleatum and 8 Veillonella spp.) was determined by agar dilution. MIC(90) values (minimum inhibitory concentration for 90% of the organisms) were 8 microg/mL for Veillonella spp., 4 microg/mL for P. intermedia, 2 microg/mL for P. buccae, 1 microg/mL for Fusobacterium spp. and 0.5 microg/mL for other Prevotella spp. Cidal activity was studied by killing curves with tinidazole and amoxicillin (alone and in combination) at concentrations similar to those achieved in crevicular fluid (41.2 microg/mL tinidazole and 14.05 microg/mL amoxicillin) against an inoculum of ca. 10(7)colony-forming units/mL of four bacterial groups, each one composed of four different strains of the following periodontal isolates: Prevotella spp., Fusobacterium spp. and Veillonella spp. (anaerobes) and one amoxicillin-susceptible Streptococcus spp. (facultative) in a proportion of 1:1:1:1. When only beta-lactamase-negative Prevotella or Fusobacterium strains were tested, significantly higher reductions were found with amoxicillin (>4 log reduction at 48 h) versus controls. The presence of beta-lactamase-positive Prevotella spp. or F. nucleatum strains rendered amoxicillin inactive (no reductions at 48 h), with no differences from controls. Amoxicillin+tinidazole produced >3 log reduction at 24h and >4 log reduction at 48 h regardless of the presence or not of beta-lactamase-positive strains. The presence in crevicular fluid of beta-lactamases produced by beta-lactamase-positive periodontal pathogens may have ecological and therapeutic consequences since it may protect beta-lactamase-negative periodontal pathogens from amoxicillin treatment. In vitro, tinidazole offered high antianaerobic activity against beta-lactamase-positive and -negative periodontal pathogens, avoiding amoxicill, Depto. de Medicina, Fac. de Odontología, TRUE, pub

The bacterium Sphingomonas paucimobilis, isolated from cinematographic films in an earlier project, was able to biodeteriorate the cellulose triacetate material (acetylation degree of 2.7). Film colonization was monitored by the indirect impedance technique and the production of carbon dioxide. The presence of ruggedness and irregularities on the surface of the film, produced when the plasticizer was extracted, accelerated the biodeterioration of the material. In contrast, cinematographic films with their layered structure made of photographic gelatine emulsion were protected and no colonization was observed. The cinematographic film without photographic emulsion reached a 5% level of biodeterioration after six weeks of incubation, confirming the possibility of biodeterioration of archival cinematographic materials if conservation conditions are not adequate. Through viscosity measurements, a decrease in relative viscosity was observed on the biodeteriorated sample, with respect to the original material, confirming a lower molecular weight as a result of enzymatic activity of S. paucimobilis. Also, using chemiluminescence, the film surface oxidation on the biodeteriorated sample was observed. This technique was very sensitive in detecting material oxidation by reactive oxygen species generated by bacteria, and could be useful to study microbiological biodeterioration in polymeric materials., Ministerio de Ciencia e Innovación (España), Filmoteca Española, Consejo Superior de Investigaciones Científicas (España), Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Penicillium expansum, a patulin producer fungus, is the most important fungus causing decay in cold stored both apples and pears. This can lead to patulin contaminated by-products. The aim of this assay was to evaluate the phenotypical and physiological variability in the population of P. expansum that cause fruit spoilage in post-harvest stages in Lleida (Spain). In total, 101 isolates of P. expansum from the 2004 and the 2005 seasons were obtained from decayed fruits. Significant differences were found in the observations from both seasons. Variability of the isolates in each season seemed to be partially explained by differences in growth in media, patulin accumulation and resistance to fungicides. Patulin production was detected in almost 100% of the isolates. Variability existing in P. expansum population could not be totally explained, but the above mentioned variables explained up to 74% of the diversity in some cases. The results obtained point to the existence of different populations of P. expansum in each season and may explain the differences in fungicide resistance observed between both seasons. The capacity to colonize apple flesh and some variables involved in fruit colonization were not a source of variation neither in each season nor when both seasons were compared. As storage rooms are cleaned and disinfected each season, this suggests that each season, the populations in storage rooms develop only from strains capable to colonize apple flesh. This may lead to rapid sporulation and spreading of spores., Gobierno de España, Gobierno de Cataluña, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The biodegradation of black and white photographic gelatin emulsion by bacteria and fungi was studied by indirect impedance technology. The presence of silver on the emulsion (black films) inhibited the microbial growth confirming the biocidal effect of the metallic silver. Biodegradation of commercial gelatines, linear and crosslinked (prepared using formaldehyde as crosslinking agent) by Bacillus subtilis was studied. The extent of crosslinking in the gelatin does not have any influence on the biodegradation rate. Through the accumulated amount of CO2 produced with bioassay time and the corresponding percentage of biodegradation reached, the relative biodegradation efficiency of the bacteria and fungi was evaluated. The six studied bacteria (five strains of Bacillus and one Staphylococcus) were effective in their ability to biodegrade the photographic emulsion with different CO2 production rates. Two of them, B. subtilis and B. megaterium showed an exponential rate of biodegradation and reaching 55% of CO2 conversion. Also, all the studied fungi, nine strains, were found to biodegrade the emulsion with Penicillium chrysogenum being the more efficient fungus showing 30% of emulsion biodegradation in 3 weeks., Ministerio de Ciencia e Innovación (España), Filmoteca Española, Consejo Superior de Investigaciones Científicas (España), Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub