Integrated selection and identification of bacteria from polluted sites for biodegradation of lipids.

Wastewater treatment plants face major social concern towards removal of problematic pollutants such as fat oils and grease (FOG). In this context, the main objective of the present work was to select natural bacterial isolates from different polluted sites and evaluate them comparatively to isolates from commercial products, for improved bioremediation strategies and bioaugmentation. In total, 196 isolates were analysed for genomic diversity by two PCR-fingerprinting methods and screened for biodegradation potential with pollutants as sole carbon source. The net area under curve (NAUC) was used for preliminary evaluation of growth ability in M9 medium supplemented with oleic acid and triolein. A principal component analysis of all NAUC data showed that natural isolates presented higher overall biodegradation ability and enabled the selection of 11 natural isolates for lipid degradation assays. Selected isolates were identified by 16S rRNA gene sequencing as members of genera with previously described degradative strains, namely, Acinetobacter (1), Aeromonas (2), Bacillus (1), Pseudomonas (1) and Staphylococcus (6). Best biodegradation results in 7-days assay of FOG content removal were 37.9% for oleic acid and 19.1% for triolein by an Aeromonas sp. isolate and a Staphylococcus cohnii isolate, respectively. A respirometry approach confirmed their higher oxygen uptake rates, although longer adaptation phases where required by the Aeromonas sp. isolate. Consequently, these isolates showed great potential for future bioaugmentation products, to promote FOG degradation, for both in situ and ex situ approaches.

Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages.

BACKGROUND: Swine brucellosis caused by B. suis biovar 2 is an emergent disease in domestic pigs in Europe. The emergence of this pathogen has been linked to the increase of extensive pig farms and the high density of infected wild boars (Sus scrofa). In Portugal and Spain, the majority of strains share specific molecular characteristics, which allowed establishing an Iberian clonal lineage. However, several strains isolated from wild boars in the North-East region of Spain are similar to strains isolated in different Central European countries. RESULTS: Comparative analysis of five newly fully sequenced B. suis biovar 2 strains belonging to the main circulating clones in Iberian Peninsula, with publicly available Brucella spp. genomes, revealed that strains from Iberian clonal lineage share 74% similarity with those reference genomes. Besides the 210 kb translocation event present in all biovar 2 strains, an inversion with 944 kb was presented in chromosome I of strains from the Iberian clone. At left and right crossover points, the inversion disrupted a TRAP dicarboxylate transporter, DctM subunit, and an integral membrane protein TerC. The gene dctM is well conserved in Brucella spp. except in strains from the Iberian clonal lineage. Intraspecies comparative analysis also exposed a number of biovar-, haplotype- and strain-specific insertion-deletion (INDELs) events and single nucleotide polymorphisms (SNPs) that could explain differences in virulence and host specificities. Most discriminative mutations were associated to membrane related molecules (29%) and enzymes involved in catabolism processes (20%). Molecular identification of both B. suis biovar 2 clonal lineages could be easily achieved using the target-PCR procedures established in this work for the evaluated INDELs. CONCLUSION: Whole-genome analyses supports that the B. suis biovar 2 Iberian clonal lineage evolved from the Central-European lineage and suggests that the genomic specialization of this pathogen in the Iberian Peninsula is independent of a specific genomic event(s), but instead driven by allopatric speciation, resulting in the establishment of a new ecovar.

Beyond Harmful: Exploring Biofilm Formation by Enterococci Isolated from Portuguese Traditional Cheeses.

This study investigated the biofilm-forming capabilities of Enterococcus isolates from Portuguese traditional cheeses with protected designation of origin (PDO) status, specifically Azeitão and Nisa. Given the absence of added starter cultures in the cheesemaking process, the characteristics of these cheeses are intrinsically linked to the autochthonous microbiota present in the raw materials and the production environment. Our findings demonstrate that all isolates possess biofilm production abilities, which are crucial for their colonization and persistence within cheese factories, thereby maintaining factory-specific microbial heritage. Through an integrated analysis utilizing principal component analysis (PCA), a direct correlation between biofilm formation and cell viability was established. Notably, these results underscore the adaptive capacity of enterococci to survive environmental fluctuations and their role in the unique characteristics of Portuguese traditional cheeses. Overall, this research enhances our understanding of the microbial dynamics in cheese production and highlights the importance of enterococci in preserving cheese quality and heritage.

Photoprotective bioactivity present in a unique marine bacteria collection from Portuguese deep sea hydrothermal vents.

Interesting biological activities have been found for numerous marine compounds. In fact, screening of phylogenetically diverse marine microorganisms from extreme environments revealed to be a rational approach for the discovery of novel molecules with relevant bioactivities for industries such as pharmaceutical and cosmeceutical. Nevertheless, marine sources deliverables are still far from the expectations and new extreme sources of microbes should be explored. In this work, a marine prokaryotic collection from four Mid-Atlantic Ridge (MAR) deep sea hydrothermal vents near the Azores Islands, Portugal, was created, characterized and tested for its photoprotective capacity. Within 246 isolates, a polyphasic approach, using chemotaxonomic and molecular typing methods, identified 23-related clusters of phenetically similar isolates with high indexes of diversity. Interestingly, 16S rRNA gene sequencing suggested the presence of 43% new prokaryotic species. A sub-set of 139 isolates of the prokaryotic collection was selected for biotechnological exploitation with 484 bacterial extracts prepared in a sustainable upscalling manner. 22% of the extracts showed an industrially relevant photoprotective activity, with two extracts, belonging to new strains of the species Shewanella algae and Vibrio fluvialis, uniquely showing UV-A, UV-B and UV-C protective capacity. This clearly demonstrates the high potential of the bacteria MAR vents collection in natural product synthesis with market applications.

Biocontrol potential of wine yeasts against four grape phytopathogenic fungi disclosed by time-course monitoring of inhibitory activities.

Grapes' infection by phytopathogenic fungi may often lead to rot and impair the quality and safety of the final product. Due to the concerns associated with the extensive use of chemicals to control these fungi, including their toxicity for environment and human health, bio-based products are being highly preferred, as eco-friendlier and safer alternatives. Specifically, yeasts have shown to possess antagonistic activity against fungi, being promising for the formulation of new biocontrol products.In this work 397 wine yeasts, isolated from Portuguese wine regions, were studied for their biocontrol potential against common grapes phytopathogenic fungal genera: Aspergillus, Botrytis, Mucor and Penicillium. This set comprised strains affiliated to 32 species distributed among 20 genera. Time-course monitoring of mold growth was performed to assess the inhibitory activity resulting from either diffusible or volatile compounds produced by each yeast strain. All yeasts displayed antagonistic activity against at least one of the mold targets. Mucor was the most affected being strongly inhibited by 68% of the tested strains, followed by Botrytis (20%), Aspergillus (19%) and Penicillium (7%). More notably, the approach used allowed the detection of a wide array of yeast-induced mold response profiles encompassing, besides the decrease of mold growth, the inhibition or delay of spore germination and the complete arrest of mycelial extension, and even its stimulation at different phases. Each factor considered (taxonomic affiliation, mode of action and fungal target) as well as their interactions significantly affected the antagonistic activity of the yeast isolates. The highest inhibitions were mediated by volatile compounds. Total inhibition of Penicillium was achieved by a strain of Metschnikowia pulcherrima, while the best performing yeasts against Mucor, Aspergillus and Botrytis, belong to Lachancea thermotolerans, Hanseniaspora uvarum and Starmerella bacillaris, respectively. Notwithstanding the wide diversity of yeasts tested, only three strains were found to possess a broad spectrum of antagonistic activity, displaying strong or very strong inhibition against the four fungal targets tested. Our results confirm the potential of wine yeasts as biocontrol agents, while highlighting the need for the establishment of fit-for-purpose selection programs depending on the mold target, the timing, and the mode of application.

Waterborne transmission of Giardia and Cryptosporidium at river beaches in Southern Europe (Portugal).

Giardia and Cryptosporidium are the most frequent enteric protozoa causing gastroenteritis in humans worldwide. Intense recreational activity at Portuguese river beaches triggered the opportunity for a 2-year seasonal survey of 19 large river basin beaches. A total of 74 samples were collected and processed according to USEPA Method 1623 to detect Cryptosporidium and Giardia (oo)cysts. Faecal indicators (thermotolerant/total coliforms, Escherichia coli, and enterococci) and physicochemical parameters were also analysed according to the EU Bath Water Directive (BWD). Results pointed to a widespread presence of these protozoa at Portuguese river beaches. The percentage of samples testing positive for Giardia and Cryptosporidium were 85 and 82% respectively, with no significant differences between wet and dry seasons (p > 0.05). Although Portuguese river beaches present a very low exposure risk for infection with Giardia and Cryptosporidium (under 10(-3)), a few particular cases revealed values over 0.2%, and were related to stormy wet events. The correlation between levels of Giardia and thermotolerant coliforms, E. coli and enterococci, was high (r ≥ 0.87, p < 0.001), suggesting the need to carry out specific procedures for the detection of Giardia and Cryptosporidium whenever the values of those faecal indicators approach the maximum allowed level of the EU BWD.

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions.

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines. The aim of this work was the transcriptional characterization of six O. oeni strains, four of them selected from distinct winemaking regions of Portugal, as candidates to malolactic starters, and two commercial malolactic starters. Using crossed assays with wines from different Portuguese winemaking regions, strain characteristic transcriptional patterns induced by each wine were analyzed based on Random Arbitrarily Primed PCR (RAP-PCR). The obtained results suggest that the starter strains showed more constrained and limited transcription profiles, whereas a high variation on the distribution of the transcription profiles was observed for the regional strains in each wine. According with our results, RAP-PCR is a useful technique for a preliminary investigation of strain behavior under different wine environmental conditions, which can be applied in field studies to monitor differential patterns of global gene expression and to select markers for the surveillance of malolactic starters performance in winemaking, as well as for quality and safety control.

Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices.

Mycobacterium bovis causes tuberculosis (TB) at the human-wildlife-livestock interface. Environmental persistence of M. bovis excreted by infected hosts may cause indirect transmission to other animals. However, methodological constrains hamper assessment of M. bovis viability and molecular signature in environmental matrices. In this work, an innovative, modular, and highly efficient single-cell workflow combining flow cytometry (FLOW), fluorescence in situ hybridization (FISH), and fluorescence-activated cell sorting (FACS) was developed, allowing detection, quantification, and sorting of viable and dormant M. bovis cells from environmental matrices. Validation with spiked water and sediments showed high efficiency (90%) of cell recovery, with high linearity between expected and observed results, both in cell viability evaluation (r2 =0.93) and FISH-labelled M. bovis cells quantification (r2 ≥0.96). The limit of detection was established at 105 cells/g of soil in the cell viability step and 102 cells/g of soil in the taxonomical labelling stage. Moreover, FACS efficiency attained noteworthy recovery yield (50%) and purity (60% viable cells; 70% taxonomically labelled M. bovis). This new methodology represents a huge step for M. bovis assessment outside the mammal host, offering the rapid quantification of M. bovis cell load and cell viability, including viable but non-culturable cells, and further downstream cell analyses after FACS. Subsequent environmental data integration with the clinical component will expand knowledge on transmission routes, promising new paths in TB research and an intervention tool to mitigate the underlying biohazard.

Genomic diversity of Oenococcus oeni from different winemaking regions of Portugal.

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium that plays an important role in the elaboration of wine. It is often added as a starter culture to carry out malolactic conversion. Given the economic importance of this reaction, the taxonomic structure of this species has been studied in detail. In the present work, phenotypic and molecular approaches were used to identify 121 lactic acid bacteria strains isolated from the wines of three winemaking regions of Portugal. The strains were differentiated at the genomic level by M13-PCR fingerprinting. Twenty-seven genomic clusters represented by two or more isolates and 21 single-member clusters, based on an 85% similarity level, were recognized by hierarchic numerical analysis. M13-PCR fingerprinting patterns revealed a high level of intraspecific genomic diversity in O. oeni. Moreover, this diversity could be partitioned according to the geographical origin of the isolates. Thus, M13-PCR fingerprint analysis may be an appropriate methodology to study the O. oeni ecology of wine during malolactic fermentation as well as to trace new malolactic starter cultures and evaluate their dominance over the native microbiota.

Molecular assessment of microbiota structure and dynamics along mixed olive oil and winery wastewaters biotreatment.

The major parcel of the degradation occurring along wastewater biotreatments is performed either by the native microbiota or by added microbial inocula. The main aim of this study was to apply two fingerprinting methods, temperature gradient gel electrophoresis (TGGE) and length heterogeneity-PCR (LH-PCR) analysis of 16S rRNA gene fragments, in order to assess the microbiota structure and dynamics during mixed olive oil and winery wastewaters aerobic biotreatment performed in a jet-loop reactor (JLR). Sequence homology analysis showed the presence of bacterial genera Gluconacetobacter, Klebsiella, Lactobacillus, Novosphingobium, Pseudomonas, Prevotella, Ralstonia, Sphingobium and Sphingomonas affiliated with five main phylogenetic groups: alpha-, beta- and gamma-Proteobacteria, Firmicutes and Bacteroidetes. LH-PCR analysis distinguished eight predominant DNA fragments correlated with the samples showing highest performance (COD removal rates of 67 up to 75%). Cluster analysis of both TGGE and LH-PCR fingerprinting profiles established five main clusters, with similarity coefficients higher than 79% (TGGE) and 62% (LH-PCR), and related with hydraulic retention time, indicating that this was the main factor responsible for the shifts in the microbiota structure. Canonical correspondence analysis revealed that changes observed on temperature and O(2) level were also responsible for shifts in microbiota composition. Community level metabolic profile analysis was used to test metabolic activities in samples. Integrated data revealed that the microbiota structure corresponds to bacterial groups with high degradative potential and good suitability for this type of effluents biotreatments.

Biosynthesis of crystalline silver and gold nanoparticles by extremophilic yeasts.

The biosynthesis of Ag and Au nanoparticles (NPs) was investigated using an extremophilic yeast strain isolated from acid mine drainage in Portugal. Three distinct studies were performed, namely, the growth of yeast strain in presence of metal ions, the use of yeast biomass for the metal nanoparticles synthesis, and of the supernatant obtained after 24-hour incubation of yeast biomass in water. The extremophilic strain under study was able to grow up to an Ag ion concentration of 1.5 mM whereas an increase of Au ion concentration over 0.09 mM caused a strong inhibitory effect. A successful route for the metal NPs synthesis was obtained using the yeast biomass. When the washed yeast cells were in contact with Ag or Au solutions, AgNPs smaller than 20 nm were produced, as for the AuNPs diameter ranged from 30 to 100 nm, as determined through transmission electron microscopy and confirmed by energy-dispersive X-ray spectra. The supernatant-based strategy provided evidence that proteins were released to the medium by the yeasts, which could be responsible for the formation and stabilisation of the Ag NPs, although the involvement of the cell wall seems fundamental for AuNPs synthesis.

Microbial Characterization of Yellow Curing Process of Codfish.

Yellow cured codfish has a typical yellow colour, distinctive taste, and low salt content due to its special curing process of the raw salted codfish involving several soaks in water of the raw salted codfish, alternated with drying steps. The purpose of this study was to assess the main functional groups of bacteria involved in this process and relate them with physicochemical properties of the product. A total of 28 codfish from Iceland were supplied by two local companies. Seven stages of the curing process were analyzed. From each of these seven stages, four fish samples were collected to carry out the microbial and physicochemical analyses (moisture, salt content, pH, total volatile basic nitrogen (TVB-N), and trimethylamine nitrogen (TMA-N)). Bacteria counts were performed using the MPN method and adequate culture media for aerobic, proteolytic, sulphite-reducing, biogenic amine, and trimethylamine-producing and ammonifying bacteria. Strains isolated from the highest dilutions with microbial growth were used to characterize the predominant bacteria. The results showed that total aerobic counts increased from 3.9 log MPN/g in raw salted codfish to 5.9 log MPN/g in the final. Proteolytic, ammonifying, and trimethylamine bacteria producers also increased to 8, 7.5, and 6.5 log MPN/g, respectively. The salt content decreases (from 17% until 8%) and moisture increases (53% until 67%) during the salted-raw-codfish soaking, favoring sulphite-reducing and biogenic amine-producing species, confirming that desalting enhances potential spoilers. The subsequent drying step benefits proteolytic, ammonifying, and trimethylamine-producing bacteria, with a corresponding non-protein-nitrogen content (TVB-N and TMA-N) increase. The dominant bacteria during yellow curing belong to the genera Staphylococcus, Psychrobacter, Pseudomonas, and Alcaligenes with a clear positive correlation between the content of Staphylococcus and Psychrobacter and TVB-N and TMA-N concentration. Staphylococcus spp. are the dominant bacteria in the steps where the product has a higher salt concentration; thus, it could be particularly useful as an indicator to control the industrially yellow curing process and could have an important role in the development of the final characteristics of this product.

Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System.

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife-livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with apparent geographic specificities, as well as the establishment of an SNP catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife-livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.

Modulation of the Wheat Seed-Borne Bacterial Community by Herbaspirillum seropedicae RAM10 and Its Potential Effects for Tryptophan Metabolism in the Root Endosphere.

Plants and their associated microbiota share ecological and evolutionary traits that are considered to be inseparably woven. Their coexistence foresees the use of similar metabolic pathways, leading to the generation of molecules that can cross-regulate each other's metabolism and ultimately influence plant phenotype. However, the extent to which the microbiota contributes to the overall plant metabolic landscape remains largely unexplored. Due to their early presence in the seed, seed-borne endophytic bacteria can intimately colonize the plant's endosphere while conferring a series of phytobeneficial services to their host. Understanding the dynamics of these endophytic communities is a crucial step toward the formulation of microbial inoculants that can modulate the functionality of the plant-associated microbiota for improved plant fitness. In this work, wheat (Triticum aestivum) roots non-inoculated and inoculated with the bacterium Herbaspirillum seropedicae strain RAM10 were analyzed to explore the impact of inoculant-endophyte-wheat interrelationships on the regulation of tryptophan (Trp) metabolism in the endosphere environment. Root inoculation with H. seropedicae led to phylum-specific changes in the cultivable seed-borne endophytic community. This modulation shifted the metabolic potential of the community in light of its capacity to modulate the levels of key Trp-related metabolites involved in both indole-3-acetic acid (IAA) biosynthesis and in the kynurenine pathway. Our results support a mode of action of H. seropedicae relying on a shift in both the composition and functionality of the seed-borne endophytic community, which may govern important processes such as root growth. We finally provide a conceptual framework illustrating that interactions among roots, inoculants, and seed-borne endophytes are critical to fine-tuning the levels of IAA in the endosphere. Understanding the outcomes of these interactions is a crucial step toward the formulation of microbial inoculants based on their joint action with seed-borne endophytic communities to promote crop growth and health in a sustainable manner.

New Insights on Streptococcus dysgalactiae subsp. dysgalactiae Isolates.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been considered a strict animal pathogen. Nevertheless, the recent reports of human infections suggest a niche expansion for this subspecies, which may be a consequence of the virulence gene acquisition that increases its pathogenicity. Previous studies reported the presence of virulence genes of Streptococcus pyogenes phages among bovine SDSD (collected in 2002-2003); however, the identity of these mobile genetic elements remains to be clarified. Thus, this study aimed to characterize the SDSD isolates collected in 2011-2013 and compare them with SDSD isolates collected in 2002-2003 and pyogenic streptococcus genomes available at the National Center for Biotechnology Information (NCBI) database, including human SDSD and S. dysgalactiae subsp. equisimilis (SDSE) strains to track temporal shifts on bovine SDSD genotypes. The very close genetic relationships between humans SDSD and SDSE were evident from the analysis of housekeeping genes, while bovine SDSD isolates seem more divergent. The results showed that all bovine SDSD harbor Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas IIA system. The widespread presence of this system among bovine SDSD isolates, high conservation of repeat sequences, and the polymorphism observed in spacer can be considered indicators of the system activity. Overall, comparative analysis shows that bovine SDSD isolates carry speK, speC, speL, speM, spd1, and sdn virulence genes of S. pyogenes prophages. Our data suggest that these genes are maintained over time and seem to be exclusively a property of bovine SDSD strains. Although the bovine SDSD genomes characterized in the present study were not sequenced, the data set, including the high homology of superantigens (SAgs) genes between bovine SDSD and S. pyogenes strains, may indicate that events of horizontal genetic transfer occurred before habitat separation. All bovine SDSD isolates were negative for genes of operon encoding streptolysin S, except for sagA gene, while the presence of this operon was detected in all SDSE and human SDSD strains. The data set of this study suggests that the separation between the subspecies "dysgalactiae" and "equisimilis" should be reconsidered. However, a study including the most comprehensive collection of strains from different environments would be required for definitive conclusions regarding the two taxa.

Genome diversity in the genera Fructobacillus, Leuconostoc and Weissella determined by physical and genetic mapping.

Pulsed-field gel electrophoresis analysis of chromosomal single and double restriction profiles of 17 strains belonging to three genera of 'Leuconostocaceae' was done, resulting in physical and genetic maps for three Fructobacillus, six Leuconostoc and four Weissella strains. AscI, I-CeuI, NotI and SfiI restriction enzymes were used together with Southern hybridization of selected probes to provide an assessment of genomic organization in different species. Estimated genome sizes varied from 1408 kb to 1547 kb in Fructobacillus, from 1644 kb to 2133 kb in Leuconostoc and from 1371 kb to 2197 kb in Weissella. Other genomic characteristics of interest were analysed, such as oriC and terC localization and rrn operon organization. The latter seems markedly different in Weissella, in both number and disposition in the chromosome. Comparisons of intra- and intergeneric features are discussed in the light of chromosome rearrangements and genomic evolution.

Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samples.

The aim of this study was to develop a PCR-based method of gene-directed multiplex PCR to rapidly identify microcystins producing cyanobacteria, regardless of their taxa, that could be applied in routine freshwater monitoring. Instead of using the amplification of only one or two mcy gene fragments, a multiplex PCR that simultaneously amplifies mcyA-cd, mcyAB, and mcyB fragments of the microcystin gene cluster was validated with DNA from 124 cyanobacterial isolates and applied in 37 environmental samples. The toxicological status of the isolates was assessed by high-performance liquid chromatography also used as the "gold standard" for the evaluation of multiplex mcy genes-based PCR, where a sensitivity of 92.3% and a specificity of 100% have been obtained. For the environmental samples, a rapid protocol for their direct use in the PCR reaction has been developed and, by using ELISA results as "gold standard" for the presence of microcystins in these samples, a sensitivity of 80% and a specificity of 100% were achieved, showing that this multiplex PCR test is a rapid, reliable, and economical way of assessing the microcystin-producing potential of cyanobacteria in freshwaters, regardless of their taxa or microcystins variant produced.

An Optimized in situ Quantification Method of Leaf H2O2 Unveils Interaction Dynamics of Pathogenic and Beneficial Bacteria in Wheat.

Hydrogen peroxide (H2O2) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H2O2 accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for in situ quantification of relative H2O2 concentrations in wheat leaves infected with the pathogenic bacterium Pseudomonas syringae pv. atrofaciens (Psa), either alone or in the presence of the beneficial bacterium Herbaspirillum seropedicae (RAM10). This protocol involved the use of 3-3'diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H2O2. Decreasing H2O2 accumulation patterns were detected at increasing distances from the site of pathogen infection, and H2O2 concentrations were different depending on the bacterial combinations tested. Notably, Psa-challenged plants in presence of RAM10 accumulated less H2O2 in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H2O2 levels in young wheat plants.

Long-term molecular surveillance provides clues on a cattle origin for Mycobacterium bovis in Portugal.

Animal tuberculosis (TB), caused by Mycobacterium bovis, is maintained in Portugal in a multi-host system, with cattle, red deer and wild boar, playing a central role. However, the ecological processes driving transmission are not understood. The main aim of this study was thus to contribute to the reconstruction of the spatiotemporal history of animal TB and to refine knowledge on M. bovis population structure in order to inform novel intervention strategies. A collection of 948 M. bovis isolates obtained during long-term surveillance (2002-2016, 15 years) of cattle (n = 384), red deer (n = 303) and wild boar (n = 261), from the main TB hotspot areas, was characterized by spoligotyping and 8 to 12-loci MIRU-VNTR. Spoligotyping identified 64 profiles and MIRU-VNTR distinguished 2 to 36 subtypes within each spoligotype, enabling differentiation of mixed or clonal populations. Common genotypic profiles within and among livestock and wildlife in the same spatiotemporal context highlighted epidemiological links across hosts and regions, as for example the SB0119-M205 genotype shared by cattle in Beja district or SB0121-M34 shared by the three hosts in Castelo Branco and Beja districts. These genomic data, together with metadata, were integrated in a Bayesian inference framework, identifying five ancestral M. bovis populations. The phylogeographic segregation of M. bovis in specific areas of Portugal where the disease persists locally is postulated. Concurrently, robust statistics indicates an association of the most probable ancient population with cattle and Beja, providing a clue on the origin of animal TB epidemics. This relationship was further confirmed through a multinomial probability model that assessed the influence of host species on spatiotemporal clustering. Two significant clusters were identified, one that persisted between 2004 and 2010, in Beja district, with Barrancos county at the centre, overlapping the central TB core area of the Iberian Peninsula, and highlighting a significant higher risk associated to cattle. The second cluster was predominant in the 2012-2016 period, holding the county Rosmaninhal at the centre, in Castelo Branco district, for which wild boar contributed the most in relative risk. These results provide novel quantitative insights beyond empirical perceptions, that may inform adaptive TB control choices in different regions.

Occurrence of putative pathogenicity islands in enterococci from distinct species and of differing origins.

Enterococci isolated from ewe's milk and cheese, clinical isolates of human and veterinary origins, and reference strains obtained from culture collections were screened for the occurrence of putative pathogenicity island (PAIs). Results obtained after PCR amplification and hybridization point toward PAI dissemination among enterococci of diverse origins (food/clinical) and species (Enterococcus faecalis/non-E. faecalis).