Use of Limestone Sludge in the Preparation of É©-Carrageenan/Alginate-Based Films.

The use of processed limestone sludge as a crosslinking agent for films based on Na-alginate and É©-carrageenan/Na-alginate blends was studied. Sorbitol was tested as a plasticizer. The produced gel formulations included alginate/sorbitol and carrageenan/alginate/sorbitol mixtures, with tested sorbitol concentrations of 0.0, 0.5 and 1.0 wt%. The limestone sludge waste obtained from the processing of quarried limestone was converted into an aqueous solution of Ca2+ by dissolution with mineral acid. This solution was then diluted in water and used to induce gel crosslinking. The necessity of using sorbitol as a component of the crosslinking solution was also assessed. The resulting films were characterized regarding their dimensional stability, microstructure, chemical structure, mechanical performance and antifungal properties. Alginate/sorbitol films displayed poor dimensional stability and were deemed not viable. Carrageenan/alginate/sorbitol films exhibited higher dimensional stability and smooth and flat surfaces, especially in compositions with 0.5 wt% sorbitol. However, an increasing amount of plasticizer appears to result in severe surface cracking, the development of a segregation phenomenon affecting carrageenan and an overall decrease in films' mechanical resistance. Although further studies regarding film composition-including plasticizer fraction, film optimal thickness and film/mold material interaction-are mandatory, the attained results show the potential of the reported É©-carrageenan/alginate/sorbitol films to be used towards the development of viable films derived from algal polysaccharides.

Nutritive Value and Bioactivities of a Halophyte Edible Plant: Crithmum maritimum L. (Sea Fennel).

Crithmum maritimum L. (sea fennel), an edible xerophyte of coastal habitats, is considered an emerging cash crop for biosaline agriculture due to its salt-tolerance ability and potential applications in the agri-food sector. Here, the nutritional value and bioactive properties of sea fennel are described. Sea fennel leaves, flowers, and schizocarps are composed of carbohydrates (>65%) followed by ash, proteins, and lipids. Sea fennel's salty, succulent leaves are a source of omega-6 and omega-3 polyunsaturated fatty acids, especially linoleic acid. Extracts obtained from flowers and fruits/schizocarps are rich in antioxidants and polyphenols and show antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermis, Candida albicans, and Candida parapsilosis. Plant material is particularly rich in sodium (Na) but also in other nutritionally relevant minerals, such as calcium (Ca), chlorine (Cl), potassium (K), phosphorus (P), and sulfur (S), beyond presenting a potential prebiotic effect on Lactobacillus bulgaricus and being nontoxic to human intestinal epithelial Caco-2 model cells, up to 1.0% (w/v). Hence, the rational use of sea fennel can bring nutrients, aroma, and flavor to culinary dishes while balancing microbiomes and contributing to expanding the shelf life of food products.

Optimized Design of Degenerate Primers for PCR Based on DNA or Protein Sequence Comparisons.

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in three different applications, including the use of specific determinants at the 3'-end, to (i) improve PCR efficiency with related sequences for members of a protein family by complete degeneration at a core box of conserved genetic information at the 3'-end with the reduction of degeneration at the 5'-end, (ii) optimize specificity of allelic discrimination of closely related DNA sequences of orthologous by 5'-end fully degenerate primers, and (iii) increase the PCR efficiency of primers by targeting DNA sequences belonging to specific phylogenetic groups, within a large and diverse gene family, allowing the use of multiplex/degenerate PCR.

Marine endophytic fungi associated with Halopteris scoparia (Linnaeus) Sauvageau as producers of bioactive secondary metabolites with potential dermocosmetic application.

Marine fungi and, particularly, endophytic species have been recognised as one of the most prolific sources of structurally new and diverse bioactive secondary metabolites with multiple biotechnological applications. Despite the increasing number of bioprospecting studies, very few have already evaluated the cosmeceutical potential of marine fungal compounds. Thus, this study focused on a frequent seaweed in the Portuguese coast, Halopteris scoparia, to identify the endophytic marine fungi associated with this host, and assess their ability to biosynthesise secondary metabolites with antioxidative, enzymatic inhibitory (hyaluronidase, collagenase, elastase and tyrosinase), anti-inflammatory, photoprotective, and antimicrobial (Cutibacterium acnes, Staphylococcus epidermidis and Malassezia furfur) activities. The results revealed eight fungal taxa included in the Ascomycota, and in the most representative taxonomic classes in marine ecosystems (Eurotiomycetes, Sordariomycetes and Dothideomycetes). These fungi were reported for the first time in Portugal and in association with H. scoparia, as far as it is known. The screening analyses showed that most of these endophytic fungi were producers of compounds with relevant biological activities, though those biosynthesised by Penicillium sect. Exilicaulis and Aspergillus chevalieri proved to be the most promising ones for being further exploited by dermocosmetic industry. The chemical analysis of the crude extract from an isolate of A. chevalieri revealed the presence of two bioactive compounds, echinulin and neoechinulin A, which might explain the high antioxidant and UV photoprotective capacities exhibited by the extract. These noteworthy results emphasised the importance of screening the secondary metabolites produced by these marine endophytic fungal strains for other potential bioactivities, and the relevance of investing more efforts in understanding the ecology of halo/osmotolerant fungi.

Corema album spp: Edible wild crowberries with a high content in minerals and organic acids.

Edible wild plants are part of the ethnobotanical and gastronomic heritage of different geographical areas. Corema album (L.) D. Don is an endemic species of the dune systems of the Atlantic coast of the Iberian Peninsula. The aerial parts of Corema album are a source of nutrients and antioxidants. The Corema album white berry (Portuguese crowberry) is rich in calcium, iron, and zinc. The plant also shows high phenolic content and antioxidant capacity associated with the leaves, fruit, and flowers. The presence of organic acids, namely phenolic acids, such as hydroxycinnamic acids, and long chain polyunsaturated fatty acids (PUFAs) omega-3 and omega-6 has also been confirmed. Toxicity studies evaluated by cell viability tests with human intestinal epithelium model cells (Caco-2) have shown that, at low concentrations, plant extracts may present beneficial effects.

Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015.

Colistin resistance genes mcr-3 and mcr-1 have been detected in an Escherichia coli isolate from cattle faeces in a Spanish slaughterhouse in 2015. The sequences of both genes hybridised to same plasmid band of ca 250 kb, although colistin resistance was non-mobilisable. The isolate was producing extended-spectrum beta-lactamases and belonged to serotype O9:H10 and sequence type ST533. Here we report an mcr-3 gene detected in Europe following earlier reports from Asia and the United States.

Strategies to Improve Efficiency and Specificity of Degenerate Primers in PCR.

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in two different applications, including the use of specific determinants at the 3'-end, to: (1) improve PCR efficiency with coding sequences for members of a protein family by fully degeneration at a core box of conserved genetic information, with the reduction of degeneration at the 5'-end, and (2) optimize specificity of allelic discrimination of closely related orthologous by 5'-end degenerate primers.

Prevalence of quinolone resistance determinants in non-typhoidal Salmonella isolates from human origin in Extremadura, Spain.

Resistance to the quinolones nalidixic acid (NAL) and ciprofloxacin (CIP) and the occurrence of quinolone resistance determinants have been investigated in 300 non-typhoidal Salmonella from human origin, isolated in the years between 2004 and 2008, in 6 hospitals within Extremadura (Spain). Salmonella Enteritidis was the major serotype found among quinolone-resistant isolates, most of which were clustered by clonal analysis to a single clone, which presented D87 or S83 substitutions in GyrA. Eleven isolates presented the non-classical quinolone resistance phenotype (resistance to CIP and susceptibility to NAL), lacking mutations in the quinolone resistance determinant region of topoisomerase genes. Among them, one Salmonella Typhimurium isolate carried a qnrS1 gene in a low-molecular-weight plasmid, pQnrS1-HLR25, identical to plasmids previously found in the UK, Taiwan, and USA. The occurrence of this genetic element could represent a risk for the horizontal transmission of quinolone resistance among Enterobacteriaceae in the Iberian Peninsula.

Co-occurrence of ACSSuT and cephalosporin resistance phenotypes is mediated by int1-associated elements in nontyphoidal Salmonella enterica from human infections in Spain.

A screening of antimicrobial resistance and its genetic determinants has been performed on 300 Salmonella enterica isolates collected during 2004-2008 from human infections in Spain. Salmonella Typhimurium and Salmonella Enteritidis were the major serotypes, which were found with similar frequencies covering 80% of the bacterial collection. Salmonella Typhimurium isolates frequently shared low susceptibility to antimicrobials of the penta-resistance phenotype (ACSSuT) and/or cephalosporin resistance. The ACSSuT profile was found closely linked to int1-associated gene cassettes, with major elements carrying DNA fragments of 1.0 Kb (aadA2 gene) plus 1.2 Kb (blaPSE-1 gene) or 2.0 Kb (aadA1 and blaOXA-1 genes). Among these, ACSSuT and cephalosporin resistances were associated in Salmonella Typhimurium isolates expressing the blaOXA gene. ß-lactamase activities were also detected from isolates carrying blaTEM, blaCMY, or blaSHV, although only the two last genes expressed extended-spectrum ß-lactamases. The clonal analysis of S. enterica strains suggests that both horizontal and vertical transfer mechanisms are involved in the wide dissemination of their antimicrobial resistance.

Dissemination of antimicrobial-resistant clones of Salmonella enterica among domestic animals, wild animals, and humans.

Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. This work focuses on the identification of Salmonella enterica clonal strains which, presenting a wide distribution potential, express resistance determinants that compromise effectiveness of the antimicrobial therapy. The screening was performed on 506 Salmonella enterica isolates from animals and humans, which were characterized by serovar and phage typing, genome macrorestriction and pulsed-field gel electrophoresis, and detection of phenotypic and genotypic traits for antimicrobial resistance. A Salmonella Enteritidis strain with strong quinolone resistance is spread on three host environments carrying one of the four variants found for the GyrA protein: (1) Asp87Tyr, the major polymorphism found in 39 Salmonella isolates from human origin and six from poultry; (2) Ser83Phe, with four isolates from human origin and one from white stork (Ciconia ciconia); and (3) Asp87Asn or (4) Asp87Gly, with two isolates each from human origins. Several Salmonella Typhimurium strains that presented int1 elements and the classically associated pentaresistance (ACSSuT) phenotype were found distributed between two host environments: domestic animals and humans, domestics and wild animals, or wild fauna plus humans. This study points out the importance of monitoring gut microbiota and its antimicrobial resistance from wildlife, in parallel to livestock animals and humans, especially for animal species that are in close contact with people.