LAB Bacteriocins Controlling the Food Isolated (Drug-Resistant) Staphylococci

Staphylococci are a group of microorganisms that can be often found in processed food and theymight pose a risk for human health. In this study we have determined the content of staphylococci in 7 different fresh goat-milk cheeses. These bacteria were present in all of them, ranging from 103 to 106 CFU/g based on growth on selective media. Thus, a set of 97 colonies was randomly picked for phenotypic and genotypic identification. They could be clustered by RAPD-PCR in 10 genotypes, which were assigned by 16S rDNA sequencing to four Staphylococcus species: Staphylococcus aureus, Staphylococcus chromogenes, S. simulans, and S. xylosus. Representative strains of these species (n = 25) were tested for antibiotic sensitivity, and 11 of them were resistant to at least one of the antibiotics tested, including erythromycin, amoxicillin-clavulanic acid and oxacillin. We also tested two bacteriocins produced by lactic acid bacteria (LAB), namely the circular bacteriocin AS-48 and the lantibiotic nisin. These peptides have different mechanism of action at the membrane level. Nevertheless, both were able to inhibit staphylococci growth at low concentrations ranging between 0.16–0.73μM for AS-48 and 0.02–0.23μM for nisin, including the strains that displayed antibiotic resistance. The combined effect of these bacteriocins were tested and the fractional inhibitory concentration index (FICI) was calculated. Remarkably, upon combination, they were active at the low micromolar range with a significant reduction of the minimal inhibitory concentration. Our data confirms synergistic effect, either total or partial, between AS-48 and nisin for the control of staphylococci and including antibiotic resistant strains. Collectively, these results indicate that the combined use of AS-48 and nisin could help controlling (pathogenic) staphylococci in food processing and preventing antibiotic-resistant strains reaching the consumer in the final products.This research was funded by the Spanish Ministry of Economy and Competitiveness (SAF2013-48971-C2-1-R project), including funds from the European Regional Development Fundings (ERDF) and the Research Group General (BIO160, UGR

El análisis de errores como herramienta para la didáctica de traducción literaria

Depto. de Filología Alemana y Filología EslavaFac. de FilologíaFALSEsubmitte

A Multiple Stakeholder Multicriteria Decision Analysis in Diabetic Macular Edema Management: The MULTIDEX‑EMD Study

Background The clinical and economic management of retinal diseases has become more complex following the introduction of new intravitreal treatments. Multicriteria decision analysis (MCDA) offers the potential to overcome the challenges associated with traditional decision-making tools. Objectives A MCDA to determine the most relevant criteria to decision-making in the management of diabetic macular edema (DME) based on the perspectives of multiple stakeholders in Spain was developed. This MCDA was termed the MULTIDEX-EMD study. Methods Nineteen stakeholders (7 physicians, 4 pharmacists, 5 health authorities and health management experts, 1 psychologist, and 2 patient representatives) participated in this three-phase project. In phase A, an advisory board defined all of the criteria that could influence DME treatment decision-making. These criteria were then screened using a discrete choice experiment (DCE) (phase B). Next, a multinomial logit model was fitted by applying the backward elimination algorithm (relevant criteria: p value = 15 letters (p value < 0.001), effect duration per administration (p value = 0.008), retinal detachment (p value < 0.001), endophthalmitis (p value = 0.012), myocardial infarction (p value < 0.001), intravitreal hemorrhage (p value = 0.021), annual treatment cost per patient (p value = 0.001), health-related quality of life (HRQoL) (p value = 0.004), and disability level (p value = 0.021). Conclusions From a multi-stakeholder perspective, the selection of an appropriate treatment for DME patients should guarantee patient safety and maximize the visual acuity improvement and treatment effect duration. It should also contribute to system sustainability by being affordable, it should have a positive impact on HRQoL, and it should prevent disability

Embedding Biomimetic Magnetic Nanoparticles Coupled with Peptide AS-48 into PLGA to Treat Intracellular Pathogens

Among the strategies employed to overcome the development of multidrug-resistant bacteria, directed chemotherapy combined with local therapies (e.g., magnetic hyperthermia) has gained great interest. A nano-assembly coupling the antimicrobial peptide AS-48 to biomimetic magnetic nanoparticles (AS-48-BMNPs) was demonstrated to have potent bactericidal effects on both Gram-positive and Gram-negative bacteria when the antimicrobial activity of the peptide was combined with magnetic hyperthermia. Nevertheless, intracellular pathogens remain challenging due to the difficulty of the drug reaching the bacterium. Thus, improving the cellular uptake of the nanocarrier is crucial for the success of the treatment. In the present study, we demonstrate the embedding cellular uptake of the original nano-assembly into THP-1, reducing the toxicity of AS-48 toward healthy THP-1 cells. We optimized the design of PLGA[AS-48-BMNPs] in terms of size, colloidal stability, and hyperthermia activity (either magnetic or photothermal). The stability of the nano-formulation at physiological pH values was evaluated by studying the AS-48 release at this pH value. The influence of pH and hyperthermia on the AS-48 release from the nano-formulation was also studied. These results show a slower AS-48 release from PLGA[AS-48-BMNPs] compared to previous nano-formulations, which could make this new nano-formulation suitable for longer extended treatments of intracellular pathogens. PLGA[AS-48-BMNPs] are internalized in THP-1 cells where AS-48 is liberated slowly, which may be useful to treat diseases and prevent infection caused by intracellular pathogens. The treatment will be more efficient combined with hyperthermia or photothermia.FEDER Operational ProgramProyectos de I + D + I, del Plan Andaluz de Investigacion, Desarrollo e Innovacion B-BIO-432-UGR20 B-BIO-268-UGR20 B-CTS-216-UGR20 A-FQM-492-UGR20Instituto de Salud Carlos III European Commission P20-00346 P20_00339 P20-00233Spanish Government PI20-01658Federation of European Microbiological Societies (FEMS) EC2019-005930-P PDC2021-121135.100 FEMS-GO-2020-20

Beneficial Shifts in the Gut Bacterial Community of Gilthead Seabream (Sparus aurata) Juveniles Supplemented with Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO)

This study analyzes the potential use of an Allium-derived compound, propyl propane thiosulfonate (PTSO), as a functional feed additive in aquaculture. Gilthead seabream (Sparus aurata) juveniles had their diet supplemented with this Allium-derived compound (150 mg/kg of PTSO) and were compared with control fish. The effects of this organosulfur compound were tested by measuring the body weight and analyzing the gut microbiota after 12 weeks. The relative abundance of potentially pathogenic Vibrio and Pseudomonas in the foregut and hindgut of supplemented fish significantly decreased, while potentially beneficial Lactobacillus increased compared to in the control fish. Shannon’s alpha diversity index significantly increased in both gut regions of fish fed with a PTSO-supplemented diet. Regarding beta diversity, significant differences between treatments only appeared in the hindgut when minority ASVs were taken into account. No differences occurred in body weight during the experiment. These results indicate that supplementing the diet with Allium-derived PTSO produced beneficial changes in the intestinal microbiota while maintaining the productive parameters of gilthead seabream juveniles.AQUAHEALTH project (Operational Programme Smart Growth 2014-2020) IDI-20170032FEDER-INNTERCONECTA-CDTI 2018, Centro para el Desarrollo Industrial, Spanish Ministry of Economy and Competitiveness ITC-20181099Programa Operativo de Empleo Juvenil (Fondo Social Europeo, Junta de Andalucia) 612

Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum

The misuse of antibiotics has led several countries to ban their use as prophylactics against bacterial diseases or as growth promoters in livestock and poultry. Phytobiotics (bioactive compounds extracted from plants) are one of the alternatives, due to their antimicrobial activity and its modulation of the gut microbiota and the improvement of productive properties. Garlic and onion extracts, rich in antimicrobial compounds, are of the most promising alternative to antibiotics. We supplemented a garlic- and onion-based product in the diet to laying hens at the beginning of their productive life. The group supplied with this product produced in one month more eggs and with bigger size. This increase in production was accompanied by changes in the bacterial community of the gut. These changes in the microbiota suggest an improvement in food digestibility, as the most important changes produced by these compounds occur in the most distal parts of the gut. The relative abundance of beneficial Lactococcus in the ileum and Lactobacillus in the cecum increased in the experimental group. Both genera are known to have beneficial effects on host. These results are very promising for the use of these compounds in poultry for short periods.Phytobiotics (bioactive compounds extracted from plants) are one of the explored alternatives to antibiotics in poultry and livestock due to their antimicrobial activity and its positive effects on gut microbiota and productive properties. In this study, we supplemented a product based on garlic and onion compounds in the diet to laying hens at the beginning of their productive life (from 16 to 20 weeks post-hatching). The experimental group showed a significant increase in the number of eggs laid and in their size, produced in one month compared to the control. This increase in production was accompanied by microbiota changes in the ileum and cecum by means of high throughput sequencing analyses. These bacterial shifts in the ileum were mainly the result of compositional changes in the rare biosphere (unweighted UniFrac), while in the cecum, treatment affected both majority and minority bacterial groups (weighted and unweighted UniFrac). These changes in the microbiota suggest an improvement in food digestibility. The relative abundance of Lactococcus in the ileum and Lactobacillus in the cecum increased significantly in the experimental group. The relative abundance of these bacterial genera are known to have positive effects on thSpanish Ministry of Science and Innovation, Consejería de Economía, Innovación, Ciencia y Empleo (Junta de Andalucía)University of Granada-CEI BioTic (Project No. P-BS-37)INTERCONECTA program (Spanish Ministry of Economy and Competitiveness

Egg Production in Poultry Farming Is Improved by Probiotic Bacteria

Antimicrobial resistance (AMR) is one of the most serious threats for human health in the near future. Livestock has played an important role in the appearance of antibioticresistant bacteria, intestinal dysbiosis in farming animals, or the spread of AMR among pathogenic bacteria of human concern. The development of alternatives like probiotics is focused on maintaining or improving production levels while diminishing these negative effects of antibiotics. To this end, we supplied the potential probiotic Enterococcus faecalis UGRA10 in the diet of laying hens at a final concentration of 108 Colony Forming Units per gram (CFU/g) of fodder. Its effects have been analyzed by: (i) investigating the response of the ileum and caecum microbiome; and (ii) analyzing the outcome on eggs production. During the second half of the experimental period (40 to 76 days), hens fed E. faecalis UGRA10 maintained egg production, while control animals dropped egg production. Supplementation diet with E. faecalis UGRA10 significantly increased ileum and caecum bacterial diversity (higher bacterial operational taxonomic unit richness and Faith’s diversity index) of laying hens, with animals fed the same diet showing a higher similarity in microbial composition. These results point out to the beneficial effects of E. faecalis UGRA10 in egg production. Future experiments are necessary to unveil the underlying mechanisms that mediate the positive response of animals to this treatment.This research was funded by the Spanish Ministry of Science and Innovation, Consejería de Economía, Innovación, Ciencia y Empleo (Junta de Andalucía), the University of Granada- CEI BioTic (Project No. P-BS-37), and the INTERCONECTA program (Spanish Ministry of Economy and Competitiveness). JP-S was funded by Junta de Andalucia (Proyectos de Excelencia 2011- RNM-8147)

Preclinical studies of toxicity and safety of the AS-48 bacteriocin

The in vitro antimicrobial potency of the bacteriocin AS-48 is well documented, but its clinical application requires investigation, as its toxicity could be different in in vitro (haemolytic and antibacterial activity in blood and cytotoxicity towards normal human cell lines) and in vivo (e.g. mice and zebrafish embryos) models. Overall, the results obtained are promising. They reveal the negligible propensity of AS-48 to cause cell death or impede cell growth at therapeutic concentrations and support the suitability of this peptide as a potential therapeutic agent against several microbial infections, due to its selectivity and potency at low concentrations. In addition, AS-48 exhibits low haemolytic activity in whole blood and does not induce nitrite accumulation in non-stimulated RAW macrophages, indicating a lack of pro-inflammatory effects. The unexpected heightened sensitivity of zebrafish embryos to AS-48 could be due to the low differentiation state of these cells. The low cytotoxicity of AS-48, the absence of lymphocyte proliferation in vivo after skin sensitization in mice, and the lack of toxicity in a murine model support the consideration of the broad spectrum antimicrobial peptideThis research was funded by the Spanish Ministry of Economy and Competitiveness (SAF2013-48971-C2-1-R, CSD2010-00065, and AGL2015-67995-C3-3-R, all including funds from the European Regional Development Funding, ERDF) and by the Research Groups (BIO160, CTS 944 and CTS 164, UGR) from Junta de Andalucía (Spain). The CIBER-EHD is funded by the Instituto de Salud Carlos III. RM-E is grateful for an FPU Grant (FPU14/01537) from the Ministry of Education, (Spain)

Analysis of the Promoters Involved in Enterocin AS-48 Expression

The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.This work was supported in part by the Ministerio de Ciencia e Innovación project BIO2008-01708, the Plan Propio from the University of Granada (Spain) and by the Research Plan Group (BIO 160)