Draft Genome Sequence of Lactococcus lactis Subsp. cremoris WA2-67: A Promising Nisin-Producing Probiotic Strain Isolated from the Rearing Environment of a Spanish Rainbow Trout (Oncorhynchus mykiss, Walbaum) Farm

Probiotics are a viable alternative to traditional chemotherapy agents to control infectious diseases in aquaculture. In this regard, Lactococcus lactis subsp. cremoris WA2-67 has previously demonstrated several probiotic features, such as a strong antimicrobial activity against ichthyopathogens, survival in freshwater, resistance to fish bile and low pH, and hydrophobicity. The aim of this manuscript is an in silico analysis of the whole-genome sequence (WGS) of this strain to gain deeper insights into its probiotic properties and their genetic basis. Genomic DNA was purified, and libraries prepared for Illumina sequencing. After trimming and assembly, resulting contigs were subjected to bioinformatic analyses. The draft genome of L. cremoris WA2-67 consists of 30 contigs (2,573,139 bp), and a total number of 2493 coding DNA sequences (CDSs). Via in silico analysis, the bacteriocinogenic genetic clusters encoding the lantibiotic nisin Z (NisZ) and two new bacteriocins were identified, in addition to several probiotic traits, such as the production of vitamins, amino acids, adhesion/aggregation, and stress resistance factors, as well as the absence of transferable antibiotic resistance determinants and genes encoding detrimental enzymatic activities and virulence factors. These results unveil diverse beneficial properties that support the use of L. cremoris WA2-67 as a probiotic for aquaculture

Evaluation of Safety and Probiotic Traits from a Comprehensive Genome-Based In Silico Analysis of Ligilactobacillus salivarius P1CEA3, Isolated from Pigs and Producer of Nisin S

Author Contributions: Conceptualization, E.M.-A., P.E.H. and J.B.; methodology, E.S., N.P., I.L., P.E.H. and J.B.; investigation, E.S., N.P., I.L., E.M.-A. and J.B.; resources, L.M.C., E.M.-A., P.E.H. and J.B.; data curation, E.S., P.E.H. and J.B.; writing—original draft preparation, E.S.; writing—review and editing, P.E.H. and J.B.; supervision, E.M.-A., P.E.H. and J.B.; project administration, J.B.; funding acquisition, L.M.C., P.E.H. and J.B. All authors have read and agreed to the published version of the manuscript.Ligilactobacillus salivarius is an important member of the porcine gastrointestinal tract (GIT). Some L. salivarius strains are considered to have a beneficial effect on the host by exerting different probiotic properties, including the production of antimicrobial peptides which help maintain a healthy gut microbiota. L. salivarius P1CEA3, a porcine isolated strain, was first selected and identified by its antimicrobial activity against a broad range of pathogenic bacteria due to the production of the novel bacteriocin nisin S. The assembled L. salivarius P1CEA3 genome includes a circular chromosome, a megaplasmid (pMP1CEA3) encoding the nisin S gene cluster, and two small plasmids. A comprehensive genome-based in silico analysis of the L. salivarius P1CEA3 genome reveals the presence of genes related to probiotic features such as bacteriocin synthesis, regulation and production, adhesion and aggregation, the production of lactic acid, amino acids metabolism, vitamin biosynthesis, and tolerance to temperature, acid, bile salts and osmotic and oxidative stress. Furthermore, the strain is absent of risk-related genes for acquired antibiotic resistance traits, virulence factors, toxic metabolites and detrimental metabolic or enzymatic activities. Resistance to common antibiotics and gelatinase and hemolytic activities have been discarded by in vitro experiments. This study identifies several probiotic and safety traits of L. salivarius P1CEA3 and suggests its potential as a promising probiotic in swine production.Ministerio de Ciencia e Innovación (España)Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer

Author Contributions: Conceptualization, E.M.-A., P.E.H. and J.B.; methodology, I.L., E.S., N.P. and A.C.; investigation, I.L., E.S. and N.P.; resources, A.C., L.M.C., P.E.H. and J.B.; writing—original draft preparation, I.L. writing—review and editing, P.E.H. and J.B.; supervision, E.M.-A. and J.B.; project administration, L.M.C., P.E.H., and J.B.; funding acquisition, L.M.C., P.E.H. and J.B. All authors have read and agreed to the published version of the manuscript.The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively.Ministerio de Ciencia e Innovación (España)Comunidad de MadridDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

Genomic and Functional Evaluation of Two Lacticaseibacillus paracasei and Two Lactiplantibacillus plantarum Strains, Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis), as Probiotics for Aquaculture

Author Contributions: Conceptualization, L.M.C. and P.P.; methodology, D.C., L.D.-F., J.F. and E.M.-A.; software, D.C. and L.D.-F.; validation, J.F. and J.B.; formal analysis, D.C., J.F. and P.E.H.; investigation, D.C., L.D.-F., J.F. and E.M.-A.; resources, J.B., P.P. and L.M.C.; data curation, E.M.- A.; writing—original draft preparation, D.C.; writing—review and editing, L.M.C., P.P. and P.E.H.; visualization, D.C., J.F., P.E.H. and E.M.-A.; supervision, L.M.C. and P.P.; project administration, L.M.C. and J.B.; funding acquisition, L.M.C. and J.B. All authors have read and agreed to the published version of the manuscript.Aquaculture plays a crucial role in meeting the increasing global demand for food and protein sources. However, its expansion is followed by increasing challenges, such as infectious disease outbreaks and antibiotic misuse. The present study focuses on the genetic and functional analyses of two Lacticaseibacillus paracasei (BF3 and RT4) and two Lactiplantibacillus plantarum (BF12 and WT12) strains isolated from a rotifer cultivation tank used for turbot larviculture. Whole-genome sequencing (WGS) and bioinformatics analyses confirmed their probiotic potential, the absence of transferable antibiotic resistance genes, and the absence of virulence and pathogenicity factors. Bacteriocin mining identified a gene cluster encoding six plantaricins, suggesting their role in the antimicrobial activity exerted by these strains. In vitro cell-free protein synthesis (IV-CFPS) analyses was used to evaluate the expression of the plantaricin genes. The in vitro-synthesized class IIb (two-peptide bacteriocins) plantaricin E/F (PlnE/F) exerted antimicrobial activity against three indicator microorganisms, including the well-known ichthyopathogen Lactococcus garvieae. Furthermore, MALDI-TOF MS on colonies detected the presence of a major peptide that matches the dimeric form of plantaricins E (PlnE) and F (PlnF). This study emphasizes the importance of genome sequencing and bioinformatic analysis for evaluating aquaculture probiotic candidates. Moreover, it provides valuable insights into their genetic features and antimicrobial mechanisms, paving the way for their application as probiotics in larviculture, which is a major bottleneck in aquaculture.Ministerio de Ciencia, Innovación y Universidades (España)Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

Antibiotic Resistance Genes, Virulence Factors, and Biofilm Formation in Coagulase-Negative Staphylococcus spp. Isolates from European Hakes (Merluccius merluccius, L.) Caught in the Northeast Atlantic Ocean

Author Contributions: Conceptualization, L.M.C., P.P. and E.M.-A.; methodology, L.D.-F., V.S., D.C., E.M.-A. and J.F.; software, L.D.-F., V.S., R.d.C. and D.C.; validation, P.P., G.I., J.F. and J.B.; formal analysis, L.D.-F., V.S., R.d.C. and J.F.; investigation, L.D.-F., E.M.-A. and D.C.; resources, J.B., P.P., G.I., P.E.H. and L.M.C.; data curation, L.D.-F.; writing—original draft preparation, L.D.-F.; writing—review and editing, E.M.-A., L.M.C. and P.P.; visualization, L.D.-F. and E.M.-A.; supervision, L.M.C., E.M.-A. and P.P.; project administration, L.M.C., P.E.H., E.M.-A. and J.B.; funding acquisition, L.M.C., J.B. and P.E.H. All authors have read and agreed to the published version of the manuscript.The indiscriminate use of antibiotics has contributed to the dissemination of multiresistant bacteria, which represents a public health concern. The aim of this work was to characterize 27 coagulase-negative staphylococci (CoNS) isolated from eight wild Northeast Atlantic hakes (Merluccius merluccius, L.) and taxonomically identified as Staphylococcus epidermidis (n = 16), Staphylococcus saprophyticus (n = 4), Staphylococcus hominis (n = 3), Staphylococcus pasteuri (n = 2), Staphylococcus edaphicus (n = 1), and Staphylococcus capitis (n = 1). Biofilm formation was evaluated with a microtiter assay, antibiotic susceptibility testing was performed using the disk diffusion method, and antibiotic resistance and virulence determinants were detected by PCR. Our results showed that all staphylococci produced biofilms and that 92.6% of the isolates were resistant to at least one antibiotic, mainly penicillin (88.8%), fusidic acid (40.7%), and erythromycin (37%). The penicillin resistance gene (blaZ) was detected in 66.6% (18) of the isolates, of which 10 also carried resistance genes to macrolides and lincosamides (mphC, msr(A/B), lnuA, or vgaA), 4 to fusidic acid (fusB), and 3 to trimethoprim-sulfamethoxazole (dfrA). At least one virulence gene (scn, hla, SCCmecIII, and/or SCCmecV) was detected in 48% of the isolates. This study suggests that wild European hake destined for human consumption could act as a vector of CoNS carrying antibiotic resistance genes and/or virulence factors.Ministerio de Ciencia, Innovación y Universidades (España)Fundação para a Ciência e a Tecnologia (Portugal)Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

Antimicrobial Activity, Genetic Relatedness, and Safety Assessment of Potential Probiotic Lactic Acid Bacteria Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis) Used as Live Feed in Fish Larviculture

2024 Descuento MDPIAquaculture is a rapidly expanding agri-food industry that faces substantial economic losses due to infectious disease outbreaks, such as bacterial infections. These outbreaks cause disruptions and high mortalities at various stages of the rearing process, especially in the larval stages. Probiotic bacteria are emerging as promising and sustainable alternative or complementary strategies to vaccination and the use of antibiotics in aquaculture. In this study, potential probiotic candidates for larviculture were isolated from a rotifer-rearing tank used as the first live feed for turbot larvae. Two Lacticaseibacillus paracasei and two Lactiplantibacillus plantarum isolates were selected for further characterization due to their wide and strong antimicrobial activity against several ichthyopathogens, both Gram-positive and Gram-negative. An extensive in vitro safety assessment of these four isolates revealed the absence of harmful traits, such as acquired antimicrobial resistance and other virulence factors (i.e., hemolytic and gelatinase activities, bile salt deconjugation, and mucin degradation, as well as PCR detection of biogenic amine production). Moreover, Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) analyses unveiled their genetic relatedness, revealing two divergent clusters within each species. To our knowledge, this work reports for the first time the isolation and characterization of Lactic Acid Bacteria (LAB) with potential use as probiotics in aquaculture from rotifer-rearing tanks, which have the potential to optimize turbot larviculture and to introduce novel microbial management approaches for a sustainable aquaculture.Ministerio de Ciencia, Innovación y Universidades (España)Fundação para a Ciência e a Tecnologia (Portugal)Comunidad de MadridEuropean CommissionMinisterio de Trabajo y Economía Social (España)Depto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpubDescuento UC

Hacia un massive online open course (MOOC) para el aprendizaje de la higiene y seguridad alimentarias

Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaFALSEsubmitte

Diseño y desarrollo de una herramienta audiovisual para la docencia virtual de la inspección veterinaria oficial de pescados y productos de la pesca en un mercado central

El objetivo global de este Proyecto de Innovación Docente es la creación de vídeos explicativos como una herramienta de aprendizaje incorporada en el Campus Virtual para mejorar el estudio sobre las actividades de higiene, inspección y control alimentario que se realizan en el Mercado Central de Pescados de Mercamadrid. La creación y el empleo de estos vídeos están dirigidos, en un principio, a los estudiantes universitarios de Grado en Veterinaria que cursan la asignatura de Higiene, Inspección y Seguridad Alimentaria. En este Proyecto se han creado vídeos explicativos que tratan sobre: (i) los controles oficiales realizados por los Técnicos Superiores Veterinarios de Mercamadrid; (ii) los riesgos sanitarios asociados al consumo de pescados, crustáceos y moluscos; (iii) la frescura del pescado; (iv) el etiquetado del pescado; (v) la identificación de especies de pescado y marisco; (vi) la prevención de fraudes en la comercialización de pescados y mariscos; y (vii) la autentificación de pescados fileteados mediante técnicas de análisis

Micromundo@ucm: research and awareness for the stealth pandemics of antibiotic resistance

Actualmente se estima que las resistencias antibióticas se cobran 1.270.000 vidas anualmente a nivel global. Es necesario contribuir desde multiples ángulos a preservar la efectividad de os antibióticos y descubrir nuevas etsrategias terapéuticas. MicroMundo es un proyecto de Aprendizaje-Servicio y Ciencia Ciudadana que pretende crear cultura científica y concienciación en cuestiones de Salud Global en los jóvenes. Se pretende que sean los más jóvenes, los estudiantes de ESO y Bachillerato, los responsables de la transmisión de ese conocimiento a la comunidad. Pero un segundo objetivo, no menos importante, es el de generar y potenciar vocaciones STEM e interés por el I+D en Biomedicina. Para conseguir estos objetivos, diversos equipos de estudiantes universitarios imparten y coordinan el proyecto en colegios e institutos de su comunidad, coordinados por sus tutores (profesores e investigadores del área de Microbiología de las Facultades de Farmacia, Medicina, Veterinaria y Biología, cubriendo los tres vértices del triángulo One Health: salud humana, animal y medioambiental). En la UCM, durante el curso 2021-22, treinta y dos equipos de han trabajado en unos treinta colegios e institutos, implicando a unos 600 estudiantes preuniversitarios en el trabajo experimental del proyecto.It is currently estimated that antibiotic resistance annually claims 1,270,000 lives globally. It is necessary to contribute from multiple angles to preserve the effectiveness of antibiotics and discover new therapeutic strategies. MicroMundo is a Service-Learning and Citizen Science project that aims to create a scientific culture and awareness of Global Health issues among young people. It is intended that the youngest, Secondary and Baccalaureate students, be responsible for the transmission of this knowledge to the community. But a second objective, no less important, is to generate and promote STEM vocations and interest in R&D in Biomedicine. To achieve these objectives, various teams of university students teach and coordinate the project in schools and institutes in their community, coordinated by their tutors (professors and researchers from the Microbiology area of ​​the Faculties of Pharmacy, Medicine, Veterinary Medicine and Biology, covering the three vertices of the One Health triangle: human, animal and environmental health). At the UCM, during the 2021-22 academic year, thirty-two teams have worked in thirty-two schools, involving some 600 pre-university students in the experimental work of the project.Depto. de Genética, Fisiología y MicrobiologíaDepto. de Microbiología y ParasitologíaDepto. de Sanidad AnimalSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de Ciencias BiológicasFac. de FarmaciaFac. de VeterinariaTRUEunpu