Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae

Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SPusp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P32 constitutive promoter, and pNZ8048c, which contains the inducible PnisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains.Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEMinisterio de Ciencia, Innovación y Universidades (MCIU)Universidad Complutense de Madrid and Banco de SantanderUniversidad Complutense de Madridpu

Immunomodulatory effects of bacteriocinogenic and non-bacteriocinogenic Lactococcus cremoris of aquatic origin on rainbow trout (Oncorhynchus mykiss, Walbaum)

11 Pág.Lactic Acid Bacteria (LAB) are a group of bacteria frequently proposed as probiotics in aquaculture, as their administration has shown to confer positive effects on the growth, survival rate to pathogens and immunological status of the fish. In this respect, the production of antimicrobial peptides (referred to as bacteriocins) by LAB is a common trait thoroughly documented, being regarded as a key probiotic antimicrobial strategy. Although some studies have pointed to the direct immunomodulatory effects of these bacteriocins in mammals, this has been largely unexplored in fish. To this aim, in the current study, we have investigated the immunomodulatory effects of bacteriocins, by comparing the effects of a wild type nisin Z-expressing Lactococcus cremoris strain of aquatic origin to those exerted by a non-bacteriocinogenic isogenic mutant and a recombinant nisin Z, garvicin A and Q-producer multi-bacteriocinogenic strain. The transcriptional response elicited by the different strains in the rainbow trout intestinal epithelial cell line (RTgutGC) and in splenic leukocytes showed significant differences. Yet the adherence capacity to RTgutGC was similar for all strains. In splenocyte cultures, we also determined the effects of the different strains on the proliferation and survival of IgM+ B cells. Finally, while the different LAB elicited respiratory burst activity similarly, the bacteriocinogenic strains showed an increased ability to induce the production of nitric oxide (NO). The results obtained reveal a superior capacity of the bacteriocinogenic strains to modulate different immune functions, pointing to a direct immunomodulatory role of the bacteriocins, mainly nisin Z.This work was supported by the Ministerio de Ciencia e Innovación (MICINN, Madrid, Spain) (projects RTI2018-094907-B-I00, and PID2020-113268RB-I00), and the Universidad Complutense de Madrid (UCM) (project FEI16/54). DC was supported by a contract from the project RTI2018-094907-B-I00. JF was supported by a FEI16/54 contract and held a predoctoral contract from UCM. LD-F was supported by a contract from the “Programa Investigo” (Ministerio de Trabajo y Economía Social, Madrid, Spain), funded by the EU (NextGenerationEU). JB was supported by the Atracción de Talento, program of the Comunidad de Madrid, Spain (2018-T1/BIO-10158).Peer reviewe

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

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

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

Listeria monocytogenes from Food Products and Food Associated Environments: Antimicrobial Resistance, Genetic Clustering and Biofilm Insights

2024 Descuento MDPIListeria monocytogenes, a foodborne pathogen, exhibits high adaptability to adverse environmental conditions and is common in the food industry, especially in ready-to-eat foods. L. monocytogenes strains pose food safety challenges due to their ability to form biofilms, increased resistance to disinfectants, and long-term persistence in the environment. The aim of this study was to evaluate the presence and genetic diversity of L. monocytogenes in food and related environmental products collected from 2014 to 2022 and assess antibiotic susceptibility and biofilm formation abilities. L. monocytogenes was identified in 13 out of the 227 (6%) of samples, 7 from food products (meat preparation, cheeses, and raw milk) and 6 from food-processing environments (slaughterhouse-floor and catering establishments). All isolates exhibited high biofilm-forming capacity and antibiotic susceptibility testing showed resistance to several classes of antibiotics, especially trimethoprim-sulfamethoxazole and erythromycin. Genotyping and core-genome clustering identified eight sequence types and a cluster of three very closely related ST3 isolates (all from food), suggesting a common contamination source. Whole-genome sequencing (WGS) analysis revealed resistance genes conferring resistance to fosfomycin (fosX), lincosamides (lin), fluoroquinolones (norB), and tetracycline (tetM). In addition, the qacJ gene was also detected, conferring resistance to disinfecting agents and antiseptics. Virulence gene profiling revealed the presence of 92 associated genes associated with pathogenicity, adherence, and persistence. These findings underscore the presence of L. monocytogenes strains in food products and food-associated environments, demonstrating a high virulence of these strains associated with resistance genes to antibiotics, but also to disinfectants and antiseptics. Moreover, they emphasize the need for continuous surveillance, effective risk assessment, and rigorous control measures to minimize the public health risks associated to severe infections, particularly listeriosis outbreaks. A better understanding of the complex dynamics of pathogens in food products and their associated environments can help improve overall food safety and develop more effective strategies to prevent severe health consequences and economic losses.Fundação para a Ciência e a Tecnologia (Portugal)Depto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpubDescuento UC

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

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: Citizen Science and Service-Learning against Antibiotic Resistance from the Academic Community

Según ha establecido la OMS y la Asamblea de las Naciones Unidas, la resistencia a los antibióticos es una de las prioridades en Salud Global para el s. XXI, al mismo nivel que la preparación frente a pandemias emergentes y el desarrollo de vacunas y tratamientos frente a tuberculosis, malaria, HIV y otras enfermedades infecciosas. El abordaje de estos problemas sólo será eficaz desde la perspectiva multidisciplinar One Health (Un Mundo: Una Salud), que integra Salud Medioambiental, Animal y Humana. Desde 2016 la UCM ha sido pionera en implicar a las comunidades universitaria y preuniversitaria en la estrategia de aprendizaje activo mediante ApS “MicroMundo” (www.ucm.es/small-world-initiative), un proyecto multidisciplinar basado en ApS en el ámbito de la Biomedicina. Se trata de una adaptación al ApS de la estrategia internacional de studentsourcing Tiny Earth (www.tinyearthnet.org). MicroMundo implica a estudiantes de ESO y Bachillerato a participar como investigadores en un proyecto real para el descubrimiento de nuevos antibióticos a partir de muestras de suelos. El objetivo final del servicio es acercar la cultura científica, la perspectiva One Health y la investigación biomédica a la sociedad. Para ello, colaboramos con una red de colegios e institutos de la Comunidad de Madrid. Un objetivo adicional es fomentar la vocación por Grados STEM y por la I+D. Para lograr estos objetivos, los estudiantes universitarios, objeto del aprendizaje, se responsabilizan de facilitar a los jóvenes estudiantes el entrenamiento y material necesarios para llevar a cabo los experimentos, así como dirigir el trabajo de investigación y diversas estrategias de divulgación hacia la comunidad. El éxito del proyecto en la UCM ha inspirado su implementación en más de 30 universidades en España y Portugal y ha merecido el Premio PRAN 2021 a la comunicación y sensibilización de la población sobre la resistencia a los antibióticos. En esta publicación presentamos la memoria final del proyecto MicroMundo@UCM realizado durante el curso 2022-23 en 33 centros educativos de la Comunidad de Madrid.Oficina de Aprendizaje-Servicio UCMConvenio MSD España-UCMDepto. de Microbiología y ParasitologíaDepto. de Genética, Fisiología y MicrobiologíaDepto. de Sanidad AnimalDepto. de Nutrición y Ciencia de los AlimentosDepto. de Bioquímica y Biología MolecularFac. de FarmaciaFac. de Ciencias BiológicasFac. de VeterinariaTRUEunpu

Integrate and learn. Building a farm-to-table blockchain

DecanatoFac. de VeterinariaFALSEsubmitte