Actividades celulolíticas, utilización de carbohidratos y producción de etanol por clostridium thermocellum

Tesis Univ. Complutense. Madrid.Fac. de VeterinariaTRUEProQuestpu

Safety assessment and molecular genetic profiling by pulsed-field gel electrophoresis (PFGE) and PCR-based techniques of Enterococcus faecium strains of food origin

Enterococcus faecium is authorized as animal probiotic in the European Union, but this species has emerged as an important cause of nosocomial infections in humans. We investigated the safety of 14 potential probiotic E. faecium strains with antimicrobial activity, previously isolated from food, following the guidance proposed by EFSA. All the enterococci were susceptible to ampicillin, and none of them harbored the genes encoding the enterococcal surface protein (esp), putative glycosyl hydrolase (hylEfm), and insertion sequence IS16. The genetic relatedness of these enterococci was determined by pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC-PCR), and restriction analysis of amplified 16S rDNA (ARDRA). PFGE analysis of SmaI patterns evidenced four subgroups, whereas RAPD and ERIC-PCR analysis gave nine and eight different subgroups, respectively. ERIC-PCR yielded the highest diversity, followed by RAPD and PFGE, while ARDRA achieved the lowest diversity. In conclusion, we demonstrated the absence of well-known enterococcal virulence markers in a collection of E. faecium strains from food, which renders them safe to be used in the food industry or as probiotics in animal production, and that ERIC-PCR is a reliable tool to be used for molecular genetic profiling of potential probiotic enterococci.Ministerio de Economía y CompetitividadComunidad de MadridEuropean Social FundDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaFALSEpu

Characterization of Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss) feed and larvae: safety, DNA fingerprinting, and bacteriocinogenicity

The use of lactic acid bacteria (LAB) as probiotics constitutes an alternative or complementary strategy to chemotherapy and vaccination for disease control in aquaculture. The objectives of this work were (1) the in vitro safety assessment of 8 Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) feed and larvae; (2) the evaluation of their genetic relatedness; (3) the study of their antimicrobial/bacteriocin activity against fish pathogens; and (4) the biochemical and genetic characterization of the bacteriocin produced by the strain displaying the greatest antimicrobial activity. Concerning the safety assessment, none of the pediococci showed antibiotic resistance nor produced hemolysin or gelatinase, degraded gastric mucin, or deconjugated bile salts. Four strains (50%) produced tyramine or putrescine, but the corresponding genes were not amplified by PCR. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting allowed clustering of the pediococci into 2 well-defined groups (68% similarity). From the 8 pediococci displaying direct antimicrobial activity against at least 3 out of 9 fish pathogens, 6 strains (75%) were identified as bacteriocin producers. The bacteriocin produced by P. acidilactici L-14 was purified, and mass spectrometry and DNA sequencing revealed its identity to pediocin PA-1 (PedPA-1). Altogether, our results allowed the identification of 4 (50%) putatively safe pediococci, including 2 bacteriocinogenic strains. ERIC-PCR fingerprinting was a valuable tool for genetic profiling of P. acidilactici strains. This work reports for the first time the characterization of a PedPA-1-producing P. acidilactici strain isolated from an aquatic environment (rainbow trout larvae), which shows interesting properties related to its potential use as a probiotic in aquaculture.Ministerio de Economía y CompetitividadComunidad de MadridSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

In vitro and in vivo evaluation of lactic acid bacteria of aquatic origin as probiotics for turbot (Scophthalmus maximus L.) farming

Turbot (Scophthalmus maximus L.) is an important commercial marine flatfish. Its production may be affected by bacterial diseases that cause severe economical losses, mainly tenacibaculosis and vibriosis, provoked by Tenacibaculum maritimum and Vibrio splendidus, respectively. An alternative or complementary strategy to chemotherapy and vaccination for the control of these diseases is the use of probiotics. In this work, we report the in vitro and in vivo potential of eight lactic acid bacteria (LAB), previously isolated from fish, seafood and fish products intended for human consumption, as turbot probiotics. Seven out of the eight LAB exerted direct antimicrobial activity against, at least, four strains of T. maritimum and V. splendidus. All LAB survived in seawater at 18 °C for 7 days, and withstood exposure to pH 3.0 and 10% (v/v) turbot bile; however, they differed in cell surface hydrophobicity (8.2–21.7%) and in their ability to adhere to turbot skin (1.2–21.7%) and intestinal (0.7–2.1%) mucus. Most of the tested strains inhibited the binding of turbot pathogens to the mucus. Leuconostoc mesenteroides subsp. cremoris SMM69 and Weissella cibaria P71 were selected based on their strong antimicrobial activity against T. maritimum and V. splendidus, good probiotic properties, and different adhesion ability to skin mucus and capacity to inhibit the adhesion of turbot pathogens to mucus. These two LAB strains were harmless when administered by bath to turbot larvae and juveniles; moreover, real-time PCR on the transcription levels of the immunity-related genes encoding IL-1β, TNF-α, lysozyme, C3, MHC-Iα and MHC-IIα in five organs (head-kidney, spleen, liver, intestine and skin) revealed the ability of these LAB to stimulate their expression in turbot juveniles, especially the non-specific immunity associated genes in mucosal tissues. Based on our results, Lc. cremoris SMM69 and W. cibaria P71 may be considered as suitable probiotic candidates for turbot farming.Ministerio de Economía y CompetitividadComunidad de MadridSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

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

Characterization of Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) feed and larvae: safety, DNA fingerprinting and bacteriocinogenicity

The use of lactic acid bacteria (LAB) as probiotics constitutes an alternative or complementary strategy to chemotherapy and vaccination for disease control in aquaculture. The objectives of this work were (1) the in vitro safety assessment of 8 Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) feed and larvae; (2) the evaluation of their genetic relatedness; (3) the study of their antimicrobial/bacteriocin activity against fish pathogens; and (4) the biochemical and genetic characterization of the bacteriocin produced by the strain displaying the greatest antimicrobial activity. Concerning the safety assessment, none of the pediococci showed antibiotic resistance nor produced hemolysin or gelatinase, degraded gastric mucin, or deconjugated bile salts. Four strains (50%) produced tyramine or putrescine, but the corresponding genes were not amplified by PCR. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting allowed clustering of the pediococci into 2 well-defined groups (68% similarity). From the 8 pediococci displaying direct antimicrobial activity against at least 3 out of 9 fish pathogens, 6 strains (75%) were identified as bacteriocin producers. The bacteriocin produced by P. acidilactici L-14 was purified, and mass spectrometry and DNA sequencing revealed its identity to pediocin PA-1 (PedPA-1). Altogether, our results allowed the identification of 4 (50%) putatively safe pediococci, including 2 bacteriocinogenic strains. ERIC-PCR fingerprinting was a valuable tool for genetic profiling of P. acidilactici strains. This work reports for the first time the characterization of a PedPA-1-producing P. acidilactici strain isolated from an aquatic environment (rainbow trout larvae), which shows interesting properties related to its potential use as a probiotic in aquaculture.Ministerio de Economia y CompetitividadComunidad de MadridUniversidad Complutense de MadridFundação para a Ciência e a Tecnologia (FCT, Portugal)European Social FundDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

Genomic Sequence of Streptococcus salivarius MDI13 and Latilactobacillus sakei MEI5: Two Promising Probiotic Strains Isolated from European Hakes (Merluccius merluccius, L.)

FEI16/54 Universidad Complutense de Madrid 2018-T1/BIO-10158 Programa Atracción del Talento de la Comunidad de MadridSimple Summary: In fish farming, diseases have commonly been fought with the abusive use of antibiotics, which have caused antibiotic (multi)resistances in bacteria. Consequently, it is necessary to explore safe and environmentally friendly alternative approaches to improve the fish health and avoid the treatment of bacterial diseases with antibiotics such as the use of probiotics. This study focuses on bioinformatic and functional analyses of the genome sequences of Streptococcus salivarius MDI13 and Latilactobacillus sakei MEI5, two Lactic Acid Bacteria (LAB) isolated from the gut of European hakes (Merluccius merluccius, L.), a highly valued marine fish for Spanish gastronomy. The potential probiotic characteristics of both bacteria, and the lack of antibiotic resistance genes and virulence were confirmed. In addition, genes encoding three antimicrobial peptides (known as bacteriocins) were identified in the genome of S. salivarius MDI13. One of these in vitro-synthesized bacteriocins (BlpK) showed antimicrobial activity against two fish pathogens (namely Lactococcus garvieae and Streptococcus parauberis). Altogether, our results suggest that S. salivarius MDI13 and L. sakei MEI5 have a strong potential as probiotics to prevent bacterial diseases in fish farming.Abstract: Frequently, diseases in aquaculture have been fought indiscriminately with the use of antibiotics, which has led to the development and dissemination of (multiple) antibiotic resistances in bacteria. Consequently, it is necessary to look for alternative and complementary approaches to chemotheraphy that are safe for humans, animals, and the environment, such as the use of probiotics in fish farming. The objective of this work was the Whole-Genome Sequencing (WGS) and bioinformatic and functional analyses of S. salivarius MDI13 and L. sakei MEI5, two LAB strains isolated from the gut of commercial European hakes (M. merluccius, L.) caught in the Northeast Atlantic Ocean. The WGS and bioinformatic and functional analyses confirmed the lack of transferable antibiotic resistance genes, the lack of virulence and pathogenicity issues, and their potentially probiotic characteristics. Specifically, genes involved in adhesion and aggregation, vitamin biosynthesis, and amino acid metabolism were detected in both strains. In addition, genes related to lactic acid production, active metabolism, and/or adaptation to stress and adverse conditions in the host gastrointestinal tract were detected in L. sakei MEI5. Moreover, a gene cluster encoding three bacteriocins (SlvV, BlpK, and BlpE) was identified in the genome of S. salivarius MDI13. The in vitro-synthesized bacteriocin BlpK showed antimicrobial activity against the ichthyopathogens Lc. garvieae and S. parauberis. Altogether, our results suggest that S. salivarius MDI13 and L. sakei MEI5 have a strong potential as probiotics to prevent fish diseases in aquaculture as an appropriate alternative/complementary strategy to the use of antibiotics.Ministerio de Ciencia, Innovación y Universidades (España)Ministerio de Trabajo y Economía Social (España)European CommissionUniversidad Complutense de MadridComunidad de MadridSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal

The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.Comisión EuropeaAgência Nacional, PortugalSpanish-Portuguese Integrated ActionMinisterio de Ciencia e InnovaciónMinisterio de Educación, Cultura y DeporteMinisterio de Ciencia y TecnologíaComunidad de MadridDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

Application of species-specific polymerase chain reaction assays to verify the labeling of quail (Coturnix coturnix), pheasant (Phasianus colchicus) and ostrich (Struthio camelus) in pet foods

Species-specific polymerase chain reaction (PCR) assays have been applied to verify the labeling of pet foods containing quail, pheasant, and ostrich. The method combines the use of quail, pheasant, ostrich, chicken, duck, pig and fish specific primers that amplify small fragments (amplicons <200 base pairs) of the mitochondrial 12S rRNA gene and a positive control primer pair that amplifies a 141 bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. The applicability of the assay was tested through the analysis of 100 commercial dog and cat food products. The reported PCR technique successfully detected the presence of the target species in 88 of the analyzed samples. However, the target species was not detected in 12 samples indicating a possible fraud in the labeling of these products. The results obtained suggest that the reported PCR method may represent a suitable tool for the detection of pet food mislabeling.Ministerio de Educación y CienciaComunidad de MadridDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture

Background. The microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for the aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate the antimicrobial/bacteriocin activity against fish pathogens, the antibiotic susceptibility, and the prevalence of virulence factors and detrimental enzymatic activities in 99 Lactic Acid Bacteria (LAB) (59 enterococci and 40 non-enterococci) isolated from aquatic animals regarded as human food. Results. These LAB displayed a broad antimicrobial/bacteriocin activity against the main Gram-positive and Gram-negative fish pathogens. However, particular safety concerns based on antibiotic resistance and virulence factors were identified in the genus Enterococcus (86%) (Enterococcus faecalis, 100%; E. faecium, 79%). Antibiotic resistance was also found in the genera Weissella (60%), Pediococcus (44%), Lactobacillus (33%), but not in leuconostocs and lactococci. Antibiotic resistance genes were found in 7.5% of the non-enterococci, including the genera Pediococcus (12.5%) and Weissella (6.7%). One strain of both Pediococcus pentosaceus and Weissella cibaria carried the erythromycin resistance gene mef(A/E), and another two P. pentosaceus strains harboured lnu(A) conferring resistance to lincosamides. Gelatinase activity was found in E. faecalis and E. faecium (71 and 11%, respectively), while a low number of E. faecalis (5%) and none E. faecium exerted hemolytic activity. None enterococci and non-enterococci showed bile deconjugation and mucin degradation abilities, or other detrimental enzymatic activities. Conclusions. To our knowledge, this is the first description of mef(A/E) in the genera Pediococcus and Weissella, and lnu(A) in the genus Pediococcus. The in vitro subtractive screening presented in this work constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture.Ministerio de Ciencia y Tecnología (AGL2009−08348-ALI)Universidad Complutense de Madrid/Banco Santander-Central Hispano (GR35/10-A)Dirección General de Universidades e Investigación, Consejería de Educación, Comunidad de Madrid (S−2009/AGR−1489)Ministerio de Ciencia e Innovación (HP2008-0070)Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu