A marine bacterium, Micrococcus MCCB 104, antagonistic to vibrios in prawn larval rearing systems

marine bacterium, Micrococcus MCCB 104, isolated from hatchery water, demonstrated extracellular antagonistic properties against Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, V. fluviallis, V. nereis, V. proteolyticus, V. mediterranei, V. cholerae and Aeromonas sp., bacteria associated with Macrobrachium rosenbergii larval rearing systems. The isolate inhibited the growth of V. alginolyticus during co-culture. The antagonistic component of the extracellular product was heat-stable and insensitive to proteases, lipase, catalase and α-amylase. Micrococcus MCCB 104 was demonstrated to be non-pathogenic to M. rosenbergii larvae

Catalysis by Enzymes immobilized on Tuned Mesoporous Silica

Mesoporous silica nanoparticles provide a non-invasive and biocompatible delivery platform for a broad range of applications in therapeutics, pharmaceuticals and diagnosis. Additionally, mesoporous silica materials can be synthesized together with other nanomaterials to create new nanocomposites, opening up a wide variety of potential applications. The ready functionalization of silica materials makes them ideal candidates for bioapplications and catalysis. These properties of mesoporous silica like high surface areas, large pore volumes and ordered pore networks allow them for higher loading of drugs or biomolecules. Comparative studies have been made to evaluate the different procedures; much of the research to date has involved quick exploration of new methods and supports. Requirements for different enzymes may vary, and specific conditions may be needed for a particular application of an immobilized enzyme such as a highly rigid support. In this endeavor, mesoporous silica materials having different pore size were synthesized and easily modified with active functional groups and were evaluated for the immobilization of enzymes. In this work, Aspergillus niger glucoamylase, Bovine liver catalase, Candida rugosa lipase were immobilized onto support by adsorption and covalent binding. The structural properties of pure and immobilized supports are analyzed by various characterization techniques and are used for different reactions of industrial applications.

Estudio de las comunidades microbianas de embutidos fermentados ligeramente acidificados mediante técnicas moleculares. Estandarización, seguridad y mejora tecnológica.

Los embutidos fermentados ligeramente acidificados son un grupo de productos tradicionales mediterráneos, caracterizados por un pH superior a 5,3. Para un control eficiente de la seguridad microbiológica de los embutidos se necesitan técnicas rápidas para la identificación y recuento de los microorganismos patógenos a estudiar. En el presente trabajo, se desarrolló una técnica para la enumeración de L. monocytogenes que combinó el método del número más probable y la identificación mediante PCR específica. Para la detección de Salmonella spp. y L. monocytogenes se desarrolló un sistema de PCR-multiplex que permitió la identificación de ambos patógenos de forma simultánea en una sola reacción. El estudio de la calidad microbiológica de los embutidos fermentados ligeramente acidificados se completó con la caracterización de las comunidades microbianas más importantes en estos productos. Se identificaron a nivel de especie los aislados de bacterias del ácido láctico (BAL), de enterococos y de cocos gram-positivos catalasa-positivos (CGC+). Posteriormente se realizó una tipificación molecular de los mismos mediante RAPD y análisis del perfil plasmídico y se estudiaron las principales características de interés higiénico-sanitario y tecnológico de las cepas. Mediante PCR se identificó Lactobacillus sakei como la especie predominante (74%), seguida por Lactobacillus curvatus (21,2%). La actividad aminoácido-descarboxilasa se asoció a la especie L. curvatus (el 66% de los aislados presentaron esta actividad). La identificación de los enterococos se realizó mediante PCR-multiplex y por secuenciación del gen sodA. Enterococcus faecium fue la especie de enterococos predominante (51,9%) seguida por Enterococcus faecalis (14,2%). Todas las cepas de E. faecalis presentaron genes asociados a factores de virulencia. E. faecalis presentó mayor resistencia a antibióticos que el resto de las especies de enterococos estudiadas. Tan sólo una cepa de E. faecium presentó el genotipo vanA (que confiere resistencia de alto nivel a la vancomicina). La identificación de los aislados de CGC+ (mediante PCR específica y amplificación de la región intergénica 16S-23S ARNr) demostró que Staphylococcus xylosus es la especie predominante en los embutidos fermentados ligeramente acidificados (80,8%). La amina biógena más común en los CGC+ fue la feniletilamina, producida por un 10,8% de aislados. Un pequeño porcentaje de aislados fueron mecA+ (4,6%), presentando además resistencia a múltiples antibióticos. El potencial enterotoxigénico de las cepas de CGC+ fue muy reducido (3,3% de los aislados), detectándose únicamente el gen entC. El estudio pormenorizado de las comunidades bacterianas de interés permitió la selección de 2 cepas de L. sakei y 2 cepas de S. xylosus con características tecnológicas e higiénico-sanitarias óptimas. Para evaluar su efectividad como cultivos iniciadores se elaboraron dos tipos de embutidos ligeramente ácidos, chorizo y fuet, inoculados con microorganismos patógenos (Salmonella spp., L. monocytogenes y S. aureus). El uso de cultivos iniciadores permitió el control de L. monocytogenes, Enterobacteriaceae y Enterococcus así como del contenido en aminas biógenas. Los recuentos de Salmonella spp. disminuyeron de forma significante durante la maduración de los embutidos, independientemente del uso de cultivos iniciadores. El uso del tratamiento de alta presión (400 MPa) en los embutidos madurados consiguió la ausencia de Salmonella spp. en los lotes tratados.

Contribution of antioxidant enzymes to toxicity assessment in fluvial biofilms

Per tal d’avaluar l’impacte de la contaminació en els ecosistemes aquàtics, aquesta tesi es centra en una aproximació multi-biomarcador en els biofilms. En complement dels biomarcadors clàssics, es va demostrar que les activitats dels enzims antioxidants (AEA): catalasa, ascorbat peroxidasa i glutatió reductasa eren biomarcardors d'estrès oxidatiu en els biofilms. Tot i que les AEA poden veure's influenciades amb la mateixa mesura per factors naturals (edat del biofilm, llum de colonització o d'exposició) i contaminants (herbicides i farmacèutics), aquestes AEA permeten entendre millor l'efecte dels contaminants. Cal remarcar que assajos de toxicitat aguda es poden utilitzar per comparar la capacitat antioxidant entre comunitats i conèixer la seva pre-exposició a l'estrès oxidatiu. Aquesta aproximació multi-biomarcador a nivell de comunitat és especialment interessant per avaluar la toxicitat dels contaminants emergents (β-blockers) sobre espècies no-diana. Per tal de millorar-la, també es va verificar la possibilitat de mesurar l'expressió gènica en biofilms.

Triazole induced drought tolerance in horse chestnut (Aesculus hippocastanum)

We determined the influence of the triazole derivatives paclobutrazol, penconazole, epixiconazole, propiconazole and myclobutanil on the drought tolerance and post drought recovery of container-grown horse chestnut (Aesculus hippocastanum L.) saplings. Myclobutanil neither conferred drought resistance, as assessed by its effects on a number of physiological and biochemical parameters, nor affected growth parameters measured after recovery from drought. Chlorophyll fluorescence (F,IF,,), photosynthetic rates, total foliar chlorophyll and carotenoid concentrations, foliar proline concentration and superoxide dismutase and catalase activities were consistently higher and leaf necrosis and cellular electrolyte leakage was lower at the end of a 3-week drought in trees treated with paclobutrazol, penconazole, epixiconazole or propiconazole than in control trees. Twelve weeks after drought treatment, leaf area and shoot, root and total plant dry masses were greater in triazole-treated trees than in control trees with the exception of those treated with myclobutanil. In a separate Study, trees were subjected to a 2-week drought and then sprayed with paclobutrazol, penconazole, epixiconazole, propiconazole or myclobutanil. Chlorophyll fluorescence, photosynthetic rate, foliar chlorophyll concentration and catalase activity over the following 12 weeks were 20 to 50% hi-her in triazole-treated trees than in control trees. At the end of the 12-week recovery period, leaf area and shoot, root and total plant dry masses were higher in triazole-treated trees than in control trees, with the exception of trees treated with myclobutanil. Application of triazole derivatives, with the exception of myclobutanil, enhanced tolerance to prolonged drought and, when applied after a 2-week drought, hastened recovery from drought. The magnitude of treatment effects was in the order epixiconazole approximate to propiconazole > penconazole > paclobutrazol > myclobutanil.

Free radicals, metals and antioxidants in oxidative stress-induced cancer

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular. ROS activation of AP-1 (activator protein) and NF-kappa B (nuclear factor kappa B) signal transduction pathways, which, in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu. Zn-SOD. Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of careinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-kappa B, AP-1 are also reviewed. 2006 Elsevier Ireland Ltd. All rights reserved.

Corynebacterium suicordis sp. nov., from pigs

Nineteen strains of Gram-positive, non-motile, non-spore-forming, catalase-positive, rod-shaped bacteria isolated from pigs were characterized by using biochemical, molecular chemical and molecular genetic methods. Two distinct groups of organisms were discerned, based on their colonial morphology, CAMP (Christie-Atkins-Munch-Petersen) reaction and numerical profile by using the API Coryne system. The first group (113 strains) gave a doubtful discrimination between Corynebacterium striatum and Corynebacterium amycolatum, whilst the second group (six strains) were identified tentatively as Corynebacterium urealyticum. Comparative 16S rRNA gene sequencing studies demonstrated that all of the isolates belonged phylogenetically to the genus Corynebacterium. The first group of organisms was highly similar to Corynebacterium testudinoris with respect to 16S rRNA gene sequences and physiological characteristics, whereas the remaining six isolates formed a hitherto unknown subline within the genus, associated with a small subcluster of species that included Corynebacterium auriscanis and its close relatives. The unknown Corynebacterium sp. was distinguished readily from these and other species of the genus by biochemical tests. Based on both phenotypic and phylogenetic evidence, it is proposed that the new isolates from pigs should be classified as a novel species, Corynebacterium suicordis sp. nov. The type strain is P81/02(T) (=CECT 5724(T) =CCUG 46963(T)).

Uruburuella suis gen. nov., sp nov., isolated from clinical specimens of pigs

Five strains of an unusual Gram-negative, catalase-positive, oxidase-positive, coccobacillus-shaped bacterium isolated from the lungs and heart of pigs with pneumonia and pericarditis were characterized by phenotypic and molecular genetic methods. On the basis of cellular morphology and biochemical criteria, the isolates were tentatively assigned to the family Neisseriaceae, although they did not appear to correspond to any recognized genus or species. Comparative 16S rRNA gene sequencing showed that the five unidentified strains were phylogenetically highly related to each other and represent a hitherto unknown subline within the family Neisseriaceae. On the basis of both phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from pigs be classified as a novel genus and species within the family Neisseriaceae, for which the name Uruburuella suis gen. nov., sp. nov. is proposed. The type strain of U. suis is 1258/02(T) (=CCUG 47806(T) =CECT 5685(T)).

Psychrobacter pulmonis sp nov., isolated from the lungs of lambs

Unusual Gram-negative, catalase- and oxidase-positive, coccus-shaped bacteria isolated from the lungs of two lambs were characterized by phenotypic and molecular-genetic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown isolates were genealogically highly related to each other (99.8% sequence similarity) and represent a novel subline within the genus Psychrobacter. The unknown bacterium was phylogenetically closely related to, but distinct from, Psychrobacter phenylpyruvicus, Psychrobacter immobilis, Psychrobacter glacincola and Psychrobacter urativorans. The novel Psychrobacter isolates were readily distinguished from all other Psychrobacter species and other Gram-negative, oxidase-positive bacteria usually responsible for lung infections in sheep by physiological and biochemical tests. Based on molecular-genetic and phenotypic evidence, it is proposed that the unknown Psychrobacter isolates from lambs be classified as Psychrobacterpulmonis sp. nov. The type strain is strain S-606(T) (= CECT 5989(T) = CCUG 46240(T)).

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1(wt)) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1(wt) in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1(wt) expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [H-3]D-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1(wt). The hSOD1-induced decline in GLT-1 protein and [H-3]D-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1(wt) in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.

Anaerotruncus colihominis gen. nov., sp. nov., from human faeces

Phenotypic and phylogenetic studies were performed on two isolates of an unidentified Gram-positive, anaerobic, non-spore-forming, rod-shaped bacterium that was isolated from human faeces. The organisms were catalase-negative, produced acetic and butyric acids as end products of metabolism and possessed a DNA G+C content of approximately 54 mol%. Comparative 16S rRNA gene sequencing demonstrated that the two isolates were related closely to each other and formed a hitherto unknown sublineage within the Clostridium leptum rRNA cluster of organisms. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium should be classified in a novel genus as Anaerotruncus colihominis gen. nov., so. nov. The type strain of Anaerotruncus colihominis is WAL 14565(T) = CCUG 45055(T) = CIP 107754(T).

Streptococcus castoreus sp nov., isolated from a beaver (Castor fiber)

A previously undescribed, Gram-positive, catalase-negative, Streptococcus-like organism originating from a European beaver (Castor fiber) was subjected to a taxonomic study. The organism displayed beta-haemolytic activity and gave a positive reaction with Lancefield group A antisera. Based on the results of biochemical testing, the organism was tentatively identified as a member of the genus Streptococcus, but it did not correspond phenotypically to any recognized species of this genus. Comparative 16S rRNA gene sequencing studies confirmed this assignment, with the bacterium forming a hitherto unknown subline within the genus. Sequence divergence values of greater than 3% from other reference streptococcal species, however, demonstrated that the unidentified coccus-shaped organism represents a hitherto unknown species. Based on phenotypic and molecular phylogenetic evidence, it is therefore proposed that the unknown organism from a beaver be classified as a novel species, Streptococcus castoreus sp. nov. The type strain is M605815/03/2(T) (=CCUG 48115(T) = CIP 108205(T)).

Streptococcus halichoeri sp nov., isolated from grey seals (Halichoerus grypus)

Phenotypic and phylogenetic studies were performed on six unidentified, Gram-positive, catalase-negative, chain-forming Streptococcus-like organisms recovered from grey seals. Biochemically the six strains were highly related to each other, but they did not appear to correspond to any recognized species of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies confirmed that phylogenetically the strains were members of the genus Streptococcus, but sequence divergence values of greater than 3 % compared with reference streptococcal species demonstrated that the organisms from seals represent a novel species. SDS-PAGE analysis of whole-cell proteins confirmed the phenotypic distinctiveness of the seal organisms. Based on biochemical criteria and molecular chemical and genetic evidence, it is proposed that the unknown organism from seals be classified as a novel species, Streptococcus halichoeri sp. nov., the type strain of which is CCUG 48324(T) (=CIP 108195(T)).

Streptococcus marimammalium sp nov., isolated from seals

Two strains of an unidentified, Gram-positive, catalase-negative, chain-forming, coccus-shaped organism recovered from seals were characterized using phenotypic and molecular taxonomic methods. Based on morphological and biochemical criteria the strains were tentatively identified as streptococci but they did not appear to correspond to any recognized species of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies showed that the strains were closely related to each other and confirmed their placement in the genus Streptococcus. Sequence divergence values of > 5 % with reference streptococcal species demonstrated the organisms from seals represent a novel species. SDS-PAGE analysis of whole-cell proteins confirmed that the two organisms were closely related to each other but were different from all currently defined streptococcal species. Based on biochemical criteria, molecular chemical and molecular genetic evidence, it is proposed that the unknown isolates from seals be assigned to a novel species of the genus Streptococcus, Streptococcus marimammalium sp. nov. The type strain is M54/01/(T) (=CCUG 48494(T)=CIP 108309(T)).

Saccharospirillum impatiens gen. nov., sp nov., a novel gamma-Proteobacterium isolated from hypersaline Ekho Lake (East Antarctica)

Five Gram-negative, motile, aerobic to microaerophilic spirilla were isolated from various depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica). The strains are oxidase- and catalase-positive, metabolize a variety of sugars and carboxylic acids and have an absolute requirement for sodium ions. The predominant fatty acids of the organisms are C-16: (1)omega7c, C-16:0 and C(18:1)omega7c, with C-10:1 3-OH, C-10:0 3-OH, C-12:0 3-OH, C-14:1 3-OH, C-14:0 3-OH and C-19:1 present in smaller amounts. The main polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmonomethylamine. The DNA base composition of the strains is 54-55 mol% G + C. 16S rRNA gene sequence comparisons show that the isolates are related to the genera Oceanospirillum, Pseudospirillum, Marinospirillum, Halomonas and Chromohalobacter in the gamma-Proteobacteria. Morphological, physiological and genotypic differences from these previously described genera support the description of a novel genus and species, Saccharospirillum impatiens gen. nov., sp. nov. The type strain is EL-105(T) (= DSM 12546(T) = CECT 5721(T)).