BACTERIOLOGY

Author: RODRÍGUEZ-AVIAL INFANTE ICIAR.
Year: 2003.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: HOSPITAL CLÍNICO SAN CARLOS.

Summary: The Streptocococcus group viridans (SGV) are a heterogeneous group of Gram-positive cocci that are part of the human microbiota. These microroganismos lacked factors viruelencia but it is the primary agent of infectious endocarditis casual and in recent years emerging as a leading cause of bacteremia in neutropenic patients. In this work we analyzed the sensitivity of SGV to antimicrobial classics, new antimicrobuianos emerging with activity against Gram-positive and have given the determinants of resistance present and influences as their presence in the activity of new drugs. Resistance to penicillin in SGV is high, with percentages above 30% and implies a decrease in the sensitivity to the other beta-lactams. Resistance to erythromycin is a 44% and 62% of resistant strains express the phenotype cMLSB with resistance to all macrolides, and clindamycin streptogramin B All this phenotype strains carrying the gene erm (B), either alone or associated with the gene mef (A). The resistance to tetracycline is also high and the primary responsibility of the resistance gene is the tet (M), although the genes tet (O) and tet (L) were also present at the SGV. The fluorquinolonas had good activity. In 10% of the strains were resistant mutations in these genes pair (C) or even (C) and gyr (A). The low level of resistance to fluoroquinolones was due to the mechanism of expulsion active antimicrobial inhibited by reserpine. The new antimicrobial presented very good activitie compared with SGV, with rates higher than 90% sensitivity. However, resistant strains are beginning to appear even before the inclusion of drugs in clinical practice. The overall sensitivity to antimicrobials is not homogenea in different species that make up the group SGV. Therefore it is necessary identification, as well as the completion of its sensitivity when clinics are isolated sample. The share the same habitat, oral cavity, with other microorganisms makes the SGV in a potential reservoir of resistant genes that can be transferred to other Streptococcus more clinical impact, as S.pneumoniae and S.pyogenes bacteria or other genres it has already been published in the literature.

Author: ALVAREZ RUBIO JUAN.
Year: 2004.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA Y ODONTOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA.

Summary: Salmonella is one of the most important food pathogens globally. Currently, the diagnosis of Salmonella is usually through conventional cultivation techniques that are slow and tedious. Moreover, the technique má sextendida for the differentiation of the isolates of Salmonella is the serotyping. This technique is based on the determination of surface antigens whose presence depends on the expression of phenotypic characters. For the taxonomic analysis of gender genotyping tools are needed that are fast, cost-effective and available to the largest possible number of laboratories. In this study we have identified three PCR techniques for the diagnosis and epidemiological characterization of Salmonella spp. First has designed a multiple PCR for diangóstico Salmonella in clinical samples of faeces. We have also designed a binary classification technique based on real-time PCR for the differentiation of genotypes within the genus Salmonella. Finally, we have designed a technique similar to MLVA but typing with the added advantage of being directed to places hipervariables of bacterial chromosome is more discriminative that the techniques described so far.

Author: JUAN FERRÉ LUCÍA DE.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA . DEPARTEMENTO DE SANIDAD ANIMAL.

Summary: In this dissertation have addressed different aspects of paratuberculosis goats, related to the development of various diagnostic techniques and molecular characterization, as well as their practical application to the study of the prevalence of paratuberculosis in the Guadarrama of the Goat the Community of Madrid, and the design of a program to fight contrd disease in a farm goats race Murciano-Granadina of Toledo. The microbiological diagnosis done in 87 samples from goats, we suggested the need to supplement the means that so far empleábamos for cultivation in our lab. Herrold with egg yolk, micobactina and Pyruvate sadist (HEYMm-PS), and Middlebrook 7Hll with micobactina (M), with the culture medium of Lowenstein-Jensen with micobactina (LJ). The inclusion of this medium has allowed us to identify 11 samples had proved negative only when it uses the media HEYMm-PS and M. This means, LJ, has been very effective in the primoaislamiento of bacteria genovar sheep. In addition to design two microbiological diagnostic techniques for detecting molecular given the problems we had in the diagnosis of isolated gene ovar sheep in muestrds caprine Of the two techniques tested, and FastDNA Spin Sequence Capture Kit for Soil (FB), the second was that offered better results and enabling the identification of all samples positive bacteriological culture. The study on the prevalence of tuberculosis and paratuberculosis perform with 28 herds of Cabra Guadarrama of the Community of Madrid, showed results that can be described as worrying given that the prevalence of bacteriological culture through paratuberculosis was 42.9%. Tuberculosis of the 10.7 '%. Mixed infections And the 21.4%. The molecular characterization of isolates Maparatuberculosis through Electrophoresis Bed Pulsado grouped pigmented isolated in the group denonúnado Type 1que is cardcteriza for its growth ex1remadamente slow and host preference for sheep. However, isolated goats were grouped cn two distinct groups. The Type II (crecedores fast), which also include isolated Maparaluberculosis dc different animal species, and the Type III (crecedores slow) that shares characteristics with both groups anteriofU1entecitados. This is the primerd description isolated home goats in the third group equivalc the intermediate group when the cardcterización with the technical analysis of genetic polymorphisms through restricciónenzimáticay detection through hybridization with the sequence insertion IS900 (lS900-RFLP). The program dc dc control and eradicate the paratubcrculosis caprína design is based principalmentc in the separation irunediaw of infants of mothers after childbirth, nutrition milk artifiCial and checks immunological pam eliminating animals reaccionantes. The implementation of the program has allowed in just two years reduce the prevalence of pamtuberculosis in the herd of a 28.7%, lm 13.4%, showing that the implementation of these measures with guidelines on the proper management reduces the exploitation the percentage of positive animals.

Author: MANTEROLA MARTIJA JOSÉ M..
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: The mycobacteria can be detected by microscopic examination, by demonstrating his DNA or rRNA after specific nucleic acid amplification, or for cultivation. For aislarlar by culture, we need clean samples with commensal flora (sputum, urine, etc..). The aim of the thesis has been to assess:-A new method for decontamination of clinical samples with C18-carboxypropilbetaína (CB-18) and compare it with the method of Tacquet-Tison. B-evaluate a system for DNA amplification by Chain Reaction the Ligasa System (LCx Abbott, USA) with another amplification of rRNA (AMTDT of Gen-Probe, USA) to detect Mycobacterium tuberculosis in biopsies pleural parafinadas. C-Compare a new liquid culture medium not radiometric (MB / BacT) with other radiometric BACTEC 12B and solid medium of LÃ ¶ wenstein-Jensen (LJ) to isolate mycobacteria. To evaluate the new method CB-18 were processed 600 samples. There were no statistically significant differences in the detection of mycobacteria by staining between the 2 methods evaluated. We isolated a 14% increase from mycobacteria after decontaminate with CB-18, a statistically significant difference both for the total of mycobacteria to environmental mycobacteria, although there was no difference to isolate M.tuberculosis. This bacillus was found 3 days after a half before decontaminate with Tacquet. We analyzed 57 biopsies pleural parafinadas (BPP) of patients with pleural tuberculosis and 17 BPP patients without TB. The sensitivity for detecting M.tuberculosis was 52.6% for the AMTDT and 63.2% for LCx. Using both techniques together will reach a sensitivity of 80.7% in the diagnosis of tuberculosis pleural and contributed a 22.8% additional diagnostic. To assess the cropping system MB / BacT is descontamiaron 600 samples per Tacquet-Tison. We isolated 79 strains of M.tuberculosis and 27 strains of mycobacteria environmental; 92%, 91% and 94% of the strains, respectively LJ, BACTEC 12B and MB / BacT. Of the samples baciloscopia-negativas were isolated significantly more M.tuberculosis in MB / BacT that BACTEC 12B. The mycobacteria grew at an average of 15 days before giving MB / BacT in LJ. We conclude that the method CB-18 is suitable as a system of decontamination, both amplification systems have an intermediate sensitivity to detect M.tuberculosis in BPP and the liquid culture medium MB / BacT is suitable for the isolation of mycobacteria from clinical samples .

Author: SALSO ORTIZ SANTIAGO.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: DEP. MICROBIOLOGÍA CLÍNICA H.CLÍNICO SAN CARLOS.

Summary: In recent years, gender Enterobacter has become a major cause of infection nosocomial.Estos agencies have caused numerous outbreaks in hospitals around the world who are often caused by strains multirresistentes.Durante two months (December 2000 to January 2001) , collected 83 isolates Enterobacter.Se determined by the sensitivity antimicrobial systems Vitek II or Wider isolated and selected those with reduced sensitivity to cephalosporins broad espectro.Mediante Test Dual Disc Synergy (DDST) determine the existence of 10 isolated producers Betalactamasas of Spread Spectrum (BLEE). they all carry out various tests for the characterization of betalactamase: Chain Reaction (PCR) Polymerase, Isoelectroenfoque (IEF) and sequencing of the gene coding for betalactamase. These tests revealed the production of a BLEE TEM-24.Mediante studies tipaje molecular determinmos the same clonal origin of all isolates studied which suggests an outbreak by E.aerogenes producer BLEE.La emergence of the colon that was described for the first once in France and subsequently has been observed in Belgium, Italis and Spain, which suggests the spread international éste.Posteriormente, repeat one years after the study and we found 3 isolates with the same clonal origin so that the outbreak had given way to a situation endemic to the instaruración and adaptation of the strain E.aerogenes producer BLEE-type TEM-24 in the middle hospitalario.El prior use of oximinobetalactámicos was the only risk factor significantly associated with the acquisition of infection E . aerogenes producer BLEE.A today, carbapenemes constitute a useful therapeutic option in the treatment of infections caused by multidrug this microorganisms.

Author: FUEYO MENDOZA JOSE MARIA.
Year: 2004.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE QUIMICA Y BIOLOGIA.

Summary: Staphylococcus aureus from different origins (nasal samples of human carriers, modified food, and milk from cows with subclinical mastitis), collected in Asturias (1996-2002) have been analyzed by conventional and molecular techniques to determine the frequency and types of genes toxins pyrogenic superantigenos (PTSAg) that carry and polymorphism of DNA in order to establish relations between genomic toxigénicas and isolations. The interrelation of the results and their comparison with published on the subject, allows highlight as relevant findings:-percentage of 31.2, 26 and 14.3 of the isolates analyzed samples collected from human, bovine and food respectively, producing one or more PTSAg-clásicas (SEA enterotoxins, SEB SEC, SED toxin and toxic shock syndrome) and carrying their genes, usually in partnership with genes from other toxins new narrative. - Different isolates contain between 0-9 genes PTSAs and between 0-2 in leucotoxinas (being negative to see all of them, Sep, seq, eta, etb and all but one for cattle and other human etd for lukPV), which were grouped in profiles of genes or genotipos-T. It is noteworthy that the 39.7 and 0% of the isolates human versus 98.8% and 50% of cattle carrying lukED and lukM respectively. This fact could be related to the role of the first purely colonizer and pathogenic of the latter. - An analysis of genomic DNA polymorphism through macrorrestricción (Smal-PFGE) discriminated to the isolates in perfiles.Smal and these were grouped into lineages (similarity coefficient greater than 0.7). Thus it was observed that the most common lineages represented by human isolates included also isolates food, but not cattle. - There was a strong relationship between genotipos-T (including genes PTSAg classic) and perfiles-Smal in the three grpos isolation and both markers with the profile of plasmids into human isolates and food. The genes associated with the grouping egc-like (seg-sei-sem-sen-seo + /-seu) and the genes lukED was pu7eden considered ubiquitous, as were present in isolates of the three groups with perfiles-Smal low similarity . - Were detected gene associations with genetic elements described above, strains de.aureus man (seb-sek-seq with SaPI3, sec-sel-sem conSaPl4 and sed-sej-ser with plásmidos-SED classic 33 -36 kb), veal (tst-sec-sel with SaPlbov), and in both groups (egc-like/lukED with vSaB). In addition, it may consider new narrative: the insertion into the cromosopa of S.aureus of grouping plasmid (sed-sej-ser), the possible location of the genes seb, sect, tst not described in genetic elements and the presence of a plasmid of 53.5 kb of genes sed-sej-ser like in two isolates of food. - We could make a first approximation of rates or lineages of S.aureus prevalientes and / or endemic in Asturias, both in human and in cattle carriers, and seconding six strains associated four outbreaks of food poisoning three of the human lineage. The results support the hypothesis that humans and cattle are reservoirs of various subpopulations of species S.aureus in possession of the grouping genomics egc-like. This work is of interest in Microbiology, Molecular Epidemiology, Public Health, Animal Health and Food Safety, and can be used as a basis for future studies on the subject at all each of these fields

Author: LEON LOMAS MARIA PILAR.
Year: 2004.
University: CANTABRIA [www.unican.es].
Place of defense: E.T.S. ING. CAMINOS, CANALES Y PUERTOS.
Place of preparation: E.T.S. ING. CAMINOS, CANALES Y PUERTOS.

Summary: Wastewater lead dissolved, among others, a large quantity of microorganisms mainly from intestinal tract of man, whose progress will depend both physical (dilution and dispersion) and bioecological factors. The objective of this thesis is the pilot study of the influence of environmental factors such as light (intensity and type of light), salinity, temeratura and pH on the behavior of the enteric bacterium Escherichia coli from fecal contamination subspecies communior and as obtaining its rate of disappearance, expressed as T90, in controlled laboratory conditions. The T90 is a parameter popuesto for the design of the construction of submarines emissaries, and represents the time in which there is a reduction of 90% in the concentration of an indicator of fecal contamination. In trials using three types of cultivation: liquid for the return and growth of the strain, solid for maintenance and other specific counting E. Coli. We tested different levels of the factors, either individually, as combined to determine the effects of synergy on the T90 E. Coli produced by the interaction between environmental factors studied by twos. The results show that the maximum survival of E. Coli and therefore the highest value of T90 is obtained in darkness, with salinity physiological (8.5 g / l), low temperature (4Â § C) and pH 7.0. The light has a dual effect decreasing the T90 while increasing your intensity for the same kind of light, such as by decreasing the wavelength, for the same light intensity. It has been tested and quantified the accused synergistic effect produced by the combination of salinity and pH, temperature and salinity, temperature and pH, as well as the intensity and type of light with the other factors, on the T90.

Author: LÓPEZ ROMALDE SONIA.
Year: 2004.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Author: MONTES LÓPEZ M. JESÚS.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE FARMACIA.

Summary: This report discusses a Doctoral integrated morphological, physiological, biochemical and genetic of 16 isolates of bacterial origin Antarctic, isolated from samples of sediment and water from snowmelt, the scientific campaigns for the years 1987 - 88, 1988-89 and 1990-91. The content of this paper begins with a historical overview and outlook for the bacterial taxonomy. It highlights the contributions of MÃ ¼ ller, Ehrengerg, Cohn, Migula, Orla-Jensen, Lehmann and Neumann, Buchanan, Bergey, Prévot, Krassilnikov, Murray, Kimura, Woese and Stackebrandt among others. As a consequence of the results it is concluded that between representatives of the Order Alteromanadales, Family Alteromanadaceae, should underscore the isolation of a representative of the genre Shewanella, NF22T, which corresponds to a new species, Shewanella livingstonensis. The remaining species of Shewanella studied correspond to Shewanella frigidimarina (NF12 and NF24). In the group of Great negative bacteria included in the Class of Gammaproteobacterias, Order Pseudomonadales and Family Moraxellaceae have been studied 11 isolates belonging to the genus Psychobacter. Two of them are new species, Psychrobacter luti (NF11T) and Psychrobacter fozzi (NF23T and EN3). The remaining species of Psychrobacter have a taxonomic profile corresponding to Psychrobacter immobilis (NF18, NF19, NF20, EN1 and EN2) and Psychrobacter glacincola (NF1, NF7 and NF8). In the group of Gram positive bacteria studied (20CMT And 20CO) include the description of a new species of the genus Paenibacillus, including naturally in the Family Paenibacillaceae, Class Bacili. This new species has been named Paenibacillus antarcticus.

Author: RUIZ RUEDA CRISTIAN.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA UNIVESIDAD DE BARCELONA.

Summary: The lipases are hidrolasas carboxilicas that break links ester of the acilglicéridos generating free fatty acids and glycerol. These enzymes are of great interest because of their high potential of biotechnology as well as they can act as virulence factors in certain infectious diseases. Therefore, the objective of this study was isolation, donation, characterization and inhibition of lipases by natural substances with interest in microbial biotechnology and infectious diseases. The first chapter is focused on isolation and characterization of the genus Bacillus new lipases to analyze the potential of biotechnology. We analyzed the system lipolítico several strains very active and with a system lipolítico unknown, and subsequently isolated, cloned and characterized the lipases lipA Bacillus megalerium CECT 370 and lipA of Bacíllus sp. BP-6, two arboxilesterasas that are of high interest because of its stability biotechnology and its biochemical and molecular properties. In chapter 2 were isolated 724 microorganisms from the soil of a forest subtroplcal, 449 of which degrade lipids and / or polysaccharides. RC-53 and RC-179, two aíslados very active strains were identified as close to the species Rhodococcus erylhropolis and Bacillus sublilis respectively. In the subsequent analysis of their system lipolítico highlighted the fact that the strain CR-53 produces a lipase that is not secreted from the few known in the genus Rhodococcus. In the third chapter we developed a new microensayo colorimetric rapid, simple and sensitive to evaluate large-scale lipase activity. That test was used to analyze the effect of several saturates about 5 lipases (Paeni) Bacillus. These lipid inhibited all lipases studied, although at low concentrations activated to the lipases not secreted, a phenomenon never before detected that could be related to the characteristics and the biological function of these enzymes. Chapter 4 was to evaluate the effect of several natural substances (saponins, flavonoids and alcaloídes) on the lipase model Candida rugosa (CRL). The beta-aescina, digitonina, (+-)- catechin and kaempferol were selected as the best candidates for clinical applications because of the high inhibition that occurred on CRL, as well as its other beneficial effects, and low toxicity. In chapter 5 were isolated, and characterized the donated lipases of pathogens Propionibacterium acnes and Helicobacler pylori. The analysis GehA of Propionibaclerium acnes P-37, a lipase which is the principal agents of acne, showed that this enzyme is well suited to the conditions in the skin. EstV (HP0739), a carboxilesterasa family V which was identified through analysis of the proteome of Helicobacler pylori 26695, is the first lipase this pathogen that has been cloned, purified and characterized. The analysis of the impact of various natural substances on GehA and EstV revealed that the beta-aescina, acid glícirrinico, (+-)- catechina and kaempferol are very promising candidates for the treatment of acne and / or ulcer because high activity antilipasa, as well as its other effects antiacné or antiúlcera and low toxicity.

Author: SIERRA I ORTIGOSA JOSEP M..
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: Quinolones are a group of synthetic antimicrobials, with a broad spectrum of action and used with great success to treat a wide variety of diseases. It is also a group in full development phase, where new molecules are continually appearing more active than existing in many cases. Unfortunately, resistance to quinolones presents among most of its molecules what is known as cross-resistance, or reduced susceptibility. For that reason and to develop new molecules within this family is important to know what are the mechanisms of resistance presented by microorganisms, in particular Gram-positive bacteria, faced with these compounds. This would allow design new molecules that are not affected by the mechanisms of resistance are already known. Another of the important parameters is known as quinolones themselves selected the emergence of resistant mutants. 1-To investigate the molecular basis of the mechanisms of resistance in Gram-positive bacteria. Specifically, S.aureus an important nosocomial pathogen, S.pneumoniae a pathogen extrahospitalario and Corynebacterium spp.un pathogen emerging opportunistic, belonging to the normal flora of the skin. 2-To study the in vitro selection of mutants resistant to quinolones in clinical isolates of S.aureus and S.pneumoniae. 3-To determine the extent of mutagenicity of the various correlacionaría fluoroquinolones and with the selection of resistant mutants.

Author: ROMERO MONTERO FRANCISCO.
Year: 2004.
University: GRANADA [www.ugr.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The memory of this thesis is based on the study of the mechanisms of action of y. Enterocolitica o9 as immunomodulating agent, since that is capable of launching a circuit suppressor of the immune response against a specific empowerment of nonspecific response. This article has introduced a new model of infection by cannulation of the animals, comrobándose its effectiveness. In turn have corroborated the effects previously observed Y. Enterocolitica as immunomodulating agent, in the previous model. These effects appeared to be due to one or more portions of the outer membrane of the bacterium. In order to determine the active fractions of Y. Enterocolitica, was obtained from the same mass, enriching the sample in outer membrane proteins, separating these proteins by molecular weight and concentration of globalization and after, were tested as mitogens, parallel studies were conducted with the LPS of the bacterium itself, as was another candidate. These experiments were repeated with various chemical and enzymatic (polymyxin, proteinaza K..). At the same time, and through separation of cell populations, attempting to characterize the people responsible for the observed facts. These processes included depleciones, separations using monoclonal antibodies conjugated with ferric pelas, mixed cropping.

Author: AVEDAÑO HERRERA RUBEN ESTEBAN.
Year: 2005.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: In this report we have carried out the characterization biochemical, serological and molecular Tenacibaculum maritimum, causative agent of the tenacibaculosis or flexibacteriosis disease, which causes major losses in fish farming. From the point of view phenotypic T. Maritimum a homogeneous group, but has antigenic heterogeneity estableciéndose different serotypes associated closely with the host. In addition, we have determined the existence of intraspecific genetic variability within the pathogen, describing the existence of two majority clonal lines, which are connected to the host of isolation and / or group infected. The study of the mechanisms of virulence associated with iron uptake of this microorganism revealed that T.maritimun possesses high affinity systems, mid and not mediated sideróforos, allowing it to use this component from the host. In addition, there is evidence that at least some of these systems works "in vivo" at the level of diagnosis. It has developed a non-lethal protocol based on the nested PCR technique, which is highly sensitive, specific and easy to apply for detecting T.maritimum, both on fish as symptomatic carriers of the disease. Studies aimed at the control and possible prevention of the disease showed that the routine use of chemotherapy in enrofloxacin in crop plants, causing a rapid emergence of strains resistant to the compound. We propose as an alternative method to prevent the occurrence of outbreaks tenacibaculosis on farms disinfecting water cultivation and surface tanks with hydrogen peroxide at a concentration of 240 ppm prior to the introduction of the fish. Epizootiological The study revealed that T.maritimum is drastically affected by the indigenous microbiota of seawater, so the water tanks crop would not be a major vector for the transmission of the disease.

Author: SORIA SORIA ELENA.
Year: 2005.
University: ALICANTE [www.ua.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: This work provides new evidence of the involvement of the topology and structure of DNA in the regulation of gene expression in haloarqueas. The results met in regulation through changes in the structure of DNA in response to the environment, both locally and in many parts of the genome of Hfx. Volcanii.

Author: Muñoz pérez del Pulgar Arantxa.
Year: 2005.
University: GRANADA [www.ugr.es].
Place of defense: Facultad de Ciencias.
Place of preparation: Facultad de Ciencias.

Summary: AS-48 is a molecule unique in several respects: it is a peptide synthesis ribosomal circular, with great stability to heat and media with high concentrations of salts, active in a wide range of pH, and that shows bactericidal activity compared to the vast majority of bacteria Gram positive and Gram negative number. Its action is exerted on the cell membrane sensitive and covers a large number of pathogenic bacteria that is transmitted through food (Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus). This activity is enhanced by chemical and physical processing even returning sensitive Gram negative bacteria, innately resistant, as Salmonella choleraesuis and Escherichia coli. His job as bioconservante food can be done by adding the bacteriocina produced by ex-situ growth of the strain in producing an adequate means, or by inoculation of food they want to protect (production on the spot).

Author: CHENOLL CUADROS MARIA EMPAR.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: INSTITUTO DE AGROQUÍMICA Y TECNOLOGÍA DE LOS ALIMENTOS.
Place of preparation: FACULTAD DE FARMACIA.

Summary: In meat products packed in vacuum or modified atmosphere growth of lactic acid bacteria (BAL) leads to the formation of lactic acid, sludges and CO2 fall pH, appearance and odor of bulging packaging, which cause the rejection of the consumer . To address the characterization of species related to the alteration of meat products, we began with the development and testing of molecular techniques based on the identification of genes RNAr for gender Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. Its implementation has revealed three new species within the genus Lactobacillus (L.rennini, L.tucceti and L.vini). It has conducted an exhaustive study of the alteration of meat products through these techniques, through the isolation and identification of 387 colonies in 18 samples of 4 meat products packed in vacuum and cooled. Following the identification scheme, 97% of the isolates were identified to genus level, and 92% at the species level. In the goods altered the dominant species was Leuconostoc mesenteroides, followed in order of abundance by L.sakei, L.plantarum and L.curvatus. In two of meat products analyzed, blood sausage and fiambre of lean adobado, samples were taken at different times of packaging and storage, with a pattern of succession kind where Lc.mesenteroides ended dominate at the time of occurrence of alteration by swelling of the package, while still below the level of detection at the time of packaging. In order to trace the source of pollution as well as analyzing the intraspecific diversity of species majority, has been conducted to characterize 211 isolated by the technique with the RAPD primers M13 and T3. Analysis of the profiles revealed characteristic patterns of species Lc.mesenteroides and L.plantarum the first M13, and L.sakei with T3. The combination of the two profiles showed that there is a intraspecific variation in diversity over storage. In addition, a profile of Lc.mesenteroides was present throughout the storage period and the two products studied. It also detected the presence of RAPD profiles of Lc.mesenteroides and L.sakei in both products, which suggests as a source of pollution the company itself. The results suggest the involvement of different species and strains on the accumulation of gas in storage lean adobado and morcilla packed in vacuum, unleashing altered by swelling pack in both products, some of the third month after the packaging. Finally, we developed a procedure for real-time PCR for the detection and quantification of Lc.mesenteroides, dominant species in the alterations by swelling. The primers are designed aimed at RNAr 23S checking their specificity in vitro in 50 strains of reference belonging to different species of BAL and 50 Lc.mesenteroides, including 3 strains of reference and asylees different RAPD profiles and meat products. The standard curves for quantitation were constructed from DNA and cell suspensions. The level of detection is 1f (0.5 equivalent genomicos) / reaction or 0.5 CFU / reaction, and the limit of quantification was estimated at 100 fg / reaction or 50 CFU / reaction. It was tested in samples of meat products, after extracting DNA through Dneasy Tissue Kit (Qiagen). The procedure Rti-PCR developed is used to detect and quantify low levels of Lc.mesenteroides in meat products, in 3 hours.

Author: OLEA VEGA FRANCISCO.
Year: 2005.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS - UNIVERSIDAD.

Summary: During the interaction between a microorganism fitopatógeno and its host plant produced a large number of physiological changes, metabolic and molecular in the host plant. We have focused on studying the changes in the metabolism of nitrogen during the infective process, as this is a central point for the metabolism of the plant and is a little studied during the interaction. As a working model we used the type compatible interaction between tomato and Pseudomonas syringae pv.tomato. Evidence from previous work developed in our laboratory that arise during the process of pathogenesis is a major mobilization process nitrogen in the tissues infected, so we focused on studying the changes that occurred on the main enzyme responsible for reasimilación the nitrogen in plants: glutamine synthetase (GS) and asparragina synthase (AS). The infected tissues were observed coordinated expression of a GS-type cytosolic (GS1) and AS together with the accumulation of their respective polypeptides. The characterization performed for AS in the genome of tomato along with existing data in the databases indicate that tomatoes there are at least two different genes for AS, patterns of speech and distinct physiological roles. The location of the expression of these enzymes and their polypeptides was performed using in situ hybridization techniques and inmunolocalización found associated expression GS1 the mesophyll cells and photosynthetic AS associated with the phloem vessels in the vascular bundles infected tissue. A similar pattern was observed for the accumulation of their respective polypeptides. To determine the physiological role of these enzymes were undertaken measures amino acid free tissue and in both exudates floemáticos found that the levels of the products of these enzymes, glutaminas and asparragina, amounted in infected tissues and in the phloem, but that their metabolic precursors, glutamate and aspartate, down only in leaflets infected and not in vascular tissue or exudates floemáticos, suggesting that the leaflets were infected conducted synthesis processes and phloem transport phenomena. The data obtained indicate that the expression of GS1 and AS in infected tissues would be responsible for reasimilación nitrogen in tissues infected in the form of glutamine and asparragina, amino acids that are transported through the phloem to other parts of the plant, allowing thus safeguard part of nitrogen that is taking place during the process of pathogenesis.

Author: MARTÍN JIMÉNEZ ROCÍO.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: Breast milk is the ideal food for newborns because it meets all their nutritional requirements, and protect against infections. As we enter this Doctoral Thesis, existing studies on the microbiology of human milk is basically restricted to the isolation and identification of pathogenic bacteria in milk banks and in cases of mastitis or neonatal infections. Therefore, it is unaware of the microbiological composition of the milk of healthy women. It was initially investigated whether this biological fluid containing lactic acid bacteria (BAL) and in fact, we could isolate milk, mammary areola skin of the chest, vagina and feces of eight healthy mothers and of the oral cavity and feces of their respective children. Here are some randomly selected individual (178 each mother-child pair) and were subjected to an analysis of their genetic profiles using RAPD technique. Subsequently, we identified those isolated, regardless of origin, they shared identical profiles. Thus, it was observed that certain strains of Lactobacillus gasseri and Enterococcus faecium were simultaneously isolate of milk samples, mammary areola, oral cavity and child beams provided by the same mother-child pair. In contrast, none of the BAL isolated from skin of the chest or vagina shared RAPD profiles with isolated from other origins. These results showed that breast milk is an important source of BAL for the neonatal intestine and that the BAL present in the milk may have an endogenous origin. The next objective was the evaluation of the potential of two strains of probiotic Lactobacillus gasseri, one of Lb. Fermentum and another Lb.salivarius, all of human milk. Overall, the results showed that Lb.gasseri CECT 5714 and CECT 5715, Lb.fermentum CECT 5716 and Lbsalivarius CECT 5713 with a potential probiotic similar or superior to Lb.rhamnosus GG, Lb.johnsonii La1 and Lb.casei imunitass. Later he studied how bacteria are part of the microbiota dominant breast milk and, in parallel, what group Lactobacillus species can be identified even if they are not dominant, by various techniques based on 16S rRNA. To do so, milk samples were collected from five mothers whose children were born by birth and five others whose infants were born by elective Caesarean section. Analysis of the profiles DGGE revealed that each mother had a specific profile, both in terms of bacteria belonging to the dominant group Lactobacillus. In bookstores clone was observed that most of the sequences were detected in BAL, streptococci, staphylococci or Escherichia coli. These results confirm that the milk of healthy women is a source of commensal bacteria for the intestine child. Finally, the evaluation of the potential of a strain of probiotic Lactobacillus coryniformis, isolated from a cheese artisan goat's milk, showed that probiotic properties were comparable to those of lactobacilli isolated from milk.

Author: FORNS FRADERA NÚRIA.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE BIOLOGÍA.
Place of preparation: FACULTAT DE BIOLOGIA (UNIVERSITAT DE BARCELONA).

Author: PRADO PLANA SUSANA.
Year: 2006.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: The cultivation of bivalve molluscs is an area of great economic and social importance in Galicia. At present, the depletion of the banks has become a natural breeding grounds in the only source of native seed, especially when it comes to oyster (Ostrea edulis) and clams (Ruditapes decussatus, Venerupis pullastra, and the species R. alochthonous philip). However, these facilities are faced with episodes of mass mortalities, resulting in many cases of bacterial infections, which hinder the smooth supply and involve economic losses. The water treatment crops through various physical and chemical processes has proved insufficient, entailing further problems such as the emergence of resistance, high cost or banning their use by the current legislation. That is why in recent years seeking alternative solutions, among them the use of probiotic bacteria. In this work we have been following the problems associated with different Galicians hatcheries, which have enabled us to identify a clear bacteria responsible for three episodes of larval mortality. The experiments demonstrate that they are capable of causing serious mortalities in cultures of larval oysters flat within the first 48 hours. In all cases have proven bacteria belonging to the genus Vibrio. The pathogen 145.98 has been identified as Vibrio neptunius, is the first description of this species as pathogenic. However, the genotypic and phenotypic characterization of the isolated 203 and 638 points to its description as a new species within the genus. In the search for potential probiotic bacteria that provide control populations of vibrios in the crops were tested amid solid 520 isolates obtained from the different compartments of the hatcheries, versus four strains witness (3 vibrios and a coconut Gram positive) . Was selected strain 154 per show maximum activity against all of them. This strain has a broad spectrum of antibacterial activity that includes pathogens aquaculture, especially of the genus Vibrio, and clinical strains of origin. It demonstrated its inhibitory activity in vitro, both in solid medium and in seawater. In vivo studies confirmed their ability to control growing populations of vibrios in the water and larval crop cultivation of microalgae used as feed for farmed. It also proved to be effective in combating bacterial infections, either arising from natural or induced in the laboratory, where used as a preventive measure. This strain, along with other isolated like she (639, 694 and 847) have been assigned to clear the kind Roseobacter gallaeciensis recently reclassified under the new gender Phaeobacter. This creation of a new genus is supported by studies carried out in this report, but not the case with its division into two species, which should be the subject of further studies. The strain 154 is a suitable candidate for use as a probiotic in larval marine crops, preventing the proliferation of opportunistic pathogens.