Tesis sobre el tema "Environmental bacterial isolates"

Cryptococcus neoformans is an encapsulated yeast responsible for severe meningoencephalitis. The importance of epidemiological studies on cryptococcosis has increased since the beginning of the AIDS epidemic. C. neoformans exists in two varieties containing four serotypes, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Locally C. neoformans var. neoformans has been associated with pigeon feces during those months having an average temperature of 64.2°F j(17.8°C) and above. Clinical and environmental isolates of C. neoformans obtained from regional hospitals and environmental samplings, respectively, have been grouped into their variety status utilizing canavanine-glycine-bromthymol blue agar. Polyclonal antisera against C. neoformans serotypes A, B, C and D were isolated from challenged rabbits. Serotyping C. neofromans isolates using the polyclonal antisera resulted in 57% (20 of 35) of the serotypes confirmed with a direct immunofluorescent assay utilizing a single monoclonal antibody (E1). Data from the immunofluorescence assay suggest all C. neoformans obtained from regional hospitals (26 of 26) and those isolated from the environment (9 of 9) belong to the A serotype group. These data have provided information leading to the origin of infection for cryptococcosis in our region, which may be beneficial to immunocompromised individuals.

This study describes the characterisation and identification of two species of sulphate-reducing bacteria isolated from marine environments. The isolate coded Ind 1 was recovered from the heavily corroded hull of an oil storage vessel moored off the Indonesian coast. An isolate, referred to as Al 1, originated from a soured oil reservoir in Alaska. Observations using microscopy (light, scanning electron and atomic force) revealed that cells were Gram-negative, rod-shaped and very motile. Physiological characterisation, analysis of the fatty acid profiles and partial and full 16S rRNA sequencing demonstrated strong similarities between the two species and members of the Desulfovibrio genus. The position of the strains within phylogenetic trees showed Al 1 clustering closely with Desulfovibrio vietnamensis. Ind 1 revealed a high degree of similarity with both Desulfovibrio gigas and Desulfovibrio gabonensis and these three strains formed a separate cluster in the delta subdivision of the Proteobacteria. However, whole-cell protein profiles and Fourier-transform infrared spectroscopy studies showed that there is enough dissimilarity between the two isolates and the remaining species of the genus Desulfovibrio to consider Al 1 and Ind 1 as new separate species. Purification, physico-chemical and spectroscopic characterisation of the key enzymes involved in the sulphate metabolism was carried out for both isolates. Nuclear magnetic resonance and electron paramagnetic resonance studies revealed that the proteins of Al 1 and Ind 1 exhibited various features in common with their counterparts from other members of the genus Desulfovibrio. In particular, proteins from Ind 1 showed many similarities with the enzymes previously described for D. gigas. Based on the obtained results, the classification of Ind 1 as Desulfovibrio indonensiensis sp. nov. and Al 1 as Desulfovibrio alaskensis sp. nov. are proposed. The overall results highlight the complexity of the relationship between cell physiology and the organisms' environmental impact.

As part of a large study aimed at searching for patterns of diversity in the genus Persephonella along the north to south geochemical gradient of the ELSC, ten novel strains of Alphaproteobacteria were isolated unexpectedly. Using defined media under microaerophilic conditions to enrich for Persephonella from chimney samples collected at the seven vent fields on the ELSC and the dilution to extinction by serial dilution method to purify cultures, a total of ten strains belonging to the Alphaproteobacteria were isolated. Two of these isolates, designate MN-5 and TC-2 were chosen for further characterization and are proposed as two new species of a novel genus to be namedThermopetrobacter. Both strains are aerobic, capable of chemoautotrophic growth on hydrogen and grow best at 55°C, pH 6 and 3.0% NaCl. Strain MN-5 is capable of heterotrophic growth on pyruvate and malate and TC-2 is only able to grow heterotrophically with pyruvate. The GC content of MN-5 is 69.1 and TC-2 is 67 mol%. GenBank BLAST results from the 16S rRNA gene reveal the most closely related sequence to MN-5 is 90% similar and the most closely related sequence to strain TC-2 is 89% similar. Sampling at a shallow marine vent on the coast of Vulcano Island, Italy in 2007 led to the isolation of a novel species of Hydrogenothermus, a genus within the Hydrogenothermaceae family. This isolate, designated NV1, represents the secondHydrogenothermusisolated from a shallow marine vent. NV1 cells are rod-shaped, approximately 1.5μm long and 0.7μm wide, motile by means of a polar flagellum and grow singularly or in short chains. Cells grow chemoautotrophically using hydrogen or thiosulfate as electron donors and oxygen as the sole electron acceptor. Growth was observed between 45 and 75°C with an optimum of 65°C (doubling time 140 min), pH 4.0-6.5 and requires NaCl (0.5-6.0% w/v). The G+C content of total DNA is 32 mol%.

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Bactérias ácido lácticas (BAL) foram isoladas do ambiente de produção de leite e avaliadas quanto ao potencial benéfico. Testes preliminares e análise por PCR foram aplicados para selecionar e identificar através de sequenciamento de rRNA 16S 15 cepas de BAL: Lactobacillus (n = 11; Lb. casei MSI1, Lb. casei MSI5, Lb. casei MRUV1, Lb. casei MRUV6, Lb. acidophilus MVA3, Lb. nagelli MSIV4, Lb. harbinensis MSI3, Lb. harbinensis MSIV2, Lb. fermentum SIVGL1, Lb. plantarum MLE5 e Lb. plantarum MSI2), Pediococcus (n = 2; P. pentosaceus MLEV8 e P. acidilactici MSI7) e Weissella (n = 2; W. paramesenteroides MRUV3 e W. paramesenteroides MSAV5). Todas as linhagens selecionadas apresentaram resistência ao baixo pH e à presença de sais biliares. O teste API ZYM foi realizado para caracterizar a atividade enzimática entre as cepas e foi observada elevada atividade β-galactosidase em 13 delas. Todas as cepas apresentaram alta taxa de sobrevivência ao suco gástrico e as condições intestinais simulados, capacidade de auto-agregação e co- agregação com micro-organismos indicadores e alta hidrofobicidade da superfície celular. A maioria das cepas foi positiva para os genes de adesão map e EFTu. Os resultados de deconjugação de sais biliares mostraram forte desconjugação para todas as cepas. Todas as cepas mostraram bons resultados para assimilar lactose. Após esta etapa de caracterização do potencial benéfico, as 15 BAL foram avaliadas quanto ao potencial de virulência e de resistência antimicrobiana. A produção de fatores de virulência (hemólise, gelatinase, lipase, desoxirribonuclease e aminas biogênicas: lisina, tirosina, histidina e a ornitina) foi avaliada por métodos fenotípicos, a 25 °C e 37 °C, bem como a resistência a 17 antibióticos. Os isolados foram também submetidos à análise de PCR para identificar a presença de 49 genes associados a fatores de virulência. Nenhuma das cepas apresentou atividade hemolítica, produção de gelatinase, lipase, desoxirribonuclease e aminas biogênicas. Das 15 cepas selecionadas, para 12 tipos de antibióticos no método de difusão em disco, todas as amostras foram resistentes à oxacilina e sulfa/trimetoprim, 14 foram resistentes a gentamicina, 11 foram resistentes a clindamicina, nove cepas foram resistentes à vancomicina, oito cepas para rifampicina, cinco foram resistentes a eritromicina, quatro foram resistentes à tetraciclina, duas cepas foram resistentes à ampicilina, uma cepa foi resistente ao cloranfenicol e nenhuma apresentou resistência ao imipenem. Para um teste quantitativo do antibiograma, 5 antibióticos em fitas Etest® (bioMérieux) foram selecionados. Todas as 15 cepas foram resistentes à vancomicina, duas para rifampicina, uma para gentamicina e uma para o cloranfenicol. Em relação aos genes relacionados com virulência, 19 dos 49 genes testados estavam presentes em algumas cepas. Após a caracterização do potencial virulento das 15 BAL, estas foram avaliadas quanto ao potencial tecnológico para aplicação na indústria de laticínios. Todas as cepas apresentaram capacidade de acidificação, atingindo valores de pH entre 0.73 e 2.11 em 24 horas: Lb. casei MRUV6 apresentou maior capacidade de acidificação (pH 2.11 após 24 h). Dez cepas foram capazes de produzir diacetil a 37 °C, com exceção de Lb. casei MSI1, Lb. harbinensis MSI3, Lb. fermentum SIVGL1, Lb. plantarum MLE5 e W. paramesenteroides MRUV3. Todas as cepas foram capazes de produzir exopolissacarídeos, e apenas duas cepas apresentaram atividade proteolítica (Lb. casei MSI5 e W. paramesenteroides MSAV5). Com base nessa caracterização, Lb. casei MRUV6 foi selecionado para produzir o leite fermentado, armazenado a 4 °C e 10 °C e monitorado até 35 dias de vida útil. As amostras foram submetidas a métodos fenotípicos e moleculares para avaliar a presença de Lb. casei MRUV6 (plaqueamento convencional e RT-PCR, verificando a expressão de gapdh, um gene housekeeping) e verificar a expressão do gene bsh, relacionado à resistência à sais biliares (RT-PCR). A população de Lb. casei MRUV6 se apresentou estável durante todo o período de armazenamento a 4 °C e 10 °C a níveis em torno de 9.9 log UFC/g e também pelo monitoramento da expressão do controle endógeno GAPDH. No entanto, o gene bsh não foi expresso durante o período de armazenamento. O estudo demonstrou o potencial uso da cepa de Lb. casei MRUV6 isolada de um ambiente lácteo para a produção de um produto lácteo fermentado e sua estabilidade durante o armazenamento a 4 °C e 10 °C. Todos os isolados do estudo apresentaram características benéficas, segurança para utilização em alimentos e potencial tecnológico para utilização na indústria de laticínios. Além disso, os mesmos podem ainda ser submetidos a estudos adicionais para avaliações in vivo e realizar a caracterização como probióticos.
Lactic acid bacteria isolated from dairy environment were evaluated for beneficial potential. Preliminary screening and PCR analysis were applied to select and identified through 16s rRNA sequencing 15 LAB strains: Lactobacillus (n = 11; Lb. casei MSI1, Lb. casei MSI5, Lb. casei MRUV1, Lb. casei MRUV6, Lb. acidophilus MVA3, Lb. nagelli MSIV4, Lb. harbinensis MSI3, Lb. harbinensis MSIV2, Lb. fermentum SIVGL1, Lb. plantarum MLE5 and Lb. plantarum MSI2), Pediococcus (n = 2; P. pentosaceus MLEV8 and P. acidilactici MSI7) and Weissella (n = 2; W. paramesenteroides MRUV3 and W. paramesenteroides MSAV5). All selected strains showed resistance to acidic pH and to presence of bile salt. API ZYM test characterized enzymatic activity of the strains and high β-galactosidase activity was observed in 13 strains. All strains presented high values for survival rate to simulated gastric and intestinal conditions, ability to auto and co-aggregate with indicators microorganisms and high cell surface hydrophobicity. Most of the strains were positive for map and EFTu beneficial genes. Strong bile salts deconjugation was applied for all strains and all strains showed good results for assimilating lactose. After this first part of the study, the 15 BAL were evaluated for potential virulence and antimicrobial resistance. The production of virulence factors (hemolysis, gelatinase, lipase, deoxyribonuclease and biogenic amines: lysine, tyrosine, histidine and ornithine) was assessed by phenotypic methods at 25 °C and 37 °C, as well as the resistance to 17 antimicrobials. The isolates were also subjected to PCR to identify the presence of 49 genes associated with virulence factors. None of the strains presented hemolytic activity or the production of gelatinase, lipase, deoxyribonuclease and tested biogenic amines. Of the 15 selected cultures, for 12 types of antibiotics in the disc diffusion method, all strains were resistant for oxacillin and sulfa/trimethoprim, 14 were resistant to gentamicin, 11 were resistant to clindamycin, nine strains were resistant to vancomycin, eight strains to rifampicin, five were resistant to erythromycin, four were resistant to tetracycline, two strains were resistant to ampicillin, one strain was resistant to chloramphenicol and none was resistant for imipenem. For a quantitative test of the antibiogram, five antibiotics were selected in Etest ® strips (bioMérieux). All 15 strains were resistant to vancomycin, two for rifampicin, one for gentamicin and one for chloramphenicol. Regarding the virulence related genes, 19 genes from 49 tested were present in some strains. Results showed that five cultures showed the presence of the int gene, four cultures showed the presence of the ant(4')-Ia gene, three cultures were positive for vanC2, cpd and tdc, two cultures for vanA, tet(K), tet(S), ermA, bcrR, mur-2ed, asa1 and ccf, and one culture was positive for vanC1, ermB, aph(3')-IIIa, aac(6’)-le-aph(2”)-Ia, bcrB and hyl. After characterizing the virulent potential of the 15 BAL, these strains were evaluated for the technological potential for application in the dairy industry. All strains presented acidification capacity, reaching pH values between 0.73 and 2.11 in 24 hours: Lb. casei MRUV6 presented the highest acidification ability (pH 2.11 after 24 h). Ten strains were able to produce diacetyl at 37 °C, except by Lb. casei MSI1, Lb. harbinensis MSI3, Lb. fermentum SIVGL1, Lb. plantarum MLE5 and W. paramesenteroides MRUV3. All strains were able to produce exopolysaccharides, and only two strains presented proteolytic activity (Lb. casei MSI5 and W. paramesenteroides MSAV5). Based on this characterization, Lb. casei MRUV6 was selected for producing fermented milk, stored at 4 °C and 10 °C and monitored until 35 days of shelf life. Samples were subjected to phenotypical and molecular methods to quantify the presence of Lb. casei MRUV6 (conventional plating and RT-PCR, by checking the expression of gapdh, a housekeeping gene) and to verify the expression of bsh gene, related to resistance to bile salts (RT-PCR). Lb. casei MRUV6 population was stable during storage period at 4 and 10 °C at levels around 9.9 log CFU/g, and by monitoring the expression of gapdh gene. However, bsh gene was not expressed during storage period. The study demonstrated the potential use of the beneficial strain Lb. casei MRUV6 isolated from a dairy environment for the production of a fermented milk product, and its stability during storage at 4 and 10 °C. All isolates from the study presented beneficial characteristics, safety for use in food and technological potential for use in the dairy industry. In addition, they may further be subjected to further studies for in vivo evaluations and characterization as probiotics.

Thesis (M.Sc.) -- University of Limpopo, 2005
Six aerobic bacterial strains [GM 10(1), GM 10 (2), GM 14, GM 15, GM 16 and GM 17] were isolated from an antimony mine in South Africa. Heavy-metal resistance and biosorptive capacities of the isolates were studied. Three of the isolates (GM 15, GM 16 and GM 17) showed different degrees of resistance to antimony and arsenic oxyanions in TYG media. The most resistant isolate GM 16 showed 90 % resistance, followed by GM 17 showing 60 % resistance and GM 15 was least resistant showing 58 % resistance to 80 mM arsenate (AsO4 3-). GM 15 also showed 90 % resistance whereas isolates GM 16 and GM 17 showed 80 % and 45 % resistance respectively to 20 mM antimonate (SbO4 3-). Arsenite (AsO2 -) was the most toxic oxyanion to all the isolates. Media composition influenced the degrees of resistance of the isolates to some divalent metal ions (Zn2+, Ni2+, Co2+, Cu2+ and Cd2+). Higher resistances were found in MH than in TYG media. All the isolates could tolerate up to 5 mM of the divalent metal ions in MH media, but in TYG media, they could only survive at concentrations below 1 mM. Also, from the toxicity studies, high MICs were observed in MH media than TRIS-buffered mineral salt media. Zn2+ was the most tolerated metal by all the isolates while Co2+ was toxic to the isolates. The biosorptive capacities of the isolates were studied in MH medium containing different concentrations of the metal ions, and the residual metal ions were determined using atomic absorption spectroscopy. GM 16 was effective in the removal of Cu2+ and Cd2+ from the contaminated medium. It was capable of removing 65 % of Cu2+ and 48 % of Cd2+ when the initial concentrations were 100 mg/l, whereas GM 15 was found to be effective in the biosorption of Ni2+ from the aqueous solutions. It was capable of removing 44 % of Ni2+ when the initial concentration was 50 mg/l. GM 17 could only remove 20 % of Cu2+ or Cd2+. These observations indicated that GM 16 could be used for bioremediation of xvi Cu2+ and Cd2+ ions from Cu2+ and Cd2+-contaminated aqueous environment, whereas GM 15 could be used for bioremediation of Ni2+.
National Research Foundation and the University of the North Research Unit

La contaminació ambiental és una de les condicions extremes que pot alterar els ecosistemes naturals i afectar tots els organismes vius. Entre els contaminants ambientals, els metalls tenen un paper rellevant degut a la seva elevada toxicitat. De fet, es pot considerar que els microorganismes que habiten zones contaminades per metalls formen freqüentment consorcis, la qual cosa els permet desenvolupar estratègies cel·lulars per sobreviure en presència de metalls evitant així els seus efectes nocius. En els darrers anys, el nostre grup de recerca ha aïllat diferents consorcis de microorganismes dels tapets microbians del delta de l’Ebre (Tarragona). Aquests consorcis estan formats per un únic microorganisme fotòtrof i diferents heteròtrofs. En la present tesi doctoral, a partir del consorci on domina la microalga Scenedesmus sp. DE2009, s’ha aïllat, caracteritzat i identificat un bacteri heteròtrof com a Ochrobactrum anthropi DE2010. En estudis preliminars, aquest bacteri ha mostrat la capacitat per fixar nitrogen atmosfèric, presentar pleomorfisme cel·lular i resistir a antibiòtics del grups dels betalactàmics. Encara que prèviament s’ha investigat l’efecte de diversos metalls pesants en el consorci Scenedesmus sp. DE2009, no se sap res sobre l’efecte d’aquests en la viabilitat cel·lular d’O. antropi DE2010 i el seu paper en la captació de metalls pesants. Els objectius de la present tesi s’han desenvolupat tenint en compte que O. anthropi DE2010 creix de manera fàcil i ràpida tant en medi líquid com sòlid, esdevenint un model adequat per a la investigació front a toxicitat per metalls pesants. A més a més, s’ha analitzat l’efecte citotòxic i la capacitat de resposta de O. anthropi DE2010 per superar l’estrès front a l’exposició a Cd, Pb(II), Cu(II), Cr(III) i Zn així com els patrons de localització cel·lular d’aquests metalls i les estratègies emprades per aquest bacteri per immobilitzar-los. Aquesta tesi s’ha organitzat en diferents capítols. Concretament, el capítol 2 correspon als articles publicats on els resultats obtinguts de la investigació realitzada es troben a les seccions de la 2.1 a la 2.4 i es discuteixen globalment en el capítol 3. Els resultats obtinguts en aquest estudi indiquen que O. anthropi DE2010 mostra una elevada resistència als cinc metalls assajats tolerant concentracions de fins a 20 mM de Zn i de 10 mM per a Cd, Pb(II), Cu(II) i Cr(III). A més, aquest bacteri té una gran capacitat per eliminar metalls del medi, captant fins a un 90% de Pb (II) i un 40% de Cr (III), ambdós a 10 mM. Analitzant la seqüencia del genoma d’O. anthropi DE2010 s’ha observat que presenta sis gens relacionats amb el metabolisme del polifosfat, i que l’anàlisi del contingut cel·lular de polyP indica que aquest bacteri el sintetitza i l’acumula de manera directament proporcional a la concentració de metall. D’altra banda, les cèl·lules d’O. anthropi DE2010 immobilitzen metalls pesants en grànuls i/o inclusions de polyP mitjançant tres patrons específics de localització subcel·lular: extracel·lularment en grànuls de polifosfat (Cu (II)); a l’espai periplasmàtic formant cristalls amb el fòsfor (Pb (II)), i intracel·lularment en inclusions de polifosfat (Pb (II), Cr (III) i Zn). Per tant, O. anthropi DE2010 genera respostes cel·lulars (estratègies de supervivència) específiques per a cada metall, com ara bioacumulació en el cas de Pb (II), Cu (II), Cr (III) i Zn, biosorció per al Cr (III) i Cd i biomineralització per al Pb (II). L’elevada resistència i la capacitat de segrestar metalls d’O. anthropi DE2010 posen de manifest el seu gran potencial com a possible agent bioremediador, especialment en zones contaminades per Pb i Cr.
La contaminación ambiental es una de las condiciones extremas que puede alterar los ecosistemas naturales y afectar a todos los organismos vivos. Entre los contaminantes ambientales, los metales tienen un papel relevante debido a su elevada toxicidad. De hecho, se puede considerar que los microorganismos que habitan zonas contaminadas por metales forman frecuentemente consorcios, lo que les permite desarrollar estrategias celulares para sobrevivir en presencia de metales evitando así sus efectos nocivos. En los últimos años, nuestro grupo de investigación ha aislado diferentes consorcios de microorganismos de los tapetes microbianos del delta del Ebro (Tarragona). Estos consorcios están formados por un único microorganismo fotótrofo y varios heterótrofos. En la presente tesis doctoral, a partir del consorcio donde domina la microalga Scenedesmus sp. DE2009, se ha aislado, caracterizado e identificado una bacteria heterótrofa como Ochrobactrum anthropi DE2010. En estudios preliminares, esta bacteria ha mostrado la capacidad para fijar nitrógeno atmosférico, presentar pleomorfismo celular y resistir a antibióticos del grupo de los betalactámicos. Aunque previamente se ha investigado el efecto de varios metales pesados en el consorcio Scenedesmus sp. DE2009, poco se conoce del efecto de estos en la viabilidad celular de O. antropi DE2010 y su papel en la captación de metales pesados. Los objetivos de la presente tesis se han desarrollado teniendo en cuenta que O. anthropi DE2010 crece de manera fácil y rápida tanto en medio líquido como sólido, lo que permite considerarlo como modelo para la investigación frente a la toxicidad por metales pesados. Además, se ha analizado el efecto citotóxico y la capacidad de respuesta de O. anthropi DE2010 para superar el estrés frente a la exposición a Cd, Pb(II), Cu(II), Cr(III) y Zn así como los patrones de localización celular de estos metales y las estrategias empleadas por esta bacteria para inmovilizarlos. Esta tesis se ha organizado en diferentes capítulos. Concretamente, el capítulo 2 corresponde a los artículos publicados donde los resultados obtenidos se encuentran en las secciones de la 2.1 a la 2.4 y se discuten globalmente en el capítulo 3. Los resultados obtenidos en este estudio indican que O. anthropi DE2010 muestra una elevada resistencia a los cinco metales ensayados tolerando concentraciones de hasta 20 mM de Zn y de 10 mM para Cd, Pb(II), Cu(II) y Cr(III). Además, esta bacteria tiene una gran capacidad para eliminar metales del medio, captando hasta un 90% de Pb (II) y un 40% de Cr (III), ambos a 10 mM. Analizando la secuencia del genoma de O. anthropi DE2010 se ha observado que presenta seis genes del metabolismo del polifosfato, y que el análisis del contenido celular de polyP indica que esta bacteria lo sintetiza y acumula de manera directamente proporcional a la concentración de metal. Por otro lado, las células de O. anthropi DE2010 inmovilizan metales pesados en gránulos y/o inclusiones de polyP mediante tres patrones específicos de localización subcelular: extracelularmente en gránulos de polifosfato (Cu (II)); en el espacio periplasmático formando cristales con el fósforo (Pb (II)), e intracelularmente en inclusiones de polyP (Pb (II), Cr (III) y Zn). Por lo tanto, O. anthropi DE2010 genera respuestas celulares (estrategias de supervivencia) específicas para cada metal, como bioacumulación en el caso de Pb (II), Cu (II), Cr (III) y Zn, biosorción para el Cr (III) y Cd y biomineralitzación para el Pb (II). La elevada resistencia y la capacidad de secuestrar metales de O. anthropi DE2010 ponen de manifiesto su gran potencial como posible agente bioremediador, especialmente en zonas contaminadas por Pb y Cr.
Environmental pollution remains as one of the extreme conditions that can disrupt the existing natural ecosystems and affect all the living organisms. Among the environmental stressors, metals play a significant role as potential toxicants. Consequently, it can be considered that the microorganisms inhabiting metal polluted areas frequently form consortia and they can develop cellular strategies to survive in the presence of heavy metals for avoid their negative effects. In the last years, our research group has isolated different consortia of microorganisms from Ebro Delta microbial mats (Tarragona). These consortia are formed by a single type of phototrophic microorganism and different heterotrophic bacteria. In the present doctoral thesis, an heterotrophic bacteria from the consortium dominated by the microalga Scenedesmus sp. DE2009, have been isolated, characterized and identified as Ochrobactrum anthropi DE2010. In preliminary studies, this bacterium showed the ability to fix atmospheric nitrogen, present cellular pleomorphism and beta-lactam antibiotic resistance. Despite the effect of some heavy metals in the microalgae consortium had previously been investigated, nothing is known about the effect of them on the cell viability of O. anthropi DE2010 and the role of this bacterium in heavy metal sequestration. The goals of this thesis have been developed taking into account the fact that O. anthropi DE2010 grows easily and fast in both liquid and solid media culture, which it becomes a suitable model for heavy metal experimental research. Thus, cytotoxic effects of heavy metals and cellular responses on O. anthropi DE2010 cultures exposed to increasing concentrations of Cd, Pb(II), Cu(II), Cr(III), and Zn, and the cellular metal localization patterns and the strategies and pathways used by this bacterium to immobilize them have extensively been analyzed. This thesis has been organized in different Chapters. Specifically, the Chapter 2 correspond to the published articles; thus, the obtained results from the research carried out are therefore in sections from 2.1 to 2.4 and are discussed globally in Chapter 3. The results of this study indicate that O. anthropi DE2010 shows high resistance to the five tested metals, supporting concentrations up to 20 mM of Zn and up to 10 mM for Cd, Pb(II), Cu(II) and Cr(III). Moreover, this bacterium has a high ability to remove metals from the environment, highlighting up to 90% for Pb(II) and 40% for Cr(III) both at 10 mM. Analyzing the sequenced genome of O. anthropi DE2010 it has been observed that it presents six genes linked to the metabolism of the polyphosphate and that the analysis of polyP content indicates that this bacterium synthesizes and accumulates it in a metal concentration-dependent manner. O. anthropi DE2010 cells immobilized heavy metals in polyP granules and/or inclusions in three metal-specific patterns for subcellular localization: extracellular in polyphosphate granules (Cu(II)); in the periplasmic space forming crystals with phosphorus (Pb(II)), and intracellular in polyphosphate inclusions (Pb(II), Cr(III) and Zn). Therefore, O. anthropi DE2010 generates specific cellular responses for each heavy metal as survival strategies, such as bioaccumulation for Pb(II), Cu(II), Cr(III) and Zn, biosorption for Cr(III) and Cd and biomineralization for Pb(II). The high resistance and the capacity to uptake metals evidenced by O. anthropi DE2010 prove its great potential as a possible candidate to bioremediate, especially Pb and Cr polluted areas.
Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologia

Chemistry
Ph.D.
Microorganisms with an ability to thrive in harsh environments are referred as “extremophiles”. With advances in biotechnology, interest has grown in the extremophile research because of their unique macromolecules’ characteristics due to their growth environments. Over last decade, researchers have isolated many extremophiles from environments like volcano, salt lakes, hydrothermal vents, deep oceans, Antarctica glaciers etc. Macromolecules of these extremophiles are responsible for their survival in extreme environments. In this research work we have isolated lipid molecules from three different microorganisms. 1) GWE1 strain, a thermophilic bacterium, isolated from dark crusty material from sterilization ovens. 2) 7L strain, a thermophilic bacterium, isolated from Chilean Copahue Volcano. 3) I1P strain, a facultative anaerobe of the family Enterobacteriaceae, recently isolated from Antarctica. Complex lipid arrangement and/or type in the cell membrane are known to affect thermostability of microorganisms and efforts were made to understand the chemical nature of the polar lipids of membrane. In this work, we extracted total lipids from cell membrane, separated them by TLC into various fractions and characterize the lipid structures of fractions with analytical tools such as 1H, 13C, 31P and 2D NMR spectroscopy, ATR-FTIR spectroscopy and MSn spectrometry. In GWE1 strain, we were able to identify glycerophosphoethanolamine, glycerophosphate, glycerophosphoglycerol and cardiolipin lipid classes and an unknown glycerophospholipid class with novel MS/MS spectra pattern. We have also noticed the presence of saturated iso-branched fatty acids with NMR spectra in individual lipid classes. In case of I1P strain, we have identified glycerophosphoglycerol, glycerophosphoethanolamine, glycerophosphate, and acyl glycerophosphoglycerol lipid classes with unsaturated fatty acids in their structure, which could be one of the many reasons for survivability at lower temperatures. In case of 7L strain, we were able to identify glycerophosphoglycerol, cardiolipin, glycerophosphoethanolamine and glycerophosphate lipid classes with saturated iso branched fatty acids. FAME analysis revealed iso-15:0 (52.29 %) and iso-17:0 (18.64 %) as major fatty acyl chains. We did not observe major difference in polar head group composition of lipid classes between thermophiles (GWE1 and 7L) compare to psychrophiles (I1P). Major difference among these three strains was in fatty acid composition of lipid molecule. Both thermophiles showed presence of lipids with long chain saturated fatty acids while I1P showed presence of lipid molecule with unsaturated fatty acid chain. Lipids made of unsaturated fatty acids have lower melting points and they introduce kink in the cell membrane structure. At lower temperatures, these effects allow membrane to maintain fluidity and its functionality, which in turn allows the microorganism to grow at lower temperature. Lipids made with saturated iso branched fatty acid chain have higher melting points and they pack together densely in cell membrane. At high temperature because of higher melting point and dense packing, membrane fluidity is not affected and this effect allows microorganism to grow at the higher temperature. We believe that change in fatty acid composition is one of the many reasons for these microorganisms to survive the extreme condition. Thermostability of the other macromolecules (DNA, enzyme) of these extremophiles is not studied in this dissertation.
Temple University--Theses

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formers that are able to spoil acidic food and beverage products through the production of guaiacol and other taint compounds, which causes a medicinal off-flavour and/or odour in the products. This thesis reports on the comparison of methods used for the isolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacol production of different strains isolated from the South African fruit processing environment. Two methods for guaiacol detection were also evaluated and compared. Three isolation methods frequently used by South African fruit processors were compared with regards to their ability to isolate a strain of A. acidoterrestris from diluted peach juice concentrate. Method 1, the International Federation of Fruit Juice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillus acidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidified potato dextrose agar (PDA); and Method 3 makes use of membrane filtration and incubation of the membrane on K agar. The IFU Method No. 12 was the most effective method for the isolation of A. acidoterrestris, with a recovery of 75.97%. These results support the use of the IFU Method No. 12 as a standard international method for the isolation and detection of species of Alicyclobacillus. Seven strains of Alicyclobacillus, including the type strains A. acidoterrestris DSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a South African fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A. acidocaldarius FB19, were analysed based on their growth characteristics and guaiacol production under optimum conditions. Strains were inoculated into BAT medium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for 7 d. All the strains had similar growth patterns, with cell concentrations increasing rapidly from 0-24 h, followed by a stabilisation around maximum cell concentrations of 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indication of spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating an increase in spores as the cell density and competition for resources increased. All the strains were able to produce guaiacol in detectable concentrations [as measured by the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess the potential to cause product spoilage. Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formers that are able to spoil acidic food and beverage products through the production of guaiacol and other taint compounds, which causes a medicinal off-flavour and/or odour in the products. This thesis reports on the comparison of methods used for the isolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacol production of different strains isolated from the South African fruit processing environment. Two methods for guaiacol detection were also evaluated and compared. Three isolation methods frequently used by South African fruit processors were compared with regards to their ability to isolate a strain of A. acidoterrestris from diluted peach juice concentrate. Method 1, the International Federation of Fruit Juice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillus acidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidified potato dextrose agar (PDA); and Method 3 makes use of membrane filtration and incubation of the membrane on K agar. The IFU Method No. 12 was the most effective method for the isolation of A. acidoterrestris, with a recovery of 75.97%. These results support the use of the IFU Method No. 12 as a standard international method for the isolation and detection of species of Alicyclobacillus. Seven strains of Alicyclobacillus, including the type strains A. acidoterrestris DSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a South African fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A. acidocaldarius FB19, were analysed based on their growth characteristics and guaiacol production under optimum conditions. Strains were inoculated into BAT medium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for 7 d. All the strains had similar growth patterns, with cell concentrations increasing rapidly from 0-24 h, followed by a stabilisation around maximum cell concentrations of 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indication of spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating an increase in spores as the cell density and competition for resources increased. All the strains were able to produce guaiacol in detectable concentrations [as measured by the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess the potential to cause product spoilage. iv The influence of temperature on the growth and guaiacol production of the Alicyclobacillus strains was also investigated and two guaiacol detection methods, the PECA and headspace gas-chromatography mass-spectrometry (HS GC-MS), were compared with regards to their ability to detect guaiacol. The strains were incubated at 25°C and 45°C for 6 d and samples analysed every 24 h. Growth of the A. acidoterrestris strains was slower at 25°C, and maximum cell concentrations were lower than at 45°C. A decrease in cell concentrations was observed in the A. acidocaldarius strains at 25°C, as this temperature is below their growth temperature range. All the strains were able to produce guaiacol at 45°C, with guaiacol only being detected once a cell concentration of 104-105 cfu.mL-1 had been reached. The maximum guaiacol concentrations detected at 45°C in the samples containing A. acidoterrestris were significantly higher than those detected in the A. acidocaldarius samples. At 25°C there was a longer lag phase before guaiacol was detected in the A. acidoterrestris samples, while no guaiacol was detected in the samples containing A. acidocaldarius. Because guaiacol is produced at ambient temperatures, cooling of products is recommended to control spoilage by A. acidoterrestris. The sensitivity of the two guaiacol detection methods also differed significantly and, therefore, the PECA is recommended for presence/absence detection of guaiacol, while HS GCMS is recommended where accurate quantification of guaiacol is required. Alicyclobacillus acidoterrestris FB2 was investigated for its ability to grow and produce guaiacol in white grape juice supplemented with vanillin at different concentrations. Alicyclobacillus acidoterrestris FB2 was inoculated into white grape juice concentrate diluted 1:10 with distilled water containing 0-500 mg.L-1 vanillin and incubated at 45°C for 6 d. Similar growth patterns were observed in all the samples, except in the sample containing 500 mg.L-1 vanillin, which had a longer lag phase of growth. Guaiacol concentrations, detected using the PECA, increased as the vanillin concentration increased, with the exception of the sample containing 500 mg.L-1 vanillin, where less guaiacol was detected than in the sample containing 250 mg.L-1 vanillin, due to growth inhibition caused by the higher vanillin concentration. A number of conditions need to be favourable for detectable guaiacol production to occur and it could, therefore, be possible to minimise or prevent guaiacol production by controlling or eliminating some of these factors. Good manufacturing practices should be employed in order to minimise contamination and, therefore, spoilage, by Alicyclobacillus species.
AFRIKAANSE OPSOMMING: Bakterieë wat aan die genus Alicyclobacillus behoort, is termo-asidofiliese spoorvormers wat suur voedsel en drank produkte kan bederf deur die produksie van guaiakol en ander bederf verbindings, wat ‘n medisinale geur en/of reuk in die produkte veroorsaak. Hierdie tesis doen verslag oor die vergelyking van metodes wat vir die isolasie van spesies van Alicyclobacillus gebruik word, sowel as die groei kenmerke en guaiakol produksie van verskillende stamme wat uit die Suid- Afrikaanse vrugte prosesseringsomgewing geïsoleer is. Twee metodes vir die deteksie van guaiakol is ook geëvalueer en vergelyk. Drie isolasie metodes wat algemeen deur Suid-Afrikaanse vrugteprosesseerders gebruik word, is vergelyk ten opsigte van hul vermoë om H A. acidoterrestris stam uit verdunde perskesap konsentraat te isoleer. Metode 1, die Internasionale Federasie van Vrugtesap Produseerders (IFU) Metode No. 12, maak gebruik van spreiplating op Bacillus acidoterrestris (BAT) agar plate; Metode 2 behels gietplating met aartappel dekstrose agar (PDA) and Metode 3 maak gebruik van membraan filtrasie en inkubasie van die membraan op K agar. Die IFU Metode No. 12 was die mees effektiewe metode vir die isolasie van A. acidoterrestris, met H sel herwinning van 75.97%. Hierdie resultate ondersteun die gebruik van die IFU Metode No. 12 as H standaard internasionale metode vir die isolasie en deteksie van spesies van Alicyclobacillus. Sewe Alicyclobacillus stamme, insluitende die tipe stamme A. acidoterrestris DSM 3922T en A. acidocaldarius DSM 446T en vyf stamme geïsoleer uit ‘n Suid- Afrikaanse vrugte prosesseringsaanleg, A. acidoterrestris FB2, FB14, FB32, FB38 en A. acidocaldarius FB19, is geanaliseer met betrekking tot hul groei kenmerke en guaiakol produksie onder optimum toestande. Stamme is in BAT medium by pH 4.00, aangevul met 100 mg.L-1 vanillin, geïnokuleer en geïnkubeer teen 45°C vir 7 d. Al die stamme het soortgelyke groeipatrone getoon, met selgetalle wat vinnig toegeneem het van 0-24 h, gevolg deur ‘n stabilisering rondom maksimum selgetalle van 105-107 kve.mL-1. Selgetalle na hitte behandeling, gemeet as H aanduiding van spoorvorming, het toegeneem tot maksimum waardes van 105-107 kve.mL-1, wat aandui dat spore toegeneem het soos die seldigtheid en kompetisie vir voedingsbronne toegeneem het. Al die stamme kon guaiakol in bespeurbare konsentrasies produseer [soos gemeet deur die peroksidase ensiem kolorimetriese bepaling (PEKB)] en besit dus die potensiaal om produkte te bederf. Die invloed van temperatuur op groei en guaiakol produksie van die Alicyclobacillus stamme is ook ondersoek en twee guaiakol deteksie metodes, die PEKB en topspasie gas-kromatografie massa-spektrometrie (TS GK-MS) is vergelyk ten opsigte van hul vermoë om guaiakol op te spoor. Die stamme is geïnkubeer teen 25°C en 45°C vir 6 d en monsters is elke 24 h geanaliseer. Groei van die A. acidoterrestris stamme was stadiger by 25°C en maksimum selgetalle was laer as by 45°C. H Vermindering in selgetalle is waargeneem in die A. acidocaldarius stamme by 25°C, aangesien hierdie temperatuur buite hul groei temperatuur grense val. Al die stamme kon guaiakol produseer by 45°C, met guaiakol deteksie wat eers H aanvang geneem het nadat H sel konsentrasie van 104-105 kve.mL-1 bereik is. Die maksimum guaiakol konsentrasies wat by 45°C in die monsters met A. acidoterrestris opgespoor is, was beduidend hoër as die konsentrasies wat in die A. acidocaldarius monsters opgespoor is. By 25°C was daar H langer sloerfase voor guaiakol opgespoor is in die A. acidoterrestris monsters, terwyl geen guaiakol opgespoor is in die monsters wat A. acidocaldarius bevat het nie. Aangesien guaiakol by kamertemperatuur geproduseer word, word verkoeling van produkte aanbeveel ten einde bederf deur A. acidoterrestris te beheer. Die sensitiwiteit van die twee guaiakol deteksie metodes het ook beduidend verskil en dus word die gebruik van die PEKB aanbeveel vir teenwoordigheid/afwesigheid deteksie van guaiakol, terwyl TS GK-MS aanbeveel word waar akkurate kwantifisering van guaiakol vereis word. Ondersoek is ingestel na die vermoë van A. acidoterrestris FB2 om te groei en guaiakol te produseer in witdruiwesap aangevul met verskillende vanillin konsentrasies. Alicyclobacillus acidoterrestris FB2 is geïnokuleer in witdruiwesap konsentraat 1:10 verdun met gedistilleerde water wat 0-500 mg.L-1 vanillin bevat het en is geïnkubeer teen 45°C vir 6 d. Soortgelyke groeipatrone is waargeneem in al die monsters, behalwe die monster wat 500 mg.L-1 vanillin bevat het, wat H langer sloerfase van groei gehad het. Guaiakol konsentrasies, soos gemeet deur die PEKB, het toegeneem soos die vanillin konsentrasie toegeneem het, met die uitsondering van die monster wat 500 mg.L-1 vanillin bevat het, waar minder guaiakol opgespoor is as in die monster wat 250 mg.L-1 bevat het as gevolg van groei inhibisie veroorsaak deur die hoër vanillin konsentrasie. H Aantal toestande moet gunstig wees vir guaiakol produksie om plaas te vind en dit kan dus moontlik wees om guaiakol produksie te minimaliseer of te voorkom deur die beheer of uitskakeling van sommige van hierdie faktore. Goeie vervaardigingspraktyke moet in plek gestel word ten einde kontaminasie en bederf deur Alicyclobacillus spesies tot H minimum te beperk.

In orthopaedic medicine, post-operative infections are associated with increased morbidity, mortality and higher health care costs. There exists a need to improve understanding of factors influencing infection rates and antimicrobial resistance. Particularly for orthopaedic surgery performed in hotter, wetter climates where the tropical microbial niche presents unique challenges. This clinical research analyses the risks of orthopaedic post-operative infections associated with weather and climatic variables. Detailed epidemiological data is presented which describes features of bacteria complicating orthopaedic surgery in North Eastern Australia and the relationship between multi-drug resistance, geographic location, season and weather. Antimicrobial prescribing recommendations are also provided.

Orientador: Márcio Garcia Ribeiro
Resumo: Escherichia coli é o principal agente de mastite clínica bovina de origem ambiental, caracterizado pela complexidade de fatores de virulência (FV). O patógeno causa sinais clínicos que variam desde alterações exclusivamente no leite (grau 1 ou leve), no quarto afetado (grau 2 ou moderado), até manifestações sistêmicas (grau 3 ou grave). No entanto, até o momento, não está estabelecido o perfil de genes deste patógeno relacionados à virulência em infecções mamárias em vacas, tampouco com a gravidade clínica dos casos. Neste cenário, o presente estudo investigou 18 genes associados com E. coli extraentérica (ExPEC), o perfil “in vitro” de motilidade swimming e swarming, e a sensibilidade/resistência aos antimicrobianos em 114 isolados de E. coli obtidos de vacas com mastite clínica com escores de gravidade 1 (45/114=39,5%), 2 (62/114=54,4%) e 3 (7/114=6,1%). Os principais genes codificadores de FV detectados foram de adesinas (fimH, 114/114=100%; ecpA, 73/114=64,0%; fimA, 36/114=31,6%), resistência ao soro (traT, 93/114=81,6%; ompT, 40/114=35,1%), sideróforos (irp2, 11/114=9,6%) e hemolisina (hlyA, 8/114=7%). Os isolados apresentaram 99,1% (113/114) de resistência in vitro a bacitracina e cloxacilina, 98,2% (112/114) a lincosamina e 54,4% (62/114) a eritromicina. Do total de isolados, 98,2% (n=112/114) foram multirresistentes pelo cálculo do índice de resistência múltipla aos antimicrobianos (IRMA). Não houve diferença estatística significante entre as medianas para motilidade ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Escherichia coli is the major pathogen involved in the etiology of bovine mastitis from the environment origin. This pathogen is characterized by a complexity of virulence factors (VF). Mammary infections by E. coli has shown a wide range of clinical signs causing changes in milk (score 1 or mild), quarters (score 2 or moderate), and systemic signs (score 3 or severe). Nevertheless, to date, the profile of the genes related to the virulence of this pathogen in mammary infections and the severity scores of the cases are not fully understood. In this scenario, a panel of 18 genes associated with extra-intestinal E. coli (ExPEC) were investigated, in addition to in vitro swimming and swarming motility profile, and antimicrobial susceptibility/resistance pattern among 114 E. coli strains isolated from cows with clinical mastitis showing severity scores 1 (45/114=39.5%), 2 (62/114=54.4%) and 3 (n=7/114=6.1%). The main genes related to VF harbored by isolates were adhesins (fimH, 114/114=100%; ecpA, 73/114=64.0%; fimA, 36/114=31.6%), serum resistance (traT, 93/114=81.6%; ompT, 40/114=35.1%), siderophores (irp2, 11/114=9.6%) and hemolysin (hlyA, 8/114=7%). Among studied isolates, 99.1% (113/114) showed in vitro resistance to bacitracin and cloxacillin, 98.2% (112/114) to lincosamin, and 54.4% (62/114) to erytromycin. Out of the total isolates, 98.2% (112/114) were considered multidrug resistant based on multiple antimicrobial resistance (MAR) index. No statistical difference was obs... (Complete abstract click electronic access below)
Doutor

Malgré l’importance des virus dans la diversité, l’adaptation et l’évolution des communautés microbiennes, la virosphère des sources hydrothermales océaniques, reste peu caractérisée.Seulement 10 virus, dont 8 bacteriovirus et 2 virus d’archées, ont été décrits à ce jour. C’est dans ce contexte que ce travail de thèse s’inscrit avec pour objectif de caractériser des archaeovirus isolés de la composante microbienne abyssale. Les études ont porté sur des virus de méthanogènes, des producteurs primaires abondants dans ces environnements hydrothermaux. MFV1, premier virus tête-queue hyperthermophile décrit, a été isolé de Methanocaldococcus fervens une méthanogènehyperthermophile issue de sédiments marins profonds. Une caractérisation fonctionnelle et génomique de ce nouveau siphovirus a été conduite.Le caractère infectieux des virions a été démontré sur des Methanocaldococcus. Le plasmide pMEFER01, porté par M. fervens, peut également être empaqueté dans les capsides virales. L’étude d’autres virus de méthanogènes hyperthermophiles hydrothermales a été amorcée. M. vulcanius produit des virions tête-queue tandis que ceux isolés de M.jannaschii ont une morphologie particulière (tigeboucles).En parallèle, l’étude d’un virus en forme de citron, infectant Thermococcus thioreducens, a permis de s’intéresser à un autre ordre archéen, bien représenté au sein des systèmes hydrothermaux marins. De façon surprenante, ce virus semble capable d’infecter des méthanogènes. La mise en évidence de nouveaux systèmes hôtes-virus mais également d’interactions avec différents éléments génétiques mobiles (plasmides, vésicules) a permis d’élargir les connaissances sur le mobilome abyssal
Despite the importance of viruses in the diversity, adaptation, and evolution of microbial communities, the virosphere of deep-sea hydrothermal vents remains poorly characterized. To date, only 10 viruses isolated from deep sea hydrothermal vents, including 8 bacterial viruses and 2 archaeal viruses, have been described. In this context, this thesis work focused on gaining insights into the viral interactions with deep-sea autotrophic archaeal component. We aimed to characterize viruses of methanogens, which are abundant primary producers in these hydrothermal environments. MFV1, the first hyperthermophilic head-tail virus described, was isolated from Methanocaldococcus fervens, a hyperthermophilic methanogen from hydrothermal sediments. A functional and genomic characterization of this new siphovirus was conducted.The infectivity of MFV1 was demonstrated on Methanocaldococcus species. The plasmid pMEFER01, carried by M. fervens, can also be packaged in viral capsids. The study of other viruses of hyperthermophilic and hydrothermal methanogens was initiated. M. vulcanius produced head-tailed virions whereas those isolated from M. jannaschii had a particular morphology (stem-loops). In parallel, the study of a lemon-shaped virus, infecting Thermococcus thioreducens, permits to take an interest on another archaeal order, which is wellrepresented in marine hydrothermal systems.Surprisingly, this virus seemed capable of infecting hyperthermophilic methanogens. The characterization of new host-virus systems but also of interactions with different mobile genetic elements (plasmids, vesicles) expanding knowledge about the abyssal mobilome

Qui riportiamo due ceppi di Burkholderia Fungorum (DBT1 e 95) e la loro capacità sia in modalità planctonici e biofilm resistere elevate concentrazioni di idrocarburi per essere utilizzata nei protocolli di biorisanamento. B. Fungorum DBT1 stato isolato da drenaggio raffineria di petrolio, mentre B. Fungorum 95 è stato isolato dai tessuti interni di una pianta pioppo ibrido coltivata in terreno contaminato da idrocarburi aromatici policiclici (IPA). Gli idrocarburi testati sono stati dibenzotiofene (DBT) come campione di tiofeni ed una miscela di IPA, e cioè: naftalene, fenantrene e pirene. Inoltre, la loro capacità di trasformare ossianioni metalloidi tossici (selenite e telluriti) a forma elementare non tossica è stata valutata. Questa trasformazione non solo sradicare i composti metalloidi tossici nella zona contaminata, ma può anche essere utilizzato per ottenere forma elementare del metalloide in forma di nanoparticelle con applicazioni in tecnologia e della medicina. I nostri risultati hanno dimostrato che entrambi i ceppi degradate alta concentrazione di dibenzotiofene e entrambe le forme di biofilm e planctonici di batteri resistito fino a 2000 mg l-1 di questo composto. Inoltre, B. Fungorum DBT1 ha mostrato riduzione della tolleranza agli IPA miscela (naftalene 2000 mg l-1, fenantrene 800 mg l-1 e pirene 400 mg l-1) come biofilm e forme planctoniche. Al contrario, la formazione di biofilm aiutato B. Fungorum 95 per resistere IPA a queste concentrazioni, mentre forma planctoniche non poteva resistere. Infatti, alta concentrazione di DBT causato la formazione di aggregazione biofilm. D'altra parte, ceppo DBT1 era in grado di ridurre 0,5 mM selenite e 0,1 mM tellurite, mentre ceppo 95 ridotta più di 1 mM selenite e 0,05 mM Tellurite a forme elementari entro 96 ore di incubazione aerobica. B. Fungorum 95 prodotti 1 mM di selenio in presenza di 2 mM selenite. nanoparticelle prodotte selenio erano sferica e lo zero accusati di diametro medio idrodinamica di 170 nm (per il ceppo 95) e 200 nm (per ceppo DBT1). Tuttavia, le nanoparticelle prodotte tellurio erano ago come e positivo accusato di diametro medio idrodinamica di 120 nm e 170 nm per i ceppi 95 e DBT1 rispettivamente. La scansione e la microscopia elettronica a trasmissione analisi hanno mostrato entrambe le nanoparticelle selenio extracellulari e intracellulari. Selenite test di attività riduzione evidenziato attivazione enzimatica citoplasmatica accettando elettrone da donatori di elettroni. Dal momento che le nanoparticelle sono verificati extracellulare ma sono prodotti all'interno delle cellule in base al test di attività riduzione selenite, sia in uscita dalla secrezione o dopo la lisi cellulare. Tuttavia, le nanoparticelle di tellurio sono prodotti e si sono verificati intracellulare dalle attività citoplasmatica. In conclusione, i risultati per la resistenza contro idrocarburi forniscono nuove prospettive sulla efficienza di utilizzo di ceppi batterici DBT-degradanti in biorisanamento dei siti contaminati che contengono un'alta concentrazione di idrocarburi aromatici poli e tiofeni e bassa concentrazione di ossianioni metalloidi di selenio e tellurio. Produzione di nanoparticelle di selenio e tellurio in condizioni aerobiche da ceppi DBT1 e 95 potrebbe essere dovuto a meccanismi di riduzione intracellulari. Queste nanoparticelle biogene di entrambi i tipi presenti dimensioni compatibili con le applicazioni mediche e tecnologiche che sono attualmente in fase di studio.
Here we report on two strains of Burkholderia fungorum (DBT1 and 95) and their ability in both planktonic and biofilm modes to resist high concentrations of hydrocarbons in order to be exploited in bioremediation protocols. In nature, bacteria often attach to surfaces by establishing biofilms. B. fungorum DBT1 was isolated from an oil refinery drainage, while B. fungorum 95 was isolated from the inner tissues of a hybrid poplar plant cultivated in a soil contaminated by polycyclic aromatic hydrocarbons (PAHs). The hydrocarbons tested were dibenzothiophene (DBT) as a sample of thiophenes and a mixture of PAHs, namely: naphthalene, phenanthrene and pyrene. Moreover, their ability to transform toxic metalloid oxyanions (namely selenite and tellurite) to non-toxic elemental form was evaluated. This transformation not only eradicate the toxic metalloid compounds in contaminated area, but also can be utilized in order to obtain elemental form of metalloid in the form of nanoparticles with applications in technology and medicine. Our results showed that both strains degraded high concentration of dibenzothiophene and both forms of biofilm and planktonic of bacteria resisted up to 2000 mg l-1 of this compound. Moreover, B. fungorum DBT1 showed reduction in tolerance to PAHs mixture (naphthalene 2000 mg l-1, phenanthrene 800 mg l-1 and pyrene 400 mg l-1) as biofilm and planktonic forms. In contrary, formation of biofilm helped B. fungorum 95 to resist PAHs in these concentrations while planktonic form could not resist. Confocal laser scanning microscopy pictures showed that by exposing biofilm to DBT and PAHs, the structure changes. In fact, high concentration of DBT caused the formation of aggregation in biofilm. On the other hand, the result of both strains behavior in the presence of metalloids showed that strain DBT1 was able to reduce 0.5 mM selenite and 0.1 mM tellurite, while strain 95 reduced more than 1 mM selenite and 0.05 mM tellurite to elemental forms within 96 hours of aerobic incubation. B. fungorum 95 produced 1 mM selenium in the presence of 2 mM selenite. Produced selenium nanoparticles were spherical and zero charged with average hydrodynamic diameter of 170 nm (for strain 95) and 200 nm (for strain DBT1). However, produced tellurium nanoparticles were needle like and positive charged with average hydrodynamic diameter of 120 nm and 170 nm for strains 95 and DBT1 respectively. Scanning and transmission electron microscopy analyses showed both extracellular and intracellular selenium nanoparticles. Selenite reduction activity test evidenced cytoplasmic enzymatic activation by accepting electron from electron donors. Since nanoparticles occurred extracellularly but they are produced intracellularly according to selenite reduction activity test, either they exit by secretion or after cell lysis. However, tellurium nanoparticles are produced and occurred intracellularly by cytoplasmic activity. In conclusion, the findings for the resistance against hydrocarbons provide new perspectives on the efficiency of using DBT-degrading bacterial strains in bioremediation of contaminated sites containing high concentration of poly aromatic hydrocarbons and thiophenes and low concentration of metalloid oxyanions of selenium and tellurium. Production of selenium and tellurium nanoparticles under aerobic conditions by strains DBT1 and 95 could be due to intracellular reduction mechanisms. These biogenic nanoparticles of both kinds present size compatible with medical and technological applications which are currently under study.

碩士
元智大學
生物科技與工程研究所
97
PHAs (polyhydroxyalkanoates) is biodegradability polymers which is synthesized by microorganisms, some bacteria will synthesize PHAs and accumulated in vivo when the environment have excess carbon source and lack of some nutrients (ex: nitrogen, phosphate or sulfate). PHB (polyhydroxybutyrate) is the most common type of PHAs which is accumulated by microorganisms in the environment. The enteric bacteria can be commonly found in many environments; however, there is no report on their ability for PHAs production. From complete genome sequences of some enteric bacteria (e.g. E. coli and Salmonella), there was also no evidence of genes related to PHS synthesis in their chromosomes. Most of these sequenced strains are from culture collection and clinical samples. During the process of screening PHA-producing microorganisms in the environment, we have isolated some Gram negative bacteria that can accumulate PHB and presumably belong to enteric bacteria according to their 16S rDNA sequences analysis. In this study, we examined if these environmental enteric bacteria can accumulate PHB.

Bioassay guided fractionation of the organic extracts obtained from cultures of several bacteria strains from the marine environment led to the isolation of twelve new and nine previously described secondary metabolites. The structures of these metabolites were determined by extensive chemical and spectroscopic analysis. Stable isotope incorporation experiments were also performed using one of the isolated strains to investigate the biosynthetic origins of the atoms in the principle active secondary metabolite. A culture of a Bacillus sp. isolated from the tissues of a marine worm collected near Loloata Island in Papua New Guinea produced a mixture of novel cyclic decapeptide antibiotics. Loloatins A (1), B (2), and C (3) were isolated and their structures were elucidated through NMR and mass spectrometric analysis. Peptides 1, 2, and 3 showed potent gram-positive antibiotic activity, including activity against drug resistant strains of Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus spp. Loloatin C (3) also showed strong Gram-negative antibiotic activity. Massetolides A - H (4 - 11), novel cyclic depsipeptides, as well as the known compound viscosin (12), were isolated from cultures of two Pseudomonas sp. isolated from a marine alga and a marine tube worm each collected near Masset Inlet, B.C. and Moira Island, B.C. respectively. Massetolide A (4) and viscosin (12) exhibited in vitro antimicrobial activity against Mycobacterium tuberculosis and Mycobacterium avium-intracellulare. The known compounds, AI77-B (13), AI77-F (14) as well as AI77-H (15), a new diastereomer of AI77-F(14), were isolated from several species of Bacillus pumilus isolated from various marine sources. AI77-B (13) exhibited cytotoxic and Gram-positive antibiotic activity. The absolute configuration of AI77-H (15) was determined by chemical modification and NMR analysis of the (R)- and (S)- a-methoxy-a-(trifluoromethyl)phenylacetate esters. Finally, stable isotope incorporation experiments were performed using a selected strain of Bacillus pumilus which demonstrated that AI77-B (13) is of mixed polyketide/amino acid biosynthetic origin.

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2011
Antibiotics are one of the most successful forms of chemotherapy and saved million of lives placing most bacterial infectious diseases under control. However, this success has been compromised the continuous selective pressure exerted by antibiotics use has resulted in multi-resistance bacteria bearing resistance mechanisms to several antibiotics. Nowadays, there is an increased recognition that not only clinical settings constitute resistance reservoirs. The soil is a vast reservoir of resistance mechanisms and their associated genes, so understanding the resistant determinants present in the soil, the soil resistome, will provide information about antibiotic resistance frequencies and also emergence of new resistance mechanisms. Quinolones are an important group of synthetic antibiotics, recently plasmid mediated quinolone resistance mechanisms were established, some originated in environmental reservoirs. In this work the main goal was to study a collection of (fluoro)quinolone resistant environmental isolates, in terms of their ciprofloxacin MIC and screen them for the presence of qnr genes. With a smaller group of isolates the phenotypic impact of ciprofloxacin in growth curves/cellular viability was studied and finally detected and characterized induced variations of cell protein profiles, to study how ciprofloxacin affects, at a molecular level these bacteria. Results showed that ciprofloxacin MIC values had similar distributions in the soil samples from the two sample types from where these strains were isolated; in terms of resistance determinants results point that a qnrS gene was found in a Comamonas testosteroni isolate. In the second part of this work, Stenotrophomonas maltophilia response to ciprofloxacin was studied, and the growth curves/cellular viabilities results showed that S. maltophilia cells tolerate/resist ciprofloxacin action when in stationary growth. Significant differences were observed between cell total protein profiles from cells exposed to ciprofloxacin and the control. That study can help understand bacterial general response to fluoroquinolones, in addition to more classical genetic mutation, and also what intracellular pathways this antibiotic might trigger.
A utilização de antibióticos permitiu salvar milhões de vidas e controlar inúmeras doenças infecciosas, contudo a eficácia destes fármacos está hoje em risco. A utilização generalizada dos antibióticos levou ao aparecimento e disseminação de diversos mecanismos de resistência aos antibióticos que comprometem a eficácia dos mesmos. As bactérias multi-resistentes estão a tornar-se um grave problema de saúde pública, pois possuem mecanismos de resistência para várias classes de antibióticos, restando assim poucos antibióticos eficazes para a sua terapêutica. Durante as últimas décadas, o estudo da resistência aos antibióticos focou-se sobretudo nos meios clínicos e em microrganismos patogénicos. Mas, recentemente, tem sido reconhecido que outros ambientes e microrganismos não patogénicos também contribuem largamente para o processo de desenvolvimento e disseminação de mecanismos de resistência. Um dos ambientes cuja importância em termos de reservatório de resistência tem sido reconhecida é o solo. É um ecossistema complexo onde os antibióticos e os seus genes de resistência sempre estiveram presentes devido aos microrganismos produtores de antibióticos. Contudo, diversas actividades antropogénicas provocam a contaminação do solo com antibióticos e microrganismos resistentes o que influencia não só a estrutura da microbiota do solo como contribui para o desenvolvimento de mecanismos de resistência aos antibióticos. Num estudo recente, em que se analisou o nível de resistência de bactérias do solo a diversas classes de antibióticos, verificou-se que a maioria delas é multi-resistente e são resistentes mesmo a antibióticos sintéticos como as quinolonas. Este e outros estudos contribuíram para o reconhecimento da importância do solo como um reservatório para a emergência de mecanismos de resistência e bactérias resistentes. Surgiu assim um novo conceito, o resistoma do solo que consiste no conjunto de determinantes de resistência presentes no solo. As quinolonas são um grupo de antibióticos sintéticos que inibe a replicação e transcrição do DNA bacteriano conduzindo à morte celular. Durante muitos anos, pensou-se que a resistência às quinolonas se devia apenas a mutações que modificassem as enzimas alvo (topoisomerases tipo II) ou activassem a expressão de bombas de efluxo. Contudo, nos últimos anos novos mecanismos de resistências às quinolonas codificados em plasmídeos têm sido identificados. Um desses mecanismos é o gene qnr, codifica uma proteína que protege o DNA e as topisomerases tipo II do efeito das quinolonas. Recentemente a proteómica tem sido utilizada como uma nova abordagem no estudo da resposta bacteriana a diversos estímulos do meio, entre os quais podemos considerar a exposição aos antibióticos. O proteoma é o conjunto de proteínas produzidas por uma bactéria num determinado momento; ao contrário do genoma, o proteoma é dinâmico e varia rapidamente em função dos diferentes estímulos do ambiente onde as bactérias se encontram. A comparação entre perfis proteicos de bactérias não expostas e expostas a antibióticos está a ser utilizada no estudo dos mecanismos de resistência e resposta das bactérias aos antibióticos ou no modo de acção dos mesmos. O objectivo geral deste trabalho foi estudar uma colecção de bactérias ambientais resistentes às (fluoro)quinolonas, isoladas de solos provenientes de dois tipos de locais, quintas de produção animal e margens do rio Tejo, onde diversos factores podem contribuir para o desenvolvimento de resistências. Primeiro, esta colecção de isolados foi caracterizada em termos da sua resistência à ciprofloxacina e foi feita uma triagem do gene qnr. Para a execução do segundo objectivo deste trabalho realizaram-se curvas de crescimento e viabilidades para estudar de que forma a ciprofloxacina afecta o crescimento de Stenotrophomonas maltophilia. Perfis protéicos totais de culturas de S. maltophilia crescidas sem ou com ciprofloxacina foram comparados para determinar variações que possam reflectir as mudanças fisiológicas desencadeadas por este antibiótico. O nível de resistência dos isolados foi estabelecido pela determinação da sua CMI (concentração mínima inibitória) para a ciprofloxacina. Comparando os valores de CMI obtidos entre os isolados provenientes dos dois tipos de locais de amostragem, margens do rio Tejo e quintas de produção de animais, verificamos que são semelhantes, sendo que a maioria das CMI é entre 1 e 8 μg/ml. Para pesquisar a presença de isolados qnr-positivos nesta colecção de bactérias ambientais efectuamos um PCR multiplex com três conjuntos de primers previamente descritos, desenhados para amplificar as principais famílias do gene qnr estudadas até agora: qnrA, qnrB e qnrS. Dos 112 isolados só para um, mais tarde identificado como Comamonas testosteroni, se obteve amplificação positiva. Contudo estes resultados apresentam alguma reserva pois a amplificação não foi totalmente específica e só estudos adicionais permitirão confirmar se este se trata ou não do primeiro gene qnr encontrado em C. testosteroni. Para a realização do segundo objectivo principal deste trabalho o grupo de estirpes em estudo foi reduzido. Primeiro, os isolados com maior CMI para a ciprofloxacina foram identificados por galerias API®. Estes 16 isolados pertencem a 4 géneros diferentes, Comamonas, Acinetobacter, Stenotrophomonas e Aeromonas. Seguidamente para os isolados seleccionados foram determinadas as CMI e os halos de inibição para alguns antibióticos da classe dos β-lactâmicos. Os resultados revelaram que os isolados S. maltophilia são os que apresentam maior nível de resistência a esta classe de antibióticos. Com base em todos os resultados obtidos previamente optou-se por utilizar os oito isolados de S. maltophilia para a realização do segundo objectivo principal deste trabalho. Primeiro foram realizadas curvas de crescimento em que diferentes quantidades de ciprofloxacina, correspondentes a 1/2MIC, MIC e 2xMIC de cada isolado, eram adicionadas a culturas de S. maltophilia durante a fase exponencial de crescimento ou durante a fase estacionária de crescimento. Em paralelo, diluições da cultura foram plaqueadas para testar como a ciprofloxacina estava a afectar a viabilidade celular. Em conjunto estes resultados permitiram verificar que as curvas de crescimento por OD não reflectem o efeito que a ciprofloxacina está a ter nas células S. maltophilia. Quando a ciprofloxacina é adicionada, durante a fase exponencial, a viabilidade celular decresce significativamente comparativamente ao controlo mas tal descida não se reflecte numa descida de OD600nm nas curvas de crescimento. A análise dos resultados obtidos quando a ciprofloxacina é adicionada a bactérias que já se encontram em fase estacionária indica que nesta fase estas toleram e/ou resistem à presença de ciprofloxacina pois o tratamento com este antibiótico não provoca morte celular. Perfis proteicos de culturas de S. maltophilia crescidas em diferentes condições foram obtidos por SDS-PAGE. Primeiro num sistema de mini géis com o qual os géis obtidos não permitiram uma suficiente discriminação do padrão mas revelaram uma clara distinção entre os perfis de células em fase exponencial ou estacionária. Visto as células de S.maltophilia serem afectadas pela ciprofloxacina, quando em crescimento exponencial, os perfis proteicos das células nestas condições foram repetidos em géis de maiores dimensões. Deste modo, foi possível obter perfis proteicos com maior resolução e comparar os perfis proteicos das células em condições controlo com as expostas à ciprofloxacina. Esta comparação revelou diferenças significativas, especialmente para o caso do isolado S. maltophilia 4 indicando que os perfis proteicos reflectem o ambiente de crescimento das bactérias e nomeadamente o stress induzido pela ciprofloxacina. O estudo mais pormenorizado destas variações do proteoma, recorrendo a técnicas como géis 2D e espectofotometria de massa, está em curso e permitirá identificar proteínas com potencial relevância nos processos de resistência ou no modo de acção da ciprofloxacina.

碩士
國立臺灣大學
海洋研究所
91
Abstract Halophilic Gram-negative bacteria are widely found in coastal zones and marine areas. They are also commonly present in the brackish water environments of estuary. In contrast, halophilic Gram-positive bacteria are rare in the microbial communities of estuary, coast and sea. Our laboratory had totally isolated 113 strains of halophilic or halotolerant bacteria, preliminarily identified as Gram-positive bacteria, from the sediment under mangroves at Tan-shui River estuary and Gau-Mei Taichung, and marine water at the coastal sites of Taiwan (Er-jen River estuary etc.) as well as Ba-dou-Zi Keelung and Shen-Ao. The propose of this study was to screen halophilic bacteria from the isolated strains and analyzed these bacteria for their diverse genera and species. There only 41 strains of 113 isolates were halophilic gram positive bacteria. According to Na+ requirement, endospore production, cell morphology, glucose fermentation and colony morphology, 41 strains were divided into 11 groups: the strains of group 1-8 required Na+ for growth, the strains of group 9-11 were not ; and only the strains of group 6 and 9 were facultatively anaerobes which were able to ferment glucose, the strains of other 9 groups were aerobe which were unable to ferment glucose. Group 1 contained two strains, which were the only group producing endospores. The cells of this bacterial group were regular rods and grew into orange circular colonies. Group 2 only included one strain, which could not ferment glucose and was characterized by its orange circular colonies and regular rod cells like group 1. However, the production of endospore was not observed in group 2, a distinction between group 1 and group 2. Group 3 only included one strain, which grew into pink circular colonies and utilized glucose as sole carbone source for growth, a distinction between group 3 and group 1-2. Group 4 contained 4 strains and group 5 contained 15 strains, which could not ferment glucose and the cells of this group were regular rod like group 1-3. However, the production of irregular gray colonies and white circular colonies separatly, a distinction between group 4-5 and group 1-3. Group 6 only included one strain, which were the only group grew into colorless circular colonies, ferment glucose and the cells of this group were irregular rods, a distinction between group 6 and group 1-5. Group 7 contained 2 strains, which the cells of this group were irregular rods, and the strain could not growthed in complex-medium whithout ultra Mg2+, a distinction between group 7 and other 10 groups. Group 8 only included one strain, which the cells of this group were irregular rods, and the only group grew into yellow colonies. Group 9 contained two strains, which the cells of this group were irregular rods, grew into white circular colonies and could not ferment glucose. Group 10 only included one strain, which were the only group grew into red circular colonies, and the cells of this group were regular rods. Group 11 contained 11 strains, which the cells of this group were regular rods,and grew into irregular gray colonies beside I strain grew into white circular colonies. The level of 16S rDNA sequence similarity revealed that the strains C1055 and J169 of Group 1 could be considered as Bacillus hwajinpoensis.The strain CS13 of Group 2 could be considered as Halobacillus trueperi. The strain B8 of Group 3, M336 of Group 7, A53 of Group 9 and A57 of Group 6 could be considered to represent a new species. The strains J149 and BS13 of Group 4, J607 and J717 of Group 5, and A2 of Group 11 Stappia aggregata the closest similary The strains A51 of Group 10 and Ba1a8 of Group 8 was identical to Rhodovulum sulfidophilum and Microbacterium ketoreductum, the closest similary. Based on the phylogenetic tree rebuilt by the 16S rDNA sequence analysis, it was revealed that the 11 groups contained at least 8 species in 8 genera. It is valuable to mention a strain in group 7, M336. Unlike other strains, it could grow neither in complex medium without added Mg2+, nor in 100％ marine water or 5% NaCl solution. In addition, it was only 94.6％ identical to 16S rDNA sequence of Devosia neptuniae, the closest similary. Therefore, there was no doubt M336 might be a new species or even a new genus. In this study, the selected strain in group 3, 4, 5, 6, 7, 9, 10 and 11 displaying Gram-positive was revealed that their closest species were all Gram-negative via 16S rDNA sequence analysis. A further study should be required to clarify why the outcome contradictory to common observation could occur.

Burkholderia phytofirmans OLGA172 is a chlorobenzoate (CBA) degrading bacterium, known to frequently lose the ability to degrade CBA in the lab. OLGA172 carries the complete set of genes for chlorocatechol degradation (an intermediate in CBA metabolism), tfdCDEF, as well as several integrases associated with DNA mobility in proximity to these genes. In this study, putative CBA degradative genes were identified in OLGA172, and an imbalance in regulation between the tfdCDEF and CBA degradative genes identified as a cause for incomplete CBA metabolism in this strain. Additionally, expression of the integrase genes was observed to occur constitutively. The role of this expression was not determined but hypothesized to be related to the phenotypic instability seen in OLGA172. Characterization of the strain was carried out with the aim to determine the ecological niche OLGA172 occupies naturally. Results of characterization are discussed in the context of evolution of the chlorocatechol degradative genes.

M. Tech. (Department of Biotechnology, Faculty of Engineering and Technology) Vaal University of Technology
The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.

Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica
A aquicultura é um dos sectores alimentares que tem crescido mais rapidamente em todo o mundo. Sendo uma atividade económica importante em muitos países contribuiu para o aumento da procura de alimentos de origem aquática. A utilização de bactérias ácido-lácticas (LAB) como probióticos constitui uma alternativa aos antibióticos e à vacinação no controlo das doenças dos peixes de aquicultura, incluindo a lactococose causada por Lactococcus garvieae. No Capítulo 1, a microbiota cultivável total (TM) e as LAB de truta arco-íris e do seu ambiente aquícola, em diferentes estados do ciclo de vida, foram isoladas e identificadas taxonomicamente. Enterobacteriaceae e Aeromonadaceae foram prevalentes na TM, enquanto o género Lactococcus foi a LAB predominante. De um total de 1620 LAB selecionadas aleatoriamente, 1159 isolados (71,5%) apresentaram atividade antimicrobiana contra os principais patógenos de peixes, incluindo 248 isolados (21,4%) que apresentaram atividade antimicrobiana contra, pelo menos, quatro patógenos de peixes. A identificação taxonómica revelou que Lactococcus lactis foi a espécie mais comum (164 isolados, 66,1%). A inocuidade, a relação genética e a actividade bacteriocinogénica de 75 estirpes de Lc. lactis com potencial probiótico foram avaliadas ao longo do Capítulo 2. Adicionalmente, a bacteriocina mais ativa contra a lactococose foi caracterizada ao nível bioquímico e genético. Dezassete estirpes (22%) produziram, pelo menos, uma amina biogénica, e 30 estirpes (40%) foram resistentes contra, pelo menos, um antibiótico. Os genes tet(K), tet(O) e tet(T) foram detetados pela primeira vez em Lc. lactis. A desconjugação dos sais biliares, produção de gelatinase e hemolisina tal como a degradação da mucina não foram observados. ERIC-PCR permitiu agrupar os lactococcos em três grupos altamente divergentes (31,0% de similaridade). Nove estirpes (12%) foram identificadas como produtoras de bacteriocinas e mediante purificação, espectrometria de massa e sequenciação de DNA da bacteriocina produzida por Lc. lactis subsp. cremoris WA2-67, foi possível identificar esta bacteriocina como nisina Z (nisZ). Trinta e quatro lactococos supostamente inócuos (45,3%) foram identificados, incluindo a estirpe bacteriocinogénica Lc. cremoris WA2-67. No Capítulo 3 foram estudadas a inocuidade, a relação genética e a atividade bacteriocinogénica de oito estirpes de Pediococcus acidilactici isoladas da ração e de larvas de truta arco-íris. Além disso, a bacteriocina produzida pela estirpe mais ativa para ser utilizada como probiótico na aquicultura foi caracterizada ao nível bioquímico e genético. Nenhum dos pediococos foi resistente aos antibióticos, produziu hemolisina ou gelatinase, degradou a mucina gástrica ou desconjugou os sais biliares, ainda que apenas quatro estirpes produziram tiramina ou putrescina. ERIC-PCR permitiu agrupar os pediococos em dois grupos bem definidos (68,0% de similaridade). Seis estirpes (75%) foram identificadas como bacteriocinogénicas e mediante purificação, espectrometria de massa e sequenciação de DNA da bacteriocina produzida por P. acidilactici L-14 foi possível identificar esta bacteriocina como pediocina PA-1. Quatro pediococos supostamente inócuos (50%) foram identificados, incluindo a estirpe bacteriocinogénica P. acidilactici L-14. No decorrer do Capítulo 4, estudou-se a inocuidade e atividade antimicrobiana contra patógenos de 64 enterococos identificados taxonomicamente isolados de truta arco-íris, ração e ambiente aquícola. Enterococcus faecium e Enterococcus hirae foram as espécies mais comuns (42,2 e 35,9%, respetivamente). Quarenta e oito estirpes (75%) foram fenotipicamente resistentes a pelo menos um antibiótico. Deste conjunto, 25 estirpes (39,1%), continham, pelo menos, um gene de resistência a antibióticos [erm(B), tet(M), tet(S), tet(K), tet(L), tet(T), vanC2 e aad(E)]. Detetou-se uma estirpe gelatinase positiva, e não se observou a produção de hemolisina, desconjugação de sais biliares e degradação de mucina. Vários genes de virulência foram detetados, incluindo gelE (46,9%), efaAfs (17,2%), agg (1,6%), e hyl (1,6%). Quarenta e oito estirpes inibiram o crescimento de, pelo menos, um dos patógenos de peixes testados, incluindo 21 (43,8%) estirpes que continham, no mínimo, um gene que codifica bacteriocina (entP, entL50A and entL50B, hirJM79, entSE-K4, entQ and entA). De um total de 17 enterococos (26,6%) considerados como supostamente inócuos, seis estirpes continham, pelo menos, um gene que codifica bacteriocina. As propriedades probióticas in vitro de três estirpes Lc. cremoris bacteriocinogénicas, a capacidade in vivo de Lc. cremoris WA2-67 para proteger a truta arco-íris contra a lactococose e o papel da produção de NisZ como um mecanismo anti-infeccioso foram analisadas no Capítulo 5. As três estipes de Lc. cremoris mostraram capacidade para sobreviver em água doce, meio ácido e bílis, e exibiram diferente hidrofobicidade da superfície celular (37,93-58,52%). A estirpe selvagem Lc. cremoris WA2-67 e o seu mutante não-bacteriocinogénico Lc. cremoris WA2-67 ΔnisZ foram administrados oralmente à truta arco-íris durante 21 dias e, posteriormente, os peixes foram infetados com Lc. garvieae pelo método de coabitação. A mortalidade observada nos peixes alimentados com a estirpe bacteriocinogénica Lc. cremoris WA2-67 (20%) foi significativamente (p <0,01) inferior à observada nos peixes tratados com a estirpe mutante (50%) e no grupo controlo (72,5%). Neste trabalho demonstrou-se que as LAB isoladas diretamente do hospedeiro, ativas contra patógenos de peixes, englobam potenciais candidatos a probióticos. Os resultados obtidos através da avaliação biotecnológica sugerem que a estirpe Lc. cremoris WA2-67 produtora de NisZ poderia ser utilizada na aquicultura para evitar a lactococose na truta arcoíris.
Aquaculture is the fastest-growing food-producing sector worldwide and an important economic activity in many countries, contributing to the increasing demand for food of aquatic origin. The use of lactic acid bacteria (LAB) as probiotics constitutes an alternative to chemotherapy and vaccination to control fish diseases in aquaculture, including lactococcosis caused by Lactococcus garvieae. In Chapter 1, the cultivable total microbiota (TM) and LAB from rainbow trout and rearing environment from selected stages of the life-cycle was isolated and taxonomically identified. Enterobacteriaceae and Aeromonadaceae were clearly prevalent in the TM while the genus Lactococcus was the predominant LAB. From a total of 1,620 randomly selected LAB, 1,159 isolates (71.5%) showed antimicrobial activity against the main fish pathogens, including 248 isolates (21.4%) that showed antimicrobial activity against, at least, four fish pathogens. The taxonomical identification revealed that Lactococcus lactis was the most common species (164 isolates, 66.1%). The safety assessment, genetic relatedness and bacteriocinogenic activity of 75 potential probiotic Lc. lactis strains were evaluated in Chapter 2. Moreover, the biochemical and genetic characterization of the bacteriocin most active against lactococcosis was performed. Seventeen strains (22%) produced, at least, one biogenic amine, and 30 strains (40%) showed resistance to, at least, one antibiotic. The genes tet(K), tet(O) and tet(T) were detected for the first time in Lc. lactis. Hemolysin and gelatinase production, mucin degradation and bile salts deconjugation were not found. ERIC-PCR allowed clustering the lactococci in three highly divergent groups (31.0% similarity). Nine strains (12%) were identified as bacteriocin producers and the purification, mass spectrometry and DNA sequencing of the bacteriocin produced by Lc. lactis subsp. cremoris WA2-67 revealed its identity to nisin Z (nisZ). Thirty four putatively safe lactococci (45.3%) were identified, including the bacteriocinogenic strain Lc. cremoris WA2-67. In Chapter 3, the safety assessment, genetic relatedness and bacteriocinogenic activity of eight Pediococcus acidilactici strains isolated from rainbow trout feed and larvae were evaluated. Furthermore, the biochemical and genetic characterization of the bacteriocin produced by the strain more interesting for being used as probiotic in aquaculture was performed. None of the pediococci showed antibiotic resistance nor produced hemolysin or gelatinase, degraded gastric mucin or deconjugated bile salts, and only four strains produced tyramine or putrescine. ERIC-PCR allowed clustering the pediococci in two well-defined groups (68.0% similarity). Six strains (75%) were identified as bacteriocin producers and the bacteriocin produced by P. acidilactici L-14 was purified, and mass spectrometry and DNA sequencing revealed its identity to pediocin PA-1. Four putatively safe pediococci (50%) were identified, including the bacteriocinogenic P. acidilactici L-14. In Chapter 4, the taxonomical identification, safety assessment and antimicrobial activity against fish pathogens of 64 enterococci isolated from rainbow trout, feed and rearing environment were evaluated. Enterococcus faecium and Enterococcus hirae were the most common species (42.2 and 35.9%, respectively). Forty-eight strains (75%) showed phenotypic resistance to, at least, one antibiotic, and from these, 25 strains (39.1%) harbored, at least, one antibiotic resistance gene [erm(B), tet(M), tet(S), tet(K), tet(L), tet(T), vanC2, and aad(E)]. Gelatinase production was detected in one strain; however, hemolysin production, bile salts deconjugation and mucin degradation were not detected. Several virulence genes were detected, including gelE (46.9%), efaAfs (17.2%), agg (1.6%), and hyl (1.6%). Forty-eight strains exerted antimicrobial activity against, at least, one of the tested fish pathogens, including 21 (43.8%) strains harboring, at least, one bacteriocin-encoding gene (entP, entL50A and entL50B, hirJM79, entSE-K4, entQ and entA). From a total of 17 enterococci (26.6%) considered as putatively safe, six strains harbored, at least, one bacteriocin-encoding gene. The in vitro probiotic properties of three bacteriocinogenic Lc. cremoris, the in vivo ability of Lc. cremoris WA2-67 to protect rainbow trout against lactococcosis infection and the role of NisZ production as an anti-infective mechanism were evaluated in Chapter 5. The three Lc. cremoris showed ability to survive/tolerate the freshwater, acidic and bile environment, and different cell surface hydrophobicity (37.93–58.52%). The wild-type NisZproducer Lc. cremoris WA2-67 and its non-bacteriocinogenic mutant Lc. cremoris WA2-67 ΔnisZ were administered orally to rainbow trout for 21 days and, afterwards, fish were challenged with Lc. garvieae by the cohabitation method. The fish fed with the bacteriocinogenic strain Lc. cremoris WA2-67 reduced significantly (p<0.01) the mortality (20%) compared to the fish treated with its non-bacteriocinogenic knockout isogenic mutant (50%) and the control (72.5%). This thesis showed that host-derived LAB active against fish pathogens comprise potential candidates as probiotics in rainbow trout farming. Our results suggest that the wild type NisZ-producer strain Lc. cremoris WA2-67 could be used in fish farming to prevent lactococcosis in rainbow trout.
Financial support provided by FCT (“Fundação para a Ciência e a Tecnologia”) and POPH-QREN/FSE (“Programa Operacional Potencial Humano-Quadro de Referência Estratégico Nacional/Fundo Social Europeu”, PhD grant SFRH/BD/62416/2009)