Dissertations / Theses on the topic 'Bacterial isolates'

Probiotics are viable nonpathogenic microbes that positively affect host health. Probiotics inhibit infection, activate immunity, and promote mucosal-barrier development. Many microbes have probiotic activity. Nonetheless, the selection of stable strains and their specific mechanism(s) of action are not fully elucidated. Bacteria from ‘Amabere amaruranu’ cultured milk from Kenya were isolated and identified by PCR sequence analysis of the 16S rRNA gene. Isolates were examined for stability to acid and bile, antimicrobial activity, mucin production, and degradation and sensitivity to antibiotics, hence their potential for probiotics. Lactobacillus isolates were acid unstable, bile-stable, nonmucinolytic, and presented antibacterial activity. L. rhamnosus cell fractions increased MUC4 and MUC3 expression in colon cells. Bacillus isolates were acid and bile stable, nonmucinolytic and lacked antimicrobial activity. In conclusion, Lactobacillus isolates that were nonmucinolytic, stable in bile, demonstrated antibacterial activity, sensitive to antibiotics, and stimulated increase MUC4 and MUC3 levels in colon cells could be potential probiotics.

The technological advances have led to faster and more cost-effective sequencing platforms, making it quicker and more affordable to generate genomic sequence data. For the study of bacterial genome, two main methods can be used, whole-genome sequencing and metagenomic shotgun sequencing, of which the first is the mostly used in the past years. As a consequence of these advances, a vast amount of data is currently available and the need of bioinformatics tools to efficiently analyse and interpret it has dramatically increased. At present, there is a great quantity of tools to use in each step of bacterial genome characterization: (1) pre-processing, (2) de novo assembly, (3) annotation, and (4) taxonomic and functional comparisons. Therefore, it is difficult to decide which tools are better to use and the analysis is slowed down when changing from one tool to another. In order to tackle this, the pipeline BACTpipe was developed. This pipeline concatenates both bioinformatics tools selected based on a previous testing and additional scripts to perform the whole bacterial analysis at once. The most relevant output generated by BACTpipe are the annotated de novo assembled genomes, the newick file containing the phylogenetic relationships between species, and the gene presence-absence matrix, which the users can then filter according to their interests. After testing BACTpipe with a set of bacterial whole-genome sequence data, 60 genes out of the 18195 found in all the Lactobacillus species analysed were classified as core genes, i.e. genes shared among all these species. Housekeeping genes or genes involved in the replication, transcription, or translation processes were identified

Thesis (S.M.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2008.
Includes bibliographical references (p. 54-59).
(cont) An increased understanding of heterotrophic bacterial strategies for acquiring nutrients and trace elements is critical for elucidating their impact on biogeochemical cycling in the ocean. It is estimated that iron is a limiting nutrient for phytoplankton growth in over 30% of the open ocean, but still little is known about bacterial strategies for iron acquisition. Siderophore (Fe ligand) production by bacteria may play a major role in influencing the bioavailability of iron in the ocean. Despite the importance of siderophores in the environment, only limited information from a select group of bacteria is available. On a cruise through the Costa Rica Dome (CRD) upwelling region in July 2005, a library of 867 isolates from five depth profiles inside and outside of the dome was obtained and screened for siderophore production using the Chrome Azurol-S (CAS) assay. Phylogenetic affiliation of 134 isolates was determined by sequencing the 16s rDNA gene, and determined that gamma proteobacteria such as Alteromonas, Pseudoalteromonas, Halomonas, and Marinobacter dominated the collection, while alpha-proteobacteria such as Roseobacter were also represented. The isolates obtained from stations in the CRD showed greater siderophore-producing capabilities between 55m and 100m while strains isolated from outside the CRD had shallower peak (-8-35m) production. Functional group determination showed that hydroxamate production dominated from 50-150m, while hydroxamate and catechol production is roughly equal in shallower waters. By characterizing the siderophores produced by these isolates and determining the genetic make-up of the population, these findings further our understanding of how heterotrophic microbes affect biogeochemical processes and the competitive nature of nutrient acquisition.
by Whitney B. Krey.
S.M.

Submitted by Alisson Mota (alisson.davidbeckam@gmail.com) on 2015-07-22T18:33:12Z No. of bitstreams: 1 Tese - Arlena Maria Guimarães Gato.pdf: 2067444 bytes, checksum: a858fc3c2c47508ee175666940ad0251 (MD5)
Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-23T14:23:02Z (GMT) No. of bitstreams: 1 Tese - Arlena Maria Guimarães Gato.pdf: 2067444 bytes, checksum: a858fc3c2c47508ee175666940ad0251 (MD5)
Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-23T14:26:09Z (GMT) No. of bitstreams: 1 Tese - Arlena Maria Guimarães Gato.pdf: 2067444 bytes, checksum: a858fc3c2c47508ee175666940ad0251 (MD5)
Made available in DSpace on 2015-07-23T14:26:09Z (GMT). No. of bitstreams: 1 Tese - Arlena Maria Guimarães Gato.pdf: 2067444 bytes, checksum: a858fc3c2c47508ee175666940ad0251 (MD5) Previous issue date: 2009-12-10
SUFRAMA - Superintendência da Zona Franca de Manaus
The flowers and ornamental plants cultivation stands out as an important agronomical activity. But despite the economical and market potential of these regional native species: helicônia, bastão, sorvetão and other plants, is necessary more basic studies about the use of advanced techniques in getting propagated material with quality assurance and good phytosanitary aspect. The objective of this study was to obtain plantlets from floral apices of Heliconia rauliniana and embryos rescue of H. marginata, identifies, inoculation of bacterial isolates and evaluate the effect of these microorganisms in the development of micropropagated plants. Established the plants micropropagation process, it was isolated from matrices root, bacterial isolates and, according to the classification criteria of activity levels of nitrogen biological fixation, phosphate solubilisation and auxin production, it was selected six Heliconia rauliniana isolates and eight of Heliconia marginata. The first experiment were inoculated in in vitro plants of H. rauliniana, the B4, B5, B8, B13, B16 and B18 bacterial isolates and on H. marginata micropropagated plants, the B1, B2, B4, B6, B7, B10, B12, B14 and B16 isolates and, then transferred to plastic boxes containing sterilized substrate Plantmax (Horticulture) and maintained under greenhouse. After 60 days of inoculation, it was realized the evaluation of root growth promotion, selecting the three isolates that showed the best results: B18, B5 and B13 (H. rauliniana) and B7, B6 and B10 (H. marginata). In the second experiment we used the B18, B5, B13 and B6, B7, B10 selected in the first experiment and inoculated in 60 plants, distributed on five treatments: control, B18, B5 and B13, cocktail x plant and control, B6, B7, B10, cocktail x plant with 12 plants per treatment. The results presented after 60 days, don’t were significant on the level of 0.05% and one good faith coefficient of 95% (ANOVA) for root growth promotion, number of leaves, plant height, fresh weight and dry weight. The plants survive was 83.3%. For microorganisms identify, it was used the analysis of the fragment about 800 bp of the 16S rDNA (Primer 27f). The sequencial analysis via Blast showed three bacterial groups: Burkholderia, Ralstonia e Enterobacteriaceae (Pantoea/Erwinia).
O cultivo de flores e plantas ornamentais tropicais destaca-se como importante atividade agrícola. Mas, apesar das potencialidades econômicas e de mercado dessas espécies nativas da região: helicônia, bastão, sorvetão e outras, são necessários mais estudos básicos sobre a utilização de técnicas avançadas na obtenção de material de propagação com garantia de qualidade e bom aspecto fitossanitário. O objetivo deste trabalho foi obter mudas micropropagadas a partir de ápice floral de Heliconia rauliniana e de resgates de embriões de H. marginata, identificação, inoculação dos isolados de bactérias e avaliar o efeito desses microrganismos no desenvolvimento das plantas micropropagadas. Estabelecido o processo de micropropagação das plantas foram isolados das raízes das matrizes, isolados bacterianos e, de acordo com os critérios de classificação dos níveis de atividades em fixação biológica de nitrogênio, solubilização de fosfato e produção de auxina, foram selecionados seis isolados da Heliconia rauliniana e oito da Heliconia marginata. No primeiro experimento, foram inoculados nas plantas micropropagadas de H. rauliniana, os isolados bacterianos B4; B5 (Enterobacter); B8; B13 (Ralstonia); B16; B18 (Enterobacter) e nas plantas micropropagadas de H. marginata, os isolados B1; B2; B4; B6 (Enterobacter); B7 (Enterobacter); B10 (Burkholderia); B12; B14 e; B16 e, posteriormente transferidas para caixas plásticas contendo substrato esterilizado PlantMax (Horticultura) e mantidas em casa de vegetação. Após 60 dias de inoculação foi realizada a avaliação de promoção de crescimento de raiz, selecionando-se os três isolados que apresentaram os melhores resultados por espécie: Heliconia rauliniana, Enterobacteriaceae (Pantoea/Erwinia), B18 e B5 Enterobacteriaceae (Pantoea/Erwinia e B13 (Ralstonia) e Heliconia marginata B7 Entereobacteriaceae (Pantoea/Erwinia), B6 Entereobacteriaceae (Pantoea/Erwinia) e B10 Burkholderia. No segundo experimento foram utilizados os isolados bacterianos B18 Enterobacteriaceae (Pantoea/Erwinia): B5 Enterobacteriaceae (Pantoea/Erwinia, B13 Ralstonia e B6, Entereobacteriaceae (Pantoea/Erwinia), B7, Entereobacteriaceae (Pantoea/Erwinia), B10, Burkholdeia selecionados no primeiro experimento e inoculados em 60 plantas, distribuídas em cinco tratamentos: controle, B18 Entereobacteriaceae (Pantoea/Erwinia: B5 Entereobacteriaceae (Pantoea/Erwinia e B13 Ralstonia, coquetel x planta e controle, B6 Entereobacteriaceae (Pantoea/Erwinia), B7 Entereobacteriaceae (Pantoea/Erwinia), B10 (Burkholderia), coquetel x planta com 12 plantas por tratamento. Os resultados apresentados após 60 dias, não foram significativos a nível de 0,05 % e um coeficiente de confiança de 95 % (A NOVA) para promoção de crescimento de raiz, número de folhas, altura de planta, peso fresco e peso seco. A sobrevivência das plantas foi de 83,3 %. Para identificação dos microrganismos foi usado o seqüenciamento de fragmento de aproximadamente 800 pb do gene 16S rDNA (“primer 27f). A análise das sequencias via Blast mostrou três grupos de bactéria: Burkholderia, Ralstonia e Enterobacteriaceae (Pantoea/Erwinia).

Mestrado em Biologia Molecular e Celular
As populações de anfíbios têm decaído ao longo dos últimos anos devido a inúmeros fatores, tais comos, a perda de habitat, a contaminação/poluição e um dos mais importantes, as doenças. Estas perdas originam também a perda de diversidade genética das espécies, podendo comprometer a sua aptidão e também capacidade de adaptação. Tendo em conta todos estes fatores, é necessário proceder à preservação das populações de anfíbios, independentemente do local em que se encontram ser contaminado, pristino, rural ou urbano. Sabendo que os anfíbios de zonas urbanas podem ser uma fonte importante para a diversidade genética da espécie e que estão expostos, tal como as populações de zonas naturais, a agentes patogénicos, sendo que normalmente são populações negligenciadas a nível de proteção, urge a necessidade de as avaliar e proteger, nomeadamente contra agentes patogénicos. De uma forma geral, esta proteção é conferida de uma forma inata por estruturas ao nível da pele, que fazem parte do seu sistema imunitário. Estas são glândulas granulares responsáveis pela produção de compostos peptídicos capazes de inibir o crescimento de agentes patogénicos. Em acréscimo, a microbiota existente na pele estimula e complementa a atividade destas secreções. Com base nestes factos, este trabalho teve como objetivos: i) avaliar de que forma fatores como as estações do ano (Primavera e Outono) e o género, podem influenciar a microbiota cultivável da pele de Pelophylax perezi de zonas urbanas, ii) avaliar se os isolados bacterianos da pele apresentam atividade antimicrobiana e iii) avaliar o potencial dos isolados bacterianos com atividade antimicrobiana enquanto possíveis agentes probióticos, na presença de um agente patogénico. Os resultados obtidos mostraram diferenças entre locais ao nível das espécies isoladas, sendo poucas as espécies comuns entre locais. Além disso, foi evidenciado que num total de 120 isolados, 19 possuíam atividade antimicrobiana face a Bacyllus aquimaris e Aeromonas salmonicida. Também se verificaram diferenças na atividade antimicrobiana entre estações do ano, existindo um maior número de espécies com atividade antimicrobiana no Outono. Dos isolados com atividade antimicrobiana, os três com maior atividade, Pseudomonas rhizosphaerae, Pseudomonas fluorescens e Bacillus mycoides foram selecionados para a segunda fase do trabalho, em que se avaliou o seu potencial enquanto possíveis agentes probióticos. Após exposição, in vivo, de girinos aos probióticos, em simultâneo com A. Salmonicida, verificou-se que estes evitavam mortalidade dos girinos, bem como diminuíam o dano peroxidativo quando comparados com os valores do agente patogénico. Dos três probióticos B. mycoides mostrou ser aquele com maior capacidade de estimular as enzimas antioxidantes, sendo o agente probiótico com os valores mais baixos de dano peroxidativo.
Amphibian populations have declined over the past few years due to numerous factors such as habitat loss, contamination / pollution and one of the most important, diseases. These losses also result in the loss of genetic diversity of the species, which may compromise their fitness and ability to adapt. Taking all these factors into account, it is necessary to preserve amphibian populations, regardless of being found in contaminated, pristine, rural or urban sites. Given that urban amphibian populations can be an important source for genetic diversity of the species and that they are exposed, such as populations of natural areas, to pathogens, there is a need for assess and protect them against pathogenic agents. Generally, this protection is conferred in an innate way by skin structures, which are part of your immune system. These are granular glands responsible for the production of peptidic compounds capable of inhibiting the growth of pathogens. In addition, the microbiota in the skin stimulates and complements the activity of these secretions. Based on these facts, this work had as objectives: i) to evaluate how factors such as seasonality (spring and autumn) and gender can influence the cultivable microbiota of Pelophylax perezi skin in urban areas; ii) assess the ability of the bacterial skin isolates to present antimicrobial activity and iii) evaluate the potential of bacterial isolates with antimicrobial activity as potential probiotic agents. The obtained results showed differences between sites at the level of the isolated species, with few common species between sites. In addition, it was evidenced that in a total of 120 isolates, 19 had antimicrobial activity against Bacyllus aquimaris and Aeromonas salmonicida. There were also differences in antimicrobial activity between seasons, with a higher number of species with antimicrobial activity in the autumn. Of the isolates with antimicrobial activity, the three with the highest activity, Pseudomonas rhizosphaerae, Pseudomonas fluorescens and Bacillus mycoides were selected for the second phase of the study, in which their potential action as probiotic agents was evaluated. After in vivo exposure of the tadpoles to the probiotics, along with A. salmonicida, these were found to decrease the mortality of tadpoles as well as to decrease the peroxidative damage, when compared to the values obtained from the exposure to the pathogen. From the three probiotics B. mycoides revealed to be the one with the greatest capacity to stimulate the antioxidant enzymes, being the probiotic agent with the lowest values of peroxidative damage.

Denitrifying biocathodes are used to treat nitrogen contaminated water resources. The present PhD dissertation have been characterized different bacterial families with a relevant role in denitrification process. Different operational conditions were used, different electron acceptors: nitrate and nitrite, and alternative electron donors: organic matter. The use of functional genes as specific target, allowed to identify Bradyrhizobiaceae as a key family in autotrophic denitrification. The dominance of this family were observed using different electron acceptors and also in nitrite and nitrous oxide reduction reactions. Five representative isolates were obtained to determine the role of this bacteria in electrotrophic processes. All of them had the ability to reduce nitrite to nitrogen gas under autotrophic conditions, besides this similar physiological characteristics, only three isolates could reduce nitrite electrochemically. The other two isolates had midpoint potentials associated to biological hydrogen production. The results suggest the interaction between different bacteria in cathodic denitrification.
Els càtodes desnitrificants s’utilitzen per tractar aigües contaminades amb compostos de nitrogen. En la present tesi doctoral, s’han identificat les famílies bacterianes amb un paper rellevant en la desnitrificació. Es van utilitzar diferents condicions d’operació, diferents acceptors d’electrons: nitrat i nitrit; i donadors d’electrons: matèria orgànica. L’ús de marcadors específics per gens funcionals, va permetre identificar la família Bradyrhizobiaceae com a clau en la desnitrificació autotròfica ja que mantenia la seva dominància utilitzant diferents acceptors d’electrons, així com també en els processos de reducció de nitrit i d’òxid nitrós. Es van aïllar cinc bacteris representatius d’aquesta família per determinar la seva capacitat electrotròfica. Tots els aïllats tenien característiques fisiològiques similars: reduir el nitrit a nitrogen gas autotròficament, però només tres aïllats podíen reduir el nitrit electroquímicament. Els altres dos, presentàven pics d’activitat electroquímica associats a la producció biològica d’hidrogen. Suggerint la interacció de diferents bacteris en la desnitrificació en els càtodes.

The Gram-negative bacterium Helicobacter pylori is found in human gastric mucosa. H. pylori, one of the most common chronic bacterial infections of humans, is present in almost half of the world population. It is associated with chronic gastritis, non-ulcer dyspepsia, gastric and duodenal ulcers, and malignant neoplasms. The aim of this study was to detect microbial candidate protein markers whose presence might be correlated with the development of four different clinical consequences of H. pylori infection, gastric ulceration [GU], duodenal ulceration [DU], non-ulcer dyspepsia [NUD] and gastritis [GI]. Eleven H. pylori isolates associated with these outcomes were analysed. The total complement of protein from these H. pylori isolates were resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and compared using PDQUEST pattern analysis software. Relationships between the isolates associated with specific disease outcomes were determined by cluster analysis.Fifty six disease specific proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Up to 1165 protein species were resolved from each H. pylori strain. Proteome analysis revealed that only 470 (40%) of the proteins detected were common to all eleven isolates. Twenty six of the 56 disease specific proteins that were selected for identification consisted of spots whose expression is altered in response to stress conditions or those that can affect H. pylori cell division and the cell membrane. The remaining 30 proteins had no known function. This study has provided further confirmation of the extensive variation that the bacterium H. pylori exhibits at the proteome level. Most significantly this study has found, through the application of cluster analysis and protein matching, that isolates do form disease groups. Comparative proteome analysis is a useful method for highlighting the extensive strain variation that H. pylori exhibits and to determine if any disease specific proteins exist.

In this study, 11 bacterial isolates from Salt Lake,Turkey were identified by using fatty acid methyl ester (FAME) analysis. They were screened for production of industrially important enzymes xylanase, cellulase, &
#945
-amylase and protease. These enzymes were characterized in terms of enzyme activity, stability, optimum temperature and optimum pH. One of the isolates was identified as Bacillus pumilus, and two of them were identified as Bacillus subtilis. Other isolates were determined to be Bacillus licheniformis. All the isolates were determined to produce xylanase. Optimum temperatures and optimum pH values of xylanases were 50-55 °
C and pH 7.0-8.0. Xylanases were quite stable up to pH 8.0 and 70 °
C. Isolates were not significant cellulase producers. Four of the isolates did not produce any cellulase enzyme and the rest produced negligible amounts of cellulase. Therefore, xylanases from the isolates were promising for pulp and paper industry, which requires cellulase free and stable xylanases. All the isolates produced appreciable quantities of &
#945
-amylase. Optimum temperatures and optimum pH values of &
#945
-amylases 60-80 °
C and pH 7.0-8.0. &
#945
-Amylases were quite stable up to pH 9.0 and 80 °
C. &
#945
-Amylases from the isolates were promising for starch processing industry, which requires &
#945
-amylases stable at high temperatures and for detergent industry, which requires &
#945
-amylases stable at alkaline pH values. Considerable protease productions were achieved by all the isolates. TTG 2 was the best protease producer with 271 U/ml. Optimum temperatures and optimum pH values of proteases were 50-60 °
C and pH 7.0-7.4. Proteases were quite stable up to pH 9.0 and 80 °
C. Proteases from the isolates were promising for detergent and leather industry, in which proteases must be stable at alkaline pH values.

The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers.
O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

Flocculation has been widely adopted as one of the most effective methods to remove colloidal particles in water or wastewater treatment. Synthetic flocculants are conventionally used because of their high flocculating efficiency and cost-effectiveness. However, they have been reported to have hazardous properties and implicated in some serious health problems including senile dementia and neuro-toxicity, as well as being recalcitrant in the environment. Consequently, efforts are being geared away from the use of synthetic flocculants in water and wastewater treatment. Hence, the need for safe and eco-friendly flocculants has become imperative. Compared with synthetic flocculants, bioflocculants have special advantages such as safety, biodegradability and harmlessness to the environment and humans; attributes which make them potential alternatives in water treatment, downstream as well as fermentation processes. In the current study, the potentials of bacterial isolates recovered from Algoa Bay in the Eastern Cape Province of South Africa for bioflocculant production were investigated. The bacterial isolates were identified by polymerase chain reaction (PCR) as belonging to the Bacillus genus. The analysis of 16S ribosomal deoxyribonucleic acid (rDNA) nucleotide sequence of isolate M72 showed 99 percent similarity to Bacillus toyonensis strain BCT-7112 and was deposited in the GenBank as Bacillus toyonensis strain AEMREG6 with accession number KP406731. Likewise, the 16S rDNA nucleotide sequences of isolates M69 and M67 showed 98 percent sequence similarity to Bacillus licheniformis strain W7 and Bacillus algicola strain QD43 respectively; and M67 isolate was subsequently deposited in the GenBank as Bacillus sp. AEMREG7 with accession number KF933697.1. The results of the nutritional requirements and fermentation conditions revealed that optimum inoculum size for REG-6 production was 4 percent (v/v), while 5 percent (v/v) and 3 percent (v/v) were most favourable for MBF-W7 and MBF-UFH production respectively. Glucose was the best carbon source for the production of bioflocculants (REG-6 and MBF-UFH) by Bacillus toyonensis AEMREG6 and Bacillus sp. AEMREG7 respectively, while maltose supported optimum bioflocculant (MBF-W7) production by Bacillus specie. Inorganic nitrogen (NH4NO3) was the favoured nitrogen source for both REG-6 and MBF-W7 production, while mixed nitrogen sources [yeast extract + urea + (NH4)2SO4] supported the maximum production of MBF-UFH. The initial medium pH for REG-6 was 5, while MBF-W7 and MBF-UFH were both maximally produced at the initial pH of 6. After a 96 h cultivation period under optimal culture conditions, 3.2 g of purified REG-6 with a maximum flocculating activity of 77 percent was recovered from 1 L fermented broth of Bacillus toyonensis AEMREG6. Yields of 3.8 g and 1.6 g pure bioflocculants with the respective highest flocculating activities of 94.9 percent and 83.2 percent were also obtained from 1 L, 72 h-fermented broths of Bacillus licheniformis and Bacillus sp. AEMREG7 respectively. Furthermore, all the three bioflocculants (REG-6, MBF-W7 and MBF-UFH), displayed thermal stability within the temperature range of 50 to 100 oC, with strong flocculating activities of over 80 percent against kaolin suspension over a wide range of pH range (3–11) and relatively low dosage requirements of 0.1-03 mg/ml in the presence of divalent cations in the treatment of kaolin clay suspension and Thyme River waters. Chemical composition analyses of the bioflocculants showed them to be glycoproteins with a predominantly polysaccharide backbones as shown by the following carbohydrate/protein (w/w) ratios: 77.8 percent:11.5 percent (REG-6); 73.7 percent:6.2 percent (MBF-W7) and 76 percent:14 percent (MBF-UFH).

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.

The stem bark of Peltophorum africanum and Bridelia micrantha are used in South Africa traditional medicine for treatment of intestinal parasites, relieve problems and human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS). The growing problem of antibiotic resistance by Helicobacter pylori the major etiological agent in gastritis, gastric cancer, peptic and gastric ulcer demands the search for novel compounds from plant based sources. This study was aimed to determine the antimicrobial activity of five solvent (ethylacetate, acetone, ethanol, methanol and water) extracts of the stem bark of P. africanum and B. micrantha on clinical strains of H. pylori in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. H. pylori strains were isolated from patients presenting with gastric related morbidities at the Livingstone Hospital, Port Elizabeth for endoscopy and confirmed following standard microbiology procedures. The plant extracts including clarithromycin were tested against 31 clinical strains of H. pylori by the agar well diffusion method. The most potent extract was evaluated by the microdilution method to determine the Minimum Inhibitory Concentration (MIC50&90), followed by the rate of kill. Preliminary phytochemical analysis was carried out. The one way ANOVA test was used to statistically analyse the results. All the extracts demonstrated anti-H. pylori activity with zone diameters of inhibition that ranged from 0 to 23 mm for the extracts and 0 to 35 mm for clarithromycin. Marked susceptibility (100%) was recorded for the ethyl acetate extract of P. africanum (P. afr. EA) and the acetone extract of B. micrantha (B. mic. A), which were statistically significant (P < 0.05) compared to all other extracts and clarithromycin. For B. micrantha ethyl acetate extract, 93.5 percent susceptibility was observed while for the control iv antibiotic, clarithromycin it was 58.1 percent. The MIC50 ranged from 0.0048 to 0.313 mg/mL for P. afr. EA, and from 0.0048 to 0.156 mg/mL for B. mic. EA; MIC90 ranged from 0.156 mg/mL to 0.625 mg/mL and 0.0048 to 2.5 mg/mL for P. afr. EA and B. mic. EA respectively. There was a significant statistical difference observed in potency of both P. afr. EA and B. mic. A compared to the two antibiotics (P < 0.05). One hundred percent killing by P. afr EA was observed at 0.05 mg/mL (½ x MIC) and 0.2 mg/mL (2 x MIC) in 66 h for strain PE466C and PE252C respectively. For B. mic. EA, 100 percent killing effect of both strains (PE430C and PE369C) was observed at 0.1 mg/mL (2 x MIC) in 66 h. Qualitative phytochemical analysis confirmed the presence of alkaloids, flavonoids, steroids, tannins and saponins in the ethyl acetate extracts of both plants, which could be a potential template of lead molecule for the design of new anti- Helicobacter pylori therapies.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: S. aureus causes serious infections in the hospital and community settings. The rate of MRSA infections are rapidly increasing worldwide. Currently, at Tygerberg hospital, approximately a third of S. aureus isolates are MRSA. This was the first epidemiological study of S. aureus conducted at Tygerberg Hospital that included prospective clinical data on patients with S. aureus bacteraemia together with spa typing of strains and the detection of the mecA and pvl genes in a multiplex PCR. Clonal cluster groups of S. aureus isolates were obtained by BURP analysis and compared to international important clones. The molecular epidemiology of hospital acquired (HA), health-care associated (HCA) and community acquired (CA) S. aureus bacteraemic strains at this hospital was examined. Lastly, repeat isolates of patients were collected to analyse any possible organism-related factors associated with persistent and recurrent bacteraemia. We investigated a total number of 113 S. aureus strains from 104 patients (70% MSSA, 30% MRSA). Repeat strains consisted of nine isolates (from 5 patients). All isolates were obtained from blood cultures collected during the period March 2008 to May 2009. Phenotypic and genotypic detection of methicillin resistance correlated well. According to the literature, most CA-MRSA strains are distinguishable from HA-MRSA strains based upon the presence of the PVL toxin. However, no CA-MRSA was detected in our study, therefore the association between HA-MRSA versus CA-MRSA strains could not be analysed. In this study, CA-MSSA was identified in 22% of all MSSA isolates versus 0% CA-MRSA. PVL positive strains were found in 22.7% of all MSSA isolates with no detection in MRSA isolates. It was noted that MRSA strains clustered in spa CC-701 and CC-012, whereas CC-002 only contained MSSA strains. Likewise HA-strains representing the majority of MRSA strains also clustered in spa CC-701 and CC-012. Forty nine spa types were identified in 89.3% of all isolates, whereas 9.7% of these strains were non-typeable. Five novel spa types were revealed. We detected a diverse number of spa-types that correlated to international clones. The most predominant spa type found in our setting was t037 (only in MRSA), followed by t891. According to the literature, t037 is associated to the Brazilian/Hungarian clone (SCCmec type III; ST 239). Our findings, as well as other South African studies, indicate that t037 has been identified in clinical strains from numerous provinces in South Africa. Interestingly, all isolates from spa type t891 were PVL positive MSSA. Bacteraemia cases were predominantly related to catheter sepsis, followed by skin and soft tissue infections (SSTI). Only one persistent bacteraemia case was identified related to a HA-SSTI. Recurrent bacteraemia cases were found in patients on dialysis for chronic renal failure and in burns patients related to intravascular catheter infections. The local epidemiology of S. aureus and the prevalence rate of different strains are important to investigate. The information provided contributes to the epidemiology of staphylococcal strains causing bacteraemia in our setting. These insights are useful for optimal diagnostic and therapeutic measures. The techniques developed can be used to identify outbreaks and recurrent infections.
AFRIKAANSE OPSOMMING: S. aureus veroorsaak ernstige infeksies in die hospitaalomgewing en in die gemeenskap. Wêreldwyd, neem metisillien-weerstandige S. aureus (MRSA) infeksies vinnig toe. Huidiglik by Tygerberg hospitaal is ongeveer ‘n derde van S. aureus isolate MRSA. Hierdie is die eerste epidemiologiese studie by Tygerberg hospitaal wat prospektiewe kliniese data van pasiënte met S. aureus bakteremie saam met spa tipering en aantoning van die mecA en pvl gene in ‘n multipleks PKR insluit. Klonale groepe (spa-CC) van MRSA en MSSA isolate is deur BURP analise verkry, en vergelyk met internasionaal belangrike klone. Die molekulêre epidemiologie van hospitaalverworwe (HA), gesondheidsorgverworwe (HCA) en gemeenskapsverworwe (CA) S. aureus bakteremie by hierdie hospitaal is ondersoek. Laastens, oorspronklike en daaropvolgende herhaal isolate is gekollekteer om moontlike organisme- faktore geassosieerd met persisterende en herhalende bakteremiese episodes te analiseer. Ons het in totaal 113 S. aureus isolate van 104 pasiënte ondersoek (70% MSSA, 30% MRSA). Nege isolate (van 5 pasiënte) was herhaal isolate. Alle isolate was afkomstig vanaf bloedkulture wat gedurende die periode Maart 2008 tot Mei 2009 gekollekteer is. Fenotipiese en genotipiese aantoning van metisillien weerstandigheid het goed gekorreleer. Volgens die literatuur kan die meeste CA-MRSA isolate van HA-MRSA isolate onderskei word op grond van die teenwoordigheid van die PVL toksien. Geen CA-MRSA is egter in ons studie gevind nie, dus kon die assosiasie tussen HA-MRSA en CA-MRSA isolate nie ondersoek word nie. CA-MSSA was in 22% van alle MSSA geidentifiseer teenoor 0% CA-MRSA. PVL is in MSSA isolate gevind (22.7% van alle MSSA) maar glad nie in MRSA nie. Dit is opgemerk dat MRSA isolate hoofsaaklik in spa CC 701 en CC-012 kloongroepe voorkom, teenoor kloongroep CC-002 wat slegs MSSA isolate bevat het. Soortgelyk het HA-isolate wat die meerderheid van MRSA isolate verteenwoordig het ook in kloongroepe 1 & 2 gegroepeer. Nege-en-veertig spa tipes is geïdentifiseer in 89.3% of alle isolate en 9.7% was nie-tipeerbaar. Vyf nuwe spa tipes is getoon. Ons het ‘n diverse aantal spa-tipes geïdentifiseer wat met internasionale klone gekorreleer het. Die mees dominante spa tipe in ons omgewing was t037 (slegs in MRSA), gevolg deur t891. Volgens die literatuur word t037 met die Brasiliaanse/Hongaarse kloon geassosieer (SCCmec tipe III; ST 239). Ons bevindings, asook ander Suid Afrikaanse studies, dui aan dat t037 in kliniese isolate vanaf talle provinsies in Suid-Afrika aangetoon is. Van belang is dat al die isolate van spa tipe t891 MSSA en PVL positief was. Bakteremiese gevalle was hoofsaaklik geassosieer met kateter-sepsis, gevolg deur vel en sagteweefsel infeksies (SSTI). Slegs een persisterende bakteremiese geval was geïdentifiseer geassosieer met HA-SSTI. Herhalende bakteremiese episodes is in pasiënte op dialise vir kroniese nierversaking en in brandwonde pasiënte met intra-vaskulêre kateter infeksies aangetoon. Die lokale epidemiologie van S. aureus en die prevalensie koers van verskillende stamme is van belang. Hierdie inligting dra by tot kennis van die epidemiologie van stafilokokkale stamme wat in ons omgewing bakteremie veroorsaak. Hierdie insigte is nuttig vir optimale diagnostiese en terapeutiese riglyne. Die tegnieke wat ontwikkel is, kan gebruik word om uitbrake en herhalende infeksies te identifiseer.

Infection with Helicobacter pylori has been associated with the pathogenesis of numerous stomach and gastroduodenal diseases that pose threats to public health. Eradicaftion of this pathogen is a global challenge due to its alarming rate of multidrug resistance. Consequently, to find an alternative treatment, the search is increasingly focused on new antimicrobial product from natural sources including honey. Honey has been used as medicine in several cultures since ancient time due to its enormous biomedical activities. Its beneficial qualities have been endorsed to its antimicrobial, antioxidant, anti-inflammatory properties added to its phytocomponents. In this study, the anti-H. pylori activity of South African honeys and their solvent extracts as well as the phytochemicals present in the two most active honeys were evaluated. Agar well diffusion test was used to investigate the antimicrobial activity of six honey varieties obtained from different locations in the country. Subsequently, the honeys were extracted with four organic solvents viz n-hexane, diethyl ether, chloroform and ethyl acetate employed in order of increasing polarity. The antibacterial activity of the different solvent extracts of each honey was evaluated by agar well diffusion; broth micro dilution and time kill assays. Different chromatographic techniques (Thin layer & column chromatography) were employed to enumerate the phytochemical constituents in the most active solvent extracts of Pure Honey (PH) and Champagne Royal Train (CRT); and were identified by gas-chromatography linked mass-spectrometry. Linalool pure compound was equally evaluated for anti-H. pylori activity in a bid to trace the antibacterial agent among the variety of compounds identified. Data were analyzed by One-way ANOVA test at 95% confidence interval. Crude honeys and their solvent extracts demonstrated potent anti-H. pylori activity with zone diameter that ranged from [16.0mm (crude) to 22.2mm (extract)] and percentage susceptibilities of test isolates between 73.3% (crude) and 93.3% (extract). The chloroform extracts of PH and CRT were most active with MIC50 in the ranges 0.01- viii 10%v/v and 0.625-10%v/v respectively, not significantly different from amoxicillin (P> 0.05); and efficient bactericidal activity (100% bacterial cells killed) at 1/2MIC and 4xMIC over different time intervals, 36-72hrs and 18-72hrs respectively. The appearance of bands on the thin layer chromatography (TLC) chromatogram spotted with the chloroform extracts of PH and CRT; and developed with hexane: ethyl acetate: acetic acid (HEA) and methanol: acetic acid: water (MAAW) solvent systems indicated the presence of compounds. Purification of the compounds contained in these extracts over silica gel column yielded numerous fractions which were evaluated for antibacterial activity and purity. PHF5 was the most active fraction with a mean MIC50 value of 1.25mg/mL. Volatile compounds belonging to different known chemical families in honey were identified in all the active fractions obtained from PH. Conversely, only four compounds were identified in the active fractions obtained from CRT hence the non volatile constituents could be of prime relevance with respect to antibacterial activity of this honey. Of novelty was the presence of thiophene and N-methyl-D3-azirdine compounds, essential precursors used for the synthesis of natural products and pharmaceuticals with vital biomedical properties. Linalool demonstrated potent inhibitory (MIC95, 0.002- 0.0313mg/mL) and bactericidal activity (0.0039-0.313mg/mL) against the test isolates. On the other hand, a significant difference was recorded (P < 0.05) in comparing the activity of linalool compound to the fractions. PH could serve as a good economic source of bioactive compounds which could be employed as template for the synthesis of novel anti-H. pylori drugs. However, further studies are needed to determine the non volatile active ingredients in PH and CRT as well as toxicological testing

Marine sediments harbour vast and diverse prokaryotic communities. With ongoing burial and ageing of respective sediment layers, however, available organic matter becomes more recalcitrant. Thus, sedimentary microorganisms face starvation and ultimately death. Nonetheless, live and active cells are present in old and deeply buried sediments, up to 111 Ma (Roussel et al., 2008). During IODP Leg 307 an organic-matter poor, cold-water, buried coral carbonate mound was sampled. Nineteen isolates, mainly Proteobacteria, were obtained from the mound and surrounding sediments. Additionally, one putative new species belonging to the genus Ornithinimicrobium (Actinobacteria) was isolated. Strains were subsequently phylogenetically and phenotypically characterised. Selected isolates and other sedimentary bacteria were subsequently subjected to anaerobic starvation-survival experiments and their responses to substrate limitation were compared to those of near-surface relatives. All strains survived long periods of starvation (incubated up to 3 years). This was confirmed by constant total cell counts and only slowly increasing proportions of dead cells (20% after one year). Culturability and FISH detectability decreased with time but radiotracer experiments conducted after starvation confirmed viability and potential metabolic activity of many strains. No significant correlations between FISH detectability and other viability measures occurred. Instead starvation time was significantly positively correlated with percentages of dead cells and inversely with culturability. Pure culture starvation experiments were complemented by a study on an estuarine, surface-sediment microbial community, which was stressed in sediment slurry sequential heating experiments. This mimicked burial and resulted in decreasing total counts, culturability, and FISH detcctability but these were still present even after heating to 90 °C. Temperatures above 42 °C were significantly correlated with the reduction of total cells and FISH detectability This project showed that marine sedimentary microbes maintain high levels of viability and culturability during long-term anaerobic starvation and during sequential heating to mimic burial this is consistent with the large cell population in sub-seafloor sediments.

This thesis reports the results obtained during the three years PhD course focused on the study of culturable bacterial endophytes of Vitis vinifera Glera and their beneficial activities. The study, part of a large project named “EndoFlorVit project” (FEARS-UE and Regione Del Veneto), aims at investigate the biodiversity and the plant growth promoting activities of culturable endophytes isolated from Glera grapevine in vineyards of Conegliano-Valdobbiadene DOCG production area. This thesis reports the results of the isolation of culturable bacterial endophytes from surface-sterilized Glera grapevine tissues. 381 culturable strains were successfully isolated from roots, shoots and leaves of Vitis vinifera Glera, sampled from six different vineyards in the Conegliano-Valdobbiadene DOCG area (Veneto, Italy). The community was investigated by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and nucleotide sequencing to identify the most representative genera of the Glera microbiome. Approximately 30% of the isolates belonged to the genus Bacillus, which was the most represented; other genera such as Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus and Agrococcus were identified. The composition of the communities isolated from different vineyards was not the same; moreover we reported that endophyte biodiversity inside plants was influenced by the season. After molecular characterization, we focused our attention to investigate the plant growth promoting abilities of the culturable strains. Using biochemical tests we assayed some of the most important and effective properties in order to investigate the physiology of these bacteria and identify some strains that could have a strong beneficial effect on plant nutrition and growth. In this work, using Carboxymethyl Cellulose degradation test, we demonstrated that 85 strains secreted cellulolytic enzymes; this trait could confer to these bacteria an advantage in plant penetration and tissues colonization. By qualitative biochemical assays, we demonstrated that many strains were able to solubilize phosphate (127 strains), produce ammonia (142 strains) and secrete siderophores (155 strains). Using the colorimetric Salkowsky assay, we determined that 17 strains produced the phytohormone Indol 3-acetic acid (IAA) ; using Arabidopsis thaliana DR5:GUS, where the β-glucuronidase reporter gene is expressed under control of a IAA-induced promoter, we demonstrated that bacterial IAA was recognised by the Arabidopsis plants and caused morphological alteration on the root architecture. It is known that IAA is not the only bacterial molecule that influence the plant growth and the root morphology. To investigate the effects of the Glera endophytes on the plant morphology we used the model system Arabidopsis thaliana co-cultured in vitro with every single strain. Morphological parameters (root length, surface and diameter) were measured by a software and statistically analysed by cluster analysis. Plants were thus clustered according with the effect of the strain on the root parameters, demonstrating the effects of the strains on roots morphology. In particular, some strains caused an enhanced root length displaying a plant growth promotion effect. By this large-scale characterization we selected two of the most promising strains, one for the putative plant growth effect (Pantoea agglomerans GL83) and one as putative biocontrol agent (Bacillus licheniformis GL174) that were transformed with a DNA cassette containing a gfp reporter gene. Using Laser Scanning Confocal Microscopy, we demonstrate the colonization of the stem endosphere of Glera cuttings 20 and 30 days after the inoculum of the fluorescent strains. In this thesis, the evidences of the colonization are reported demonstrating that Pantoea agglomerans GL83 and Bacillus licheniformis GL174 are true Glera endophytes able to colonize cuttings when re-inoculated. After we demonstrated that B. licheniformis GL174 is a true endophyte of Glera, we investigated the biocontrol abilities of the strain. Results of antagonism tests against plant pathogenic fungi are shown, demonstrating that the strain is able to reduce and inhibit the mycelia growth of the grapevine pathogens Phaeoacremonium aleophilum, Phaeomoniella spp., Botryosphaeria spp., Botrytis cinerea and for the more generic plant pathogens Sclerotinia sclerotiorum and Phytophthora infestans. After that, we demonstrated by PCR and DNA sequencing that GL174 has the operons coding for lipopeptide synthetase enzymes and that the strain produced cyclic lipopeptide belonging to surfactin and lichenysin families. These molecules with antimicrobial effects were identified and characterized by mass spectrometric analysis and the results are reported and discussed in this thesis. The genome of the strain was thus sequenced to better investigate the strain and the sequences were preliminary analysed identifying the presence of many genes coding for lytic enzymes. The production of lipopeptides, the inhibition of fungal growth and the ability to colonize inner tissues of Glera indicated GL174 as a good candidate for biocontrol. Another aspect of endophyte-plant symbiosis that is not well explained is the ecology of these bacterial strains and the interactions between different bacterial species in the rhizosphere and inside plants are poorly described. Bacteria-bacteria interactions are likely to be an important factor that defines the composition of the endophyte community. The study of these interactions is essential to understand plant-bacteria relationship; moreover, the study of how the native community of rhizobacteria and endophytes may change after the inoculation of other bacteria is important for a safe and aware use of commercial biocontrol or bio-fertilizer products containing endophytes. A preliminary ecological study is presented in this thesis: some ecological aspects of endophyte of grapevine, endophytes of other plant species and some bacteria commercialized as beneficial strains, called “biofector strains” were analysed using tomato, a model plant for agriculture and horticulture. In this work, we demonstrated that these strains were able to colonize tomato plants and the population densities of the diverse tissues sampled are reported in the Chapter number 5. This work, that is still ongoing, aims to evaluate the impact of these endophytic strains investigating if the inoculum of the bacteria on tomato plants leads to a different endophytic community in comparison to uninoculated plants. This study is essential to unravel the effects of the bio effector strains on natural endophytic populations of plants: from peeled stems of all the inoculated plants the total DNA was extracted; this material will be used as template for the 16S rDNA amplification of all the endophytes present in the plants. Many sets of primers are being tested to select the best combination for this approach. The amplicons will be sequenced and analysed to determine if the community of endophytes has been changed by the inoculum of the external endophytic bio-effector strain. In conclusion, the results presented in this thesis are an overview of the composition of the endophytic community of Glera plants cultivated in the Conegliano-Valdobbiadene DOCG area. The isolation of the culturable strains has provided a large collection of bacteria that, during the PhD course, was characterized investigating plant growth promoting activities and bacteria effects on plant morphology considering different mechanisms underlying plant-microbe interactions. The evidence obtained in this work describes a clear and novel background to understand Glera endophytes biology and ecology and, when confirmed in planta by field trials, will permit the selection of some efficient strains to use as safe endophytic bio-fertilizers and biocontrol agents, for a sustainable production of Glera grapes.
Questo lavoro di tesi presenta e discute i risultati sperimentali ottenuti durante il corso di dottorato in Biologia Evoluzionistica presso la Scuola di Dottorato di Bioscienze e Biotecnologie dell’Università degli Studi di Padova. Questa ricerca, parte di un progetto più ampio denominato “EndoFlorVit” (FEARS-UE e Regione del Veneto), ha come scopo la caratterizzazione molecolare e lo studio delle proprierà di promozione della crescita vegetale e di bio-controllo di batteri endofiti isolati da piante di Vitis vinifera di cultivar Glera, coltivate nell’area di produzione del Prosecco di Conegliano-Valdobbiadene DOCG. Le comunità di microrganismi isolate sono state studiate utilizzando la tecnica ARDRA (Amplified Ribosomal DNA Restriction Analysis) e il sequenziamento di porzioni del 16S rDNA identificando che circa il 30% dei ceppi isolati appartengono al genere Bacillus, il quale risulta essere il più rappresentato nelle piante campionate. Altri generi a cui appartengono numerosi ceppi isolati sono Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus e Agrococcus. La composizione delle comunità endofite isolate da differenti piante non è uniforme: esse variano nei differenti vigneti e sono inoltre influenzate dalla stagionalità. Oltre alla descrizione dei ceppi isolati, in questo lavoro di tesi sono presentati e discussi i risultati dello studio delle proprietà di promozione della crescita vegetale dei ceppi isolati. Utilizzando saggi biochimici sono state investigate alcune delle principali attività benefiche che hanno un effetto di miglioramento della nutrizione vegetale. Mediante il test di degradazione della carbossimetil-cellulosa sono stati identificati 85 ceppi capaci di secernere enzimi degradanti la cellulosa: questa capacità può conferire ai ceppi che la esprimono un vantaggio nella colonizzazione dei tessuti vegetali facilitando loro il processo di penetrazione nei tessuti della pianta. Attraverso saggi biochimici qualitativi è stato possibile dimostrare che numerosi ceppi sono in grado di solubilizzare il fosfato insolubile (127 ceppi), produrre ammoniaca (142 ceppi) e secernere siderofori (155 ceppi). Inoltre, utilizzando il saggio di Salkowski, è stato dimostrato che 17 ceppi batterici producono l’ormone vegetale Acido 3-indolacetico (IAA). Per investigare l’effetto dei ceppi produttori di IAA sulla fisiologia e morfologia della pianta è stato utilizzata la pianta modello Arabidopsis thaliana DR5:GUS, una linea mutante esprimenti l’enzima β-glucuronidasi sotto controllo di un promotore indotto da IAA. Utilizzando questo sistema sperimentale è stato dimostrato che l’IAA prodotto dai batteri viene riconosciuto dalle piante di Arabidopsis e causa alterazioni alla morfologia e architettura radicale. È noto tuttavia che l’IAA non è l’unica molecola batterica che influenza la crescita vegetale e la morfologia vegetale. In tal senso, è stato valutato l’effetto di ciascun ceppo isolato sulla pianta Arabidopsis thaliana Col-0 wild tipe. Tre parametri morfologici della radice (lunghezza superficie e diametro) sono considerati e analizzati statisticamente mediante cluster analysis. Le piante quindi sono state raggruppate secondo gli effetti che i batteri hanno provocato sull’apparato radicale assegnando in questo modo a ciascun ceppo l’effetto corrispondente. In questo modo è stato dimostrato che alcuni ceppi hanno causato allungamento della radice, un effetto ascrivibile come promozione della crescita. Da questa caratterizzazione ed analisi su larga scala dei ceppi isolati sono stati selezionati due ceppi particolarmente promettenti per la promozione della crescita vegetale e per il biocontrollo: Pantoea agglomerans GL83 e Bacillus licheniformis GL174. Questi due ceppi sono stati trasformati geneticamente con un costrutto contenente il gene che esprime la proteina fluorescente GFP. Utilizzando tecniche di microscopia confocale è stato dimostrato che entrambi i ceppi fluorescenti sono in grado di ricolonizzare talee di vite Glera quando inoculate e che persistono all’interno dei tessuti del fusto dopo 20 e 30 giorni dopo l’inoculo. In questa tesi quindi sono presentate le evidenze sperimentali che questi due ceppi, Pantoea agglomerans GL83 e Bacillus licheniformis GL174, sono veri endofiti di vite Glera e risultano quindi interessanti per le loro proprietà benefiche. Dopo aver confermato che GL174 è endofita della vite Glera, il ceppo è stato investigato per evidenziarne alcune capacità utili per il biocontrollo dei patogeni. In questo lavoro di tesi sono presentati i risultati di saggi di antagonismo in vitro nei quali il ceppo in esame ha effetto di inibizione della crescita del micelio di alcuni funghi patogeni della vite (Phaeoacremonium aleophilum, Paeomoniella spp., Botryosphaeria spp., Botrytis cinerea) e di due patogeni più generalisti (Sclerotinia sclerotiorum e Phytophtora infestans). Lo studio del ceppo GL174 si è successivamente focalizzato sulla capacità del batterio di produrre i lipopeptidi ciclici, una classe di molecole con forte attività antimicrobica e surfattante. Per prima cosa, attraverso PCR e sequenziamento del DNA, è stata identificata la presenza nel genoma batterico degli operoni codificanti per alcune lipopepide sintetasi, gli enzimi deputati alla sintesi di queste molecole. La produzione di lipopeptidi è stata successivamente dimostrata utilizzando tecniche di spettrometria di massa; le quali hanno permesso di identificare le molecole prodotte e di ricostruirne la struttura chimica. La produzione di queste molecole e la capacità inibitoria di funghi patogeni rendono il ceppo GL174 un buon candidato come agente di biocontrollo nella coltivazione della vite e di altre specie economicamente rilevanti. L’ ecologia dei batteri endofiti è un tema che ancora non è stato del tutto investigato all’interno dello studio dell’interazione tra piante ed endofiti. Inoltre, le interazioni che avvengono a livello di rizosfera ed endosfera non sono ancora ben descritte ma evidenze sperimentali suggeriscono che esse siano un fattore importante nella definizione della composizione delle comunità endofite. Lo studio di come un inoculo batterico esogeno può modificare la composizione della comunità nativa di endofiti è essenziale per un uso consapevole di formulati commerciali a base di batteri endofiti. In questa tesi viene quindi presentato un lavoro preliminare che analizza l’ecologia di ceppi isolati da Glera, ceppi isolati da altre specie vegetali, e batteri commercializzati come biostimolanti. La colonizzazione di piante di pomodoro, pianta modello per lo studio delle specie orticole, da parte di questi ceppi microbici è stata dimostrata e quantificata per radici, fusto e foglie di piante coltivate per 3 e 5 settimane. Questo lavoro ha come scopo inoltre l’analisi delle comunità di endofiti di queste piante inoculate per confrontarne la composizione con piante non inoculate. Lo studio, che è ancora in corso, ha comportato l’estrazione del DNA totale della endosfera di porzioni di fusto; da questo DNA saranno amplificati i 16S rDNA dei batteri endofiti presenti e sequenziati con tecniche di NGS. In conclusione, i risultati presentati in questa tesi descrivono la composizione delle comunità di endofiti coltivabili isolate da piante di vite Glera della zona del Prosecco Conegliano-Valdobbiadene DOCG. L’isolamento di tali batteri ha fornito una larga collezione di ceppi batterici, le cui proprietà benefiche che promuovono la crescita vegetale e gli effetti dei batteri sulla morfologia radicale di Arabidopsis thaliana sono stati analizzati e presentati criticamente coinvolgendo più aspetti importanti nell’interazione pianta-endofiti. I risultati ottenuti in questo lavoro descrivono le proprietà di alcuni ceppi isolati da Glera che, quando confermato da prove sperimentali in campo, potranno essere utilizzati in sicurezza come agenti endofiti di biofertilizzazione e/o biocontrollo nella produzione dell’uva Glera e di altre specie vegetali economicamente importanti.

Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates.
Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013

Made available in DSpace on 2014-06-11T19:25:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T20:13:38Z : No. of bitstreams: 1 paro_mlc_me_araca.pdf: 3376248 bytes, checksum: 3cb7c10029858de2655c594466804a68 (MD5)
As doenças infecciosas representam uma das principais causas de perda precoce dos dentes, podendo levar a seqüelas graves. Os microrganismos normalmente envolvidos nessas patologias quase sempre pertencem a microbiota autóctone da cavidade bucal e, quase invariavelmente, são de baixa virulência. Assim, o objetivo desse estudo foi avaliar a susceptibilidade a antimicrobianos de bactérias anaeróbias obrigatórias e anaeróbias facultativas isoladas de processos infecciosos da cavidade bucal, procurando verificar a existência de padrões de susceptibilidade à fármacos nas diferentes espécies e gêneros microbianos. As amostras microbianas foram obtidas de 4 casos de osteomielite crônica da mandíbula, 3 lesões periapicais refratárias ao tratamento endodôntico, 30 infecções endodônticas, 7 casos de periodontite agressiva localizada e 2 casos de periodontite agressiva generalizada. O isolamento dos microbiano foi realizado em meios de cultura seletivos e a identificação dos isolados foi realizada de acordo com suas características morfológicas e bioquímico-fisiológicas. Os isolados, uma vez identificados, foram mantidos em nitrogênio líquido (- 196 oC). Nos testes de susceptibilidade, empregou-se o método de diluição em ágar e o meio de cultura empregado foi o ágar infuso de cérebro coração acrescido de extrato de levedura. Os resultados evidenciaram resistência natural dos anaeróbios facultativos ao metronidazol e níveis moderados de resistência às penicilinas, enquanto a cefoxitina, a associação de amoxicilina/ácido clavulânico e imipenem foram quase que universalmente eficazes. A lincomicina e a clindamicina também se mostraram eficazes, particularmente sobre os anaeróbios obrigatórios. O principal mecanismo de resistência aos b-lactâmicos foi a produção de compostos capazes de degradar essas drogas.
Infections diseases represent one of the major causes of early tooth loss, and they can lead to sequels. The microorganisms involved oftenly in these pathologies belong to oral microflora and almost all of them are of virulence potential. Thus, the objective of this study was to evaluate the susceptibility to antimicrobials of obligate and facultative anaerobes recovered from infections in head and neck area, trying to verify the existence of susceptibility patterns to those drugs the different species and microbial goods. Microbials samples were obtained of 4 cases of chronic osteomyelitis, 3 refractary periapical lesions, 30 endodontics infections, 7 cases of localized aggressive periodontitis, 2 cases of generalized aggressive periodontitis. The isolation microbial was accomplished in selective culture means and the identification of the isolated ones was accomplished in agreement with its morphologic and biochemical-physiologic characteristics. The isolates, after identification, were maintained in liquid nitrogen (- 196°C). The susceptibility tests, were carried out throught na agar dilution method and the culture medium employed was brain heart infusion agar supplemented with yeast extract. The results evidenced natural resistance of facultative anaerobes to metronidazole and moderate levels of resistance to penicillin, while cefoxitin, amoxycillin/clavulanic acid and imipenem were almost universally effective, particularly on obligate anaerobes. The main mechanisms of resistance to b-lactams was the production of compounds capable to destroy these drugs.

Objectives: The aims of this study was to investigate the RC microbiota in CCF canine teeth in the domestic dogs (Canis familiaris) and cheetahs (Acinonyx jubatus), identify the possible factors related to the presence of aerobic or anaerobic bacteria and evaluate and evaluate antibiotic susceptibility of bacteria isolated. Animals: Thirty nine animals suffering from CCF of their canine teeth were included in this study, of which 20 were dogs and 19 were cheetahs. Procedures: Evaluation of the oral cavity of animals while under general anaesthesia was performed and those without necrotic pulps or those that had received antibiotic therapy in the previous two weeks were excluded. Microbial samples were taken from 63 RC of which 27 were from dogs and 36 were from cheetahs. Strict anaerobic and aerobic techniques were used in parallel for plating, incubation and identification of the bacteria isolated in this manner. In an attempt to evaluate the sensitivity of the culture media and anaerobic technique used, additional samples were collected after the samples for bacterial isolation had been taken from the last eight pulps. These comprised those from six cheetahs and two dogs and were analysed using culture techniques and an initial screening with the 16S rRNA-specific PCR. Results: • Dogs: A total of 49 cultivable isolates were recovered belonging to 19 different bacterial species and 13 different genera. Individual RC yielded a maximum of four bacterial species. Of the bacterial isolates, 4.08 % were strict anaerobes, being represented by Clostridium acetobulitycum (2.04 %) and Prevotella melalinogenica (2.04 % ). The incidence of aerobic bacteria and facultative anaerobic bacteria in this study were 18.36 % and 77.56 %respectively of all the bacterial isolates. Of these Pasteurella multocida ( 10.20 % ), Corynebacterium spp. (10.20 %), Moraxella spp. (8.17 %), Bacillus spp. (6.12 %), Aeromonas salmonicida (6.12 %), Escherichia coli (6.12 %) and Pseudomonas aeruginosa (6.12 %) were the bacteria most frequently isolated. In summary, the RC microflora was found to be predominantly Gram negative facultative anaerobic microorganisms. The antibiotic agents that showed the highest efficacy in vitro against the different bacteria isolates were Enrofloxacin (85.21 % ), Gentamicin (92.39 %), Chloramphenicol (89.13 %). • Cheetahs: A total of 59 cultivable isolates, belonging to 19 different microbial species and 13 different genera were recovered from 36 RC sampled. Thirty-two (54.49 %) of the cultivable isolates were Gram positive while 27 (45.71 %) were Gram negative. Individual root canals each yielded a maximum of six species. Four RC had no cultivable bacteria. The bacterial micro flora recovered from the RC of the animals showed a higher number of facultative anaerobes (62.72 % of all the bacterial isolates). Aerobic isolates were 28.81 %, and strict anaerobes 8.47 % of all the isolates. The latter species comprised Clostridium sordelli (5.08 % ), and Clostridium septicum (3.38 % ). The species with the highest isolation frequency were Bacillus spp. (22.13 %), Pasteurella multocida (10.16 %), Corynebacterium spp. (8.47 %), Enterococcus spp. (8.47 %), Moraxella spp. (8.47 %) and Pseudomonas aeruginosa (5.25 %). In summary, the bacteria isolated from the RC were Gram positive facultative anaerobic bacteria. The antibiotics, which showed the highest efficacy in vitro against the different bacteria isolates, were Enrofloxacin (91.96 %), Gentamicin (86.37 %) and Orbifloxacin (86.28 %). • Nucleic Acid-Base detection: In dogs, Gram negative and Gram positive bacterial species were equally represented. Anaerobic bacterial species predominated at 83.3 % (5/6) of the species detected. On the other hand, in cheetahs, the bacterial species isolated by the PCR method showed a prevalence of anaerobic bacteria (60.8 %, 14/23), while facultative anaerobes were isolated in 30.2 % (7 /23) of cases and aerobic bacteria in 8.6 % (2/23). Conclusions and Clinical Relevance: This study has indicated that the microbial flora in any single infected RC is much more diverse than it has been shown using cultural techniques alone and can contain potentially uncultivable bacterial species. Some of these species may represent potentially new phylotypes, which may be involved in endodontic infections and ultimatelyin periradicular periodontitis, and should therefore be considered in any future studies involved in defining endodontic pathogens. Copyright
Dissertation (MSc)--University of Pretoria, 2012.
Companion Animal Clinical Studies
restricted

Dissertação de Mestrado Integrado em Medicina Veterinária
Embora os cateteres endovenosos sejam indispensáveis nos dias de hoje, ao providenciar de forma segura um fácil acesso vascular, constituem um risco acrescido para o paciente. Com efeito, os cateteres endovenosos podem ser colonizados por bactérias e provocar as chamadas infecções sanguíneas provocadas por cateteres (“CR-BSI”, Catheter Related BloodStream Infections). As CR-BSI têm sido implicadas no aumento da mortalidade e morbilidade em unidades de cuidados intensivos de pequenos animais. De entre os factores de virulência relevantes, encontram-se a capacidade de colonização e antibiorresistência das bactérias colonizadoras, ambos directamente afectados pela capacidade de formação de biofilmes por parte destas bactérias. Este importante factor de virulência, para além de permitir o estabelecimento de comunidades bacterianas sobre o cateter, promove a resistência a antibióticos e a evasão bacteriana às defesas do hospedeiro. O objectivo deste trabalho foi avaliar a presença de dois factores de virulência em bactérias isoladas a partir de cateteres endovenosos obtidos no Hospital Escolar da Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa: a capacidade de formação de biofilmes e o perfil de susceptibilidade a agentes antimicrobianos. As bactérias isoladas nos cateteres pertenciam à microbiota normal do hospedeiro ou do ambiente e o género mais representativo neste estudo foi Staphylococcus spp. A maioria das bactérias isoladas foram resistentes a pelo menos três antibióticos e os princípios activos que demostraram menor incidência de resistências foram a amoxicilinina associada ao ácido clavulânico, a gentamicina e a cefotaxima. A maioria das bactérias isoladas (62,5%) foram capazes de, in vitro, expressar biofilmes em menos de 72 horas. Foi encontrada uma correlação positiva significativa entre a formação de biofilmes e a antibiorresistência.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The deterioration of harvested sugarcane as a result of bacterial growth causes major losses of sucrose and a build-up of exopolysaccharides (EPS). Polysaccharides present during production increase the massecuite viscosity, which negatively influences evaporation and crystallisation. In this study 38 culturable EPSproducing bacteria were isolated from milled sugarcane. Analysis of the EPS showed the ubiquitous presence of glucose, however, 14 polysaccharides also contained mannose, fructose or galactose. In vitro treatment using Chaetomium erraticum dextranase to evaluate is effectiveness indicated that 37 of the EPS were hydrolysed to some extent. There were 21 polysaccharides that were only partially digested. The capacity of the isolates to produce EPS on different sugars indicated a correlation between sucrose and polysaccharide formation in 37 isolates. The results indicate there are more species involved in EPS production than previously thought as well as the presence of non-dextran polysaccharides.
AFRIKAANSE OPSOMMING: Bakteriële groei veroorsaak ‘n afname in gehalte, sukrose en ‘n verhoging in die hoeveelheid van eksternepolisakkeriede (EPS). Die verhoogde konsentrasie van polysakkariede gedurende die verwerkingsprosses veroorsaak ‘n verhoging in “massecuite” viskositeit. Hierdie verskynsel het ‘n nadelige uitwerking op die verdamping en kristalvorming van die produk. In gemaalde skuikerriet was 38 groeibare EPS-produserende bakterieë geisoleer. Die geanaliseerde EPS van hierdie bogenoemde bakterieë was daar in almal glukose teenwoordig. In 14 van hulle was mannose, fruktose en galaktose ook gevind. Die in vitro effektiwieteit van Chaetomium erraticum dekstranase op die EPS het gewys dat 37 het tot ‘n mate gehidroliseer maar 21 was net gedeeltelik verteer. As gevolg van die bo-genoemde resultate was daar gevind dat sukrose was ‘n noodsaaklike subtraat vir EPS produksie in die geisoleerde bakterieë. In hierdie studie was bevestig ‘n groter verskiedenheid EPS-produserende bakterieë gevind was en dat hulle assosiasie aan sukierriet prossering meer kompleks is as wat vooreen gedink was.

A murcha bacteriana, causada por Ralstonia solanacearum, afeta principalmente as solanáceas, destacando-se as culturas da batata, berinjela, jiló, pimentão e tomate. No presente trabalho foi conduzida a caracterização molecular de isolados de R. solanacearum e sua possível relação com características relacionadas à morfologia, bioquímica, patogenicidade, agressividade e distribuição geográfica. Foram utilizados 51 isolados pertencentes à biovar 2, sendo 9 provenientes de berinjela e 42 de batata, coletados em diversas regiões brasileiras. A análise molecular permitiu separar os isolados em quatro grupos distintos de padrões de bandas para os iniciadores BOX e ERIC, e em cinco para o iniciador REP. Não foi encontrada relação dos grupos de isolados caracterizados molecularmente com tamanho de colônias, ocorrência de mutantes, produção de melanina, capacidade de colonização do sistema radicular e resistência a antibióticos/fungicidas. A identificação de isolados de batata, como biovar 2-A, e de berinjela, como biovar 2-T, com base em teste bioquímico do uso de trealose, foi confirmadas pela análise molecular. Não houve variação de agressividade entre os isolados inoculados em batata e berinjela, exceção feita ao isolado avirulento CNPH-65. Portanto, isolados das biovares 2-A e 2-T podem infectar estas duas hospedeiras com a mesma intensidade sob altas temperaturas. Para todos os isolados, o desenvolvimento da população bacteriana foi significativamente maior no sistema radicular de plantas das cultivares suscetíveis, tanto para batata como para berinjela. No entanto, dentro de cada cultivar, os isolados se comportaram de maneira semelhante, não sendo possível fazer distinção entre os mesmos. A tentativa de se associar grupos de isolados caracterizados molecularmente com os locais de origem revelou alguns aspectos interessantes. O grupo I agregou somente isolados do Paraná. No grupo II ficaram isolados da Bahia, Distrito Federal e do Paraná. No Grupo III, foram reunidos todos os isolados de berinjela e um único de batata, sendo todos procedentes do Distrito Federal. O grupo IV, de forma semelhante ao grupo II, reuniu isolados de locais diversos como Paraná, Goiás, Rio Grande do Sul e Distrito Federal. Portanto, nos grupos I e III parece haver uma tendência de relação entre grupamento molecular e local de origem, enquanto que para os grupos II e IV, isolados de características genéticas similares são provenientes de locais distintos, apontando considerável diversidade genética do patógeno.
The bacterial wilt disease caused by Ralstonia solonacearum affects mainly the solanaceous species, specially potato, eggplant, peppers, tomato and brazilian gilo (Solanum gilo). This work reports the molecular characterization of R. solanacearum biovar 2 isolates and the possible relationship of this molecular data with other characteristics related to morphology, biochemistry, pathogenicity, aggressiveness and geographical distribution. Fifty-one biovar 2 isolates were studied, 9 isolated from eggplant and 42 from potato, all of them collected from different regions of Brazil. According to the molecular analysis, the isolates were clustered in four different groups, with distinct band patterns to the primers BOX and ERIC, and five groups to the primers REP. There was no relationship between the groups clustered through molecular analyses and phenotypic characteristics, such as colony size, presence of mutants, melanin presence, capability of root system colonization and antibiotic/fungicide resistance. The identification of potato isolates as the biovar 2-A, and the eggplant isolates as biovar 2-T, based on biochemical tests using trealose were confirmed with the molecular analyses. There was no variation of aggressiveness in the isolates inoculated on potato an eggplant, except the avirulent isolate CNPH-65. Consequently, isolates of biovars 2-A and 2-T are able to infect both hosts with the same aggressiveness under high temperatures. The population of all isolates developed in significant levels at the root system of susceptible cultivars of both hosts, potato and eggplant. However, considering each cultivar tested, there was no difference between isolates. Interesting results were observed when the isolates clustered based on molecular data were associated with the geographical region of their collection. The group I clustered only the isolates collected in Paraná. The group II clustered the isolates collected in Bahia, Federal District and some in Paraná. The group III clustered all isolates from eggplant and only one of potato, all of them collected in the Federal District. The group IV, as the group II, clustered isolates from different regions, like Paraná, Goiás, Rio Grande do Sul and Federal District. These results suggest a relationship between the isolates clustered through molecular analysis in the groups II and III and their geographical region of collection. The isolates clustered in the same way, with similar genetic background in the groups II and IV, were however collected in different regions, showing the great genetic variation of this pathogen.

Thesis (M.S.)--Georgia Southern University, 2009.
"A thesis submitted to the Graduate Faculty of Georgia Southern University in partial fulfillment of the requirements for the degree Master of Science." Directed by Laura B. Regassa. ETD. Includes bibliographical references (p. 64-69) and appendices.

Sorghum plants excrete phenolic acids which reduce subsequent crop yields. These acids accumulate in field soil by combining with soil and clay particles to form stable complexes which remain until degraded by bacterial metabolism. The amount of phenolic acids in soil samples were obtained by gas chromatography measurements, while Azotobacter populations were obtained by plate counts in 40 sorghum field samples from Denton County, Texas. One can conclude that increasing the Azotobacter population in the soil increased the degradation rate of phenolic acids proportionally. It is proposed that seed inoculation will introduce selected strains of Azotobacter into the soil. The presence of Azotobacter should increase crop size in subsequent plantings.

Dissertação de Mestrado Integrado em Medicina Veterinária
O presente estudo pretende verificar a possível associação entre a doença periodontal e a doença cardiovascular, avaliando a presença e diversidade de Enterococcus spp. na gengiva e no coração de cães com doença periodontal. Através de métodos fenotípicos e moleculares identificaram-se 117 isolados como pertencentes ao género Enterococcus e avaliou-se a sua diversidade pela técnica de PCR-fingerprinting. Selecionaram-se 46 isolados representantes, 39 identificados como E. faecalis, 7 como E. faecium e 2 permaneceram por identificar. Para estimar o potencial de patogenicidade avaliaram-se os isolados quanto à suscetibilidade a antimicrobianos e à presença de fatores de virulência. Todos os isolados mostraram resistência à clindamicina; para a tetraciclina e a gentamicina as percentagens foram acima dos 50% e para os restantes antimicrobianos mantiveram-se abaixo desse valor. Na pesquisa de fatores de virulência 43% dos isolados revelaram-se β-hemolíticos e 23% gelatinase positivos. Para os genes de virulência pesquisados detetaram-se percentagens acima dos 50% para gelE, efaAfs, ebpA, ebpB, ebpC e gls24 e abaixo desse valor para agg, esp, efaAfm, cylA, acm e ace. Não se verificou associação entre a doença periodontal e a endocardite bacteriana, mas foi possível verificar a presença de bactérias de importância clínica disseminadas pela boca e coração de cães em níveis relativamente elevados, sendo de suma importância prosseguir estudos no sentido de melhor compreender esta possível associação.
ABSTRACT - Characterization of Enterococcus spp. isolated from the mouth and heart of dogs with periodontal disease - The present study investigated the possible association between periodontal and cardiovascular disease, evaluating the presence and diversity of Enterococcus spp. in the gum and heart of dogs with periodontal disease. Phenotypic and molecular methods were used, yielding a total of 117 isolates identified as Enterococcus spp., evaluated for diversity by PCR-fingerprinting. 46 representative isolates were selected, 39 of which identified as E. faecalis, 7 as E. faecium and two remained unidentified. To estimate the potential pathogenicity of the isolates, they were evaluated for susceptibility to antimicrobial agents and the presence of virulence factors. All isolates showed resistance to clindamycin; for tetracycline and gentamicin percentages were above 50% and for the remaining antimicrobials remained below this value. In search of virulence factors 43% of the isolates proved to be β-hemolytic and 23% gelatinase positive. For the virulence genes surveyed, percentages observed were above 50% for gelE, efaAfs, ebpA, ebpB, ebpC and gls24 and below this value for agg, esp, efaAfm, cylA, acm and ace. It was not possible to establish an association between periodontal disease and bacterial endocarditis, but it was possible to verify the presence of bacteria of clinical importance disseminated through the mouth and heart of dogs at relatively high levels. It is extremely important to continue studies to better understand this possible association.

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%.

Os psitacídeos estão entre as espécies de aves mais apreendidas e encaminhadas aos centros de triagem animal em São Paulo. Também são comumente mantidos em ambiente doméstico como aves de estimação. A manutenção destas aves em cativeiro pode representar um risco zoonótico e contribuir para a propagação das estirpes de enterobactérias multirresistentes, como Klebsiella spp. produtora de beta-lactamase de espectro estendido (ESBLs), que podem interferir no tratamento de infecções nosocomiais em humanos. O objetivo deste estudo foi identificar e caracterizar estirpes de Klebsiella spp. isoladas de secreções respiratórias de 46 psitácideos doentes, determinando a virulência e o perfil de resistência a 15 antimicrobianos. Dentre as 19 estirpes de Klebsiella spp. isoladas, 16 (16/19) foram identificadas como Klebsiella pneumoniae, e três (3/19) foram identificadas como Klebsiella oxytoca. O perfil de suscetibilidade aos antimicrobianos demonstrou alta resistência para ampicilina (89,5%), e o perfil de virulência demonstrou uma alta prevalência dos genes fimH (94,7%), kpn (89,4%), uge (84,2%) e irp-2 (78,9%). Três estirpes de K. pneumoniae foram positivas para produção de beta-lactamase de espectro estendido. Estas estirpes foram classificadas nos sequence types (STs) ST15, ST147 e ST307. Esses três grupos clonais representam os principais responsáveis por surtos de infecções hospitalares por K. pneumoniae no mundo. No entanto, esse é o primeiro relato desses clones como causadores de doença em aves. Esses dados indicam a ocorrência de K. pneumoniae produtora de CTX-M-15 e CTX-M-8 em psitacídeos cativos e confirmam o potencial zoonótico e antropozoonótico do agente, destacando a relevância clínica para humanos e animais.
Psittacine birds are among the most seized bird species that are sent to animal sorting centers in São Paulo. They are also commonly kept in the domestic environment like pet birds. The maintenance of these birds in captivity may represent a zoonotic risk and contribute to the propagation of strains of multiresistant enterobacteria, such as Klebsiella spp. beta-lactamase extended-spectrum (ESBLs), which may interfere in the treatment of nosocomial infections in humans. The objective of this study was to identify and characterize strains of Klebsiella spp. isolated from respiratory secretions of 46 diseased psittacines, determining virulence and resistance profile to 15 antimicrobials. Among the 19 strains of Klebsiella spp. isolated, 16 (16/19) were identified as Klebsiella pneumoniae, and three (3/19) were identified as Klebsiella oxytoca. The antimicrobial susceptibility profile demonstrated high resistance to ampicillin (89.5%), and the virulence profile demonstrated a high prevalence of fimH (94.7%), kpn (89.4%), uge (84.2% %) and irp-2 (78.9%). Three strains of K. pneumoniae were positive for extended-spectrum beta-lactamase production. These strains were classified in sequence types (STs) ST15, ST147 and ST307. These three clonal groups represent the main responders for outbreaks of K. pneumoniae nosocomial infections worldwide. However, this is the first account of these clones as causing disease in birds. These data indicate the occurrence of K. pneumoniae producing CTX-M-15 and CTX-M-8 in captive parrots and confirm the zoonotic and anthropozoonotic potential of the agent, highlighting the clinical relevance for humans and animals.

Made available in DSpace on 2016-08-17T18:39:42Z (GMT). No. of bitstreams: 1 4412.pdf: 1461979 bytes, checksum: 8e5362be1a8593a0acafb4ba8014c92e (MD5) Previous issue date: 2011-08-29
Financiadora de Estudos e Projetos
The searching for new fuel sources has motivated new studies which aim to produce the second-generation of ethanol, also called 2G . However, the costs to produce that fuel are still high because the sugarcane bagasse is not considered a residue any more, but a subproduct that can generate income to the industries that produce it, being a vapor energy form to the company or an electricity source to sell. The use of the sugar cane bagasse to produce 2G ethanol requires a significant amount of lignocellulolitic enzymes. The main aim of this study is to reduce the cost of the production of these enzymes by means of studying isolated lignocellulolitic bacterium from Stenochironomus larva. The goal is to evaluate its productivity in several submersed fermentation processes and surface fermentations. Submersed fermentations were conducted using sugar cane bagasse and soy meal as substrates and different concentrations were applied in culture media. A total of 11 isolated enzymes were evaluated initially, and re-selected at each new stage of the process. The enzyme cultures were conducted by the reduction sugars quantification and the results enabled us to demonstrate that the bacteria Sphingobium and Pseudomonas were the most productive when compared to the others. Our results have highlighted that those bacteria were strongly influenced by the addition of different organic nitrogen sources in the fermentation culture. From the introduction of those components, the enzyme activity became higher and this fact was corroborated by the index of 4.36 U/mg protein for Xilanase, 0.12 U/mg protein for FPase and 2.31 U/mg protein for CMCase.
A busca por novas fontes de combustíveis tem feito com que diferentes estudos surgissem com o objetivo de produzir etanol de segunda geração, conhecido como etanol 2G. Os custos para se produzir esse combustível ainda são elevados, uma vez que o bagaço de cana não é mais considerado um resíduo, mas sim um subproduto que gera renda para a usina que o detém, seja na forma de energia (vapor) ou na venda de energia elétrica. Para se conseguir utilizar o bagaço de cana a fim de produzir etanol 2G é necessário uma grande quantidade de enzimas lignocelulolíticas. Visando a redução do custo de produção dessas enzimas para que o processo de produção do etanol 2G se torne viável, o presente trabalho tem o objetivo de estudar diferentes isolados bacterianos lignocelulolíticos de larvas de Stenochironomus, pretendendo avaliar seu rendimento na produção dessas enzimas. Para se realizar essa avaliação, fermentações submersas foram realizadas utilizando o bagaço de cana e farelo de soja como substrato indutor e diferentes concentrações de meios de cultivo. Um total de 11 isolados foram avaliados inicialmente e selecionados a cada nova etapa. A partir dos resultados dos ensaios enzimáticos, que foi realizado através da quantificação de açúcares redutores, foi demonstrado que as bactérias Sphingobium e Pseudomonas se destacaram na produção dessas enzimas em relação às demais bactérias inicialmente testadas. Os resultados deste trabalho evidenciou que essas bactérias foram fortemente influenciadas pela adição de diferentes fontes de nitrogênio orgânico em seu meio de cultivo para as fermentações, o que gerou uma maior atividade enzimática obtendo índices de 4,36 U/mg de proteína para Xilanase, 0,12 U/mg de proteína para FPAse e 2,31 U/mg de proteína para CMCase, a partir da introdução desses componentes aos meios.