A utilização de técnicas moleculares têm facilitado o estudo de comunidades bacterianas complexas no ambiente. O presente trabalho teve como objetivo utilizar a técnica DGGE para avaliação dos efeitos do aumento da temperatura, causado pela queima de um canavial, na estrutura da comunidade bacteriana de solo, com ênfase ao grupo dos actinomicetos. Foram coletadas amostras de solo em diferentes profundidades, antes e depois da queima, e dados físico-químicos e climáticos associados. O DNA da comunidade bacteriana foi amplificado utilizando conjunto de primers específicos para o Domínio Bacteria e para o grupo de actinomicetos, e os produtos de amplificação analisados por DGGE. Resultados obtidos para as populações de actinomicetos não foram conclusivos. Apesar da variação dos parâmetros físico-químicos do solo provocadas pela queima, os padrões de bandas obtidos com os primers para o Domínio Bacteria, apresentaram-se uniformes. Sendo assim, nas condições de estudo deste trabalho, os resultados obtidos não revelaram alterações na estrutura da comunidade bacteriana do solo de canavial depois da queima. / The utilization of molecular techniques has facilitated the study of complex bacterial communities in the environment. The present study aimed at using DGGE technique to evaluate the effect of temperature variation, caused by sugar-cane plantation burn, in the soil bacterial community structure emphasizing the actinomycete group. Soil samples from different depths, were collected before and after the bum, as well as physical-chemical and climatic associated data. The bacterial community DNA was amplified using a specific primer set and the amplification products analyzed by DGGE. The results obtained for the actinomycete populations were not conclusive. Despite the variation of the soil parameters caused by the burn, the band patterns obtained used in this study were uniform. Therefore, under the conditions used in this study, the results obtained did not show any alteration in the structure of soil bacterial community associated with sugar-cane plantation after the burn.

Endophytic microorganisms were screened for hemicellulases production using plate assays and liquid cultivations. Two strains were selected and used in further studies. Aspergillus niger DR02 strain and Annulohypoxylon stigyum DR47. In A.niger fed-batch submerged cultivation approaches were developed using liquor from hydrothermal sugar cane pretreatment, and maximum xylanase activities obtained were 458.1 U/mL for constant fed-batch mode. For A. stygium DR47 media optimization and bioreactor cultivation using citrus bagasse and soybean bran were explored and revealed a maximum production of 6.26 U/mL of pectinase and 10.13 U/mL of β-glucosidase. Improved hemicellulase production was also done by genetic modifications at carbon catabolic repression levels in A. niger. Mass spectrometric studies were done in enzymatic extracts produced and contributed to understand the enzymatic supplementation of Celluclast 1.5L.In addition the extracts produced were tested for an enzyme formulation and that was able to increase sugar cane bagasse hydrolysis. / Microrganismos endofíticos foram selecionados para a produção de hemicelulases através de testes em placa e de cultivo líquido. Duas linhagens foram selecionadas para posteriores cultivos, Aspergillus niger DR02 e Annulohypoxylon stigyum DR47. Para o fungo A.niger batelada alimentada em cultivo submerso com licor do tratamento hidrotérmico do bagaço de cana de açúcar permitiram obtenção de máxima atividade de xilanase de 458,1 U/mL. Para a linhagem A. stygium DR47 otimização do cultivo utilizando bagaço cítrico e farelo de soja foram explorados e revelaram produção de 6,26 U/mL de pectinase e 10,13 U/mL de β-glucosidase. Melhoramento da produção de hemicelulase foi realizado com modificações genéticas ao nível de repressão catabólica de carbono em A. niger. Estudos de espectrometria de massas foram realizados nos extratos enzimáticos produzidos e contribuíram para entender a suplementação da Celluclast 1.5L. Além disso, os extratos produzidos foram testados para uma formulação que foi capaz de aumentar a hidrólise do bagaço de cana de açúcar.

Discoveries of new natural antibiotics are now relatively rare, therefore the construction of strains producing hybrid substances seems to be a very promising opportunity to gain new interesting biologically active compounds. This work is part of a larger project focused on the preparation of new biologically active substances derived from the antibiotic lincomycin. Lincomycin is composed of saccharide (MTL) and amino acid (propylhygric acid) moieties condensed by amide bond. Various modifications of amino acid moiety, especially of the side alkyl chain, are known to improve the antibiotic properties of final molecule. The bottleneck of biosynthesis of such modified compounds is the condensing enzyme NDL-synthetase, and especially its A-domain, which, similarly to nonribosomal peptide synthetases (NRPS), specifically recognizes and activates the amino acid precursor. In this work a set of degenerate primers for PCR searching of NRPS A-domains was proposed and the conditions of PCR reaction were optimized. In the first step a collection approximately 800 isolates of soil actinomycetes will serve as a source of genetic information for search of interesting NRPS A-domains, applicable for the construction of hybrid biosynthetic clusters. The isolates of this collection have been also characterized taxonomically...

As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. / Dissertation / Doctor of Philosophy (PhD) / Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target.

Actinobactérias estão presentes nos mais diversos ambientes e são conhecidas por produzirem compostos com aplicabilidade, principalmente, na área médico-farmacológica e agronômica. Nos últimos anos, as pesquisas com microorganismos de ambientes pouco explorados têm aumentado significativamente, a fim de isolar novas espécies e novos compostos bioativos eficazes no controle de patógenos resistentes. Nesse contexto, a exploração de ilhas desponta como uma alternativa promissora. Ilhas oceânicas apresentam um habitat particular, com características diferentes do continente, muitas vezes abrigando organismos endêmicos. Neste estudo foram realizados o isolamento e o estudo químico e biológico de actinobactérias isoladas das ilhas de Alcatrazes e Palmas, localizadas no litoral norte de São Paulo. Amostras de solo rizosférico, folhas, raízes e pecíolo foram coletadas das plantas Anthurium alcatrazense e Anthurium urvilleanum. O material coletado foi inoculado em três meios de cultivo, resultando no isolamento de 23 actinobactérias, que foram identificadas majoritariamente como pertencentes ao gênero Streptomyces, com exceção de um isolado, pertencente ao gênero Amycolatopsis. Os isolados foram avaliados quanto à presença de genes de policetídeos (PKS-I) e peptídeos não ribossomais (NRPS), e foram também cultivados em quatro meios de cultivo, para explorar a produção de metabólitos secundários. Os extratos foram particionados e avaliados frente a bactérias patogênicas e fungos fitopatogênicos. Extratos de seis linhagens inibiram o crescimento de pelo menos um destes patógenos: Burkholderia cepacia, Escherichia coli, Staphylococcus aureus, Staphylococcus schleiferi, Rhizoctonia solani e Sclerotium rolfsii. Todos os isolados positivos no bioensaio, foram positivos para uma das vias biossintéticas (PKS-I ou NRPS). O isolado identificado como Streptomyces fulvissimus (A32), foi selecionado para investigação dos metabólitos secundários. Este é o primeiro relato do isolamento de actinobactérias das ilhas de Alcatrazes e Palmas, bem como do estudo do potencial antimicrobiano destes micro-organismos. / Actinobacteria are present in the most diverse environments and are known to produce compounds with applicability, mainly in the medical-pharmacological and agronomic area. In recent years, research with microorganisms from poorly explored environments has increased significantly in order to isolate new species and new bioactive compounds effective in the control of resistant pathogens. In this context, the exploration of islands emerges as a promising alternative. Oceanic islands have a particular habitat, with different characteristics of the continent, often harboring endemic organisms. In this study were carried out the isolation and chemical and biological study of actinobacteria isolated from the Palmas and Alcatrazes islands, north coast of São Paulo. Samples of rhizospheric soil, leaves, roots and petiole were collected from plants Anthurium alcatrazense and Anthurium urvilleanum. The collected material was inoculated in three culture media, resulting in the isolation of 23 actinobacteria, which were identified mainly as belonging to the genus Streptomyces, with the exception of one isolate belonging to the genus Amycolatopsis. The isolates were evaluated for the presence of polyketide (PKS-I) and non-ribosomal peptides (NRPS), in addition, were cultured in four culture media to explore the production of secondary metabolites. The extracts were partitioned and evaluated against pathogenic bacteria and phytopathogenic fungi. Extracts from six isolates inhibited the growth of at least one of these pathogens: Burkholderia cepacia, Escherichia coli, Staphylococcus aureus, Staphylococcus schleiferi, Rhizoctonia solani and Sclerotium rolfsii. All the positive isolates in the bioassay were positive for one of the biosynthetic pathways (PKS-I or NRPS). The isolate identified as Streptomyces fulvissimus (A32) was selected for investigation of secondary metabolites. This is the first report of the isolation of actinobacteria from the Alcatrazes and Palmas islands, as well as the study of the antimicrobial potential of these microorganisms.

Metabólitos secundários produzidos por actinobactérias são uma inesgotável fonte de compostos com potentes atividades biológicas e estruturas intrínsecas. O desenvolvimento em instrumentação analítica tem contribuído significantemente para acelerar o processo de identificação e caracterização desses metabólitos bioativos. Sem dúvida alguma, a espectrometria de massas (MS) e o seu acoplamento com técnicas de separação, especialmente a cromatografia líquida (UHPLC-MS), tem sido reconhecida como a técnica mais eficiente em análises de produtos naturais. Nesta dissertação foi explorado o potencial da espectrometria de massas como ferramenta analítica para a identificação e caracterização estrutural de fitotoxinas produzidas por actinobactérias isoladas da rizosfera de plantas da caatinga. Foram produzidos noventa extratos de actinobactérias, dos quais quinze apresentaram alguma atividade para o bioensaio da Lemna minor e seis apresentaram atividade para o bioensaio da Chlorella vulgaris. Os extratos brutos ativos das actinobactérias Caat 7-38, Caat 8-6 e Caat 5-29 foram selecionados para caracterização dos compostos ativos, os quais foram isolados empregando o fracionamento guiado por bioensaios. No extrato bruto Caat 7-38, a actinomicina D foi identificada como fitotoxina, ao passo que para o extrato bruto Caat 8-6, foi possível inferir a atividade fitotóxica à presença do griseorhodin A. Já para o extrato bruto Caat 5-29, o composto identificado com atividade fitotóxica apresenta uma estrutura inédita, provavelmente pertencente à classe das anguciclinonas. Foi realizado ainda um estudo para avaliar o efeito da adição de terras raras ao meio de cultivo da actinobacteria Caat 7-38. Para os meios de cultivos contendo neodímio e, principalmente, lantânio ocorreu uma superprodução da actinomicina D, indicando assim, o grande potencial da aplicação das terras raras nos estudos de micro-organismos. / Secondary metabolites produced by actinomycetes are an inexhaustible source of compounds with potent biological activities and intrinsic structures. The development analytical instrumentation has contributed significantly to accelerate the identification and characterization of these bioactive metabolites. Undoubtedly, mass spectrometry (MS) and its coupling with separation techniques, especially liquid chromatography (UHPLC-MS) has been recognized as the most \"efficient\" technique in natural product analysis. In this work was explored the potential of mass spectrometry as an analytical tool for identification and structural characterization of phytotoxins produced by actinomycetes isolated from the rhizosphere of plants from the Caatinga biome. Ninety actinomycetes extracts were produced, of which fifteen showed some activity for the bioassay with Lemna minor and six showed activity for the bioassay with Chlorella vulgaris. The crude active extract of actinomycetes Caat 7-38, Caat 8-6 and Caat 5-29 were selected to characterize the active compounds, which were isolated using bioassay-guided fractionation. In the crude extract Caat 7-38, actinomycin D was identified as phytotoxin, while for crude extract Caat 8-6, it was possible to infer phytotoxic activity to the presence of griseorhodin A. For the crude extract Caat 5-29, the compound identified with phytotoxic activity presents a new structure, probably belonging to the class of anguciclinones. A study to evaluate the effect of addition of rare earths to the culture medium of actinobacteria Caat 7-38 was also carried out. To the culture medium containing neodymium and especially lanthanum occurred overproduction of actinomycin D, thus indicating the great potential of application of rare earths in the studies of microorganisms.

Saccharothrix algeriensis est une bactérie filamenteuse capable de produire différentes molécules de la famille des dithiolopyrrolones présentant des activités antibiotiques et anticancéreuses. Ce travail porte sur l’analyse macroscopique et la modélisation du comportement dynamique de cette bactérie en vue de concevoir et d’optimiser un procédé de bioproduction de thiolutine. Les expériences menées en fermenteur de deux litres ont permis de décrire le comportement macroscopique de Sa. algeriensis. Sur substrat mixte (extrait de levure et glucose), la bactérie présente une croissance diauxique. La production de thiolutine, découplée de la croissance, est provoquée par l’épuisement du substrat préférentiel (extrait de levure). L’analyse des bilans carbone, azote et rédox nous a conduit à considérer deux phénomènes physico-chimiques qui n’étaient pas pris en compte dans le modèle proposé par Strub (2008) : l’entrainement de l’ammonium dans la phase gazeuse et l’adsorption des ions ammonium et des acides aminés sur la biomasse. Les données réconciliées sur la base des bilans élémentaires sont utilisées comme support pour la construction d’un modèle stœchio-cinétique. Différentes approches de modélisation des cinétiques et de représentation des bascules métaboliques ont été testées afin de retenir un schéma stœchio-cinétique apparent cohérent avec les données réconciliées. Cette étape de discrimination a été réalisée à l’aide de la toolbox ExOptim, développée en partenariat avec AgroParisTech. Cet outil, alliant une méthode d’optimisation robuste tout en limitant les temps de calcul, permet de prendre en compte les erreurs de mesure dès la mesure initiale et de reparamétrer indépendamment les paramètres du modèle et les données expérimentales. Les cinétiques réactionnelles sont représentées de manière satisfaisante par l’association du modèle de Monod et la loi logistique. Les seize paramètres du modèle sont identifiés sur sept expériences indépendantes. Le modèle finalement obtenu est ensuite testé sur cinq nouvelles expériences aux conditions initiales différentes. Ce modèle permet de prédire raisonnablement les cinétiques de croissance et de production de thiolutine. Toutefois, son utilisation reste limitée à des conditions initiales proches de celles de cette étude. / Saccharothrix algeriensis is a filamentous bacterium that produces several dithiolopyrrolone compounds with antibiotic and antitumor properties. This study focuses on the macroscopic analysis and modelling of the dynamic behaviour of this bacterium, in order to design and optimize the bioproduction process of thiolutin. Experiments were conducted in a two-litres bioreactor to characterise the macroscopic behaviour of Sa. algeriensis. On a medium containing several substrates (yeast extract and glucose), the bacterium shows a diauxic growth. Thiolutin production, which appears to be dissociated from growth, is triggered by the preferential substrate (yeast extract) depletion. The analysis of the carbon, nitrogen and redox balances led us to consider two physicochemical phenomena, which were not taken into consideration in the model proposed by Strub (2008): ammonium stripping in gaseous phase and adsorption of ammonium ions and amino acids on biomass. The data, reconciled on the basis of elementary balances, were used for the construction of a stoichio-kinetic model. Different modelling of kinetic and metabolic switches were tested in order to select a stoichio-kinetic model consistent with the reconciled data. This discrimination step was carried out using the ExOptim toolbox, developed in partnership with AgroParisTech. This toolbox combines a robust optimisation method and a reduced computation time. The experimental biases are taken into account from the first measurement and model parameters and experimental data reconditioned independently. The reaction kinetics are well represented by association of the Monod model and logistic laws. The sixteen model parameters were estimated from seven experiments. Finally, the model was validated by five additional experiments using different initial conditions. The kinetics of growth and thiolutin production are reasonably well predicted. However, the model is limited to initial conditions close to those of this study.

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No. of bitstreams: 1 Dissertação - Nélly Mara Vinhote da Silva.pdf: 662011 bytes, checksum: 008fdae35511497291f0cd0b8ca08a91 (MD5) Previous issue date: 2008-03-11 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / The study of habitats that have been few explored with purpose of obtaining composts with biotechnological interest that has been produced by the Amazonian’s biological diversity it’s a strategy that will allow the discovery of important bioactive principles. In microbial biodiversity, the actinomycetes represents the most important bacterial group. They have a filamentous organization that occur in great varieties of substrata and present a great application in pharmaceutical industry. The scope of this work was to analyze taxonomically the actinomycetes associated at lichens and to determine the capability of these bacteria for antibiotic producing. Ten samples of foliose lichens were collected in area Federal University of Amazonas (UFAM), South Section. For the isolation of actinomycetes were utilized the culture media Yeast Extract-Malt Extract Agar–Starch (ISP-2A), Casein-Starch Agar (CAA), Raffinose-Histidin Agar (RHA) and Water Agar (AA), added with antifungics. Were isolated 71 actinomycetes associated to foliose lichens. The isolated were tested for antimicrobial activity against eight microorganisms-test by the techniques with cultivation in solid medium and broth culture. Among the actinomycetes tested by solid medium 80% shown antimicrobial activity, mainly against Aspergillus niger, Candida albicans and Staphylococcus aureus. In assay by broth cultivation 79% of the actinomycetes inhibited the growth of microorganisms-test, although the higher activities were against Mycobacterium smegmatis and Staphylococcus aureus. The isolated antimicrobial activity varied from moderate (halo=13 at 18 mm) to high (halo=19 at 35 mm) activity. It was observed that 68% of isolated presented high antimicrobial activity. The identification of the actinomycetes was done by the macro and micromorphological determination, physiologic tests and by the determination of aminoacids from the cell wall, and most of them belonging to genus Streptomyces. The microorganisms were preserved by freezing at -20 oC and by preservation of actinomycetes colony directly in water (Castellani’s method). / O estudo de habitats pouco explorados com a finalidade de obter biocompostos de interesse biotecnológicos produzidos pela diversidade biológica da Amazônia é uma estratégia que permitirá a descoberta de importantes princípios bioativos. Dentre a biodiversidade microbiana, destacam-se os actinomicetos, que são um grupo de bactérias de organização filamentosa que ocorrem em uma grande variedade de substratos e apresentam múltiplas aplicações na indústria farmacêutica, principalmente no que tange a produção de antimicrobianos. Este trabalho teve como escopo analisar taxonomicamente os actinomicetos associados a liquens e determinar a capacidade destas bactérias em produzir antibióticos. Foram coletadas dez amostras de liquens folhosos das árvores presentes na área do Campus Universitário, Setor Sul, da Universidade Federal do Amazonas (UFAM). Para o isolamento dos actinomicetos foram utilizados os meios de cultivo: Ágar Extrato de Levedura-Extrato de Malte-Amido (ISP-2A), Ágar Caseína-Amido (CAA), Ágar Rafinose-Histidina (RHA) e Ágar-Água (AA), suplementados com antifúngicos. Como resultados foram isolados 71 actinomicetos associados aos liquens. Os isolados foram testados quanto à caracterização da atividade antimicrobiana contra oito microrganismos-teste, através de técnicas em meio sólido e em meio líquido. Das linhagens de actinomicetos testados, 80% apresentaram atividade antimicrobiana em meio sólido, principalmente contra Aspergillus niger, Candida albicans e Staphylococcus aureus. No ensaio em meio líquido, 79% das linhagens foram capazes de inibir o crescimento dos microrganismos-teste, sendo que as maiores atividades foram detectadas contra Mycobacterium smegmatis e Staphylococcus aureus. A atividade antimicrobiana dos isolados variou de moderada (halo=13 a 18 mm) a alta (halo=19 a 35 mm) atividade. Observou-se que 68% dos isolados em meio sólido apresentaram alta atividade antimicrobiana frente aos microrganismos-teste. A identificação dos actinomicetos se deu em nível de gênero, através da determinação da micromorfologia, testes fisiológicos e da determinação de aminoácidos da parede celular, sendo a grande maioria pertencente ao gênero Streptomyces. Os microrganismos foram preservados por congelamento a -20 oC e através da técnica de preservação em água (método de Castellani).

Metabólitos secundários produzidos por actinobactérias são uma inesgotável fonte de compostos com potentes atividades biológicas e estruturas intrínsecas. O desenvolvimento em instrumentação analítica tem contribuído significantemente para acelerar o processo de identificação e caracterização desses metabólitos bioativos. Sem dúvida alguma, a espectrometria de massas (MS) e o seu acoplamento com técnicas de separação, especialmente a cromatografia líquida (UHPLC-MS), tem sido reconhecida como a técnica mais eficiente em análises de produtos naturais. Nesta dissertação foi explorado o potencial da espectrometria de massas como ferramenta analítica para a identificação e caracterização estrutural de fitotoxinas produzidas por actinobactérias isoladas da rizosfera de plantas da caatinga. Foram produzidos noventa extratos de actinobactérias, dos quais quinze apresentaram alguma atividade para o bioensaio da Lemna minor e seis apresentaram atividade para o bioensaio da Chlorella vulgaris. Os extratos brutos ativos das actinobactérias Caat 7-38, Caat 8-6 e Caat 5-29 foram selecionados para caracterização dos compostos ativos, os quais foram isolados empregando o fracionamento guiado por bioensaios. No extrato bruto Caat 7-38, a actinomicina D foi identificada como fitotoxina, ao passo que para o extrato bruto Caat 8-6, foi possível inferir a atividade fitotóxica à presença do griseorhodin A. Já para o extrato bruto Caat 5-29, o composto identificado com atividade fitotóxica apresenta uma estrutura inédita, provavelmente pertencente à classe das anguciclinonas. Foi realizado ainda um estudo para avaliar o efeito da adição de terras raras ao meio de cultivo da actinobacteria Caat 7-38. Para os meios de cultivos contendo neodímio e, principalmente, lantânio ocorreu uma superprodução da actinomicina D, indicando assim, o grande potencial da aplicação das terras raras nos estudos de micro-organismos. / Secondary metabolites produced by actinomycetes are an inexhaustible source of compounds with potent biological activities and intrinsic structures. The development analytical instrumentation has contributed significantly to accelerate the identification and characterization of these bioactive metabolites. Undoubtedly, mass spectrometry (MS) and its coupling with separation techniques, especially liquid chromatography (UHPLC-MS) has been recognized as the most \"efficient\" technique in natural product analysis. In this work was explored the potential of mass spectrometry as an analytical tool for identification and structural characterization of phytotoxins produced by actinomycetes isolated from the rhizosphere of plants from the Caatinga biome. Ninety actinomycetes extracts were produced, of which fifteen showed some activity for the bioassay with Lemna minor and six showed activity for the bioassay with Chlorella vulgaris. The crude active extract of actinomycetes Caat 7-38, Caat 8-6 and Caat 5-29 were selected to characterize the active compounds, which were isolated using bioassay-guided fractionation. In the crude extract Caat 7-38, actinomycin D was identified as phytotoxin, while for crude extract Caat 8-6, it was possible to infer phytotoxic activity to the presence of griseorhodin A. For the crude extract Caat 5-29, the compound identified with phytotoxic activity presents a new structure, probably belonging to the class of anguciclinones. A study to evaluate the effect of addition of rare earths to the culture medium of actinobacteria Caat 7-38 was also carried out. To the culture medium containing neodymium and especially lanthanum occurred overproduction of actinomycin D, thus indicating the great potential of application of rare earths in the studies of microorganisms.

No / Propionibacterium acnes is the target of antimicrobial treatments for acne vulgaris. Acquired resistance to erythromycin, clindamycin and tetracyclines has been reported in strains from diverse geographical loci, but the molecular basis of resistance, via mutations in genes encoding 23S and 16S rRNA, respectively, has so far only been elucidated for isolates from the U.K. The study set out to determine whether similar or different resistance mechanisms occur in resistant P. acnes isolates from outside the U.K. The phenotypes and genotypes of 73 antibiotic-resistant strains of P. acnes obtained from the skin of acne patients in the U.K., U.S.A., France, Germany, Australia and Japan were compared. Antibiotic susceptibilities were determined by minimum inhibitory concentration (MIC) measurements, and polymerase chain reaction and DNA sequencing were used to identify mutations in genes encoding rRNA. Most erythromycin-resistant isolates (MIC90¿ 512 ¿g mL¿1) were cross-resistant to clindamycin but at a much lower level (MIC90¿ 64 ¿g mL¿1). As in the U.K., resistance to erythromycin was associated with point mutations in 23S rRNA in 49 of 58 strains. An A¿G transition at Escherichia coli equivalent base 2058 was present in 24 strains. This gave a unique cross-resistance phenotype against a panel of macrolide, lincosamide and type B streptogramin antibiotics. Two further point mutations (at E. coli equivalent bases 2057 and 2059) were identified (in three and 22 isolates, respectively) and these were also associated with specific cross-resistance patterns originally identified in isolates from the U.K. However, nine of 10 erythromycin resistant-strains from Germany did not exhibit any of the three base mutations identified and, in six cases, cross-resistance patterns were atypical. Consistent with previous U.K. data, 34 of 38 tetracycline-resistant strains carried a base mutation at E. coli 16S rRNA equivalent base 1058. Tetracycline-resistant isolates displayed varying degrees of cross-resistance to doxycycline and minocycline, but isolates from the U.S.A. had higher MICs for minocycline (4¿16 ¿g mL¿1) than isolates from other countries and, in particular, Australia. All the P. acnes isolates resistant to one or more of the commonly used antiacne antibiotics were sensitive to penicillin, fusidic acid, chloramphenicol and the fluoroquinolone, nadifloxacin. All but one isolate (from the U.K.) were sensitive to trimethoprim. This study shows that 23S and 16S mutations identified in the U.K. conferring antibiotic resistance in P. acnes are distributed widely. However, resistant strains were isolated in which mutations could not be identified, suggesting that as yet uncharacterized resistance mechanisms have evolved. This is the first report of high-level resistance to minocycline and is of concern as these strains are predicted to be clinically resistant and are unlikely to remain confined to the U.S.A. Epidemiological studies are urgently required to monitor how resistant strains are selected, how they spread and to ascertain whether the prevalence of resistance correlates with antibiotic usage patterns in the different countries.