Instead of planktonic growth in nature, many species of bacteria form biofilm to survive in harsh conditions. Although many chronic bacterial infections are caused by bacterial species in a biofilm lifestyle, previous research has focused on studying antibiotic resistance in planktonic growth. Here we used a modified MBEC assay, i.e. biofilm growth on pegs, to determine Escherichia coli biofilm inhibitory concentrations (BIC) of ciprofloxacin, streptomycin and rifampicin and to study the minimal selective concentration (MSC) for ciprofloxacin in E. coli biofilm. We could observe high inhibitory concentrations for all antibiotics in the biofilm pre-formed in media without antibiotics compared to the biofilm formed in antibiotics. We also show preliminary result indicating that sublethal concentrations of ciprofloxacin lead to the selection of ciprofloxacin resistant mutants in biofilm and that the selection level is lower than what was observed in planktonic growing E. coli. With more knowledge in how the biofilm formation precedes in different antibiotic settings, the treatment for chronic biofilm infections used today could be evaluated and changed so that the infections could be eradicated.

Em concentrações subletais, agentes antimicrobianos modulam a expressão gênica bacteriana, sendo que o conjunto de genes que é modulado depende tanto da cepa bacteriana, como da natureza do agente antimicrobiano. Neste trabalho, avaliamos o perfil de expressão gênica de Xylella fastidiosa cepa 9a5c em resposta ao tratamento por até 60 minutos com dose subletal do antibiótico estreptomicina. Esta é uma cepa virulenta, originalmente isolada de laranjeiras com sintomas de clorose variegada dos citros. A hibridização de microarranjos de DNA representando 2608 das 2848 sequências codificadoras (CDS) previamente anotadas no genoma desta cepa, revelou que 136 CDS apresentaram expressão gênica diferencial em resposta à exposição à estreptomicina, sendo que destas 109 foram negativamente moduladas e 27 positivamente moduladas. Realizamos, também, ensaios de PCR quantitativo precedido de transcrição reversa (RTqPCR) de 21 CDS para confirmar a modulação observada na análise global da expressão gênica. O perfil de expressão gênica de X. fastidiosa em resposta à estreptomicina foi analisado de forma integrada com outros perfis de expressão gênica desta bactéria. Entre as CDS positivamente moduladas, destacamos aquelas codificadoras das chaperoninas GroEL e GroES, que estão associadas a resposta de choque térmico, e CDS associadas à tradução, tais como proteínas ribossomais e fatores de tradução. Interessantemente, a exposição à estreptomicina induz a expressão da CDS que codifica poligalacturonase, que é um fator de virulência em algumas cepas de X. fastidiosa. Por outro lado, o tratamento com estreptomicina promoveu a modulação negativa de CDS relacionadas à formação e manutenção de biofilme ao contrário do observado quando estas bactérias foram submetidas ao tratamento com gomesina, um peptídeo antimicrobiano. O conjunto destas observações sugere que a exposição à dose subletal de estreptomicina possa promover um fenótipo de maior virulência, contrariamente ao efeito previamente observado com a gomesina. Neste trabalho, também descrevemos o pirosequenciamento do genoma da cepa J1a12 de Xylella fastidiosa, que exibe fenótipo menos virulento em citros e tabaco em relação à cepa 9a5c. A comparação da sequência genômica destas duas cepas confirma diferenças anteriormente observadas utilizando-se microarranjos de DNA e destaca genes potencialmente importantes para virulência de Xylella fastidiosa. / At sublethal concentrations, antimicrobials compounds modulate bacterial gene expression and the gene set that is modulated depends not only on the bacterial strain but also on the nature of antimicrobial agent. In this study, we evaluated changes in gene expression profile of Xylella fastidiosa strain 9a5c exposed up to 60 min to sublethal concentration of streptomycin. This a virulent strain originally isolated from orange trees with symptoms of citrus variegated chlorosis. Hybridization of DNA microarrays representing 2,608 out of 2848 coding sequences (CDS) previously annotated in strain 9a5c genome revealed 136 CDS differentially expressed upon streptomycin treatment. Of which 109 were down-regulated and 27 up-regulated. Differential expression for a subset of 21 CDS was further evaluated by reverse transcriptionquantitative PCR (RT-qPCR). In addition, we performed an integrated analysis of the gene expression profile of X. fastidiosa in response to streptomycin along with other gene expression profiles available for this bacterium. Among the up-regulated CDS, we highlight those encoding chaperonins GroEL and GroES, which are associated with heat shock response, and those CDS related to translation, such as ribosomal proteins and translation factors. Interestingly, exposure to streptomycin induces the expression of a CDS encoding polygalacturonase, which is a virulence factor for some X. fastidiosa strains. Furthermore, treatment with streptomycin down-regulates some CDS related to biofilm formation oppositely to treatment with gomesin, an antimicrobial peptide. Together, these observations suggest that exposure to sublethal dose of streptomycin might promote a higher virulent phenotype, in contrast to the effect previously observed with gomesin. In the present work, we also describe the pyrosequencing of J1a12 genome, a X. fastidiosa strain that exhibits a less virulent phenotype in citrus and tobacco if compared to strain 9a5c. A comparison of genome sequences of these two strains confirms differences previously observed using DNA microarrays and highlights important genes for virulence of X. fastidiosa.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-24T11:51:36Z No. of bitstreams: 1 Marcelo Rodrigues Figueira de Mello.pdf: 590787 bytes, checksum: 0b21179fd04d627436888523d18d7338 (MD5) / Made available in DSpace on 2017-03-24T11:51:36Z (GMT). No. of bitstreams: 1 Marcelo Rodrigues Figueira de Mello.pdf: 590787 bytes, checksum: 0b21179fd04d627436888523d18d7338 (MD5) Previous issue date: 2009-02-16 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The production of Chinese cabbage (Brassica pekinensis) may be limited by occurrence of diseases, among which the soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc). The effect of inducers and antibiotics on the disease control was studied in laboratory, greenhouse and field. In the first paper acibenzolar-S-metil (ASM) (0.025 g L-1), Ecolife® (2 mL L-1), Agro-Mos® (2 mL L-1) and calcium oxide (0.22 g L-1) were evaluated for in vitro bacterial growth inhibition; epidemiological components, incidence (INC), incubation period (PI), disease final severity (SEVF), diasease index (IDO) and area under disease curve progress (AUPDC); enzymatic activity of peroxidase and polyphenoloxidase and physiological cost of induction. Under greenhouse and field conditions inducers were sprayed seven days after transplant, and seven days after induction strain Pcc120 was inoculated by pricking, except for enzymatic activity and physiological cost experiments. In vitro Pcc120 was not inhibited by any tested product. The INC was not reduced in any experiment. In greenhouse SEVF was reduced by 47.5% by Agro-Mos® and ASM which also reduced IDO by 35.1 and 45.3% respectively and AUDCP. Induced resistance was confirmed by negative antibiosisagainst pathogen and increase of peroxidase and polyphenoloxidase activities in treated plants. In field only ASM confirmed greenhouse results by reducing disease intensity. There was no physiological cost for plants sprayed with ASM or Agro-Mos®. In the second paper it was evaluated the in vitro sensibility of Pcc to bactericides, and the effect of Mycoshield® (oxitetracycline 20%) at 3.0 and 1.5 g L-1, and yeasts (Rh1 and Rh2 - Rhodotorula spp. and Sc1 - Saccharomyces cerevisae at 108 cel mL-1) to control the disease in greenhouse and field. Plants were sprayed with Mycoshield® and yeasts seven days after transplant and inoculated seven days and 12 h after treatment, respectively. In all experiments INC, PI, SEVF, IDO and AUDPC were evaluated. In vitro 40 Pcc isolates were resistant to copper sulfate and sensitive to oxitetracycline, streptomycin, oxitetracycline+ streptomycin and oxitetracycline + copper sulfate all at 0.2 g L-1. Six Pcc isolates were significantly more inhibited by MycoshieldÒ than by Agri-Micina® (oxitetracycline 1.5% + streptomycin 15%), but there was no inhibition by Kasumin® (kasugamicin 2%). In greenhouse MycoshieldÒ at 3.0 g L-1 reduced SEVF and IDO until 47.4 and 19.0%. The yeast Sc1 reduced SEVF and AUDCP till 27.6 and 39.3% respectively, while Rh1 reduced AUDCP until 33.5%. In field MycoshieldÒ reduced IDO, SEVF and AUDCP by 14.4; 15.5 and 28.9% respectively; while Rh1 reduced IDO by 8.8% and Sc1diminished the AUDCP by 15.7%. The reduction of disease intensity and low physiological cost of induction indicate the potentiality of ASM in an integrate management program of Chinese cabbage soft rot. On the other hand MycoshieldÒ and yeasts showed low efficiency for controlling disease in field. / A produção de couve-chinesa (Brassica pekinensis) pode ser limitada pela ocorrência de doenças, dentre as quais se destaca a podridão-mole causada por Pectobacterium carotovorum subsp. carotovorum (Pcc). O efeito de indutores e antibióticos no controle desta doença foi estudado em laboratório, casa de vegetação e campo. No primeiro trabalho, foram testados acibenzolar-S-metil (ASM) (0,025 g/L), Ecolife® (2 mL/L), Agro-Mos® (2 mL/L) e óxido de cálcio (0,22 g/L). Foram avaliados, a inibição do crescimento bacteriano “in vitro”; os componentes epidemiológicos, incidência (INC), período de incubação (PI), severidade final da doença (SEVF), índice de doença (IDO) e área abaixo da curva de progresso da doença (AACPD); a atividade das enzimas peroxidase e polifenoloxidase e o custo fisiológico da indução. Em casa de vegetação e campo os indutores foram pulverizados sete dias após o transplante e sete dias após a indução, o isolado Pcc120 foi inoculado por picada, exceto nos experimentos de atividade enzimática e custo fisiológico. Pcc120 não foi inibido “in vitro” por nenhum dos produtos testados. Não houve redução da INC em nenhum dos experimentos. Em casa de vegetação, a SEVF da doença foi reduzida em 47,5% pelos tratamentos Agro-Mos® e ASM, os quais também, respectivamente, reduziram o IDO em 35,1% e 45,3% e propiciaram as menores AACPD. A resistência induzida foi evidenciada pela ausência de antibiose dos dois produtos contra Pcc e pelo aumento das atividades da peroxidase e polifenoloxidase. Em campo, apenas o ASM confirmou os resultados de casa de vegetação reduzindo a intensidade da doença. Não houve custo fisiológico para as plantas com aplicação do ASM ou Agro-Mos®. No segundo trabalho avaliou-se a sensibilidade “in vitro” de Pcc a bactericidas, o efeito de Mycoshield® (oxitetraciclina 20%) nas dosagens de 3,0 e 1,5 g/L, e das leveduras (Rh1 e Rh2 - Rhodotorula spp. e Sc1 - Saccharomyces cerevisae a 108 cel/mL) no controle da doença em casa de vegetação e em campo. As plantas foram pulverizadas com Mycoshield® e leveduras sete dias após o transplante, e inoculadas sete dias e 12 h após o tratamento, respectivamente. Foram avaliados INC, PI, SEVF,IDO e AACPD. In vitro, 40 isolados de Pcc testados apresentaram resistência ao sulfato de cobre e sensibilidade a oxitetraciclina, estreptomicina, oxitetraciclina+estreptomicina e oxitetraciclina+sulfato de cobre, todos na concentração de 0,2 g/L. Seis isolados de Pcc foram significativamente mais inibidos por MycoshieldÒ do que por Agri-Micina® (oxitetraciclina 1,5% + estreptomicina 15%), não sendo inibidos por Kasumin® (casugamicina 2%). Em casa de vegetação, o MycoshieldÒ na dosagem 3,0 g/L reduziu a SEVF e o IDO em até 47,4 e 19%; já a levedura Sc1 reduziu a SEVF e a AACPD em até 27,6 e 39,3%, respectivamente, enquanto Rh1 reduziu a AACPD em até 33,5. Em campo, o MycoshieldÒ reduziu o IDO, a SEVF e a AACPD em respectivamente 14,4; 15,5 e 28,9%; enquanto que Rh1 reduziu o IDO em 8,8% e Sc1 reduziu a AACPD em 15,7%. A redução da intensidade da doença e o baixo custo fisiológico da indução indicam a potencialidade do uso do ASM em um programa de manejo integrado da podridão-mole em couve-chinesa. No entanto,o MycoshieldÒ e as leveduras apresentaram baixa eficiência para o controle da doença em campo.

No description available.

Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.

La sclérose systémique (SSc) est une maladie auto-immune chronique incurable caractérisée par une présentation clinique complexe et hétérogène. Notre laboratoire a développé un modèle murin de fibrose pulmonaire et cutanée qui est induit par l’immunisation répétitive avec des cellules dendritiques chargées avec des peptides de la topoisomérase I, et qui partage de nombreuses caractéristiques avec la SSc humaine. Premièrement, nous avons caractérisé la maladie expérimentale quant à sa persistance à long terme (objectif 1) et son caractère progressif (objectif 2). Une cascade de coagulation dérégulée est impliquée dans le développement de la fibrose dans la SSc. La thrombine, un médiateur clé de la coagulation, semble contribuer à ce processus. Deuxièmement, nous avons étudié l’efficacité d’un inhibiteur de la thrombine, i.e. dabigatran, dans ce modèle (objectif 3). Le microbiote intestinal semble jouer un rôle déterminant dans plusieurs pathologies, y compris les maladies auto-immunes. Troisièmement, nous avons évalué l’effet de la manipulation du microbiote des souris par l’administration de streptomycine (objectif 4). Les souris immunisées développent une maladie persistante et la fibrose observée est précédée d’une phase inflammatoire. Le dabigatran aggrave la fibrose pulmonaire et cutanée lorsqu’administré durant la période inflammatoire et n’a aucun effet protecteur durant la phase fibrotique. La manipulation du microbiote par la streptomycine aggrave l’atteinte pulmonaire lorsque l’antibiothérapie est donnée en début de vie et exacerbe l’atteinte cutanée lorsqu’administrée à l’âge adulte. Notre modèle expérimental représente donc un outil important pour évaluer différentes approches thérapeutiques pour la SSc de par sa persistance et son caractère progressif. En se basant sur nos résultats, le dabigatran ne semble pas constituer un choix thérapeutique adéquat pour traiter la fibrose chez les patients atteints de SSc. L’exposition à la streptomycine à certaines périodes de la vie affecte différentiellement le développement et les manifestations cliniques de la maladie expérimentale. / Systemic sclerosis (SSc) is an incurable and chronic autoimmune disease characterized by a complex and heterogeneous clinical presentation. Our laboratory has developed a mouse model of lung and skin fibrosis that shares many features with human SSc, and is induced by repeated immunization with dendritic cells loaded with peptides of topoisomerase I. First, the long term persistence (objective 1) and progressive nature (objective 2) of this experimental disease model was characterized. A dysregulated coagulation cascade is implicated in the development of fibrosis in SSc. Thrombin, a key mediator of coagulation, appears to contribute to this process. Next, the efficacy of dabigatran, a thrombin inhibitor, to ameliorate lung and skin fibrosis was studied in this model (objective 3). Intestinal microbiota appears to play a key role in several diseases including autoimmune diseases. Finally, the effect of manipulating gut microbiota by administration of streptomycin on disease pathogenesis was evaluated in this model (objective 4). Immunized mice developed persistent fibrosis that was preceded by an inflammatory phase. Dabigatran aggravated pulmonary and skin fibrosis when administered during the inflammatory period and was not protective when given during the fibrotic phase. Manipulation of intestinal microbiota by streptomycin aggravated lung fibrosis when it was given early in life and exacerbated skin disease when administered in adulthood. Our model of experimental SSc with progressive and persistent disease represents an important tool to evaluate different therapeutic approaches for SSc. Furthermore, our results caution against the use of dabigatran as a therapeutic option to treat fibrosis in patients with SSc. Exposure to streptomycin for certain periods of life differentially affects the development and clinical manifestations of experimental SSc.

Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.