Estudos anatômicos e histológicos sôbre a sub-família Triatominae (Heteropterareduviidae): parte XVIII: sistema traqueal do Triatoma infestans

O presente trabalho consta de um estudo sõbre a topografia do sistema traqueal em Triatoma infestans. Com esta finalidade empregaram-se diversas técnicas de coloração por infiltração, além de introduzir-se uma modificação no método de Gäbler. Foram usados vários fixadores e feitas muitas Lâminas histológicas para a comprovação dos resultados. É apresentado, na parte referente a considerações gerais sôbre o sistema, uma citação de alguns autores que mais se dedicaram ao estudo do aparelho respiratório nos insetos. O estudo sôbre a distribuição do sistema traqueal abrange ambos os sexos, porém, é limitado a exemplares adultos. O assunto é abordado em 3 seções: 1) traquéias da região cefálica; 2) traquéias da região torácica; e 3) traquéias da região abdominal. O percurso das traquéias da região cefálica é acompanhado e estudado em todo o trajeto, o mesmo acontecendo com as do tórax e do abdômen. A disposição do sistema em estudo, na região torácica, apresenta-se bem amis complexa e com inúmeras ramificações, que se destinam aos diversos órgãos desta região. É estudada, também, a distribuição dos ramos traqueais nas pernas e nas asas do inseto. É evidenciado um rompimento na continuidade do sistema traqueal, na altura do 1º para o 2º estígmas abdominais. Supõe-se que êste fato esteja ligado ao desaparecimento do 1º esternito e a migração do esclerito contendo o 1º estígma abdominal, para a região dorsal do inseto. Devido a êste fenômeno, o sistema traqueal do Triatoma infestans apresenta-se diferente, funcionando como se fôsse dos sistemas independentes entre si. As diferenças, o número e a disposição das traquéias na região abdominal é abordado amplamente, assim como, realçada a formação e função dos citados sacos traqueais.

Ueber eine Besondere Muskelform von Barypenthus sp. (Trichoptera)

O autor descreve uma modificação da musculatura pleural do tricóptero Barypenthus sp. A musculatura é caracterizada pela formação de tendões secundários hipodérmicos. Êstes constam de feixes de tonofibrilas que, de um lado, entram em contacto com feixes de fibras musculares e, de outro, com a cutícula. O músculo, assim, é fixado ao esqueleto cutâneo, não só pelas inserções nas suas extremidades, como em vários outros pontos do seu comprimento. A contração desta musculatura condiciona um aumento imediato da pressão interna do corpo, sendo esta necessária para modificar a forma do esqueleto, possibilitando a abertura de um órgão glandular, situado nos tergitos dos 5.º e 6.º segmentos abdominais.

Dietary modulation of plasma antioxidant deficiencies: effect of mediterranean diet

Estudi elaborat a partir d’una estada al National Research Institute for Food and Nutrition, Itàlia, des de novembre del 2006 fins a febrer del 2007. La capacitat antioxidant total (TAC) en plasma pot ser un bon biomarcador del estat antioxidant dels humans. Prenent les mostres de dos projectes diferents de recerca s’ha mesurat la TAC mitjançant el FRAP (ferric reductant antioxidant potencial) i el TRAP (total radical-trapping antioxidant parameter ). D’una banda el PREDIMED, és un estudi prospectiu aleatoritzat i controlat, amb una cohort d’ individus sense patología vascular coneguda, però amb un alt risc de patir-la. En aquest es valora la utilitat d’una intervenció dietética del tipus mediterrània en la prevenció primària de la malaltia cardiovascular. L’altre és el de biodisponibilitat en humans dels metabòlits dels polifenols presents en els solubles de cacau, un estudi crònic (28 dies) on es vol mesurar la influència de la llet en l’absorció dels polifenols del cacau, en voluntaris amb elevat risc de sofrir patologia cardiovascular.

Estudos anatômicos e histológicos, principalmente do aparelho genital de Bulimulus corumbaensis Pilsbry, 1897: (Gastropoda, Pulmonata, Bulimulidae)

No trabalho apresentado os autores fazem um estudo sôbre a estrutura anatômica e histológica do aparelho genital de Bulimulus corumbaensis Pilsbry, 1897, considerando também a descrição da concha, mandíbula, rádula e câmara paleal. Esta espécie, que era conhecida apenas pelos seus caracteres conchiológicos, apresenta como caracteres importantes para a sua diagnose a morfologia e a estrutura das seguintes partes: 1 - Conchas de adultos com comprimento variando entre 19 mm e 29 mm. Muito característico nesta espécie é sua ornamentação com faixas axiais translúcidas e opacas. As primeiras variam de incolores a castanhas com tôdas as tonalidades. Devido a isto, conforme a intensidade da pigmentação e a freqüência das faixas translúcidas, a coloração da concha fica entre o branco e o castanho. Nas conchas totalmente brancas, existem faixas axiais translúcidas (desprovidas de pigmentação castanha) e faixas branco opacas intercaladas. As conchas intensamente castanhas, apresentam esta tonalidade pela proximidade das faixas castanhas translúcidas. 2 - Ovotestis constiuído por numerosos grupos de folículos bem individualizados, variando nos exemplares dissecados de 4 a 6. 3 - "Talon" aproximadamente três vêzes menor que o canal hermafrodita, apresentando-se dividido em dois tubos. O menos, situado no lado oposto à entrada do canal hermafrodita, histològicamente, é constituído por um único tupo de luz ampla. O maior, situado entre o canal hermafrodita e o tubo acima referido, microscòpicamente mostra 7 túbulos, que em alturas diferentes vão desembocar no tubo menor. O canal hermafrodita, por sua vez, desemboca um pouco mais abaixo. 4 - A bainha muscular do pênis, que é bem desenvolvida, é atravessada aproximadamente na metade do seu comprimetno pelo canal deferente. 5 - Porção proximal do falus apresentando 5 glândulas tubulosas ramificadas, distribuídas do seguinte modo: posteriormente são em número de 4, dispostas envolvendo a luz central do pênis e nela desembocando em alturas variáveis. A outra glândula, anteriormente situada, tem numerosos septos e resulta de uma divisão da luz central do pênis, com modificação do epitélio que se tornou glandular. 6 - A inserção do músculo retrator no flagelo é subterminal.

Microanatomia e citologia das glândulas pençonhentas de Rhninocricus Padbergii (Diplopoda)

No presente trabalho, descreve-se a estrutura microanatômica e citológica, bem como a função das glândulas laterais de um Diplópode, Rhinocricus padbergii. Chegamos aos seguintes resultados principais: 1 — Do ponto de vista anatômico o, o aparelho glandular representa uma invaginação complicada do integumento. O epitélio glandular é uma formação homóloga a hipoderme, fato provado pela presença de um revestimento cuticular e de pigmentos nas células de todo o aparelho. O sistema compõe-se de uma vesícula glandular, de um canal condutor e de um dispositivo de fechamento. 2 — Os detalhes da construção do aparelho glandular inteiro apresentamos na figura 1. Todo o complexo possui apenas um forte músculo para o movimento do aparelho de fechamento; seu antagonista é uma região elástica do próprio aparelho de fechamento que funciona à maneira de mola em virtude de numerosas dobras grudadas. 3 — A hipoderme glandular possui uma membrana basal muito fina, sendo porem reforçada, secundàriamente, por uma membrana celular mais forte. 4 — A expulsão de secreção através da abertura externa ("poro glandular") dá-se por meio de aumento da pressão no interior da cavidade geral do corpo (contrações generalizadas da musculatura inteira do corpo) e pela pressão da borda posterior do segmento anterior, exercida sobre a vesícula glandular devido a contração dos troncos da musculatura longitudinal. 5 — A respeito da função das células glandulares diferenciamos quatro estágios: a) Fase I: Na zona media da célula formam-se concentrações de secreção difusas. Os mitocôndrios localizam-se, quase exclusivamente, sobre a face basal da célula. b) Fase II: As concentrações de secreção difusas tornam-se mais densas; as esferas de secreção aumentam, gradativamente, de diâmetro e localizam-se em vacúolos, em forma de fendas, no protoplasma. Os mitocôndrios aumentam de número, distribuindo-se sobre todo o interior da célula. c) Fase III: Os vacúolos pequenos confluem em alguns grandes, , preenchendo-se com esferas de secreção, maiores e menores. Os mitocôndrios deslocam-se em direção à zona apical da célula, encontram-se porém, ainda, também em número elevado no protoplasma. d) Fase IV: Os vacúolos juntam-se, formando um só vacúolo grande que ocupa mais do que a metade do volume da célula e que é preenchida por esferas de secreção. Os mitocôndrios encontram-se agora quase exclusivamente na face apical da célula. 6 - Durante a formação das esferas de secreção ocorre no seu interior um acondensação secondária, que se inicia no centro de cada esfera, e que pode ser comparada com o mesmo fato observado nas esferas de secreção das células lipócrinas das glândulas salivares de Aedes scapularis. 7 - A secreção não é de natureza lipóide. 8 - A expulsão da secreção da célula é processo micro-apócrino. 9 - As esferas de secreção no interior da célula e a secreção contida na vesícula glandular têm uma composição química diferente. Baseando-se na migração dos mitocôndrios (veja os itens 5 a-d dêste resumo) conclui-se que, antes ou durante sua passagem através da face apical da célula, a secreção sofre uma modificação química por ação enzimática.

Ciclo "L" em Proteus vulgaris: I - massas nucleares em esferoplastos induzidos pela penicilina

Esferoplastos foram obtidos em Proteus vulgaris, cultivado em meio sintético simples, tendo como agente indutor a penicilina, em diferentes concentrações. Estabelecida a dosagem ótima, de sensibilidade da bactéria ao antibiótico, para obtenção de "grandes corpos" do Ciclo "L", definidos, morfologicamente, como esferoplastos, foi feita a coloração de massas nucleares, em diversas fases do crescimento bacteriano. A colheita do material foi feita por impessão em lamínula, usando-se ácido ósmico como fixador, seguindo-se hidrólise clorídrica, com aquecimento, e coloração pela fucsina. Dos resultados conseguidos ficou evidenciado: 1º - a posssibilidade de obtenção de "grandes corpos" em Proteus vulgaris em um meio sintético simples, a base de sais minerais e glicose; 2º - que houve uma concentração ótima de penicilina para surgir o efeito indutor de esferoplasto, na fase logarítmica de crescimento; 3º - a facilidade de coloração de massas nucleares nos esferoplastos em vários períodos de crescimento; 4º - a reversão ao tipo morfológico normal, depois de 24 horas de crescimento no meio com o antibiótico; 5º - modificação no crescimento do Proteus vulgaris em superfície de agar, com dose mínima de penicilina formando colônias localizadas.

Role of secretory immunoglobulin A and secretory component in the protection of mucosal surfaces.

The contribution of secretory immunoglobulin A (SIgA) antibodies in the defense of mucosal epithelia plays an important role in preventing pathogen adhesion to host cells, therefore blocking dissemination and further infection. This mechanism, referred to as immune exclusion, represents the dominant mode of action of the antibody. However, SIgA antibodies combine multiple facets, which together confer properties extending from intracellular and serosal neutralization of antigens, activation of non-inflammatory pathways and homeostatic control of the endogenous microbiota. The sum of these features suggests that future opportunities for translational application from research-based knowledge to clinics include the mucosal delivery of bioactive antibodies capable of preserving immunoreactivity in the lung, gastrointestinal tract, the genito-urinary tract for the treatment of infections. This article covers topics dealing with the structure of SIgA, the dissection of its mode of action in epithelia lining different mucosal surfaces and its potential in immunotherapy against infectious pathogens.

Host-microorganism interactions in lung diseases.

Until recently, the airways were thought to be sterile unless infected; however, a shift towards molecular methods for the quantification and sequencing of bacterial DNA has revealed that the airways harbour a unique steady-state microbiota. This paradigm shift is changing the way that respiratory research is approached, with a clear need now to consider the effects of host-microorganism interactions in both healthy and diseased lungs. We propose that akin to recent discoveries in intestinal research, dysbiosis of the airway microbiota could underlie susceptibility to, and progression and chronicity of lung disease. In this Opinion article, we summarize current knowledge of the airway microbiota and outline how host-microorganism interactions in the lungs and other tissues might influence respiratory health and disease.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).

Epithelium-Intrinsic NAIP/NLRC4 Inflammasome Drives Infected Enterocyte Expulsion to Restrict Salmonella Replication in the Intestinal Mucosa.

The gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/β and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogen's intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.

Early antibiotic administration affects the gut barrier function and the immune response to oral antigen in suckling rats

Abstract: The increasingly high hygienic standards characterizing westernized societies correlate with an increasingly high prevalence of allergic disease. Initially based on these observations, the hygiene hypothesis postulates that reduced microbial stimulation during infancy impairs the immune system development and increases the risk of allergy. Moreover, there is increasing evidence that the crosstalk existing between the intestine and the resident microbiota is crucial for gut homeostasis. In particular, bacterial colonization of the gut affects the integrity of the gut barrier and stimulates the development of the gut associated immune tissue, both phenomena being essential for the immune system to mount a controlled response to food antigens. Therefore, alterations in the microbial colonization process, by compromising the barrier homeostasis, may increase the risk of food allergy. In this context, antibiotic treatment, frequently prescribed during infancy, affects gut colonization by bacteria. However, little is known about the impact of alterations in the colonization process on the maturation of the gut barrier and on the immunological response to oral antigens. The objective of this work was to determine the impact of a commercial antibiotic preparation employed in pediatric settings on the gut barrier status at the critical period of the suckling/weaning transition and to evaluate the physiological consequences of this treatment in terms of immune response to food antigens. We established an antibiotic-treated suckling rat model relevant to the pediatric population in terms of type, dose and route of administration of the antibiotic and of changes in the patterns of microbial colonization. Oral tolerance to a novel luminal antigen (ovalbumin) was impaired when the antigen was introduced during antibiotic treatment. These results paralleled to alterations in the intestinal permeability to macromolecules and reduced intestinal expression of genes coding for the major histocomptatibility complex II molecules, which suggest a reduced capacity of antigen handling and presentation in the intestine of the antibiotic-treated animals. In addition, low luminal IgA levels and reduced intestinal expression of genes coding for antimicrobial proteins suggest that protection against pathogens was reduced under antibiotic treatment. In conclusion, we observed in suckling rats that treatment with abroad-spectrum antibiotic commonly used in pediatric practices reduced the capacity of the immune system to develop tolerance. The impact of the antibiotic treatment on the immune response to the antigen-was likely mediated by the alterations of the gut microbiota, through impairment in the mechanisms of antigen handling and presentation. This work reinforces the body of data supporting a key role of the intestinal microbiota modulating the risk of allergy development and leads us to propose that the introduction of new food antigens should be avoided during antibiotic treatment in infants. Résumé: L'augmentation du niveau d'hygiène caractérisant les sociétés occidentales semble être fortement corrélée avec l'augmentation des cas d'allergie dans ces pays. De cette observation est née l'hypothèse qu'une diminution des stimuli microbiens pendant l'enfance modifie le développement du système immunitaire augmentant ainsi le risque d'allergie. En ce sens, un nombre croissant de données indiquent que les interactions existant entre l'intestin et les bactéries résidantes sont cruciales pour l'équilibre du système. En effet, la présence de bactéries dans l'intestin affecte l'intégrité de sa fonction de barrière et stimule le développement du système immunitaire intestinal. Ces deux paramètres étant essentiels à la mise en place d'une réponse contrôlée vis à vis d'un antigène reçu oralement, toute modification du processus naturel de colonisation compromettant l'équilibre intestinal pourrait augmenter le risque d'allergie. Les traitements aux antibiotiques, fréquemment prescrits en pédiatrie, modifient de façon conséquente le processus de colonisation bactérienne. Cependant peu de données existent concernant l'impact d'une altération du processus de colonisation sur la maturation de la barrière intestinale et de la réponse immunitaire dirigée contre un antigène. L'objectif de ce travail était de déterminer l'impact d'un antibiotique commercial et employé en pédiatrie sur l'état de la barrière intestinale au moment critique du sevrage et d'évaluer les conséquences physiologiques d'un tel traitement sur la réponse immune à un antigène alimentaire. Nous avons mis en place un modèle de rats allaités, traités à l'antibiotique, le plus proche possible des pratiques pédiatriques, en terme de nature, dose et voie d'administration de l'antibiotique. Nous avons constaté que l'établissement de la tolérance orale à un nouvel antigène (l'ovalbumine) est altéré quand celui-ci est donné pour la première fois au cours du traitement antibiotique. Ces résultats coïncident avec une diminution de la perméabilité intestinale aux macromolécules, ainsi qu'avec une diminution de l'expression des gènes codant pour les molécules du complexe majeur d'histocomptatibilité de classe II, suggérant une modification de l'apprêtement et de la présentation de l'antigène au niveau intestinal chez les rats traités à l'antibiotique. De plus, un faible taux d'IgA et une diminution de l'expression des gènes codant pour des protéines antimicrobiennes, observés après l'administration d'antibiotique, laissent à penser que la protection contre un pathogène est diminuée lors d'un traitement antibiotique. En conclusion, nous avons observé qu'un traitement antibiotique à large spectre d'activité, couramment utilisé en pédiatrie, réduit la capacité d'induction de la tolérance orale chez le rat allaité. L'impact du traitement antibiotique sur la réponse immune semble induite par l'altération de la flore intestinale via son effet sur les mécanismes d'apprêtement et de présentation de l'antigène. Ce travail renforce l'ensemble des données existantes qui accorde à la flore intestinale un rôle clef dans la modulation du risque de développement d'allergie et nous amène à recommander d'éviter l'introduction d'un nouvel aliment lorsqu'un enfant est traité aux antibiotiques.

Dibenzofuran degradation by Sphingomonas wittichii RW1 under environmental stresses

Sphingomonas wittichii is a gram-negative Alpha-proteobacterium, capable of degrading xenobiotic compounds such as dibenzofuran (DBF), dibenzo-p-dioxin, carbazole, 2-hydroxybiphenyl or nitro diphenyl ether herbicides. The metabolism of strain RW1 has been the subject of previous studies and a number of genes involved in DBF degradation have been characterized. It is known that RW1 posseses a unique initial DBF dioxygenase (encoded by the dxnAl gene) that catalyzes the first step in the degradation pathway. None of the organisms known to be able to degrade DBF have a similar dioxygenase, the closest match being the DBF dioxygenase from Rhodococcus sp. with an overall amino acid similarity of 45%. Genes participating in the conversion of the metabolite salicylate via the ortho-cleavage pathway to TCA cycle intermediates were identified as well. Apart from this scarce information, however, there is a lack of global knowledge on the genes that are involved in DBF degradation by strain RW1 and the influence of environmental stresses on DBF-dependent global gene expression. A global analysis is necessary, because it may help to better understand the behaviour of the strain under field conditions and suggest improvements for the current bioaugmentation practice. Chapter 2 describes the results of whole-genome analysis to characterize the genes involved in DBF degradation by RW1. Micro-array analysis allowed us to detect differences in gene transcription when strain RW1 was exposed to DBF. This was complemented by ultra-high throughput sequencing of mutants no longer capable of growing on salicylate and DBF. Some of the genes of the ortho-cleavage pathway were induced 2 to 4 times in the presence of DBF, as well as the initial DBF dioxygenase. However two gene clusters, named 4925 and 5102 were induced up to 19 times in response to DBF induction. The cluster 4925 is putatively participating in a meta-cleavage pathway while the cluster 5102 might be part of a gentisate pathway. The three pathways, ortho-cleavage, meta-cleavage and gentisate pathway seem to be active in parallel when strain RW1 is exposed to DBF, presenting evidence for a redundancy of genes for DBF degradation in the genome of RW1. Chapter 3 focuses on exploiting genetic tools to construct bioreporters representative for DBF degradation in RW1. A set of basic tools for genetic manipulation in Sphingomonas wittichii RW1 was tested and optimized. Both plasmids and mini-transposons were evaluated for their ability to be maintained in RW1 with or without antibiotic selection pressure, and for their ability to lead to fluorescent protein expression in strain RW1 from a constitutive promoter. Putative promoter regions of three of the previously found DBF-induced genes (Swit_4925, Swit_5102 and Swit_4897-dxnAl) were then used to construct eg/^-bioreporters in RW1. Chapter 4 describes the use of the constructed RW1-based bioreporter strains for examining the expression of the DBF degradation pathway genes under microcosm conditions. The bioreporter strains were first exposed to different carbon sources in liquid culture to calibrate the egfp induction. Contrary to our expectations from micro-array analysis only the construct with the promoter from gene cluster 4925 responded to DBF, whereas the other two constructs did not show specific induction with DBF. The response from the bioreporters was subsequently tested for sensitivity to water stress, given that this could have an important impact in soils. Exposure to liquid cultures with decreasing water potential, achieved by NaCl or PEG addition to the growth media, showed that eGFP expression in RW1 from the promoter regions 4925 and 5102 was not directly influenced by water stress, but only through an overall reduction in growth rate. In contrast, expression of eGFP from the dxnAl or an uspA promoter was also directly dependent on the extent of water stress. The RW1 with the 4925 construct was subsequently used in soil microcosms to evaluate DBF bioavailability to the cells in presence or absence of native microbiota or other contaminated material. We found that RW1 could grow on DBF added to soil, but bioreporter expression suggested that competition with native microbiota for DBF intermediates may limit its ability to proliferate to a maximum. Chapter 5 describes the results from the experiments carried out to more specifically detect genes of RW1 that might be implicated in water stress resistance. Hereto we created transposon mutagenesis libraries in RW1, either with a classical mini-Tn5 or with a variant that would express egfp when the transposon would insert in a gene induced under water stress. Classical mutant libraries were screened by replica plating under high and low water stress conditions (achieved by adding NaCl to the agar medium). In addition, we screened for smaller microcolonies formed by mutants in agarose beads that could be analized with flow cytometry. A number of mutants impaired to grow on NaCl-supplemented media were recovered and the transposon insertion sites sequenced. In a second procedure we screened by flow cytometry for mutants with a higher eGFP production after exposure to growth medium with higher NaCl concentrations. Mutants from both libraries rarely overlapped. Discovered gene functions of the transposon insertions pointed to compatible solute synthesis (glutamate and proline), cell membrane synthesis and modification of cell membrane composition. The results obtained in the present study give us a more complete picture of the mechanisms of DBF degradation by S. wittichii RW1, how it reacts to different DBF availability and how the DBF catabolic activity may be affected by the conditions found in contaminated environments. - Sphingomonas wittichii est une alpha-protéobactérie gram-négative, capable de dégrader des composés xénobiotiques tels que le dibenzofurane (DBF), la dibenzo-p-dioxine, le carbazole, le 2-hydroxybiphényle ou les herbicides dérivés du nitro-diphényléther. Le métabolisme de la souche RW1 a fait l'objet d'études antérieures et un certain nombre de gènes impliqués dans la dégradation du DBF ont été caractérisés. Il est connu que RW1 possède une unique dioxygénase DBF initiale (codée par le gène dxnAl) qui catalyse la première étape de la voie de dégradation. Aucun des organismes connus pour être capables de dégrader le DBF n'a de dioxygénase similaire. L'enzyme la plus proche étant la DBF dioxygénase de Rhodococcus sp. avec 45% d'acides aminés conservés. Les gènes qui participent à la transformation du salicylate en métabolites intermédiaires du cycle de Krebs par la voie ort/io-cleavage ont aussi été identifiés. Outre ces informations lacunaires, il y a un manque de connaissances sur l'ensemble des gènes impliqués dans la dégradation du DBF par la souche RW1 ainsi que l'effet des stress environnementaux sur l'expression génétique globale, en présence du DBF. Une analyse globale est nécessaire, car elle peut aider à mieux comprendre le comportement de la souche dans les conditions de terrain et de proposer des améliorations pour l'utilisation de la bio-augmentation comme technique de bio-remédiation. Le chapitre 2 décrit les résultats de l'analyse du génome pour caractériser les gènes impliqués dans la dégradation du DBF par RW1. Une analyse de micro-arrays nous a permis de détecter des différences dans la transcription des gènes lorsque la souche RW1 a été exposée au DBF. L'analyse a été complétée par le criblage à ultra-haut débit de mutants qui n'étaient plus capables de croître avec le salicylate ou le DBF comme seule source de carbone. Certains des gènes de la voie ortho-cleavage, dont la DBF dioxygénase initiale, ont xî été induits 2 à 4 fois, en présence du DBF. Cependant, deux groupes de gènes, nommés 4925 et 5102 ont été induits jusqu'à 19 fois en réponse au DBF. Le cluster 4925 participe probablement dans une voie de meta-cleavage tandis que le cluster 5102 pourrait faire partie d'une voie du gentisate. Les trois voies, ortho-cleavage, meta-cleavage et la voie du gentisate semblent être activées en parallèle lorsque la souche RW1 est exposée au DBF, ce qui représente une redondance de voies pour la dégradation du DBF dans le génome de RW1. Le chapitre 3 se concentre sur l'exploitation des outils génétiques pour la construction de biorapporteurs de la dégradation du DBF par RW1. Un ensemble d'outils de base pour la manipulation génétique dans Sphingomonas wittichii RW1 a été testé et optimisé. Deux plasmides et mini-transposons ont été évalués pour leur capacité à être maintenu dans RW1 avec ou sans pression de sélection par des antibiotiques, et pour leur capacité à exprimer la protéine fluorescente verte (eGFP) dans la souche RW1. Les trois promoteurs des gènes Swit_4925, Swit_5102 et Swit_4897 (dxnAl), induits en réponse au DBF, ont ensuite été utilisés pour construire des biorapporteurs dans RW1. Le chapitre 4 décrit l'utilisation des souches biorapportrices construites pour l'analyse de l'expression des gènes de la voie de dégradation du DBF dans des microcosmes avec différents types de sols. Les souches biorapportrices ont d'abord été exposées à différentes sources de carbone en cultures liquides afin de calibrer l'induction de la eGFP. La construction avec le promoteur du gène 4925 a permis une réponse au DBF. Mais contrairement à nos attentes, basées sur les résultats de l'analyse des micro-arrays, les deux autres constructions n'ont pas montré d'induction spécifique au DBF. La réponse des biorapporteurs a ensuite été testée pour la sensibilité au stress hydrique, étant donné que cela pourrait avoir un impact important dans les microcosmes. La diminution du potentiel hydrique en culture liquide est obtenue par addition de NaCl ou de PEG au milieu de croissance. Nous avons montré que l'expression de la eGFP contrôlée par les promoteurs 4925 et 5102 n'était pas directement influencée par le stress hydrique, mais seulement par une réduction globale des taux de croissance. En revanche, l'expression de la eGFP dépendante des promoteurs dxnAl et uspA était aussi directement dépendante de l'ampleur du stress hydrique. La souche avec la construction 4925 a été utilisée par la suite dans des microcosmes avec différents types de sols pour évaluer la biodisponibilité du DBF en présence ou absence des microbes indigènes et d'autres composés contaminants. Nous avons constaté que RW1 pouvait se développer si le DBF a été ajouté au sol, mais l'expression de la eGFP par le biorapporteur suggère que la compétition avec la microbiota indigène pour les métabolites intermédiaires du DBF peut limiter sa capacité à proliférer de manière optimale. Le chapitre 5 décrit les résultats des expériences réalisées afin de détecter spécifiquement les gènes de RW1 qui pourraient être impliquées dans la résistance au stress hydrique. Ici on a crée des bibliothèques de mutants de RW1 par transposon, soit avec un mini-Tn5 classique ou avec une variante qui exprime la eGFP lorsque le transposon s'insère dans un gène induit par le stress hydrique. Les bibliothèques de mutants ont été criblées par la méthode classique de repiquage sur boîtes, dans des conditions de stress hydrique élevé (obtenu par l'addition de NaCl dans les boîtes). En outre, nous avons criblé des micro¬colonies dans des billes d'agarose qui ont pu être analysées par cytométrie de flux. Un certain nombre de mutants déficients à croître sur des milieux supplémentés avec du NaCl ont été isolés et les sites d'insertion du transposon séquencés. Dans une deuxième procédure nous avons criblé par cytométrie de flux des mutants avec une production de eGFP supérieure, après exposition à un milieu de croissance avec une concentration élevée de NaCl. Les mutants obtenus dans les deux bibliothèques n'étaient pas similaires. Les fonctions des gènes où se trouvent les insertions de transposons sont impliqués dans la synthèse de solutés compatibles (glutamate et de la proline), dans la synthèse de la membrane cellulaire et dans la modification de la composition de la membrane cellulaire. Les résultats obtenus dans la présente étude nous donnent une image plus complète des mécanismes de dégradation du DBF par S. wittichii RW1, comment cette souche réagit à la disponibilité du DBF et comment l'activité catabolique peut être affectée par les conditions rencontrées dans des environnements contaminés.

Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenothypical gentamicin resistance amplification (frequencies of 10-3 to 10-5, compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromossomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells.

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer's patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c(+)CD11b(+)CD8(-) dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.

Colite à Clostridium difficile: nouvelles recommandations de prise en charge [Clostridium difficile infections: update on new European recommendations].

Clostridium difficile infections: update on new European recommandations While metronidazole and vancomycin have been the only drug options to date for the treatment of C. difficile infection, new therapeutic approaches with promising results have recently emerged for the treatment of the first episode and relapses. Fidaxomicin is a new macrocyclic antibiotic more active against C. difficile and with a narrow spectrum allowing preservation of the intestinal microbiota. While having the same efficacy as vancomycin for the treatment of the first episode, this agent is associated with a lower rate of relapse. The highest relapse-free cure rate is achieved through fecal microbiota transplantation, which should be considered for patients with multiple relapses.