CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Serratia marcescens se encontra largamente distribuÃda na natureza, mas tem emergido nos Ãltimos anos como um importante patÃgeno nosocomial resistente a diversos antimicrobianos. Este estudo teve como objetivo verificar a susceptibilidade de isolados ambientais, orais e extra-orais de Serratia marcescens a diferentes antibiÃticos e avaliar a terapia fotodinÃmica antimicrobiana na reduÃÃo do crescimento bacteriano em culturas de cÃlulas planctÃnicas e biofilme. O teste de susceptibilidade antimicrobiano E-test foi realizado para as 55 cepas e o TFA para as 30 cepas mais resistentes aos antimicrobianos testados. O efeito antimicrobiano do azul de o-toluidina associado com 4,72 J cm-2 de luz emitida por um diodo (LED) foi avaliado. Antes e apÃs os tratamentos, os inÃculos bacterianos foram analisados com consideraÃÃo do nÃmero de unidades formadoras de colÃnias. Considerando o perfil antimicrobiano observamos que das 55 cepas analisadas, 13 (23,63%) apresentaram resistÃncia à doxiciclina, mas apenas um (1,81%) isolado apresentou resistÃncia ao ciprofloxacino, outro à tobramicina e outro à cefotaxima; 24 (43,63%) cepas apresentaram sensibilidade intermediÃria à doxiciclina, todas foram sensÃveis ao imipenem e a maioria foi sensÃvel ao ciprofloxacino, à tobramicina e à cefotaxima. A anÃlise estatÃstica demonstrou nÃo haver diferenÃas significativas no perfil de resistÃncia das amostras de diferentes origens em relaÃÃo as drogas DX, CT e IP. Considerando a resistÃncia a CI, as amostras ambientais foram significativamente mais resistentes do que as amostras orais e extra-orais. Para a droga TM, as amostras orais foram significantemente mais sensÃveis do que as demais amostras. A irradiaÃÃo das culturas planctÃnicas e biofilmes na ausÃncia de TBO (L+C-), a incubaÃÃo com TBO sozinho (L-C+) e o grupo controle nÃo tratado (L-C-) nÃo apresentou efeitos significativos na viabilidade das cepas de S. marcescens estudadas (p < 0,05). DecrÃscimos significativos na viabilidade bacteriana foram observados somente quando cultura planctÃnica e biofilme de cepas ambientais, orais e extra-orais de S. marcescens foram expostas ao azul de orto toluidina e luz LED ao mesmo tempo (L+C+). ReduÃÃes significativas nas contagens bacterianas foram observadas pela Terapia FotodinÃmica Antimicrobiana com variaÃÃo de 10-11 a 10-7. A associaÃÃo de TBO e LED, com densidade de energia de 4,72 J cm-2 , foi efetivo na reduÃÃo da viabilidade bacteriana em cepas ambientais, orais e extra-orais de S. marcescens podendo ser uma ferramenta biotecnolÃgica Ãtil no controle da resistÃncia bacteriana. / Serratia marcescens is widely distributed in nature, but has emerged in the last years as important nosocomial pathogen with resistance of many antimicrobial drugs. This study aimed to verify the susceptibility of Serratia marcescens isolates from environment, from oral infections and from extra-oral infections to different antibiotics and evaluate the antimicrobial effect of photodynamic antimicrobial therapy as biotechnology tools reducing bacterial growth in planktonic cells and biofilm. E-test were performed for fifty-five strains and the PACT for the thirty strains more resistant to antimicrobials tested. The antimicrobial effect of toluidine blue O, associated with 4,72 J cm-2 of a light-emitting diode , was evaluated. Before and after the treatments, bacterial inocula were analysed with regard to the number of colony- forming units. For antimicrobials, we observed that the 55 strains analyzed, 13 (23.63%) were resistant to doxycycline, but only one (1.81%) isolate showed resistance to ciprofloxacin, another to tobramycin and another to cefotaxime, 24 ( 43.63%) strains had intermediate sensitivity to doxycycline, all were sensitive to imipenem and most were sensitive to ciprofloxacin, tobramycin and cefotaxime Statistical analysis showed no significant differences in resistance of samples of different origins for drugs DX, CT, and IP. Considering the resistance to CI, the environmental samples were significantly more resistant than samples oral and extra-oral. For the drug TM, the oral samples were significantly more sensitive than the other samples. The irradiation of planktonic and biofilm cultures in the absence of TBO (L+S-), incubation with TBO alone (L-S+) and untreated control group (L-S-) had no significant effect on the viability of strains of S. marcescens studied (p <0.05). Significant decreases in bacterial viability was observed only when planktonic and biofilm culture of environmental strains, oral and extra-oral S. marcescens were exposed to toluidine blue O and LED light at the same time (L+S+). Significant reductions in bacterial counts were observed by antimicrobial photodynamic therapy ranging from 10-11 to 10-7.The association of TBO and light, with energy density 4,72 J cm-2, was effective in reducing the viability of bacterial strains in environmental, oral and extra-oral S. marcescens and can be a useful biotechnological tool in the control of bacterial resistance.

by Yuen Pui-yee, Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 213-221). / Acknowledgments --- p.i / Abstract --- p.ii / List of Tables --- p.ix / List of Figures --- p.xi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Problems of Pollution From Textile Industries --- p.1 / Chapter 1.2 --- Current Treatment Methods of Wastewater from Textile Industries --- p.5 / Chapter 1.3 --- Adverse Effects of Dyes on the Environment --- p.11 / Chapter 1.4 --- Classification of Dyes --- p.16 / Chapter 1.5 --- Azo Dyes --- p.17 / Chapter 1.6 --- Metabolisms of Azo Dyes in Microbial and Animal Systems --- p.21 / Chapter 1.7 --- "Toxicity, Mutagenicity and Carcinogenicity of Azo Dyes" --- p.31 / Chapter 1.8 --- Removal of Azo Dyes --- p.35 / Chapter 1.8.1 --- Biological Methods --- p.35 / Chapter 1.8.2 --- Physico-chemical Methods --- p.49 / Chapter 1.9 --- Purposes of Study --- p.50 / Chapter 2. --- Objectives --- p.53 / Chapter 3. --- Materials and Methods --- p.54 / Chapter 3.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-Dimethyl-p-phenylene diamine-degrading Microbial Isolates" --- p.54 / Chapter 3.1.1 --- "Isolation of Methyl Red-degrading Microbial Isolates from Dye- containing Wastewater, Activated Sludge and Soil" --- p.54 / Chapter 3.1.2 --- Selection of Methyl Red-degrading Microbial Isolates --- p.56 / Chapter 3.1.3 --- "Enrichment of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria from Dye-containing wastewater, Activated Sludge and Soil" --- p.59 / Chapter 3.1.4 --- "Isolation of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.60 / Chapter 3.1.5 --- Selection of N，N-Dimethyl-p-phenylene diamine-degrading Bacteria --- p.60 / Chapter 3.1.6 --- "Identification of the Selected Methyl Red-degrading and N,N- Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.61 / Chapter 3.1.7 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.63 / Chapter 3.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.64 / Chapter 3.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.64 / Chapter 3.2.2 --- Change of UV-Vis Spectra of Methyl Red and N，N-Dimethyl-p- phenylene diamine at Different pH and Matrixes --- p.64 / Chapter 3.2.3 --- "UV-Vis Spectra and Standard Curves of Methyl Red, N，N- Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.66 / Chapter 3.2.4 --- "HPLC separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.67 / Chapter 3.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.68 / Chapter 3.3.1 --- "Monitoring of Percentage of Methyl Red Cleaved, Degradation Value of N,N-Dimethyl-p-phenylene diamine and o- Aminobenzoic acid, and Growth of Selected Methyl Red- degrading Bacteria by Spectrophotometric Analysis " --- p.68 / Chapter 3.3.2 --- Study of Degrading Products of Methyl Red by Selected Methyl Red-degrading Isolates --- p.71 / Chapter 3.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.73 / Chapter 4. --- Results --- p.74 / Chapter 4.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-dimethyl-p-phenylene diamine-degrading Microbial Isolates " --- p.74 / Chapter 4.1.1 --- "Isolation of Methyl Red-degrading Microbial Isolates from Dye- containing Wastewater, Activated Sludge and Soil " --- p.74 / Chapter 4.1.2 --- Selection of Methyl Red-degrading Microbial Isolates --- p.79 / Chapter 4.1.3 --- "Enrichment of N,N-dimethyl-p-phenylene diamine-degrading Bacteria from Dye-containing Wastewater, Activated Sludge and Soil " --- p.85 / Chapter 4.1.4 --- "Isolation of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.85 / Chapter 4.1.5 --- "Selection of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.90 / Chapter 4.1.6 --- "Identification of the Selected Methyl Red-degrading and N,N- Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.90 / Chapter 4.1.7 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.94 / Chapter 4.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.94 / Chapter 4.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.94 / Chapter 4.2.2 --- "Change of UV-Vis Spectra of Methyl Red and N,N-Dimethyl-p- phenylene diamine at Different pH and Matrixes " --- p.108 / Chapter 4.2.3 --- "UV-Vis Spectra and Standard Curves of Methyl Red, N，N- Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.123 / Chapter 4.2.4 --- "HPLC Separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.129 / Chapter 4.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.138 / Chapter 4.3.1 --- "Monitoring of Percentage of Methyl Red Cleaved and Degradation Value of N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid and Growth of Selected Methyl Red- degrading Bacterial Isolates by Spectrophotometric Analysis " --- p.138 / Chapter 4.3.2 --- Study of Degradation Products of Methyl Red by Selected Methyl Red-degrading Isolates by HPLC --- p.175 / Chapter 4.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.175 / Chapter 5. --- Discussion --- p.181 / Chapter 5.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-dimethyl-p-phenylene diamine-degrading Microbial Isolates " --- p.181 / Chapter 5.1.1 --- "Isolation and Selection of Methyl Red-degrading Microbes from Dye-containing Wastewater, Activated Sludge and Soil " --- p.181 / Chapter 5.1.2 --- "Isolation and Selection of N,N-Dimethyl-p-phenylene diamine- degrading Microbial Isolates from Dye-containing Wastewater, Activated Sludge and Soil " --- p.183 / Chapter 5.1.3 --- Identification of the Selected Methyl Red-degrading and N，N- Dimethyl-p-phenylene diamine-degrading Bacteria --- p.185 / Chapter 5.1.4 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.185 / Chapter 5.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.186 / Chapter 5.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid in 0.05 M phosphate buffer and 0.2MHC1 " --- p.186 / Chapter 5.2.2 --- "Change of UV-Vis Spectra of Methyl Red and N,N-Dimethyl-p- phenylene diamine at Different pH and Matrixes " --- p.187 / Chapter 5.2.3 --- "Change of UV-Vis Spectra of N,N-Dimethyl-p-phenylene diamine in Different Matrixes at Different pH " --- p.187 / Chapter 5.2.4 --- "UV-Vis Spectra and Standard Curve of Methyl Red, N,N- dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.188 / Chapter 5.2.5 --- "HPLC Separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.189 / Chapter 5.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.190 / Chapter 5.3.1 --- Effect of Glucose --- p.194 / Chapter 5.3.2 --- Effect of Ethanol --- p.196 / Chapter 5.3.3 --- Effect of Ammonium Sulphate --- p.198 / Chapter 5.3.4 --- Effect of Yeast Extract --- p.199 / Chapter 5.3.5 --- Effect of Phosphate Buffer (pH 7) --- p.200 / Chapter 5.3.6 --- Effect of pH --- p.201 / Chapter 5.3.7 --- Effect of Temperature at Static and Shaking Conditions --- p.203 / Chapter 5.3.8 --- Study of Degradation Products of Methyl Red by Selected Methyl Red-degrading Isolates by HPLC Analysis --- p.206 / Chapter 5.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.207 / Chapter 6. --- Conclusion --- p.209 / Chapter 7. --- References --- p.213 / Chapter 8. --- Appendix 1: Composition of Media --- p.222 / Appendix 2: Composition of Buffers --- p.225 / Appendix 3 --- p.228

Submitted by Tatiana Oliveira (tsilva@icict.fiocruz.br) on 2012-06-02T21:21:04Z No. of bitstreams: 1 daniele_p_castro_ioc_bp_0003_2009.pdf: 3267511 bytes, checksum: 7b658c7e6da7e158039b1155918729e9 (MD5) / Made available in DSpace on 2012-06-02T21:21:04Z (GMT). No. of bitstreams: 1 daniele_p_castro_ioc_bp_0003_2009.pdf: 3267511 bytes, checksum: 7b658c7e6da7e158039b1155918729e9 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / Nesta tese, foram investigados fatores que interferem no desenvolvimento dos parasitas, Trypanosoma cruzi e T. rangeli, no inseto-vetor, Rhodnius prolixus, com a administração de substâncias extraídas de Physalis angulata, fisalinas, bem como fatores relacionados a microbiota bacteriana intestinal do inseto, como Serratia marcescens. As fisalinas B, D, F e G administradas junto ao sangue alimentar não alteraram a fisiologia de ninfas de 5° estádio de R. prolixus, entretanto quando inoculados com T. rangeli ou Enterobacter cloacae b12 causaram alta mortalidade. Nos insetos tratados com fisalina B e infectados com parasita observou-se diminuição na formação de microagregados de hemócitos e produção de óxido nítrico. As fisalinas B e F reduziram a atividade de lisozima e a fisalina D reduziu a atividade antibacteriana, ambas de insetos inoculados com bactéria. Insetos tratados com as fisalinas B, D, F e G exibiram reduç§es significativas na atividade fagocitária. O tratamento dos insetos com as fisalinas B, F e G e inoculação de bactéria ocasionou menor contagem na formação de microagregados de hemócitos e o tratamento com as fisalinas B e F menor n·mero de hemócitos circulantes na hemolinfa. As atividades de fagocitose e microagregação de hemócitos inibidas pelo tratamento oral com fisalina B foram revertidas pela inoculação de ácido. O tratamento dos insetos com fisalina B não alterou a atividade da PLA2, porém aumentou a atividade de PAF-AH. Noutro enfoque da interação inseto-parasita, foram estudados os efeitos citotóxicos da S. marcescens. Ensaios in vitro, de incubação da bactéria com T. cruzi ou T. rangeli juntamente com o carboidrato D-manose (0,2M) resultaram na proteção dos parasitas da adesão e da lise de S. marcescens (variantes SM365 e RPH) numa relação dose dependente. Entretanto, a D-manose não interferiu na atividade hemolítica das variantes SM365 e RPH de S. marcescens. Enquanto com estas variantes a adesão das bactérias ao parasita ocorria em apenas alguns segundos, a variante DB11 não aderiu e não lisou o parasita. A adesão bacteriana é densa e compacta e ocorre por finos filamentos que com o tempo se desenvolve formando biofilme em volta da superfície do parasita lisado. Portanto, concluímos que dentre os fatores que interferem na interação entre inseto e T. cruzi e T. rangeli estudados, podemos citar as fisalinas e S. marcescens por suas atividades imunosupressoras e lítica contra os parasitas, respectivamente. Enquanto a fisalina B atua diminuindo os níveis de análogo de PAF (iPAF) na hemolinfa e elevando os níveis de PAF-AH, com ação inibitória sobre o sistema imune de R. prolixus, a bactéria S. marcescens lisa parasitas resultando em formação de biofilme, os quais regulam, direta ou indiretamente, o desenvolvimento do parasita no inseto vetor / In this thesis, we investigated factors affecting the development of the parasite, Trypanosoma cruzi and T. rangeli in the insect vector, Rhodnius prolixus, with the administration of substances extracted from Physalis angulata, physalins as well as factors related to intestinal bacterial microbiota of the insect, such as Serratia marcescens. Physalins B, D, F and G given by the blood feeding did not alter the physiology of fifth instar nymphs of R. prolixus, however, when inoculated with T. rangeli or Enterobacter cloacae b12 caused high mortality. In insects treated with physalin B and infected with parasites observed decrease in the formation of microaggregates of hemocytes and production of nitric oxide. Physalins B and F decreased the activity of lysozyme and physalin D reduced the antibacterial activity of both insects inoculated with bacteria. Insects treated with physalins B, D, F and G § s exhibited significant reduction in phagocytic activity. Treatment of insects with physalins B, F and G and inoculation caused lower counts of bacteria in the formation of hemocyte microaggregates and treatment with physalins B and F minor · n number of circulating hemocytes in the hemolymph. The activities of hemocyte phagocytosis and microaggregation inhibited by treatment with oral physalin B were reversed by the injection of acid Treatment of insects with physalin B did not affect PLA2 activity, but increased the activity of PAF-AH. In another approach to insect-parasite interaction, we studied the cytotoxic effects of S. marcescens. In vitro assays, incubation of bacteria with T. or T. cruzi rangeli together with the carbohydrate D-mannose (0.2 M) resulted in protection of parasites adhesion and lysis of S. marcescens (SM365 and RPH variants) in a dose dependent. However, the D-mannose did not affect the hemolytic activity of SM365 and RPH variants of S. marcescens. While these variants with bacteria from adhering to the parasite occurred in only a few seconds, the variant has not adhered DB11 and not lysed the parasite. The bacterial adhesion is dense and compact and thin filaments occurs with time forming biofilm grows around the surface of the parasite lysate. Therefore, we conclude that among the factors that influence the interaction between insect and T. and T. cruzi rangeli studied, we can cite physalins and S. marcescens for their immunosuppressive and lytic activities against the parasites, respectively. While physalin B acts by decreasing the levels of PAF analogue (IPAF) in the hemolymph and raising levels of PAF-AH, with an inhibitory effect on the immune system of R. prolixus bacteria S. marcescens smooth parasites resulting in biofilm formation, which regulate, directly or indirectly, the development of the parasite in the insect vector.Desfazer edições

Une partie conséquente de mon travail a été d'effectuer un crible génétique en utilisant une bibliothèque de mutants générés par insertion aléatoire de Tn5-Sm, un minitransposon bactérien. Le crible a été réalisé dans un contexte défini: celui de mouches-hôtes auxquelles manquait le gène Eater, lequel code un récepteur de phagocytose (Kocks et al., 2005). Dans ces mouches, l'infection n'est plus contrôlée dans l'hémocoele par les hémocytes et les drosophiles mutantes succombent rapidement à une bactériémie. Plusieurs phénotypes bactériens étaient attendus à l'issue de ce crible. Une première catégorie de phénotype prévisible était une virulence accrue, par exemple si les bactéries mutantes devenaientcapables de traverser plus rapidement ou efficacement la barrière intestinale conséquemment à la perte d'un régulateur négatif. Un deuxième type de phénotype attendu était une virulence atténuée pouvant s'expliquer de plusieurs manières: 1- perte de résistance à l'environnement existant dans le lumen intestinal (enzymes digestives et lysozyme, radicaux libres et peptides antimicrobiens induits au niveau de l'épithélium intestinal dans le cadre d'une réponse immunitaire locale de l'hôte); 2- incapacité à traverser la matrice péritrophique; 3-incapacité à envahir les cellules épithéliales (adhésion, pénétration); 4- incapacité à résister aux défenses intracellulaires potentielles; 5- incapacité à sortir du côté basal des entérocytes 6- incapacité à proliférer dans l'hémolymphe ou perte de la résistance à l'action de la réponse immunitairesystémique qui est, quant à elle, fortement induite en l'absence de phagocytose, laquelle empêche chez les mouches sauvages la prolifération des bactéries ayant traversé la paroi intestinale. [...] Dans le cadre d'une infection intestinale, les mouches sauvages (et imd) succombaient en six jours alors que, de manière surprenante, les mouches mutantes de la voie Toll périssaient plus lentement, une situation opposée à celle du modèle de la piqûre septique. Quelques bactéries sont capables de traverser la paroi intestinale mais sont incapables de proliférer à moins que la réponse cellulaire ait été préalablement bloquée. L'épithélium intestinal apparaissait normal à la dissection et la presque totalité des bactéries ingérées étaient tuées dans l'intestin. Après avoir exclu l'hypothèse d'une toxine sécrétée dans le surnageant des bactéries adsorbées sur le filtre sur lequel viennent se nourrir les mouches, nous avons testé l'hypothèse qu'une suractivation de la réponse immunitaire était à l'origine du décès des mouches. La génétique mettant hors de cause les peptides antimicrobiens, la voie Toll n'étant apparemment pas activée dans l'épithélium intestinal, nous avons alors étudié laréponse oxydative induite par l'ingestion de bactéries (Ha et al., 2009), laquelle est capable de tuer les mouches lorsqu'elle n'est pas régulée correctement. Là-aussi, le résultat s'est avéré négatif. En fin de compte, j'ai pu établir que la mort des mouches était due à un état de famine, confirmé par des mesures des réserves métaboliques. Mes travaux ont permis d'établir un nouveau rôle de la voie Toll dans la résistance à la famine, en présence ou absence d'infection, qui sera peut-être à mettre en relation avec un rôle métabolique de la voie Toll consistant à bloquer la voie de réponse à l'insuline lors d'une infection. En conclusion, mes travaux permettent de mieux comprendre les relations hôte-pathogènequi s'établissent lors d'une infection intestinale. / For the systematic study of bacteri al virulence factors, we initially planned to screen the 12,000 mutant strains of the miniTn5-Sm tranposon-induced mutant bank in a wild-type S. marcescens strain Db10. Phagocytosis-deficient eater mutant flies (Kocks et al., 2005) were used in this screen to isolate the bacterial strains mutated for virulence factors and the genes responsible for crossing the gut barrier. In the eater mutant background, flies succumb to septicemia caused by the rapid proliferation of the bacteria in the hemolymph. Out of 1348 mutant strains screened, 58 candidate mutants have been isolated. Only 20% percent of the potential mutant strains displayed an increased virulence indicating that there are very few factor(s) that negatively control the virulence program of the bacterium. The fly survival phenotypes induced by the candidate mutants isolated in the first round of screen were retested. Only those bacterial strains that were consistent with the phenotype were chosen for the molecular identification of the transposon insertion sites using one primer PCR (Karlyshev et al., 2000). Once the genes impaired in each case had been identified, they were knocked off in the S. marcescens Db10 by site specific plasmid insertion mutagenesis. A mutant strain with the transposon inserted into the fliR gene, a component of the type III flagellar protein export system, exhibited attenuation of virulence. The plasmid insertionmutant strain generated to interrupt the gene fliR reproduced the fly survival phenotyp, indicating that the fliR gene is important for the virulence of S. marcescens. The fliR mutants are able to cross the peritrophic matrix, functionally similar to the human mucus. The bacteria were found in the vicinity of the epithelial cells but were not able to efficiently invade the intestinal epithelium as compared to the wild-type strain. Consequently lower titer of FliR mutants was found in the hemolymph. The inefficiency of the FliR mutants to invade cells was also confirmed in ex-vivo assay using insect cells.I thus demonstrated that the fliR gene which is important in the motility apparatus is also required by S. marcescens for the crossing of the epithelial barrier of D. melanogaster.[...]A strong oxidative response is triggered by D. melanogaster in the midgut against commensals and pathogens (Ha et al., 2009). In order to check whether the strong oxidative immune response is eventually killing the flies themselves, hydrogen peroxide was chemically neutralized in the midgut during the S. xylosus A. oral infection. No difference inthe fly survivals was observed with or without neutralization of the oxidative response indicating that over-production of reactive oxygen species (ROS) does not seem to be responsible for the fly death caused by a very low number of bacteria. Flies could efficiently survive to killed bacteria and filtered supernatant solution from overnight bacterial culture indicating that they do not die to the toxins released by the bacteria. Most surprisingly MyD88-, the Toll pathway-, mutant flies were surviving better to S. xylosus A. oral infection. A series of experiments lead us to the finding that the flies actually succumbed to starvation when orally infected with S. xylosus and that the MyD88 is required for the starvation susceptibility in microbiota-mediated manner. In conclusion my work has lead us to the better understanding of the host-bacterial interactions in the intestine.

O cuidado a saúde incorpora continuamente, novas tecnologias relacionadas a produtos e processos que podem trazer riscos, especialmente, quando não possuem embasamento técnico-científico. Ampolas de plástico são amplamente utilizadas no preparo de injetáveis, no entanto, a contaminação biológica das soluções na sua abertura é ainda questionável. Sabe-se que o risco de infecção tem etiologia multifacetada envolvendo aspectos complexos da microbiota endógena e das condições ambientais. O objetivo do estudo é contribuir para com a segurança microbiológica da abertura de ampolas com base no procedimento de desinfecção e, assim, minimizar os riscos de contaminação biológica no preparo de injetáveis. Trata-se de um experimento de laboratório que permitiu avaliar a esterilidade do conteúdo das ampolas e, consequentemente produziu evidencias acerca da segurança microbiológica no preparo de injetáveis. Para determinação se a abertura de ampolas possibilita veiculação bacteriana para as soluções utilizaram-se dois métodos de desinfecção do gargalo um com suabe e outro com algodão ambos umedecidos em álcool a 70%. Das 120 ampolas de plástico com água esterilizada 60 tiveram seus gargalos contaminados intencionalmente com Serratia marcescens (ATTCC 14756) e outra metade com Staphylococcus aureus resistente à meticilina (MRSA) (ATTCC 43300) na ordem de 106 UFC/mL. Na abertura das respectivas ampolas utilizaram-se os princípios e o rigor de assepsia em termos de higiene das mãos e uso de luvas esterilizadas. Na avaliação da positividade das culturas uma alíquota da solução de cada ampola foi pipetada em caldo nutriente e incubada a 35ºC por 14 dias. A fricção dos gargalos das ampolas com suabe ou bolas de algodão embebidas em 3 ml de álcool a 70% não foi eficaz na redução da contaminação do conteúdo destas ampolas. Evidencia-se que houve maior contaminação nas ampolas, intencionalmente contamindas com Serratia marcescens, que receberam desinfecção com suabe 19 (63,3%) comparado as ampolas 15 (50%) que foram desinfetadas com bolas de algodão embebidas em álcool. As ampolas contaminadas com Staphylococcus aureus resistente à meticilina independentemente de utilizar suabe ou bolas de algodão embebidas em álcool, a contaminação do conteúdo das ampolas foi alta 24 (80%) e 18 (60%), respectivamente. Das 60 (100%) ampolas contaminadas com Serratia marcescens 34 (56,7%) apresentaram contaminação da água destilada e, das 60 (100%) ampolas contaminadas com Staphylococcus aureus resistente à meticilina, 42 (70%) apresentaram contaminação. A elucidação do processo de contaminação do conteúdo de ampolas de plástico durante sua abertura é urgente, especialmente considerando a possibilidade do contato da solução com o meio externo e vice- versa. Consta-se que a temática carece de mais investimentos de pesquisa dado a relevância do procedimento de desinfecção na redução da carga microbiana. / The health care incorporates continuously new technologies related to products and administration processes that may pose risks, especially when there is no technical- scientific basis. Plastic ampoules are widely used in the preparation of injectables, however, biological contamination in solutions at its opening is still questionable. It is known that the risk of infection presents a multifaceted etiology involving complex aspects of endogenous microbiota and environmental conditions. The present investigation was carried out in order to contribute to the microbiological safety of opening ampoules based on disinfection procedure and thereby minimize the risk of biological contamination in the preparation of injectables. This is a laboratory experiment that allowed to evaluate the sterility of ampoules´ contents and consequently produced evidences regarding the microbiological safety in the preparation of injectables. To determine whether the opening of ampoules allows the carrying of bacteria into the solutions it was used two methods of ampoule neck disinfection, one with cotton balls and another with cotton swab both soaked with 70% alcohol. Of the 120 plastic ampoules containing sterile water, 60 had the ampoules necks intentionally contaminated with Serratia marcescens (ATTCC 14756) and the other half with methicillin-resistant Staphylococcus aureus (MRSA) (ATTCC 43300) of the order of 106 CFU/mL. At the opening of respective ampoules it was used the principles of strict asepsis and rigor in terms of hand hygiene and use of sterile gloves. In the evaluation of positive cultures an aliquot of solution from each ampoule was pipetted in nutrient broth and incubated at 35 °C for 14 days. Rub the ampoules necks with swab or cotton balls soaked with 70% alcohol in 3 ml was not effective in decreasing contamination of contents of those ampoules. It is evident that there were more contamination in ampoules intentionally contaminated with Serratia marcescens which received disinfection with swabs 19 (63.3%) if compared ampoules disinfected with cotton balls soaked in alcohol 15 (50%). Ampoules contaminated with methicillin-resistant Staphylococcus aureus neither swab nor cotton balls soaked in alcohol was effective, contamination of the contents of the ampoules 24 was high (80%) and 18 (60%), respectively. Of the 60 (100%) ampoules contaminated with Serratia marcescens 34 (56.7%) had distilled water contaminated, and from 60 (100%) ampoules contaminated with methicillin-resistant Staphylococcus aureus, 42 (70%) were contaminated. The elucidation of contamination process of contents of plastic ampoules during its opening is an urgent need, especially considering the possibility of contact of the solution with the external environment and vice versa. The evidence suggests that the issue needs more research investments given the relevance of the disinfection procedure in decreasing microbial load.

L’étude des interactions hôte-pathogène permettra de mieux comprendre les bases des maladies infectieuses. Durant ma thèse, j’ai étudié les interactions entre l’organisme modèle Drosophila melanogaster et la bactérie pathogène à Gram négatif Serratia marcescens (S.m). Cette bactérie est capable de tuer les mouches en moins de 24 heures une fois introduite directement dans l’hémolymph. Au contraire, les mouches peuvent survivre plusieurs jours après avoir ingéré du Serratia malgré les dommages à l’épithélium intestinal qui en résulte. Le travail de ma thèse a mené à comprendre la virulence des vésicules de la membrane externe purifiés de S.m et injectés dans la mouche. En plus, nous avons mis en évidence deux gènes qui jouent un rôle dans la virulence de la bactérie en infection intestinale, notamment dans la capacité de S.m à endommager les cellules intestinales. Enfin, nous avons identifié plusieurs gènes impliqués dans un mécanisme de résilience de la drosophile aux infections intestinales par S.m. / The study of host-pathogen interactions will provide a better understanding of the basics of infectious diseases. During my thesis, I studied the interactions between the model organism Drosophila melanogaster and the Gram-negative pathogen Serratia marcescens (S.m). This bacterium is capable of killing flies in less than 24 hours once introduced directly into the hemolymph. On the contrary, flies can survive several days after ingesting Serratia despite he resulting damages to the intestinal epithelium. The work of my thesis led to an understanding of the virulence of the outer membrane vesicles purified from S.m and injected into the fly. In addition, we have identified two genes that play a role in the virulence of the bacterium in the intestinal infection model, particularly in the ability of S.m to damage the intestinal cells. Finally, we have identified several Drosophila genes involved in a resilience mechanism to intestinal infections by S.m.

O cuidado a saúde incorpora continuamente, novas tecnologias relacionadas a produtos e processos que podem trazer riscos, especialmente, quando não possuem embasamento técnico-científico. Ampolas de plástico são amplamente utilizadas no preparo de injetáveis, no entanto, a contaminação biológica das soluções na sua abertura é ainda questionável. Sabe-se que o risco de infecção tem etiologia multifacetada envolvendo aspectos complexos da microbiota endógena e das condições ambientais. O objetivo do estudo é contribuir para com a segurança microbiológica da abertura de ampolas com base no procedimento de desinfecção e, assim, minimizar os riscos de contaminação biológica no preparo de injetáveis. Trata-se de um experimento de laboratório que permitiu avaliar a esterilidade do conteúdo das ampolas e, consequentemente produziu evidencias acerca da segurança microbiológica no preparo de injetáveis. Para determinação se a abertura de ampolas possibilita veiculação bacteriana para as soluções utilizaram-se dois métodos de desinfecção do gargalo um com suabe e outro com algodão ambos umedecidos em álcool a 70%. Das 120 ampolas de plástico com água esterilizada 60 tiveram seus gargalos contaminados intencionalmente com Serratia marcescens (ATTCC 14756) e outra metade com Staphylococcus aureus resistente à meticilina (MRSA) (ATTCC 43300) na ordem de 106 UFC/mL. Na abertura das respectivas ampolas utilizaram-se os princípios e o rigor de assepsia em termos de higiene das mãos e uso de luvas esterilizadas. Na avaliação da positividade das culturas uma alíquota da solução de cada ampola foi pipetada em caldo nutriente e incubada a 35ºC por 14 dias. A fricção dos gargalos das ampolas com suabe ou bolas de algodão embebidas em 3 ml de álcool a 70% não foi eficaz na redução da contaminação do conteúdo destas ampolas. Evidencia-se que houve maior contaminação nas ampolas, intencionalmente contamindas com Serratia marcescens, que receberam desinfecção com suabe 19 (63,3%) comparado as ampolas 15 (50%) que foram desinfetadas com bolas de algodão embebidas em álcool. As ampolas contaminadas com Staphylococcus aureus resistente à meticilina independentemente de utilizar suabe ou bolas de algodão embebidas em álcool, a contaminação do conteúdo das ampolas foi alta 24 (80%) e 18 (60%), respectivamente. Das 60 (100%) ampolas contaminadas com Serratia marcescens 34 (56,7%) apresentaram contaminação da água destilada e, das 60 (100%) ampolas contaminadas com Staphylococcus aureus resistente à meticilina, 42 (70%) apresentaram contaminação. A elucidação do processo de contaminação do conteúdo de ampolas de plástico durante sua abertura é urgente, especialmente considerando a possibilidade do contato da solução com o meio externo e vice- versa. Consta-se que a temática carece de mais investimentos de pesquisa dado a relevância do procedimento de desinfecção na redução da carga microbiana. / The health care incorporates continuously new technologies related to products and administration processes that may pose risks, especially when there is no technical- scientific basis. Plastic ampoules are widely used in the preparation of injectables, however, biological contamination in solutions at its opening is still questionable. It is known that the risk of infection presents a multifaceted etiology involving complex aspects of endogenous microbiota and environmental conditions. The present investigation was carried out in order to contribute to the microbiological safety of opening ampoules based on disinfection procedure and thereby minimize the risk of biological contamination in the preparation of injectables. This is a laboratory experiment that allowed to evaluate the sterility of ampoules´ contents and consequently produced evidences regarding the microbiological safety in the preparation of injectables. To determine whether the opening of ampoules allows the carrying of bacteria into the solutions it was used two methods of ampoule neck disinfection, one with cotton balls and another with cotton swab both soaked with 70% alcohol. Of the 120 plastic ampoules containing sterile water, 60 had the ampoules necks intentionally contaminated with Serratia marcescens (ATTCC 14756) and the other half with methicillin-resistant Staphylococcus aureus (MRSA) (ATTCC 43300) of the order of 106 CFU/mL. At the opening of respective ampoules it was used the principles of strict asepsis and rigor in terms of hand hygiene and use of sterile gloves. In the evaluation of positive cultures an aliquot of solution from each ampoule was pipetted in nutrient broth and incubated at 35 °C for 14 days. Rub the ampoules necks with swab or cotton balls soaked with 70% alcohol in 3 ml was not effective in decreasing contamination of contents of those ampoules. It is evident that there were more contamination in ampoules intentionally contaminated with Serratia marcescens which received disinfection with swabs 19 (63.3%) if compared ampoules disinfected with cotton balls soaked in alcohol 15 (50%). Ampoules contaminated with methicillin-resistant Staphylococcus aureus neither swab nor cotton balls soaked in alcohol was effective, contamination of the contents of the ampoules 24 was high (80%) and 18 (60%), respectively. Of the 60 (100%) ampoules contaminated with Serratia marcescens 34 (56.7%) had distilled water contaminated, and from 60 (100%) ampoules contaminated with methicillin-resistant Staphylococcus aureus, 42 (70%) were contaminated. The elucidation of contamination process of contents of plastic ampoules during its opening is an urgent need, especially considering the possibility of contact of the solution with the external environment and vice versa. The evidence suggests that the issue needs more research investments given the relevance of the disinfection procedure in decreasing microbial load.

Nos últimos anos a incidência de infecções causadas por bactérias Gram-negativas multirresistentes aumentou expressivamente. Esse fenômeno não foi acompanhado pelo desenvolvimento de novos antimicrobianos, resultando em infecções cuja opção de tratamento é a combinação de antimicrobianos. Acinetobacter baumannii, Pseudomonas aeruginosa e Serratia marcescens são importantes agentes de infecções relacionadas à assistência à saúde (IRAS), e capazes de adquirir resistência a várias classes de antimicrobianos, como os carbapenêmicos e polimixinas. Os testes que avaliam sinergismo in vitropodem ser uma ferramenta importante na escolha do tratamento antibiótico adequado para infecções causadas por microrganismos multirresistentes. O objetivo deste estudo foi avaliar métodos de sinergismo in vitroque possam ser utilizados na rotina de laboratórios de microbiologia clínica como alternativa ao método considerado padrão ouro (time-kill). Foram selecionados para o estudo 48 isolados Gram-negativos não fermentadores (20 A. baumannii e 28 P. aeruginosa) e 14 fermentadores (S. marcescens) do banco de cepas do LIM-54 com diferentes mecanismos de resistência identificados por PCR e sequenciamento total do genoma. Foram realizados, concentração inibitória mínima dos antimicrobianos e avaliação do sinergismo pelos métodos time-kill (TK), disco aproximação (DA) e CIM:CIM razão.A concordância dos métodos foi avaliada por teste de kappa e os resultados discordantes foram classificados em tipos de erros baseado no FDA.Os isolados apresentaram alta proporção de resistência a meropenem e 7 isolados de A. baumanniiapresentaram resistência a colistina. A combinação de colistina com fosfomicina ou meropenem apresentou elevado efeito sinérgico para os isolados de A. baumanniiresistentes a colistina pelo DA e TK. Por outro lado, a combinação de fosfomicina-meropenem apresentou concordância boa pelo teste de kappa e baixo número de erros entre os métodos TK e DA para todos os isolados de A. baumannii. Para os isolados de P. aeruginosa não foram detectados efeitos sinérgicos pelos métodos DA e CIM:CIM razão.O método CIM:CIM razão apresentou alta concordância com o TK entre os isolados de A. baumannii resistentes a colistina. A combinação ceftazidima-avibactam com meropenem apresentou elevado efeito sinérgico para os isolados de S. marcescensportadores do gene blaKPC-2 pelo DA e TK. O método de DA apresentou uma boa correlação com o TK para a combinação de fosfomicina-meropenem e ceftazidima-avibactam-meropenem, com baixa porcentagem de erros menores, podendo ser utilizado para avaliar sinergismo in vitro para essas combinações. Não foi identificado no estudo efeito antagônico, portanto, foram encontrados somente erros menores / In recent years, the incidence of infections caused by multiresistant Gram-negative bacteria has increased. This event has not been accompanied by the development of new antimicrobials, resulting in infections which treatment option is the combination of antimicrobials. Acinetobacter baumannii, Pseudomonas aeruginosa and Serratia marcescens are important causesof Hospital Acquired Infection (HAI), and able to acquire resistance to multiple classes of antimicrobials, such ascarbapenems and polymyxins. Synergism testing may be an important tool in choose the appropriate antibiotic treatment. The aim of this study was to evaluate in vitro synergism methods that can be used in a clinical microbiology laboratory as an alternative to the method considered the gold standard (time-kill).For the study were selected 48 non-fermenting (20 A. baumannii and 28 P. aeruginosa)and 14 fermenting(S. marcescens) Gram-negativeisolates from LIM-54strains bench with different resistance mechanisms identified by PCR and total genome sequencing.Minimum antimicrobial inhibitory concentration and synergism evaluation by time-kill (TK), disk approximation (DA) and MIC: MIC ratio were performed. Agreement of the methods was evaluated by kappa test and the discordant results were classified in types of errors based on the FDA. The isolates showed high proportion of resistant to meropenem and 7A. baumannii isolates showed resistance to colistin. The combination of colistin with fosfomycin or meropenem showed high synergistic effect for colistin-resistant A. baumannii isolates by DA and TK.On the other hand, the combination of fosfomycin-meropenem showed good concordance by kappa test and low number of errors between TK and DA for all A. baumannii isolates.For P. aeruginosa isolates no synergistic effects were detected by DA and MIC: MIC ratio.The method MIC: MIC ratio showed high concordance with the TK among the colistin-resistant A. baumannii isolates.The combination ceftazidime-avibactam with meropenem showed high synergistic effect for the isolates of S. marcescenscarryingblaKPC-2 gene by DA and TK. The DA showed a good agreement with TK for the combination of fosfomycin-meropenem and ceftazidime-avibactam-meropenem, with a low percentage of minor errors, and could be used to evaluate synergism in vitro for these combinations

The potential value of the chitin biomass (e.g. food waste) is recently considered being ignored by landfill. Chitin can be a potential cheap carbon source for converting into value-added chemicals by microorganisms. Serratia marcescens is a chitinolytic bacterium that harbors endogenous chitinase systems. With goals of characterzing S. marcescens chitinolytic capabilities and applying S. marcescens to chemical production from chitin, my dissertation main content includes five chapters: 1) Chapter 1 highlights background information of chitin source, S. marcescens and potential metabolic engineering targets using chitin as a substrate; 2) Chapter 2 demonstrates that ChiR is a key regulator in regulating 9 chitinase-related genes in S. marcescens Db11 and manipulation of chiR can be a useful and efficient genetic target to enhance chitin utilization; 3) Chapter 3 reports the production of N-acetylneuraminic acid (Neu5Ac) from chitin by a bottom-up approach of engineering the nonconventional chitinolytic bacterium, Serratia marcescens, including native constitutive promoter characterization and transcriptional and translational pathway balancing; 4) Chapter 4 describes improvement of S. marcescens chitinolytic capability by an adaptive evolution approach; 5) Chapter 5 elucidates S. marcescens intracellular metabolite profile using a constraint-based genome-scale metabolic model (iSR929) based on genomic annotation of S. marcescens Db11. Overall, the dissertation work is the first report of demonstrating the concept of chitin-based CBP using S. marcescens and the computational model and genetic molecular tools developed in this dissertation are valuable but not limited to design-build-test of S. marcescens for contributing to the field of biological science and metabolic engineering applications.

Une partie conséquente de mon travail a été d'effectuer un crible génétique en utilisant une bibliothèque de mutants générés par insertion aléatoire de Tn5-Sm, un minitransposon bactérien. Le crible a été réalisé dans un contexte défini: celui de mouches-hôtes auxquelles manquait le gène Eater, lequel code un récepteur de phagocytose (Kocks et al., 2005). Dans ces mouches, l'infection n'est plus contrôlée dans l'hémocoele par les hémocytes et les drosophiles mutantes succombent rapidement à une bactériémie. Plusieurs phénotypes bactériens étaient attendus à l'issue de ce crible. Une première catégorie de phénotype prévisible était une virulence accrue, par exemple si les bactéries mutantes devenaientcapables de traverser plus rapidement ou efficacement la barrière intestinale conséquemment à la perte d'un régulateur négatif. Un deuxième type de phénotype attendu était une virulence atténuée pouvant s'expliquer de plusieurs manières: 1- perte de résistance à l'environnement existant dans le lumen intestinal (enzymes digestives et lysozyme, radicaux libres et peptides antimicrobiens induits au niveau de l'épithélium intestinal dans le cadre d'une réponse immunitaire locale de l'hôte); 2- incapacité à traverser la matrice péritrophique; 3-incapacité à envahir les cellules épithéliales (adhésion, pénétration); 4- incapacité à résister aux défenses intracellulaires potentielles; 5- incapacité à sortir du côté basal des entérocytes 6- incapacité à proliférer dans l'hémolymphe ou perte de la résistance à l'action de la réponse immunitairesystémique qui est, quant à elle, fortement induite en l'absence de phagocytose, laquelle empêche chez les mouches sauvages la prolifération des bactéries ayant traversé la paroi intestinale. [...] Dans le cadre d'une infection intestinale, les mouches sauvages (et imd) succombaient en six jours alors que, de manière surprenante, les mouches mutantes de la voie Toll périssaient plus lentement, une situation opposée à celle du modèle de la piqûre septique. Quelques bactéries sont capables de traverser la paroi intestinale mais sont incapables de proliférer à moins que la réponse cellulaire ait été préalablement bloquée. L'épithélium intestinal apparaissait normal à la dissection et la presque totalité des bactéries ingérées étaient tuées dans l'intestin. Après avoir exclu l'hypothèse d'une toxine sécrétée dans le surnageant des bactéries adsorbées sur le filtre sur lequel viennent se nourrir les mouches, nous avons testé l'hypothèse qu'une suractivation de la réponse immunitaire était à l'origine du décès des mouches. La génétique mettant hors de cause les peptides antimicrobiens, la voie Toll n'étant apparemment pas activée dans l'épithélium intestinal, nous avons alors étudié laréponse oxydative induite par l'ingestion de bactéries (Ha et al., 2009), laquelle est capable de tuer les mouches lorsqu'elle n'est pas régulée correctement. Là-aussi, le résultat s'est avéré négatif. En fin de compte, j'ai pu établir que la mort des mouches était due à un état de famine, confirmé par des mesures des réserves métaboliques. Mes travaux ont permis d'établir un nouveau rôle de la voie Toll dans la résistance à la famine, en présence ou absence d'infection, qui sera peut-être à mettre en relation avec un rôle métabolique de la voie Toll consistant à bloquer la voie de réponse à l'insuline lors d'une infection. En conclusion, mes travaux permettent de mieux comprendre les relations hôte-pathogènequi s'établissent lors d'une infection intestinale.