Le fer est un élément important de plusieurs métabolismes et fonctions physiologiques. Il possède la propriété de gagner ou de perdre facilement un électron, passant ainsi de la forme ferreuse (FeII) à la forme ferrique (FeIII), et inversement. C'est cette propriété qui lui confère un rôle primordial dans les phénomènes d'oxydations et de réductions biologiques. Bien qu'il soit très abondant dans la croûte terrestre, le fer est très peu soluble en milieu aérobie et à pH physiologique et est de ce fait est très peu biodisponible. La concentration de FeIII libre dans l'environnement est d'environ 10-18 M, bien trop basse pour les bactéries pathogènes qui exigent une concentration de l'ordre de 10-5 à 10–7 M pour établir et maintenir une infection. Pour contourner le problème de la faible disponibilité de fer, les bactéries ont développé différents mécanismes d'acquisition de ce métal. Le mécanisme le plus répandu implique la synthèse et la sécrétion des sidérophores, ayant une très forte affinité pour leFeIII. Après leur sécrétion, les sidérophores chélatent le Fe3+ dans le milieu extracellulaire et le transportent au travers de la membrane externe via les récepteurs TonB-dépendants (RTBDs). Les RTBDs sont également impliqués dans le transport d'autres molécules comme l'hème. Durant cette thèse nous nous sommes intéressés à l'étude structurale des RTBDs de différentes bactéries à Gram-négatif et aussi au devenir, au niveau du périplasme, de la ferri-pyoverdine, après son transport à travers la membrane externe via le RTBD FpvA chez P. aeruginosa. Pour les études structurales, nous nous sommes intéressés à 4 RTBDs (ShuA, SuxA, FauA et FetA). Nous avons défini et optimisé un protocole de surexpression, de purification et de cristallisation pour les RTBDs. Le protocole mis en place est rapide, efficace et reproductible. Il nous a permis de purifier, de cristalliser rapidement et de collecter des données de diffraction pour 4 RTBDs. La structure de ShuA a ensuite été résolue à 2,6 Å, celle de FauA à 2,3 Å de résolution par le Dr D. Cobessi. Celle de FetA est actuellement en cours d'affinement à 3,2 Å. Pour les études fonctionnelles, nous nous sommes intéressés à l'implication des gènes du cluster fpvCDEFGHJK dans l'acquisition du fer par la voie Pvd chez P. aeruginosa. Ce cluster est conservé chez toutes les espèces de Pseudomonas produisant la Pvd, il est organisé en 2 opérons, fpvCDEF et fpvGHJK, séparés par 32 paires de bases. Les résultats obtenus pendant cette thèse, suggèrent l'implication des protéines FpvCDEF dans la dissociation périplasmique de la ferri-pyoverdine par réduction du FeIII en FeII. En effet, la mutation de ces gènes abolie complètement le transport du fer par la Pvd. Le phénotype sauvage a été restauré par l'addition du DTT, suggérant l'implication de ses protéines dans la dissociation périplasmique de la ferri-pyoverdine par réduction du FeIII en FeII. Nous avons également montré que la protéine FpvG est une protéine périplasmique dont l'expression est régulée par les niveaux de fer. Les études de spectrométrie de masse et de dosage des métaux ont montré que cette protéine était également capable de lier directement le fer. Enfin, l'étude des interactions protéine-protéine a montré une interaction entre les protéines FpvG et FpvA, confirmant l'implication de la protéine FpvG dans le transport du fer par la Pvd. FpvG serait probablement impliquée dans la prise en charge du fer dans le périplasme après sa dissociation de la Pvd et assurant ensuite son transport vers le cytoplasme via le transporteur ABC FpvHJ. L'ensemble de ces résultats a permis également de montrer que le transport du fer via la Pvd implique des mécanismes très différents de ceux qui sont décrits précédemment pour les sidérophores utilisés par E. coli.

In recent decades, most countries of the world have experienced a shortage of water and increase its rate of consumption. Today, every country in the world are interested in this problem by trying to find alternatives to address this shortage. One solution is reuse greywater (GW) for irrigation after treatment. GW is all water generated from Household except toilet water. The risks associated with the reuse of these waters are the presence of pathogens that can infect humans, animals and plants. In this thesis focused on studying treatment by slow sand filtration and the survival of representatives of pathogens, such as E. Coli, P. aeruginosa , E. Faecalis and Bacteriophage MS2 which could be found in the greywater. The study factors was a physico-chemicals factors such as; temperature (6±2,23±2,42±2°c), salinity (1.75 and 3.5% Nacl), oxygen (aerobic and anaerobic condition), nutrient ( rich media , 50%: 50% salt and poor media ), light with photocatalysis ( UV and Visible lights) and slow sand filter (Egyptian desert sand and swimming pool sand). A combination of high temperature, sunlight and photocatlysis are mainly responsible for the rapid decline of bacteria and MS2 coliphage. Slow sand filter have clearly less influence on the survival of bacteria in the greywater, but it effective to decline turbidity and COD for short times.

Background: Gram-negative resistance is a growing problem worldwide. It is generally believed that rates of resistant bacteria within a hospital are a function of antibiotic use, resistant organisms brought into the hospital, infection control efforts, and underlying severity of patient illness. The relative contribution of each to a particular resistance phenotype is unclear. P. aeruginosa is responsible for many hospital acquired infections and it may become resistant to carbapenems. In addition, newer threats to the future utility of the carbapenems are carbapenemase-producing K. pneumoniae Purpose: To determine if there is an association between the volume and composition of antibiotic use, geography, severity of illness and rates of carbapenem-resistant P. aeruginosa and K. pneumoniae. Methods: This is a retrospective ecological longitudinal investigation within the University HealthSystem Consortium affiliated academic medical centers. Antibiotic use data between January 1, 2006 and December 31, 2009 were obtained from billing records and reported as days of therapy per 1000 patient days (DOT/1000 PD), in addition to hospital characteristics (e.g. geographical location, bed size, case mix index). “Whole house” antibiograms were obtained to determine rates and proportions of carbapenem-resistant P. aeruginosa (CR-PA) and carbapenem resistant K. pneumoniae (CR-KP). Also, CR-KP isolation was generated as a binary outcome. Generalized estimating equations (GEE) were used to model CR-KP and CR-PA. Results: CR-KP rates (1000PDs) increased from 0.07 in 2006 to 0.15 in 2009 (P= 0.0118) and CR-KP proportions increased from 1.3% in 2006 to 3.1% in 2009 (0.0003) within 40 hospitals over 2006-2009. However, CR-PA rates and proportions were stable over the same period. Geographical location, carbapenems use, and antipseudomonal penicillins use were significantly associated with CR-KP isolation. Thus, for every ten DOT/1000 PDs increase in carbapenem use, the odds of CR-KP isolation increased by 42% (P=0.0149). In contrast, for every ten DOT/1000 PDs increase in antipseudomonal penicillin use, the odds of CR-KP isolation decreased by 14%. However, there was no significant model to explain CR-PA rates and proportions. Conclusion: Carbapenems, antipseudomonal penicillins, and geographical location were identified as risk factors associated with CR-KP isolation. These findings emphasize the challenges associated with the treatment of multidrug- gram-negative bacteria.

La saprolégniose est une maladie fongique causée par le champignon aquatique Saprolegnia sp. qui affecte les poissons sauvages et ceux provenant des piscicultures. L’apparition de touffes cotonneuses semblables à de la ouate de couleur blanche à grise est souvent la première indication de l’infection. Ce saprophyte ubiquitaire se nourrit habituellement des œufs de poissons morts, mais peut se propager rapidement aux œufs sains causant la mort de ces derniers. La saprolégniose est souvent une infection secondaire, mais des souches virulentes peuvent facilement se développer sur les salmonidés ayant subi un stress ou une mauvaise manipulation. De grandes pertes économiques associées à la saprolégniose sont rapportées chaque année à travers le monde surtout dans l’industrie de la pisciculture. Jusqu’en 2002, le contrôle de la saprolégniose pouvait se faire par l’utilisation du vert de malachite, un colorant organique ayant une grande activité antifongique. Malheureusement, cette molécule a été bannie à cause de ses propriétés cancérigènes. Aucun composé aussi efficace n’est actuellement disponible pour traiter les infections de la saprolégniose. Des molécules ou extraits naturels ayant un potentiel antifongique ont donc été testés à l’aide de deux techniques (par graines de chanvre et par cylindre d’agar). Les molécules d’un extrait de propolis (cire de ruches d’abeilles) démontrant de l’activité anti-Saprolegnia ont été identifiées. De plus, une bactérie, Pseudomonas aeruginosa, pouvant être retrouvée dans le même environnement que Saprolegnia sp. a démontré un effet antagoniste au champignon. Une molécule de signalisation intercellulaire produite par P. aeruginosa, 4-hydroxy-2-heptylquinoline (HHQ), a été identifiée comme responsable de l’effet antagoniste contre Saprolegnia sp. / Saprolegniosis, a fungal disease which affects wild and farm fish, is caused by the water mold Saprolegnia sp. Visible cotton-like white or grey patches on fish skin are often the first sign of infection. This ubiquitous saprophyte infects dead fish eggs and then spreads to healthy eggs resulting in major losses due to mortalities. Saprolegniosis is often a secondary infection; however some virulent strains are known to cause primary infection on injured or stressed salmonids. Important economic losses in aquaculture are reported every year worldwide due to saprolegniosis. Infection by Saprolegnia sp. was well controlled with the use of an organic dye with great antifungal efficacy; malachite green. Unfortunately, the use of malachite green was banned in 2002 worldwide due to its carcinogenic and toxicological effects. Up until now, no new treatment as effective as malachite green has been discovered. Natural extracts or molecules with potential antifungal properties have also been tested using two techniques (with hemp seeds and with agar cylinders). Molecules from propolis extracts (beeswax), active against Saprolegnia sp. have been identified. Furthermore, culture supernatant of the bacteria Pseudomonas aeruginosa was found to be able to inhibit the growth of Saprolegnia. The intercellular signal molecule produced by P. aeruginosa 4-hydroxy-2-heptylquinoline (HHQ) was identified as the active compound against Saprolegnia.

During colonization of the cystic fibrosis airway Pseudomonas aeruginosa converts from non-mucoid to a mucoid phenotype, characterized by the production of the exopolysaccharide alginate. Alginate production has been shown to enhance survival by promoting biofilm formation, evading complement killing, and resisting phagocytosis. The mechanism by which alginate protects P. aeruginosa from phagocytosis is unclear. To investigate the role of alginate in the inhibition of phagocytosis, a human monocytic cell line (THP-1) and a murine alveolar macrophage cell line (MH-S) were used to determine the effects of alginate on macrophage binding, signaling, and phagocytosis. Phagocytosis assays using the mucoid cystic fibrosis clinical isolate FRD1, and its non-mucoid isogenic algD mutant FRD1131, revealed that alginate inhibits opsonic and non-opsonic phagocytosis. The inhibitory effect of alginate production is intrinsic to the bacteria as exogenous alginate was unable to protect non-mucoid FRD1131 from phagocytosis. Decreased binding of FRD1 compared to FRD1131 was also demonstrated by using the actin polymerization inhibitor cytochalasin D to inhibit phagocytosis. Furthermore, studies using blocking antibodies to CD11b and CD14 found that both of these receptors were important for the phagocytosis of FRD, and it is likely that these receptors are blocked by alginate. Alginate production by P. aeruginosa may reduce lipid raft formation, however, it was not found to affect acid sphingomyelinase activity, which is important for ceramide formation within the lipid raft. Decreased binding led to decreased signaling in macrophages demonstrated by reduction in level and alteration in kinetics of phosphorylation of AKT and ERK1/2 kinases. Signaling pathway inhibitors revealed that PI3K, but not MEK, activation was critical for phagocytosis of P. aeruginosa. Despite altered intracellular signaling in murine macrophages, both mucoid and non-mucoid P. aeruginosa induced similar levels of IL-8 and MIP-2 from human and murine macrophages, respectively. By understanding the pathways involved in mediating efficient phagocytosis of clinical isolates, it may be possible to develop a treatment to promote clearance by the resident alveolar macrophages. These experiments may serve as a model to evaluate the effectiveness of such treatments. This approach also provides valuable insight into previously unknown mechanisms of phagocytosis of P. aeruginosa.

Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, <i>Stenotrophomonas maltophilia</i> and <i>Pseudomonas aeruginosa</i>, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in<i> S. maltophilia</i>, isogenic deletion of phosphoglycerate mutase (<i>gpmA</i>) and two chaperone-usher pilin subunits, <i>S. maltophilia</i> fimbrae-1 (<i>smf-1</i>) and<i> cblA</i>, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δ<i>smf-1</i> and Δ<i>cblA</i> showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in <i>P. aeruginosa</i>, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when <i>polB</i>, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in <i>S. maltophilia</i> and genetic switches during the acute infection lifestyle in <i>P. aeruginosa</i>.

This dissertation thesis focuses on the removability of algal organic matter (AOM) by coagulation during water treatment and also on the influence of AOM on the coagulation of other substances present in source water. Special emphasis is put on the description of coagulation mechanisms. The effectiveness of AOM removal by coagulation was investigated by coagulation tests performed with optimized doses of coagulants (aluminium or ferric sulphate) under different pH values. Peptides and proteins contained in cellular organic matter of cyanobacterium Microcystis aeruginosa were used in the experiments since they have been previously reported to disturb the coagulation process. Moreover, peptides and proteins underwent coagulation experiments together with kaolin particles, representing clay particles in turbid waters, in both the presence and absence of coagulants to investigate the effect of AOM on the coagulation of turbid waters. To enable the description of coagulation mechanisms, AOM were characterised in terms of charge, functional groups, molecular weight and ability to form dissolved complexes with coagulant metals. The experimental results demonstrated that the removability of peptides and proteins is greatly dependent on pH value and on the properties of the involved particles or molecules....

Microcistinas são potentes hepatotoxinas e promotoras de tumores encontradas em águas doces que causam riscos à saúde pública e, portanto, representam um sério problema para as estações de tratamento de água. O gênero de cianobactéria Microcystis é o mais conhecido produtor dessas toxinas e também o mais comumente encontrado formando florações em reservatórios de água usados para abastecimento público. Entretanto, algumas bactérias são capazes de utilizar essas toxinas como fonte de carbono o que pode contribuir para a sua remoção da água. Neste estudo foi avaliado o potencial de degradação da microcistina-XR (MCYST-XR) por bactérias heterotróficas isoladas de um sistema de abastecimento público de água da cidade de Piracicaba-SP. A cianotoxina MCYST-XR avaliada foi isolada da linhagem Microcystis aeruginosa NPLJ-4 e purificada. Culturas puras de 35 bactérias isoladas do sistema de abastecimento de água foram testadas. Para isso, cada bactéria foi inoculada em meio mínimo de sais contendo 60 g mL-1 de MCYST-XR purificada e após 144 horas a degradação da toxina foi avaliada por análise em LC-MS/MS. Os picos indicando a massa da microcistina-XR (m/z 1037) não foram detectados no meio de cultura de seis bactérias, as quais foram identificadas pelo sequenciamento quase completo do gene de RNAr 16S como pertencentes aos gêneros Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. e Acinetobacter sp. Este é o primeiro relato de uma linhagem de Acinetobacter capaz de degradar MCYST. A cinética de biodegradação da MCYST-XR mostrou redução de 80% em 72 horas, período em que as bactérias apresentaram o máximo crescimento. A presença do gene mlrA codificante da enzima microcistinase envolvida no metabolismo da microcistina foi avaliada nos genomas dos seis isolados bacterianos usando iniciadores de PCR específicos. Fragmentos de aproximadamente 800 pb foram amplificados em dois isolados, Bacillus sp.e Microbacterium sp. Ensaios de toxicidade utilizando o cládocero Daphnia similis foram realizados para verificar a efetiva remoção da MCYST-XR e a não formação de subprodutos tóxicos na via de biodegradacão. Os resultados negativos dos bioensaios após 48 horas indicaram a ausência de subprodutos tóxicos na via da degradação. / Microcystins are potent hepatotoxins and tumor promoters found in freshwaters that cause public health risks and thus represent a serious problem for water treatment plants. The cyanobacterium genus Microcystis is the most known toxin-producer and the most common bloom-forming in water reservoirs used for public supply. However, some bacteria are able to use these toxins as carbon source, which can contribute to its removal from water. This study assessed the potential for degradation of microcystin-XR (MCYST-XR) by heterotrophic bacteria isolated from a public water supply system of the city of Piracicaba-SP. The cyanotoxin MCYSTXR evaluated was isolated from Microcystis aeruginosa strain NPLJ-4 and purified. Pure cultures of 35 bacteria isolated from the water supply system were tested. For this, each bacterium was inoculated in minimal medium salts containing 60 g mL-1 of purified MCYST-XR and after 144 hours the toxin degradation was evaluated by LC-MS/MS analysis. The peaks indicating the mass of microcystin-XR (m/z 1037) were not detected in the culture medium of six bacteria, which were identified by almost complete sequencing of the 16S rRNA gene as belonging to the genera Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. and Acinetobacter sp. This is the first report of an Acinetobacter strain able to degrade MCYST. The kinetic of the MCYST-XR biodegration showed 80% reduction in 72 hours, period in which the bacteria showed the maximum growth. The presence of the mlrA gene encoding the microcystinase enzyme involved in the metabolism of microcystin was evaluated in the genomes of the six bacterial isolates using specific PCR primers. Fragments of approximately 800 bp were amplified in two isolates, Bacillus sp. and Microbacterium sp. Toxicity assays using the cladoceran Daphnia similis were conducted to verify the effective removal of MCYST-XR and the non formation of toxic by-products in the biodegradation pathway. The negative results of the bioassays after 48 hours indicated absence of toxic by-products in the biodegradation pathway.

Microcistinas são potentes hepatotoxinas e promotoras de tumores encontradas em águas doces que causam riscos à saúde pública e, portanto, representam um sério problema para as estações de tratamento de água. O gênero de cianobactéria Microcystis é o mais conhecido produtor dessas toxinas e também o mais comumente encontrado formando florações em reservatórios de água usados para abastecimento público. Entretanto, algumas bactérias são capazes de utilizar essas toxinas como fonte de carbono o que pode contribuir para a sua remoção da água. Neste estudo foi avaliado o potencial de degradação da microcistina-XR (MCYST-XR) por bactérias heterotróficas isoladas de um sistema de abastecimento público de água da cidade de Piracicaba-SP. A cianotoxina MCYST-XR avaliada foi isolada da linhagem Microcystis aeruginosa NPLJ-4 e purificada. Culturas puras de 35 bactérias isoladas do sistema de abastecimento de água foram testadas. Para isso, cada bactéria foi inoculada em meio mínimo de sais contendo 60 g mL-1 de MCYST-XR purificada e após 144 horas a degradação da toxina foi avaliada por análise em LC-MS/MS. Os picos indicando a massa da microcistina-XR (m/z 1037) não foram detectados no meio de cultura de seis bactérias, as quais foram identificadas pelo sequenciamento quase completo do gene de RNAr 16S como pertencentes aos gêneros Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. e Acinetobacter sp. Este é o primeiro relato de uma linhagem de Acinetobacter capaz de degradar MCYST. A cinética de biodegradação da MCYST-XR mostrou redução de 80% em 72 horas, período em que as bactérias apresentaram o máximo crescimento. A presença do gene mlrA codificante da enzima microcistinase envolvida no metabolismo da microcistina foi avaliada nos genomas dos seis isolados bacterianos usando iniciadores de PCR específicos. Fragmentos de aproximadamente 800 pb foram amplificados em dois isolados, Bacillus sp.e Microbacterium sp. Ensaios de toxicidade utilizando o cládocero Daphnia similis foram realizados para verificar a efetiva remoção da MCYST-XR e a não formação de subprodutos tóxicos na via de biodegradacão. Os resultados negativos dos bioensaios após 48 horas indicaram a ausência de subprodutos tóxicos na via da degradação. / Microcystins are potent hepatotoxins and tumor promoters found in freshwaters that cause public health risks and thus represent a serious problem for water treatment plants. The cyanobacterium genus Microcystis is the most known toxin-producer and the most common bloom-forming in water reservoirs used for public supply. However, some bacteria are able to use these toxins as carbon source, which can contribute to its removal from water. This study assessed the potential for degradation of microcystin-XR (MCYST-XR) by heterotrophic bacteria isolated from a public water supply system of the city of Piracicaba-SP. The cyanotoxin MCYSTXR evaluated was isolated from Microcystis aeruginosa strain NPLJ-4 and purified. Pure cultures of 35 bacteria isolated from the water supply system were tested. For this, each bacterium was inoculated in minimal medium salts containing 60 g mL-1 of purified MCYST-XR and after 144 hours the toxin degradation was evaluated by LC-MS/MS analysis. The peaks indicating the mass of microcystin-XR (m/z 1037) were not detected in the culture medium of six bacteria, which were identified by almost complete sequencing of the 16S rRNA gene as belonging to the genera Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. and Acinetobacter sp. This is the first report of an Acinetobacter strain able to degrade MCYST. The kinetic of the MCYST-XR biodegration showed 80% reduction in 72 hours, period in which the bacteria showed the maximum growth. The presence of the mlrA gene encoding the microcystinase enzyme involved in the metabolism of microcystin was evaluated in the genomes of the six bacterial isolates using specific PCR primers. Fragments of approximately 800 bp were amplified in two isolates, Bacillus sp. and Microbacterium sp. Toxicity assays using the cladoceran Daphnia similis were conducted to verify the effective removal of MCYST-XR and the non formation of toxic by-products in the biodegradation pathway. The negative results of the bioassays after 48 hours indicated absence of toxic by-products in the biodegradation pathway.

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