Indiana University-Purdue University Indianapolis (IUPUI) / The patients with Cystic fibrosis (CF), an inherent genetic disorder, suffer from chronic bacterial infection in the lung. In CF, modification of epithelial cells leads to alteration of the lung environment, such as inhibition of ciliary bacterial clearance and accumulation of thickened mucus in the airways. Exploiting these conditions, opportunistic pathogens like Pseudomonas aeruginosa cause lifelong persistent infection in the CF lung by forming into antibiotic-resistant aggregated communities called biofilms. Airway infections as well as inflammation are the two major presentations of CF lung disease. P. aeruginosa strains isolated from CF lungs often contain mutations in the mucA gene, and this mutation results in higher level expression of bacterial polysaccharides and toxic lipoproteins. In a previous work, we have found a putative lipoprotein gene (PA4326) which is overexpressed in antibiotic-induced biofilm formed on cultured CF-derived airway cells. In the current work, we speculated that this particular putative lipoprotein affects cellular cytotoxicity and immune-stimulation in the epithelial cells. We found that mutation of this gene (ΔPA4326) results in reduced airway cell killing without affecting other common virulence factors.Moreover, we observed that this gene was able to stimulate secretion of the proinflammatory cytokine IL-8 from host cells. Interestingly, we also found that ΔPA4326 mutant strains produced less pyocyanin exotoxin compared to the wild type. Furthermore, our results suggest that PA4326 regulates expression of the pyocyanin biosynthesis gene phzM, leading to the reduced pyocyanin phenotype. Overall, these findings implicate PA4326 as a virulence factor in Pseudomonas aeruginosa. In the future, understating the molecular interplay between the epithelial cells and putative lipoproteins like PA4326 may lead to development of novel anti-inflammatory therapies that would lessen the suffering of CF patients.

Corynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to develop because they use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets. We have found that grape seed extract provides resistance to five different AB toxins: diphtheria toxin (DT), P. aeruginosa exotoxin A (ETA), ricin, Shiga toxin, and cholera toxin (CT). To identify individual compounds in grape seed extract that are capable of inhibiting the activities of these AB toxins, we screened twenty common phenolic compounds of grape seed extract for anti-toxin properties. Three compounds inhibited DT, four inhibited ETA, one inhibited ricin, and twelve inhibited CT. Additional studies were performed to determine the mechanism of inhibition against CT. Two compounds inhibited CT binding to the cell surface and even stripped bound CT off the plasma membrane of a target cell. Two other compounds inhibited the enzymatic activity of CT. We have thus identified individual toxin inhibitors from grape seed extract and some of their mechanisms of inhibition against CT. This work will help to formulate a defined mixture of phenolic compounds that could potentially be used as a therapeutic against a broad range of AB toxins.

In our pervious study, the ethanol extracts from Eupatorium fortunei Turcz at concentrations of 200 ÷ 500 mg L-1 significantly inhibited the growth of Microcystis aeruginosa, which is the most common species, responsible for toxic cyanobacteria blooming in fresh water. o-Coumaric acid (or 2-hydroxy-cinnamic acid) isolated from E. fortunei was tested its growth-inhibitory effect on M. aeruginosa and Chlorella vulgaris at the concentrations of 1.0, 10.0 and 100.0 mg L-1 in the 96- hour experiment by the optical density and the analytical method of chlorophyll a concentration. Results indicated that the compound strongly affected towards M. aeruginosa at the concentration of 100.0 mg L-1 with the inhibition efficiency (IE) values of 76.76 % and 84.66 %, respectively while those for C. vulgaris were lower just of 60.59%, and 74.53 %, respectively. The obtained data demonstrated that two methods were highly consistent and o- coumaric acid was more toxic to M. aeruginosa than C. vulgaris at all tested concentrations (p<0.05). The images of M. aeruginosa and C. vulgaris cells under the light microscope clearly showed the damage of these cells under the attck of o-coumaric acid. Although o-coumaric compound was widely demonstrated antibacterial properties in previous reports, to the best of our knowledge, our study was the first report about effect of o- coumaric acid on the growth of M. aeruginosa and C. vulgaris. / Những nghiên cứu trước đây đã chỉ ra rằng cao chiết etanol từ cây Mần tưới Eupatorium fortune Turcz tại dải nồng độ 200 ÷ 500 μg mL-1 ức chế mạnh sinh trưởng của Microcystis aeruginosa, - loài phổ biến nhất gây nên sự bùng nổ tảo độc trong hệ sinh thái nước ngọt. o-Coumaric axit (hay 2-hydroxy-cinnamic axit) phân lập từ E. fortune được tiến hành đánh giá ảnh hưởng lên sinh trưởng của hai loài M. aeruginosa và Chlorella vulgaris tại ba nồng độ là 1.0, 10.0 and 100.0 mg L-1 trong thời gian 96 giờ thực nghiệm theo phương pháp đo mật độ quang và phân tích hàm lượng chlorophyll a. Kết quả nghiên cứu cho thấy sau 96 giờ phơi nhiễm tại nồng độ 100 mg L-1 hoạt chất ức chế mạnh tới M. aeruginosa với giá trị ức chế sinh trưởng (IE) tương ứng là 76.76 và 84.66%. Giá trị IE đối với C.vulgaris ghi nhận thấp hơn chỉ là 60.65 và 74.53%, tương ứng. Hai phương pháp phân tích trên có tính nhất quán cao và o-coumaric ức chế sinh trưởng lên loài M.aeruginosa mạnh hơn so với loài C.vulgaris tại tất cả các nồng độ nghiên cứu (p<0.05). Ảnh chụp các tế bào M.aeruginosa và C.vulgaris dưới kính hiển vi điện tử đã chứng minh những tổn thương của tế bào dưới tác động của o-coumaric axit. Mặc dù o- coumaric cho thấy đặc tính chống khuẩn cao trong các công bố trước đây, nhưng theo hiểu biết của chúng tôi đây là nghiên cứu đầu tiên công bố về ảnh hưởng của hoạt chất o-coumaric axit lên sinh trưởng của M. aeruginosa và C. vulgaris.

Thesis (MScEng)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Biosurfactants are microbially produced molecules that show excellent surface-active properties. Bacillus subtilis ATCC 21332 produces the biosurfactant, surfactin, which exhibits antimicrobial activity against bacteria as well as fungi. Although antimicrobial activity has been exhibited by a number of bacterially produced biosurfactants, notably the rhamnolipid from the pathogen Pseudomonas aeruginosa, the GRAS status B. subtilis makes the use of this organism preferable for large scale bioprocesses. The objectives of this study were to: (1) evaluate the effect of different nutrient conditions on growth and surfactin production; (2) evaluate the growth of B. subtilis ATCC 21332 and associated surfactin production on a hydrocarbon substrate; (3) evaluate the antimicrobial activity of surfactin against Mycobacterium aurum, and (4) to establish whether active growth of B. subtilis ATCC 21332 and associated surfactin production can be extended during fed-batch culture. B. subtilis ATCC 21332 was grown on low-nitrate; phosphate-limited and nutrient rich media with glucose as substrate during shake flask culture. Nitrate, phosphate, glucose and surfactin were quantified by HPLC analyses and growth via CDW and optical density measurements. Growth and surfactinproduction were further evaluated during shake flask cultureon a hydrocarbon substratereplacing the glucose in the nutrient rich medium with an equivalent amount of n-hexadecane. The antimicrobial activity was quantified by growth inhibition of M. aurum. Bioreactor batch and fed-batch studies were conducted to evaluate growth and surfactin production under controlled conditions. The fed-batch experiments included four constant dilution rate (D=0.40h-1; D=0.15h-1; D=0.10h-1 and D=0.05h-1) and two constant feed rate (F=0.40L/h and F=0.125L/h) fed-batch strategies. The nutrient rich medium was used for these experiments and also as the feed medium for fed-batch experiments. A CDW of 12.6 g/L was achieved in the nutrient rich medium during shake flask culture and was 2.5- and 1.6-fold higher than that achieved in the phosphate-limited medium and the lownitrate medium respectively. A surfactin concentration of 652 mg/L was achieved in the nutrient rich medium, while a maximum surfactin concentration of 730 mg/L was achieved in the phosphate-limited medium. A surfactin concentration of only 172 mg/L was achieved in the low-nitrate medium. Subsequently, growth and surfactin production were evaluated on n-hexadecane as sole carbon source. After inoculation, the CDW did not increase over a period of 119 h, which indicated that B. subtilis ATCC 21332 was unable to utilize n-hexadecane for growth and surfactin production. The maximum CDW (27 g/L) and maximum surfactin concentration (1737 mg/L) achieved in the bioreactor batch experiments were 2.1- and 2.6-fold higher respectively than that achieved in the nutrient rich medium during shake flask experiments. These results served as a benchmark for further fed-batch experiments. During the fed-batch phase of the D=0.40h-1 experiment, the biomass further increasedby 9 g/h, which was 3.5-, 3.1- and 5.3-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Similarly, the biomass increased by 10.7 g/h during the fed-batch phase of the F=0.40L/h experiment, which was 4.6-fold higher than that of the F=0.125L/h experiment. The average rate of surfactin production was 633 mg/h during the fed-batch phase of the D=0.40h-1 experiment, 29.4-, 5.4- and 34.2-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Analogously, the average rate of surfactin production (544 mg/h) of the F=0.40L/h experiment was 9.4 fold higher than that of the F=0.125L/h experiment. The antimicrobial assay showed that surfactin inhibits M. aurum growth. An inhibition zone diamater of 4mm was measured at a surfactin concentration of 208 mg/L, which linearly increased to 24mm at a surfactin concentration of 1662 mg/L. High feed flow rate strategies achieved higher rates of biomass increase and surfactin production and will thus decrease the production time required for large scale surfactin production.The antimicrobial activity of surfactin against M. aurum indicates that this biosurfactant has the potential to be used against M. tuberculosis, and as such has the potential to be used in the medical industry to reduce the spread of this, and other deadly diseases. / AFRIKAANSE OPSOMMING: Biosurfaktante is oppervlak-aktiewe molekules wat deur sekere mikro-organismes geproduseer word. Bacillus subtilis ATCC 21332produseer ‘n biosurfaktant genaamd surfactin, wat antimikrobiese eienskappe toon teen bakterieë sowel as fungi.Menige bakterieël geproduseerde biosurfaktante toon antimikrobiese eienskappe, vernaam die rhamnolipied van die patogeen Pseudomonas aeruginosa, maar die algemene veiligheids-status van B. subtilis gee voorkeur aan hierdie organisme vir grootskaalse bioprosesse. Die doelwitte van hierdie studie was: (1) om die effek van verskillende medium samestellings (in terme van voedingstowwe) ten opsigte van bakteriële seldigtheid en surfactin-produksie te evalueer; (2) om die bakteriële seldigtheid van B. subtilis ATCC 21332 en geassosieerde surfactin produksie vanaf ‘n alkaan-substraat te evalueer; (3) om die antimikrobiese aktiwiteit van surfactin teen Mycobacterium aurum te evalueer; (4) om vas te stel of die aktiewe groei van B. subtilis ATCC 21332 en geassosieerde surfactin-produksie gedurende voer-lot kultuur verleng kan word. B. subtilis ATCC 21332 was op lae-nitraat; fosfaat-beperkte en voedingstofryk-media met glukose as substraat in skudflesse gekultiveer. Nitraat, fosfaat, glukose en surfactin was deur hoëdruk vloeistofchromatografie gekwantifiseer en die seldigtheid deur middel van seldroëmassa en optiese digtheid metings. Verder was die groei van B. subtilis, en geassosieerde surfactin produksie, vanaf ‘n alkaan-substraat in skudflesse ge-evalueer deur die glukose in die voedingstofryke medium met ‘n ekwivalente hoeveelheid van n-heksadekaan te vervang. Die antimikrobiese aktiwiteit van surfactin was deur die geїnhibeerde groei van M. aurum gekwantifiseer. Bioreaktor lot en voer-lot studies was uitgevoer om die groei en surfactin produksie onder beheerde toestande te evalueer. Die voer-lot eksperimente het vier konstante verdunningstempos (D=0.40h-1; D=0.15h-1; D=0.10h-1 en D=0.05h-1) en twee konstante voertempos (F=0.40L/h and F=0.125L/h) ingesluit. Die voedingstofryke medium was vir hierdie eksperimente en ook as die voermedium vir dievoer-lot eksperimente gebruik. ‘n Seldigtheid van 12.6 g/L is bereik gedurende skudfleskultuur in die voedingstofryk-media en was 2.5- en 1.6-voud hoër as die seldigthede wat in die fosfaat-beperkte en lae-nitraat media bereik is. ‘n Surfactin konsentrasie van 652 mg/L is bereik in die voedingstofryke medium, terwyl ‘n maksimum surfactin konsentrasie van 730 mg/L in die fosfaat-beperkte medium bereik is. ‘n Surfactin konsentrasie van slegs 172 mg/L is in die lae-nitraat medium bereik.Hierna was bakteriële seldigtheid en surfactin produksie geuvalueer met slegs n-heksadekaan as die enigste koolstof bron. Die bakteriële seldigtheid het geen verandering getoon na inokulasie nie, wat aangedui het dat B. subtilis ATCC 21332 nie die vermoë beskik om n-heksadekaan vir groei en surfactin produksie te gebruik nie. Die maksimum seldigtheid (27 g/L) en maksimum surfactin konsentrasie (1737 mg/L) bereik in die bioreaktor lot eksperimente was 2.1- en 2.6-voud hoër onderskeidelik as dit bereik in die voedingstofryke medium gedurende skudfles eksperimente. Hierdie resultate dien as ‘n basis vir verdere voer-lot eksperimente. Gedurende die voer-lot fase van die D=0.40h-1 het die biomassa verder verhoog teen 9 g/h, wat 3.5-, 3.1- en 5.3-voud hoër was as dit van die D=0.15h-1, D=0.10h-1 en D=0.05h-1 eksperimente onderskeidelik. Die biomassa het soortgelyk tydens die voer-lot fase van die F=0.40L/h eksperiment teen 10.7 g/h verhoog, wat 4.6-voud hoër was as dit van die F=0.125L/h eksperiment. Die gemiddelde tempo van surfactin produksie was 633 mg/h gedurende die voer-lot fase van die D=0.40h-1 eksperiment, 29.4-, 5.4- en 34.2-voud hoër vergeleke met die voer-lot fases van die D=0.15h-1, D=0.10h-1en D=0.05h-1 eksperimente onderskeidelik. Die gemiddelde tempo van surfactin produksie (544 mg/L) was soortgelyk 9.4-voud hoër gedurende die voer-lot fase van die F=0.40L/h eksperimente, vergeleke met die die F=0.125L/h eksperiment. Die antimikrobiese toetse van surfactin teen M. aurum het positief getoets, wat aandui dat surfactin die groei van hierdie organisme inhibeer. ‘n Inhibisie sone deursnee van 4mm was gemeet teen ‘n surfactin konsentrasie van 208 mg/L, wat lineêr verhoog het tot 24 mm teen ‘n surfactin konsentrasie van 1662 mg/L. Hoë voertempo strategieë het hoër biomassa verhogingstempos en surfactin produksie tempos getoon en sal dus die produksietyd aansienlik verkort tydens grootskaalse surfactin produksie. Die antimikrobiese aktiwiteit van surfactin teen M. aurum toon dat hierdie biosurfaktant die vermoë het om gebruik te word teen M. tuberculosis. Daarom het surfactin die potensiaal om gebruik te word in die mediese industrie om die verspreiding van Tuberkulose, en ander dodelike patogene, te voorkom.

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>.