Pseudomonas aeruginosa (PA) est une bactérie Gram négatif, pathogène opportuniste, impliquée dans un grand nombre d’infections nosocomiales. Cette bactérie est aussi le principal micro-organisme responsable des surinfections broncho-pulmonaires chez les patients atteints de mucoviscidose. Cette prééminence est due en partie à la capacité de PA à former des biofilms, ce qui lui confèrent une résistance exceptionnelle aux antimicrobiens. Au sein de notre laboratoire, une analyse protéomique différentielle a permis de démontrer, en 2004, l’existence d’un protéome spécifique lorsque la bactérie se développe en mode biofilm. Parmi les protéines spécifiquement exprimées en mode biofilm, la protéine hypothétique PA3731 a été plus particulièrement étudiée. Cette protéine est impliquée dans la formation de biofilm, la production de rhamnolipides, la résistance à la tobramycine et la mobilité de type « swarming ». Des recherches bioinformatiques ont montré que le gène pA3731 appartient à un cluster de 4 gènes allant de pA3729 à pA3732 (système BAC), qui pourraient être impliqués dans l’élaboration et/ou la régulation d’un même système protéique. Cette hypothèse a constitué le point de départ de ce travail de thèse. La présente étude a permis de confirmer l’implication du système BAC dans la formation du biofilm, la résistance aux antibiotiques et la production de rhamnolipides chez PA. Les études protéomiques ont mis en évidence l’implication de ce système dans l’expression de la pompe MexEF-OprN, de la porine OprD, et dans la régulation du Quorum Sensing. Des études intéractomiques, menées en parallèle, ont montré une forte interaction entre la protéine PA3731 et PA3732. Ces études ont également permis de valider une forte interaction entre ces protéines et les rhamnolipides. L’ensemble de ces résultats nous permettent d’avancer une hypothèse quant à l’implication du système BAC dans le transport des rhamnolipides vers le milieu extracellulaire. / Pseudomonas aeruginosa (PA) is a Gram-negative bacterium, opportunistic pathogen, involved in a large number of nosocomial infections. This microorganism is also the main infectious agent involved in bronchopulmonary infections in cystic fibrosis patients. This pre-eminence is partly due to the ability of PA to form biofilms, which confers to the bacterial cells an increased resistance to antibiotics. In our laboratory, a differential proteomic analysis allowed to demonstrate in 2004, the existence of a specific proteome when the bacterium grows in the biofilm mode. This study allowed identifying about 40 proteins, specifically accumulated when bacteria adhere to a surface. Among these proteins, the hypothetical protein PA3731 has been particularly investigated. This protein is involved in the biofilm formation, the rhamnolipids production, the resistance to tobramycin and the swarming mobility. Bioinformatic research showed that the pA3731 gene belongs to a cluster of 4 genes ranging from pA3729 to pA3732 (BAC system), which could be involved in the development and / or regulation of the same protein system. This hypothesis was the starting point of this thesis work. The present study confirmed the involvement of the BAC system in the biofilm formation, the antibiotic resistance and the rhamnolipid production in PA. Proteomic studies highlighted the implication of this system in the expression of the MexEF-OprN pump and that of the OprD porin, and in the regulation of Quorum Sensing. Interactomic investigations, conducted in parallel, showed a strong interaction between PA3731 and PA3732 proteins. These studies have also pointed out a strong interaction between these proteins and rhamnolipids. All these results suggest that the BAC system could play a major role in the transfer of rhamnolipids to the extracellular environment.

Les infections en biofilm, notamment de dispositifs médicaux, mettent fréquemment en échec les traitements antibiotiques, imposant le retrait du matériel. Pseudomonas aeruginosa s’est imposé comme le pathogène-type des infections en biofilm. Pour explorer les déterminants de l’échec du traitement antibiotique en biofilm, un modèle de biofilm in vitro à P. aeruginosa exposé à des doses supra-inhibitrices d’antibiotiques a été développé. En culture planctonique, une bithérapie deciprofloxacine et d’amikacine permettait de prévenir la sélection de mutants résistants pour des souches de P. aeruginosa de sensibilité diminuée à la ciprofloxacine ou à l’amikacine par surexpression d’efflux. En biofilm, l’association de la ciprofloxacine et de l’amikacine, administrées simultanément ou séquentiellement, n’était pas supérieure aux monothérapies, permettant une réduction bactérienne, mais pas d’éradication complète du biofilm. Quelles que soient les souches (sauvages ou exprimant un efflux) et l’antibiotique, l’échec microbiologique en biofilm était lié à la sélection de cellules persistantes, tolérantes aux antibiotiques. La ciprofloxacine induisait des modifications importantes de la structure du biofilm avec une réduction considérable des exopolysaccharides, composants majeurs de la matrice. L’étude transcriptomique de gènes potentiellement impliqués dans la persistance suggérait que l’activation précoce de la réponse stringente pourrait être une des voies principales de la tolérance en biofilm sous ciprofloxacine. Enfin, la présence de « small colony variants » au sein du biofilm, dotés d’une capacité accrue de formation de biofilm, témoignait de la diversité des populations en biofilm. Ces travaux participent ainsi à une meilleure compréhension des mécanismes d’échappement aux antibiotiques de P. aeruginosa en biofilm. / Biofilm device-related infections can lead to antibiotic failure requiring frequent removal of medical device. Pseudomonas aeruginosa has emerged as the typical pathogen for biofilm infections. To explore the determinants of antibiotic failure in biofilm, an in vitro P. aeruginosa biofilm model exposed to suprainhibitory antibiotic concentrations was developed. In planktonic culture, the ciprofloxacin and amikacin combination prevented the selection of resistant mutants in ciprofloxacin and amikacinlow-level resistant P. aeruginosa strains overexpressing efflux. In biofilm, the ciprofloxacin and amikacin combination, used simultaneously or sequentially, didn’t show superior effects compared to monotherapies. Despite an initial bacterial reduction, biofilm eradication was not obtained. Regardless of wild-type or efflux strains and antibiotic regimen used, antibiotic failure was related to the selection of antibiotic-tolerant cells named “persisters”. Ciprofloxacin induced significant alterations in the biofilm structure, notably a considerable reduction in the exopolysaccharides of the matrix. The transcriptomic analysis of genes, potentially involved in persistence, suggested that early activation of the stringent response might be one of the main pathways for ciprofloxacin tolerance in biofilm. Finally, the emergence of "small colony variants" within the biofilm, characterized by enhanced ability to form biofilm, attested to biofilm heterogeneity. This work therefore contributes to a better understanding of how P. aeruginosa biofilms escape antibiotic.

Les voies respiratoires des patients atteints de mucoviscidose sont colonisées par de nombreux pathogènes, parmi lesquels la bactérie P. aeruginosa est prédominante. Après des épisodes répétés d’infections du tractus respiratoire principalement liées à P. aeruginosa, les patients développent une insuffisance pulmonaire associée à un déclin du statut clinique et à une aggravation du pronostic puisqu’elle est souvent responsable du décès de ces patients. Les infections chroniques à P. aeruginosa affectent 80 à 90 % des patients atteints de mucoviscidose et, une fois ces infections installées, les associations actuelles d’antibiotiques sont incapables d’éradiquer la bactérie des voies aériennes de ces patients. La chronicité des infections est liée au développement de la bactérie sous un mode de vie particulier, le biofilm. Les bactéries s’assemblent en communautés complexes et organisées, entourées par la sécrétion d’une matrice extracellulaire polymérique. Ce mode de vie procure aux bactéries présentes dans le biofilm un environnement dense et protecteur, augmentant la résistance du pathogène au système immunitaire de l’hôte et aux antibiotiques conventionnels. <p><p>Dans la première partie de notre travail, nous avons caractérisé différentes souches de P. aeruginosa, comprenant des souches de référence et des souches cliniques isolées des expectorations de patients atteints de mucoviscidose. Les propriétés d’adhésion, de développement des biofilms, de mobilité, de production de rhamnolipides, l’activité protéolytique et la production d’acylhomosérine lactones se sont avérées très différentes au sein des souches. De plus, les caractéristiques phénotypiques des souches ne constituaient pas une valeur prédictive de la sensibilité des bactéries à un antibiotique, soulignant la nécessité d’étudier un panel large de souches pour caractériser l’effet d’un agent antimicrobien.<p><p>Dans la lutte pour combattre les infections et l’apparition de souches multirésistantes, de nouvelles stratégies thérapeutiques sont développées. Les céragenines sont une famille de molécules synthétisées dans le but de mimer la structure amphipatique des peptides antimicrobiens responsable de leur activité bactéricide importante. Contrairement à ces derniers, les céragenines maintiennent leur activité dans des conditions physiologiques. <p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p>Dans la deuxième partie de ce travail, nous avons étudié l’effet d’un composé antimicrobien appartenant à la famille des céragenines, le CSA-13, sur les différentes souches de P. aeruginosa. Nous avons confirmé le potentiel bactéricide du CSA-13 sur des cultures planctoniques de P. aeruginosa. Nous avons démontré qu’une concentration très faible et non cytotoxique de CSA-13 (10 fois inférieure à la CMI), inhibait la formation d’un biofilm de 3 souches de P. aeruginosa sur les 8 testées. L’étude du potentiel zêta des souches nous a permis de proposer un mécanisme basé sur des interactions électrostatiques pour expliquer l’action préventive du CSA-13 sur le développement du biofilm. Une concentration plus importante de CSA-13 a éradiqué l’entièreté d’un biofilm âgé de 24 h pour 7 des 8 souches étudiées. Six souches ont été évaluées dans un biofilm mature et toutes ont répondu au composé avec des concentrations croissantes ou un temps d’exposition du composé au biofilm plus important. Aucune résistance au CSA-13 n’est apparue durant le traitement. L’usage de la microscopie confocale à balayage laser a confirmé la rapidité et l’efficacité d’action du CSA-13 sur un biofilm robuste et complexe de P. aeruginosa par visualisation dans le temps et dans l’espace de l’effet du CSA-13 sur le biofilm. L’ensemble des observations de ce travail nous a permis de conclure que 7 sur les 8 souches de P. aeruginosa étaient sensibles au CSA-13, soit à un stade initial de la formation du biofilm, soit après maturation du biofilm. Ces résultats soulignent le potentiel thérapeutique important, envers tous les stades de formation et de développement du biofilm, de composés à structure amphiphile comme le CSA-13, avec une face cationique favorisant les interactions avec les membranes bactériennes chargées négativement et une face hydrophobe contribuant à la perturbation de ces membranes. <p><p>Le traitement de référence actuel envers les infections à P. aeruginosa, chez les patients souffrant de mucoviscidose, consiste en l’administration par inhalation de tobramycine commercialisée sous le médicament TOBI®. Nous avons investigué l’intérêt d’une administration combinée de l’aminoglycoside avec le CSA-13. Un bénéfice évident de la combinaison de CSA-13 et de tobramycine est apparu dans cette étude aussi bien sur biofilm jeune que mature. Dans certaines conditions, le CSA-13 semblait même prévenir la résistance à la tobramycine. Il sera cependant indispensable de concevoir des expériences in vivo pour confirmer l’intérêt du CSA-13 ou d’une co-administration de CSA-13 et de la tobramycine dans le traitement d’infections chroniques à P. aeruginosa chez des patients atteints de mucoviscidose.<p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p><p>Nos études in vitro sur cellules eucaryotes humaines ont mis en évidence une toxicité membranaire et mitochondriale provoquée par le CSA-13 lors de l’administration de concentrations importantes. L’association du CSA-13 avec l’acide pluronique F-127 a permis de réduire significativement la toxicité du composé sur les membranes. Cependant, l’association n’a pas diminué les effets délétères exercés par le CSA-13 sur l’activité mitochondriale. Les études devront donc se poursuivre afin d’affiner la compréhension du mécanisme d’action des céragenines et de pouvoir déceler des dérivés moins toxiques. L’évaluation de l’activité in vivo du composé devrait nous éclairer quant à la fenêtre thérapeutique utilisable en clinique.<p><p>\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Es gibt keine bekannte Lungenerkrankung, die eine so frühe, chronische und intensive Entzündungsreaktion hervorruft, wie sie in den Atemwegen von Patienten mit Mukoviszidose (CF) auftritt. CF ist die häufigste tödliche autosomal-rezessiv vererbte Krankheit in der kaukasischen Bevölkerung, die durch eine Mutation im CFTR-Gen (Cystic Fibrosis Transmembrane Conductance Regulator) verursacht wird, dass für das CFTR-Protein kodiert. Defekte in diesem Protein führen zu einer epithelialen Dysfunktion und betreffen mehrere Organe, aber die Lungenpathologie ist für über 85% der Morbidität und Mortalität bei CF verantwortlich. Die CF-Lungenpathologie konzentriert sich auf die Wechselwirkungen zwischen Wirt und Erreger, wobei die CFTR-Dysfunktion Infektionen begünstigt und die Infektionen in Verbindung mit einer dysfunktionalen Immunantwort einen anhaltenden Entzündungskreislauf in Gang setzen. Dieser Teufelskreis aus Infektion und Entzündung führt schließlich zu Lungenschäden, Atemversagen und letztlich zum Tod. Die vorherrschende Infektion bei Mukoviszidose ist die durch P. aeruginosa, wobei im europäischen Durchschnitt 41% der erwachsenen Patienten infiziert sind. Bemerkenswert ist, dass das Mikrobiom der Atemwege bei Mukoviszidose polymikrobieller Natur ist. Da frühere Studien einen positiven Zusammenhang zwischen einer hohen Mikrobiom-Diversität und einer verbesserten Lungenfunktion bei Mukoviszidose festgestellt haben, wurde die Hypothese aufgestellt, dass bestimmte Kommensalen vor einer Infektion mit P. aeruginosa in den CF-Atemwegen schützen könnten. Um dies genauer zu untersuchen wurden 105 kommensale Isolate aus 32 verschiedenen Arten von Sputumproben von Patienten mit Mukoviszidose isoliert und mit einem fluoreszierenden P. aeruginosa PA01-mcherry-Stamm auf antagonistische Wirkungen bei direkten Erreger-Kommensalen-Interaktionen untersucht. Diese Isolate wurden zusätzlich auf immunmodulatorische Effekte bei Kommensal-Wirt-Pathogen-Interaktionen, unter Verwendung von BEAS-2B-Bronchialepithelzelllinien, untersucht. Ausgewählte Isolate mit schützender Wirkung wurden anschließend auf immunmodulatorische Effekte unter Verwendung von CFBE41o ΔF508-Zellen und einem natürlicheren Lungen-Präzisionsschnitt-Modell (PCLS) untersucht und die produzierten Zytokine mittels ELISA sowie mit einem Multiplex-Zytokin-Assay gemessen. Genexpressionsanalysen wurden zudem mittels qRT-PCR durchgeführt. Die zugrundeliegenden Mechanismen wurden mittels transkriptomischer Analysen, Vergleiche der gesamten Genomsequenz (WGS) und mechanischer Studien einschließlich Stoffwechselanalysen mittels Hochleistungsflüssigkeitschromatographie untersucht. Es konnte gezeigt werden, dass ausgewählte Streptokokken-Kommensal-Isolate, insbesondere Vertreter von S. mitis, S. oralis, S. cristatus, S. gordonii, S. sanguinis und S. parasanguinis, starke antagonistische Effekte auf das Wachstum von P. aeruginosa in direkten Kokulturen haben. Ausgewählte Vertreter von S. mitis, S. oralis und S. cristatus verhinderten zudem das Wachstum anderer klinischer und nicht-klinischer Isolate von P. aeruginosa, sowie anderer typischer Mukoviszidose-Erreger wie Staphylococcus aureus, Burkholderia spp., Achromobacter xylosoxidans, Proteus mirabilis, Haemophilus influenzae, Stenotrophomonas maltophilia, Enterococcus faecalis und Klebsiella pneumoniae. Eine wirksame Mukoviszidose-Therapie sollte nicht nur die Infektion, sondern auch die damit einhergehende bösartige Entzündung bekämpfen. Im Gegensatz zu den Mitgliedern der gram-negativen Neisseria spp., die die IL-8-Produktion bei einer Monoinfektion signifikant stimulierten, taten dies alle gram-positiven Kommensalen-Isolate nicht. Ausgewählte Kommensalen regulierten auch die P. aeruginosa PA01- und LPS-induzierte Produktion mehrerer entzündlicher Zytokine in menschlichen Atemwegsepithelzellen (BEAS-2B sowie CFBE41o ΔF508 und korrigierte wtCFTR) und in PCLS der Maus. Diese Ergebnisse wurden auch durch Genexpressionsanalysen bestätigt, was darauf hindeutet, dass die Immunmodulation möglicherweise durch eine veränderte TLR-Signalübertragung vermittelt wird. Transkriptomische Analysen nach Koinfektion von S. mitis Isolat 4 (SM4) und PA01 auf PCLS zeigten eine signifikante Runterregulation von Entzündungsreaktionen wie mTOR und Toll-like-Rezeptor-Signalen. Ein WGS-Vergleich zeigte, dass mehr als die Hälfte der am stärksten angereicherten Genfunktionen bei hemmenden Streptokokken-Isolaten für den Kohlenhydrat-Transport und -Stoffwechsel verantwortlich waren, während sie bei den nicht hemmenden Streptokokken-Isolaten unter den am stärksten angereicherten Genfunktionen fehlten. Mechanische Untersuchungen zeigten, dass der glykolytische Signalweg für die antipseudomonische Wirkung entscheidend ist und dass hemmende Kommensalen hemmende Wirkungen vermitteln, indem sie den pH-Wert ihrer Wachstumsmedien < 5,0 senken und Acetat > 0,2 mg/ml produzieren. Es wurde nachgewiesen, dass Acetat signifikante immunmodulatorische Effekte gegen PA01- und LPS-induzierte Entzündungsreaktionen in BEAS-2B und PCLS vermittelt. Zusammenfassend lässt sich sagen, dass ausgewählte kommensale Bakterien Schutzwirkungen in den Atemwegen von Mukoviszidose-Patienten herbeiführen, indem sie Acetat produzieren, das antipseudomonale und immunmodulatorische Wirkungen vermittelt. Einserseits direkt, indem es durch Bakterien- und Wirtszellen diffundiert und so unmittelbare Auswirkungen hat, als auch indirekt, indem es Wirtszellen dazu anregt, Bakterien effizient zu beseitigen und Entzündungen zu kontrollieren. Da die Verwendung ganzer Bakterien als Probiotika bei immungeschwächten Patienten beispielsweise bei Mukoviszidose mit einigen Herausforderungen verbunden ist, stellt die Verwendung von bakteriellen Metaboliten wie Acetat eine sicherere, einfachere und praktischere Alternative dar.:List of Abbreviations (i) Table of Contents (iv) 1. SUMMARY (1) 1.1 Zusammenfassung (1) 1.2 ABSTRACT (3) 2. INTRODUCTION (5) 2.1 Cystic fibrosis (5) 2.2 Development of the CF lung pathology (6) 2.3 The immune response (8) 2.3.1 Innate and adaptive immunity (8) 2.3.2 Toll-like receptors (TLRs) (9) 2.4 Inflammation in CF (11) 2.4.1 Neutrophils in CF (11) 2.4.2 Macrophages in CF (12) 2.4.3 Eicosanoid metabolites in CF (12) 2.4.4 Chemokines in CF (12) 2.5 Airway sampling for microbiome studies (13) 2.6 CF airway microbiome (14) 2.6.1 The healthy lung microbiome (14) 2.6.2 Pathogenic bacterial members of the CF microbiome and pulmonary exacerbations (15) 2.6.3 Pseudomonas aeruginosa in CF (16) 2.6.4 Anaerobic CF microbiota (17) 2.6.5 Fungal CF microbiota (17) 2.6.6 Virus CF microbiota (18) 2.6.7 Commensal-pathogen interactions in CF (18) 2.7 CFTR modulators (18) 2.8 Human epithelial cell lines and murine precision-cut lung slices (PCLS) as in vitro model systems (19) 2.9 Next-generation sequencing (NGS) in CF microbiome studies (20) 2.10 Objectives of this study (21) 3. MATERIALS AND METHODS (23) 3.1 Materials (23) 3.1.1 Devices and Instruments (23) 3.1.2 Software (24) 3.1.3 Consumables (25) 3.1.4 Chemicals, Reagents, Media, and Antibiotics (26) 3.1.5 Kits (29) 3.1.6 Buffers, Media, and Solutions (30) 3.1.7 qPCR Primers (32) 3.1.8 Cell lines (33) 3.1.9 Mouse strains (33) 3.1.10 Bacteria isolates (34) 3.2 Methods (37) 3.2.1 Isolation, identification, and storage of isolates (37) 3.2.2 Pathogens-Commensals direct cocultures (38) 3.2.3 HPLC of conditioned media from bacterial isolates (40) 3.2.4 Cell-Pathogen-Commensal cocultures (41) 3.2.5 PCLS cocultures (42) 3.2.6 RNA extraction, cDNA preparation, and quantitative RT-PCR (43) 3.2.7 RNA Sequencing and Transcriptome analysis (45) 3.2.8 Bacteria DNA extraction and Whole Genome Sequencing (46) 3.2.9 Biochemistry (47) 3.2.10 Statistical analyses (49) 4. RESULTS (50) 4.1 Analysis of direct commensal-pathogen interactions (50) 4.1.1 Several streptococcal isolates inhibit the growth of P. aeruginosa with inter- and intra-species variability in the antipseudomonal effect (51) 4.1.2 Further commensal isolates that do not inhibit the growth of P. aeruginosa (54) 4.1.3 The lack of antipseudomonal effect by noninhibitory isolates is not due to insufficient cell numbers (54) 4.1.4 Fungal CF isolates in this study do not possess antipseudomonal effects (56) 4.1.5 SCAPEs (Selected Commensals with strong Anti-Pseudomonal Effects) also inhibit other P. aeruginosa strains (58) 4.1.6 SCAPEs inhibit other non-pseudomonal pathogenic CF isolates (60) 4.1.7 Inhibitory effects mediated by SCAPEs do not extend to the fungal CF isolates in this study (63) 4.2 Analysis of commensal-host-pathogen interactions using human bronchial epithelial cell lines (63) 4.2.1 Some commensal isolates are able to modulate PA01-induced IL-8 release in BEAS-2B cells (64) 4.2.2 Commensal-mediated IL-8 modulation in BEAS-2B cells is not due to PA01 growth inhibition (67) 4.2.3 Selected commensal isolates also modulate LPS-induced IL-8 release in BEAS-2B cells (68) 4.2.4 Selected S. mitis isolates also modulate IL-8 release in BEAS-2B cells induced by other CF P. aeruginosa isolates (68) 4.2.5 Selected commensal isolates modulate PA01-induced IL-8 release in CFBE41o cells (70) 4.2.6 Protective commensals need to be metabolically active to exert immunomodulatory effects (72) 4.2.7 Hydrogen peroxide produced by peroxide-producing Streptococcus spp. affects the viability of human bronchial epithelial cells (72) 4.2.8 Selected peroxide-producing Streptococcus spp. possess immunomodulatory activity when peroxide-induced cell death is prevented (75) 4.3 Analysis of commensal-host-pathogen interactions using mouse PCLS (80) 4.3.1 PCLS is more resilient against peroxide-induced loss of viability (80) 4.3.2 Selected S. mitis isolates modulate PA01-induced inflammatory response in mouse PCLS (82) 4.3.3 Immunomodulation of PA01-induced response by SM4 in PCLS is not due to active PA01 growth inhibition (84) 4.4 Analysis of the underlying mechanisms behind the streptococcal-mediated effects via transcriptome and whole genome sequencing (84) 4.4.1 Transcriptomic analyses show that SM4 downregulates signalling pathways involved in PA01-induced inflammatory responses in mouse PCLS (84) 4.4.2 Whole genome sequence comparison shows that in inhibitory commensals, most of their genes are involved in carbohydrate transport and metabolism (87) 4.5 Uncovering the mechanisms behind the observed streptococcal-mediated antipseudomonal effects (89) 4.5.1 Conditioned medium (CM) from SCAPEs inhibits the growth of P. aeruginosa and other typical CF pathogens (89) 4.5.2 Inhibitory activity of SCAPEs CM is neither heat sensitive nor proteinaceous (91) 4.5.3 Iron competition and the arginolytic pathway are not responsible for the observed inhibitory effects (91) 4.5.4 Peroxide production may contribute but does not play a major role in the antipseudomonal effects (94) 4.5.5 Several members of Streptococcus spp. mediate antipseudomonal effects via the glycolytic pathway (94) 4.5.6 Low pH plays a major role in the observed inhibition (97) 4.5.7 SCAPEs and other selected commensal isolates can mediate antipseudomonal effects by simultaneously lowering the pH and secreting acetate (98) 4.5.8 Extracellular addition of 0.5 mg/ml acetate at pH 5.0 inhibits the growth of P. aeruginosa (100) 4.5.9 Other SCFAs like propionate and butyrate at pH 5.0 also inhibit P. aeruginosa isolates (102) 4.5.10 Acetate has better antipseudomonal activity than propionate and butyrate (103) 4.6 Commensals may mediate their protective effects via acetate production (104) 4.6.1 SCFAs modulate PA01- and LPS-induced IL-8 release in BEAS-2B cells (104) 4.6.2 SCFA levels used are well below cell toxicity levels (105) 4.6.3 Acetate modulates PA01 and LPS-induced immune response in mouse PCLS (107) 5. DISCUSSION (110) 5.1 SCAPEs mediate inhibitory effects in direct commensal-pathogen interactions against P. aeruginosa and other typical CF pathogens (110) 5.1.1 Members of Streptococcus spp. mediate inter- and intra-species variability in their antipseudomonal effects (111) 5.1.2 SCAPEs inhibit other clinical and nonclinical P. aeruginosa strains as well as other typical CF pathogens (113) 5.2 Selected commensals modulate PA01- and LPS-induced inflammatory response in human airway epithelial cells and mouse PCLS (115) 5.2.1 The gram-positive commensal isolates in this study do not significantly stimulate inflammatory response in human bronchial epithelial cells and mouse PCLS (115) 5.2.2 Selected gram-positive commensal isolates modulate P. aeruginosa-triggered inflammatory response in BEAS-2B cells with inter- and intra-species variation (117) 5.2.3 Selected commensal isolates modulate P. aeruginosa-triggered inflammatory response in CFBE41o ΔF508 (120) 5.2.4 Selected S. mitis isolates modulate P. aeruginosa-induced inflammatory response in mouse PCLS (121) 5.3 Commensals exert protective effects against P. aeruginosa infection via acetate production (124) 5.3.1 Conditioned medium (CM) from selected commensal isolates need to be acidic to mediate inhibition of growth of P. aeruginosa and other typical CF pathogens (125) 5.3.2 The glycolytic pathway is important for streptococcal-mediated antipseudomonal effects (127) 5.3.3 Commensal bacteria mediate growth inhibitory effects by simultaneously lowering the pH and producing acetate (128) 5.3.4 Acetate modulates PA01- and LPS-induced inflammation in bronchial epithelial cells and PCLS (131) 5.4 Conclusions and Outlook (134) 6. DECLARATIONS (158) 6.1 Statement of Authorship (158) 6.2 Declaration of compliance (160) 7. Acknowledgements (161) / There is no known lung disease that causes such a very early, chronic, and intense inflammatory reaction as seen in the airways of patients with cystic fibrosis (CF). CF is the most common lethal autosomal recessive genetic condition in the Caucasian population caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding for the CFTR protein. Defects in this protein result into epithelial dysfunction and affect several organs but lung pathology accounts for over 85% of CF morbidity and mortality. The CF lung pathology centers on the host-pathogen interactions where CFTR dysfunction predisposes to infections and the infections coupled with a dysfunctional immune response drive a sustained inflammatory cycle. This vicious cycle of infection and inflammation ultimately results in lung damage, respiratory failure and then, death. The most predominant infection in CF is by P. aeruginosa with an overall European average of 41.0% of adult patients infected. Of note, airway microbiome in CF is of polymicrobial nature. Given that previous studies have established positive correlations between a high microbiome diversity and improved lung function in CF, it was hypothesized that certain commensals may be protective against P. aeruginosa infection in the CF airways. Therefore, 105 commensal isolates from 32 different species were isolated from sputum samples of patients with CF and screened for antagonistic effects in direct pathogen-commensal interactions using a fluorescent P. aeruginosa PA01-mcherry strain. These isolates were also screened for immunomodulatory effects in commensal-host-pathogen interactions using BEAS-2B bronchial epithelial cell lines. Selected isolates with protective effects were subsequently screened for immunomodulatory effects using CFBE41o ΔF508 cells and a more natural precision-cut-lung-slices (PCLS) model and the produced cytokines were measured via ELISA as well as via a multiplex cytokine assay. Gene expression analyses were also conducted via qRT-PCR. Underlying mechanisms were explored via transcriptomic analyses, whole genome sequence (WGS) comparisons, and mechanistic studies including metabolic analyses via high-performance liquid chromatography. It was demonstrated that selected streptococcal commensal isolates, especially members belonging to S. mitis, S. oralis, S. cristatus, S. gordonii, S. sanguinis, and S. parasanguinis, mediate potent antagonistic effects against the growth of P. aeruginosa in direct cocultures. Selected members from S. mitis, S. oralis, and S. cristatus also prevented the growth of other P. aeruginosa clinical and nonclinical isolates as well as other typical CF pathogens including Staphylococcus aureus, Burkholderia spp., Achromobacter xylosoxidans, Proteus mirabilis, Haemophilus influenzae, Stenotrophomonas maltophilia, Enterococcus faecalis, and Klebsiella pneumoniae. An effective CF therapy should not only address infection but the accompanying vicious inflammation as well. Unlike the members of the gram-negative Neisseria spp. which significantly stimulated IL-8 production in mono-infection, all the gram-positive commensal isolates did not. Selected commensals also modulated P. aeruginosa PA01- and LPS-induced production of several inflammatory cytokines in human airway epithelial cells (BEAS-2B as well as CFBE41o ΔF508 and corrected wtCFTR) and in mouse PCLS. These findings were also confirmed via gene expression analyses indicating that the immunomodulation may be mediated by altered TLR signalling. Transcriptomic analyses after co-infection of S. mitis isolate 4 (SM4) and PA01 on PCLS revealed a significant downregulation of inflammatory responses such as mTOR and toll-like receptor signalling. WGS comparison showed that over half of the most enriched gene functions in inhibitory streptococcal isolates were responsible for carbohydrate transport and metabolism but were absent among the most enriched gene functions for the noninhibitory streptococcal isolates. Mechanistic investigations demonstrated that the glycolytic pathway was essential for antipseudomonal effects and that inhibitory commensals mediate inhibitory effects by lowering the pH of their growth media < 5.0 and producing acetate > 0.2 mg/ml. Acetate was demonstrated to mediate significant immunomodulatory effects against PA01- and LPS-induced inflammatory response in BEAS-2B and PCLS. In conclusion, selected commensal bacteria induce protective effects in the CF airway by producing acetate, which mediates antipseudomonal and immmunomodulatory activities both directly by diffusing across bacterial and host cells to mediate direct effects as well as indirectly by stimulating host cells to clear bacteria efficiently and control inflammation. Given that the use of whole bacteria as probiotics in immunocompromised patients like in CF possesses several challenges, the use of bacterial metabolites like acetate presents a safer, easier, and more practical alternative.:List of Abbreviations (i) Table of Contents (iv) 1. SUMMARY (1) 1.1 Zusammenfassung (1) 1.2 ABSTRACT (3) 2. INTRODUCTION (5) 2.1 Cystic fibrosis (5) 2.2 Development of the CF lung pathology (6) 2.3 The immune response (8) 2.3.1 Innate and adaptive immunity (8) 2.3.2 Toll-like receptors (TLRs) (9) 2.4 Inflammation in CF (11) 2.4.1 Neutrophils in CF (11) 2.4.2 Macrophages in CF (12) 2.4.3 Eicosanoid metabolites in CF (12) 2.4.4 Chemokines in CF (12) 2.5 Airway sampling for microbiome studies (13) 2.6 CF airway microbiome (14) 2.6.1 The healthy lung microbiome (14) 2.6.2 Pathogenic bacterial members of the CF microbiome and pulmonary exacerbations (15) 2.6.3 Pseudomonas aeruginosa in CF (16) 2.6.4 Anaerobic CF microbiota (17) 2.6.5 Fungal CF microbiota (17) 2.6.6 Virus CF microbiota (18) 2.6.7 Commensal-pathogen interactions in CF (18) 2.7 CFTR modulators (18) 2.8 Human epithelial cell lines and murine precision-cut lung slices (PCLS) as in vitro model systems (19) 2.9 Next-generation sequencing (NGS) in CF microbiome studies (20) 2.10 Objectives of this study (21) 3. MATERIALS AND METHODS (23) 3.1 Materials (23) 3.1.1 Devices and Instruments (23) 3.1.2 Software (24) 3.1.3 Consumables (25) 3.1.4 Chemicals, Reagents, Media, and Antibiotics (26) 3.1.5 Kits (29) 3.1.6 Buffers, Media, and Solutions (30) 3.1.7 qPCR Primers (32) 3.1.8 Cell lines (33) 3.1.9 Mouse strains (33) 3.1.10 Bacteria isolates (34) 3.2 Methods (37) 3.2.1 Isolation, identification, and storage of isolates (37) 3.2.2 Pathogens-Commensals direct cocultures (38) 3.2.3 HPLC of conditioned media from bacterial isolates (40) 3.2.4 Cell-Pathogen-Commensal cocultures (41) 3.2.5 PCLS cocultures (42) 3.2.6 RNA extraction, cDNA preparation, and quantitative RT-PCR (43) 3.2.7 RNA Sequencing and Transcriptome analysis (45) 3.2.8 Bacteria DNA extraction and Whole Genome Sequencing (46) 3.2.9 Biochemistry (47) 3.2.10 Statistical analyses (49) 4. RESULTS (50) 4.1 Analysis of direct commensal-pathogen interactions (50) 4.1.1 Several streptococcal isolates inhibit the growth of P. aeruginosa with inter- and intra-species variability in the antipseudomonal effect (51) 4.1.2 Further commensal isolates that do not inhibit the growth of P. aeruginosa (54) 4.1.3 The lack of antipseudomonal effect by noninhibitory isolates is not due to insufficient cell numbers (54) 4.1.4 Fungal CF isolates in this study do not possess antipseudomonal effects (56) 4.1.5 SCAPEs (Selected Commensals with strong Anti-Pseudomonal Effects) also inhibit other P. aeruginosa strains (58) 4.1.6 SCAPEs inhibit other non-pseudomonal pathogenic CF isolates (60) 4.1.7 Inhibitory effects mediated by SCAPEs do not extend to the fungal CF isolates in this study (63) 4.2 Analysis of commensal-host-pathogen interactions using human bronchial epithelial cell lines (63) 4.2.1 Some commensal isolates are able to modulate PA01-induced IL-8 release in BEAS-2B cells (64) 4.2.2 Commensal-mediated IL-8 modulation in BEAS-2B cells is not due to PA01 growth inhibition (67) 4.2.3 Selected commensal isolates also modulate LPS-induced IL-8 release in BEAS-2B cells (68) 4.2.4 Selected S. mitis isolates also modulate IL-8 release in BEAS-2B cells induced by other CF P. aeruginosa isolates (68) 4.2.5 Selected commensal isolates modulate PA01-induced IL-8 release in CFBE41o cells (70) 4.2.6 Protective commensals need to be metabolically active to exert immunomodulatory effects (72) 4.2.7 Hydrogen peroxide produced by peroxide-producing Streptococcus spp. affects the viability of human bronchial epithelial cells (72) 4.2.8 Selected peroxide-producing Streptococcus spp. possess immunomodulatory activity when peroxide-induced cell death is prevented (75) 4.3 Analysis of commensal-host-pathogen interactions using mouse PCLS (80) 4.3.1 PCLS is more resilient against peroxide-induced loss of viability (80) 4.3.2 Selected S. mitis isolates modulate PA01-induced inflammatory response in mouse PCLS (82) 4.3.3 Immunomodulation of PA01-induced response by SM4 in PCLS is not due to active PA01 growth inhibition (84) 4.4 Analysis of the underlying mechanisms behind the streptococcal-mediated effects via transcriptome and whole genome sequencing (84) 4.4.1 Transcriptomic analyses show that SM4 downregulates signalling pathways involved in PA01-induced inflammatory responses in mouse PCLS (84) 4.4.2 Whole genome sequence comparison shows that in inhibitory commensals, most of their genes are involved in carbohydrate transport and metabolism (87) 4.5 Uncovering the mechanisms behind the observed streptococcal-mediated antipseudomonal effects (89) 4.5.1 Conditioned medium (CM) from SCAPEs inhibits the growth of P. aeruginosa and other typical CF pathogens (89) 4.5.2 Inhibitory activity of SCAPEs CM is neither heat sensitive nor proteinaceous (91) 4.5.3 Iron competition and the arginolytic pathway are not responsible for the observed inhibitory effects (91) 4.5.4 Peroxide production may contribute but does not play a major role in the antipseudomonal effects (94) 4.5.5 Several members of Streptococcus spp. mediate antipseudomonal effects via the glycolytic pathway (94) 4.5.6 Low pH plays a major role in the observed inhibition (97) 4.5.7 SCAPEs and other selected commensal isolates can mediate antipseudomonal effects by simultaneously lowering the pH and secreting acetate (98) 4.5.8 Extracellular addition of 0.5 mg/ml acetate at pH 5.0 inhibits the growth of P. aeruginosa (100) 4.5.9 Other SCFAs like propionate and butyrate at pH 5.0 also inhibit P. aeruginosa isolates (102) 4.5.10 Acetate has better antipseudomonal activity than propionate and butyrate (103) 4.6 Commensals may mediate their protective effects via acetate production (104) 4.6.1 SCFAs modulate PA01- and LPS-induced IL-8 release in BEAS-2B cells (104) 4.6.2 SCFA levels used are well below cell toxicity levels (105) 4.6.3 Acetate modulates PA01 and LPS-induced immune response in mouse PCLS (107) 5. DISCUSSION (110) 5.1 SCAPEs mediate inhibitory effects in direct commensal-pathogen interactions against P. aeruginosa and other typical CF pathogens (110) 5.1.1 Members of Streptococcus spp. mediate inter- and intra-species variability in their antipseudomonal effects (111) 5.1.2 SCAPEs inhibit other clinical and nonclinical P. aeruginosa strains as well as other typical CF pathogens (113) 5.2 Selected commensals modulate PA01- and LPS-induced inflammatory response in human airway epithelial cells and mouse PCLS (115) 5.2.1 The gram-positive commensal isolates in this study do not significantly stimulate inflammatory response in human bronchial epithelial cells and mouse PCLS (115) 5.2.2 Selected gram-positive commensal isolates modulate P. aeruginosa-triggered inflammatory response in BEAS-2B cells with inter- and intra-species variation (117) 5.2.3 Selected commensal isolates modulate P. aeruginosa-triggered inflammatory response in CFBE41o ΔF508 (120) 5.2.4 Selected S. mitis isolates modulate P. aeruginosa-induced inflammatory response in mouse PCLS (121) 5.3 Commensals exert protective effects against P. aeruginosa infection via acetate production (124) 5.3.1 Conditioned medium (CM) from selected commensal isolates need to be acidic to mediate inhibition of growth of P. aeruginosa and other typical CF pathogens (125) 5.3.2 The glycolytic pathway is important for streptococcal-mediated antipseudomonal effects (127) 5.3.3 Commensal bacteria mediate growth inhibitory effects by simultaneously lowering the pH and producing acetate (128) 5.3.4 Acetate modulates PA01- and LPS-induced inflammation in bronchial epithelial cells and PCLS (131) 5.4 Conclusions and Outlook (134) 6. DECLARATIONS (158) 6.1 Statement of Authorship (158) 6.2 Declaration of compliance (160) 7. Acknowledgements (161)

Chronic Pseudomonas aeruginosa infections play an important role in the progress of lung disease in patients suffering from cystic fibrosis (CF). Recent studies indicate that polymicrobial microbiome profiles in the airway are associated with less inflammation. Thus, the hypothesis was raised that certain commensal bacteria might protect the host from inflammation. We therefore performed a screening study with commensals isolated from CF airway microbiome samples to identify potential beneficial commensals. We isolated more than 80 aerobic or facultative anaerobic commensal strains, including strains from genera Streptococcus, Neisseria, Actinomyces, Corynebacterium, Dermabacter, Micrococcus and Rothia. Through a screening experiment of co-infection in human epithelial cell lines, we identified multiple commensal strains, especially strains belonging to Streptococcus mitis, that reduced P. aeruginosa triggered inflammatory responses. The results were confirmed by co-infection experiments in ex-vivo precision cut lung slices (PCLS) from mice. The underlying mechanisms of the complex host-pathogen-commensal crosstalk were investigated from both the host and the bacterial sides with a focus on S. mitis. Transcriptome changes in the host in response to co-infection and mono-infection were evaluated, and the results indicated that several signalling pathways mediating inflammatory responses were downregulated by co-infection with S. mitis and P. aeruginosa compared to P. aeruginosa mono-infection, such as neutrophil extracellular trap formation. The genomic differences among S. mitis strains with and without protective effects were investigated by whole genome sequencing, revealing genes only present in the S. mitis strains showing protective effects. In summary, through both in vitro and ex vivo studies, we could identify a variety of commensal strains that may reduce host inflammatory responses induced by P. aeruginosa infection. These findings support the hypothesis that CF airway commensals may protect the host from inflammation.

In der vorliegenden Untersuchung wurde der Postantibiotische Effekt (PAE) von Amikacin und Ceftazidim alleinig und in Kombination beider Antibiotika nach einmaliger und mehrfacher Exposition bei Pseudomonas aeruginosa bestimmt. Es wurden verschiedene Stämme mit unterschiedlicher Resistenz gegen Amikacin und Ceftazidim untersucht. Die MHK-Werte bewegten sich für Ceftazidim zwischen < 0,25 µg/ml bis 4 µg/ml und für Amikacin zwischen < 2 µg/ml bis 32 µg/ml. Der PAE lag nach der einmaligen Inkubation von Amikacin bei 0 bis 2,65 Stunden, von Ceftazidim bei 0 bis 2,78 Stunden und in der Kombination beider bei 2,4 bis 5,37 Stunden. Außerdem ergab die mehrmalige Exposition bei Amikacin nach der 1. Inkubation einen PAE von 0,75 bis 2,25 Stunden und stieg auf 2,3 bis 3,5 Stunden nach der 3. Inkubation an; bei Ceftazidim von 1,1 bis 2,18 Stunden Anstieg auf 1,18 bis 2,5 Stunden und bei der Kombination von Amikacin und Ceftazidim von 2,3 bis 3,75 Stunden war ein geringer Abfall auf 1,25 bis 3,25 Stunden zu verzeichnen. Es wurde ein Zusammenhang zwischen dem PAE und der MHK beobachtet: je höher die Resistenz, desto kürzer der PAE. Die Dosierungsintervalle wurden so gewählt, daß sie der klinisch üblichen dreifach Applikation pro Tag entsprachen. Aus unseren Untersuchungen läßt sich theoretisch eine einmalige Applikation pro Tag für Aminoglykoside und eine Kombinationstherapie Aminoglykosid plus Beta-Lactamantibiotika ableiten; vor allem auch, daß bei Mehrfachapplikation der PAE nicht kürzer wird. Für Ceftazidim erscheint eine Dauerinfusion bei fehlendem PAE sinnvoll. / In the current study we examinated the postantibiotic effect (PAE) of the antimicrobial agents amikacin and ceftazidime in vitro. We analyzed the PAE using both agents alone and in combination and after once and several expositions of different isolates of Pseudomonas. Different resistant stains of Pseudomonas were explored versus amikacin and ceftazidime. The observed minimum inhibitory concentration values (MIC) for ceftazidime ranged from < 0.25µg/ml to 4.0µg/ml and for amikacin from < 2.0µg/ml to 32µg/ml. After unique incubation the PAE of amikacin ranged from 0 to 2.65 hours while ceftazidime ranged from 0 to 2.78 hours. In combination of both we observed PAEs between 2.4 to 5.37 hours. A several exposition of amikacin showed after the first incubation PAEs between 0.75 to 2.25 hours increasing after third incubation from 2.3 to 3.5 hours whereas ceftazidime ranged from 1.1 to 2.18 hours and 1.18 to 2.5 hours. Both antimicrobial agents in combination had PAEs between 2.3 and 3.75 hours after first incubation and decreased low after third incubation between 1.25 and 3.25 hours. There is significant correlation to be seen between PAE and MIC-values: the higher resistance is, the shorter PAE will become.

La fibrose kystique est une maladie autosomale récessive multisystémique se manifestant principalement au niveau du système respiratoire et digestif. En raison d’une altération de la protéine régulatrice CFTR, l’équilibre ionique chez les cellules de l’hôte est déficient. Il en résulte, entre autres, une sécrétion et une accumulation d’un mucus pulmonaire très consistant. L’évacuation du mucus étant très fastidieuse chez les patients atteints de fibrose kystique, l’élimination des particules et bactéries des voies respiratoires est inadéquate. Cet aspect de la maladie participe grandement à sa progression, car l’établissement d’infections bactériennes chroniques contribue principalement à l’effet délétère sur la santé respiratoire des patients atteints de fibrose kystique. La colonisation du mucus pulmonaire par les bactéries pathogènes est le sujet de nombreuses recherches extensives dans le contexte de la fibrose kystique. La présence d’une diversité microbienne dans les voies respiratoires est connue depuis longtemps, mais avec les techniques récentes d’analyse moléculaire, il a été constaté que celle-ci peut présenter une très grande richesse qui était préalablement non détectée par les méthodes d’isolement classiques. Les interactions et comportements adoptés par les populations polymicrobiennes du microbiome FK dans la progression de la maladie sont maintenant de grand intérêt. La virulence des principaux pathogènes en FK a préalablement et exhaustivement été décrite de façon spécifique. Parmi les plus notoires, comme Staphylococcus aureus, Pseudomonas aeruginosa et Burkholderia cepacia, certains utilisent des systèmes de communication cellulaire élaborés («quorum sensing») dans le but de réguler leur virulence. De nouvelles voies de recherche sont construites sur la suggestion qu’une population polymicrobienne peut être perçue comme un seul agent infectieux. Notre étude a été conduite afin de documenter les possibles réponses interespèces entre S. aureus et P. aeruginosa suite à l’échange de molécules sécrétées dans le cadre du «quorum sensing». Pour ce faire, nous avons caractérisé un éventail d’interactions existantes entre des isolats cliniques des deux pathogènes d’intérêt. Nous avons également développé un modèle nous permettant de mieux comprendre l’impact de la coexistence sur leur pathogenèse. Inévitablement, l’étude vise à définir l’instance de la présence simultanée des deux bactéries dans l’établissement d’infections chroniques chez les patients atteints de fibrose kystique ainsi que sur leur santé respiratoire. Cette étude tente ultimement de contribuer à l’effort visant à comprendre l’importance des interactions polymicrobiennes dans un contexte infectieux dans le but de trouver de nouveaux indices quant à l’élaboration de stratégies thérapeutiques.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2009. / Surfactants are compounds that reduce interfacial surface tension, resulting in detergency, emulsifying, foaming and dispersing properties. Surfactants produced via biochemical processes (biosurfactants) form a niche market with their low toxicity, biodegradability and high specificity attributes. Biosurfactants have recently received considerable attention owing to their potential as biomedical molecules. In this study a knowledge base was established for the development of a process which produces biosurfactants for use as antimicrobial agents. Specifically, rhamnolipid biosurfactants were produced from Pseudomonas aeruginosa and tested for antimicrobial activity against target organisms. Accurate and reproducible analyses for the quantification of rhamnolipids and antimicrobial activity were developed. The amount of rhamnolipid was determined indirectly by measuring the rhamnose concentration. A novel HPLC method as well as an orcinol colorimetric method were developed for rhamnose measurement. In order to obtain accuracy with the orcinol method it was found that samples must be extracted at least three times prior to the analysis. An examination of literature on rhamnolipid production showed that many studies used colorimetric methods without extraction. Antibacterial activity was quantified by zone clearing around wells of supernatant in soft agar containing the target organism Mycobacterium aurum. This target organism is especially important in a South African context, since it is used to indicate possible susceptibility of tuberculosis to antibiotics. This method was developed for antibacterial testing, after a standard disk diffusion method proved to be ineffective. Antifungal activity of rhamnolipids was evaluated against the fungus Botrytis cinerea, by growing a lawn of fungus on a plate and adding rhamnolipid. The factors influencing rhamnolipid production were studied by growing different Pseudomonas aeruginosa strains from the ATCC culture collection, namely ATCC 9027 and ATCC 27853 as well as a locally isolated strain under different media conditions. The initial focus was on production of biosurfactants in media containing glucose as substrate. Alkanes were subsequently investigated as an alternative substrate, since they are readily available in South Africa as byproducts from the petrochemical industry. The rhamnolipids produced from the culture collection strains were evaluated for their antibacterial activity against Mycobacterium aurum. A number of key factors were identified which were important for the development of a rhamnolipid production process. Of critical importance were the media conditions. Good production was achieved on glucose media containing a phosphate limitation, pH buffering around neutral pH and a high carbon concentration (2 % carbon). When Pseudomonas aeruginosa ATCC 9027 was cultured on this medium (a minimal salts phosphate limited medium with a Tris buffer), it produced 1.31 g/l rhamnose, equivalent to 4.0 g/l rhamnolipid. This rhamnolipid concentration is 2.7-fold higher that of 1.47 g/l reported in the literature with the same strain (cultured on a different phosphate limited medium The particular strain also proved to be a factor which influenced the yield of rhamnolipids. A rhamnose concentration of 0.43 g/l was obtained with Pseudomonas aeruginosa ATCC 27853 grown on MSM+Tris medium, compared to 1.31 g/l produced by Pseudomonas aeruginosa ATCC 9027 on the same medium. The most promising strain and medium, Pseudomonas aeruginosa ATCC 9027 and MSM+Tris medium, were evaluated under controlled conditions in an instrumented bioreactor. Nearly double the rate of growth and production were obtained in the bioreactor, indicating that production time can be shortened considerably under controlled conditions. However, when compared to shake flask studies, only a 4 % increase in growth and a 5 % increase in rhamnolipid production were achieved in the bioreactor, indicating that the yield was limited by the media components or process conditions. With media containing hexadecane as sole carbon source, negligible rhamnolipid production was achieved. Slow growth was observed and the stationary phase had not been reached even after 2 weeks of growth. It was shown that in glucose media rhamnolipid production only commenced in the stationary phase. Since the stationary phase was not reached during growth on hexadecane, rhamnolipids, which are known to increase the availability of alkanes through emulsification and solubilisation, could not be produced. A strategy was devised to accelerate growth on alkane media. A dual substrate medium containing both glucose and hexadecane was investigated. It was hypothesised that growth would be promoted by glucose leading to rhamnolipid production, which would then increase the uptake of hexadecane. Rhamnolipid was produced in the dual substrate experiments, but the hexadecane uptake was still poor. This was suggested to be due to the exposure of the cells to glucose in the inoculum or test flask, which hampered the ability of the cells to utilise hexadecane. It was reasoned that the ability to utilise hexadecane was determined by the cell hydrophobicity, which was influenced by the exposure to hydrophilic or hydrophobic substrates. Rhamnolipids from Pseudomonas aeruginosa ATCC 9027 and ATCC 27853 were shown to have antibacterial activity against Mycobacterium aurum. The largest zone of clearing of 45 mm was obtained with 4 g/l rhamnolipid from Pseudomonas aeruginosa ATCC 9027. The activity was shown to be directly related to the rhamnolipid concentration, highlighting the importance of maximising the biosurfactant yield when developing a process for the production of rhamnolipids as antimicrobial agents. Antifungal activity tests against Botrytis cinerea were inconclusive. Future studies should expand the antimicrobial application of rhamnolipids by testing their activity against a larger range of target organisms. In order to maximise the rhamnolipid yield in future studies, a fed batch process is proposed which would increase the cell density thereby increasing rhamnolipid production and prolonging the stationary phase, which was found to be the phase associated with rhamnolipid production. Different feeding strategies should be investigated, depending on the kinetics of substrate consumption. It is desirable to feed the smallest volume of substrate that is necessary with a high concentration in order to keep the dilution rate low and maximise the product concentration. A factorial design is recommended for this purpose. Further studies with alkanes as carbon source should be conducted using strains that have been maintained and cultured on media containing alkanes as sole carbon source. Alternative biosurfactant producing strains should also be investigated, which have higher natural cell hydrophobicities.

Patients with cystic fibrosis who are infected with the pathogen Pseudomonas aeruginosa have shown favorable responses to the drug azithromycin (AZM). This drug works in an anti-inflammatory capacity, improving clinical outcomes and improving quality of life in this population. The drug has also been shown to affect macrophage polarization by shifting these cells away from an inflammatory phenotype toward an alternatively activated anti-inflammatory phenotype. The full impact of this phenotypic change is not well understood in the context of the response to P. aeruginosa infection, or the overall immune response in cystic fibrosis. To understand how the AZM-polarized macrophage affects other types of cells, we utilized a co-culture in vitro system, with macrophages and fibroblasts incubating together. In this system, we determined that AZM causes upregulation of the pro-fibrotic mediator transforming growth factor-β as well as the extracellular matrix (ECM) protein fibronectin. The mediator of ECM turnover, matrix metalloproteinase (MMP)-9 was upregulated in this system as well. In an in vivo model of P. aeruginosa infection, MMP- 9 and fibronectin were increased in the bronchoalveolar lavage 7 days post-infection in mice that were treated with AZM. This was accompanied by a decrease in damage to the lung tissue, determine by histological examination. To determine if these changes would continue in human subjects with cystic fibrosis, a clinical study was done in this population. Subjects with AZM treatment had decreased TGF-β levels, but no differences in MMP-9 or fibronectin. Interestingly, correlations between certain fibrotic mediators and inflammatory cytokines, specifically interleukin -1β, were different in subjects with AZM treatment compared to subjects without AZM therapy. Together, these data indicate that AZM alters the fibrotic response from the macrophages, as well as the interaction of the inflammatory response and fibrosis development.

Rhamnolipids are surface-active molecules produced by Pseudomonas aeruginosa as congener mixtures. They are considered “green” alternatives to synthetic surfactants used in many applications. Optimizing yield and controlling congener distribution are necessary steps for successful commercialization. Studies have noted that vegetable oils, composed of a mixture of fatty acids, increase rhamnolipid yield. The physiological explanation for this is not yet understood. Furthermore the exact effects of various fatty acid components in the oils on rhamnolipid production have not been reported. The first part of the dissertation was to investigate rhamnolipid biosynthesis when fatty acid substrates are present. A combination of stable isotope tracing and gene expression assays were used to identify rhamnolipid lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis. Result suggests that an octanoyl-CoA intermediate of β-oxidation is diverted from β-oxidation to de novo fatty acid synthesis via a “bypass route”, and is incorporated into either a 2-carbon or a 4 carbon β-ketoacyl- ACP, which can then be recognized by the RhlA enzyme for the biosynthesis of rhamnolipid lipid moiety. The second part of the dissertation focuses on studying how fatty acid substrates of different chain length (C₁₂ to C₂₂) and saturation (C(18:1) and C(18:2)) affect rhamnolipid yield, carbon conversion rate, and congener distribution. Results showed that stearic acid significantly increased rhamnolipid yield. A positive linear correlation between the mass percent of stearic acid used and the carbon conversion rate was observed. For all treatments, the RhaC₁₀C₁₀ was the most abundant and RhaC₁₀C(12:1) was the least abundant of the major congeners produced. However, the relative amounts of RhaC₁₀C₈ and RhaC₁₀C₁₂ congeners were dependent on several factors. In general, fatty acid substrates with relatively short chain length (C₁₂ and C₁₄), the unsaturated fatty acid C(18:2), and longer cultivation times resulted in a higher RhaC₁₀C₈/ RhaC₁₀C₁₂ ratio. The studies presented here demonstrate that the medium composition, in particular the organic substrate component, can affect rhamnolipid biosynthesis, yield, and congener distribution. Furthermore, this work presents evidence that C₁₈ fatty acids as co-substrates increase rhamnolipid yield by draining rhamnolipid intermediates directly from the β-oxidation pathway.