Identifica la prevalencia de genes que codifican carbapenemasas de tipo metalo β-lactamasas (MBL) en aislados clínicos de P. aeruginosa. Analiza 76 aislados clínicos de P. aeruginosa resistentes a Ceftazidima y “no sensibles” (intermedio o resistentes) a Imipinem y/o Meropem colectados en el Hospital Militar Central de enero a setiembre del 2016. Muestras de secreciones respiratorias, heridas, orinas y hemocultivos de pacientes hospitalizados son procesadas en el Laboratorio de Microbiología. Determina la sensibilidad antimicrobiana por el método de disco de difusión según los criterios del Clinical and Laboratory Standards Institute. Realiza la detección fenotípica de MBL por el test de sinergia de doble disco con imipenem, meropenem y EDTA. La detección genotípica se realiza amplificando por PCR multiplex, los genes blaIMP y blaVIM, mientras que para el gen blaNDM se hizo PCR convencional. Obtiene fenotípicamente 25 de 76 pruebas positivas para MBL, 24(31.58%) de las cuales se confirmaron genéticamente, encontrando el gen blaIMP (23/24, 95.83%) y el gen blaVIM (1/24, 4.17%). La sensibilidad de la prueba fenotípica es del 100% y la especificidad del 98%. Concluye que el 31.58% de los aislamientos clínicos de P. aeruginosa recuperados de pacientes hospitalizados en el HMC, presentan MBL, siendo el gen blaIMP el más prevalente. / Tesis

Phytohormone spielen eine zentrale Rolle in der Regelung normalen Wachstums, der Entwicklung und der Mitwirkung an Abwehrmechanismen in Pflanzen. Allgemein betrachtet können Phytohormone in zwei Klassen unterteilt werden – in solche, die in Beziehung zu Stressreaktionen stehen und in jene, die das Wachstum begünstigen. Salizylsäure, und Jasmonsäure sind in erster Linie an der Stressresonanz, Ethylen, Auxine, Cytokinine (CKs) und Gibberilline an Entwicklungsprozessen beteiligt. In den letzten Jahrzehnten wurde den Phytohormonen aus diesem Betrachtungswinkel starke Aufmerksamkeit gewidmet und heute stehen ihre wechselseitigen Beeinflussungen im Fokus. Die Tatsache, dass Pflanzenpathogene ein hormonelles Ungleichgewicht an der Wirtspflanzen-Pathogen Schnittstelle bedingen und es begleitend zu physiologischen Veränderungen kommt, wird dabei als Werkzeug für Erforschungen in Pflanzengeweben genutzt. Abgesehen von der bekannten Bedeutung, die Cytokinine für Wachstum und Entwicklung haben, sind sie bisher am meisten vernachlässigt worden und eher als Konsequenz denn als Grund von Pathogeninfektionen angesehen worden. Die Ergebnisse dieser Arbeit basieren auf der Hyphothese, dass erhöhte Gehalte an CKs die Pflanzen mit einer Resistenz gegen hemibiotrophe Pathogene ausstatten. In diesem Zusammenhang wurden transgenetische Pflanzen untersucht, in welchen das bakterielle Gen IPT überexpremiert wurde. Kontrolliert wurde die Expression durch einen pathogen-induzierbaren, einen tetracyclin-induzierbaren oder durch einen wachstumsabhängigen Promotor. Für die weitere Validierung der an den transgenetischen Pflanzen gewonnenen Ergebnisse wurden unterschiedliche Cytokinin von abgeschnittenen Tabakblätter aufgenommen. Alle transgenetischen Ansätze und exogen applizierten Cytokiningaben zeigten ähnliche verringerte Krankheitsanzeichen. Diese Art der Resistenz wurde im Weiteren mit verschiedenen zellulären, biochemischen, mikrobiellen Techniken sowie durch Signalwirkungstests fundiert. Die Gehalte von SA und JA blieben unverändert, während die Expression des Gens PR1 in Proben mit erhöhtem Cytokiningehalt stark hoch reguliert wurde. Darüber hinaus konnte eine verringerte Akkumulation von ROS in IPT exprimierenden Blättern gegenüber der entsprechende Kontrolle beobachtet wurden. Zusätzlich konnte weder ein direkter Effekt im Wachstum von P. syringae pv. tabaci noch die Präsenz von antimikrobiellen Peptiden in Cytokinin-angereicherten Extrakten festgestellt werden. Interessanterweise ist die verstärkte Akkumulation von Phytoalexinen bei erhöhtem CKs-Status der Pflanze als ein mögliches Anzeichen für die Gefährdung durch die Ausbreitung von Pathogenen belegt. Im Gegensatz dazu konnten wir keine Wachstumsverlangsamung für Sclerotinia sclerotiorum in Blättern mit erhöhten CKs-Gehalten feststellen. Neben der Wirt-Pathogen Interaktion im Hinblick auf erhöhte CK-Gehalte wurden die Auswirkungen eines modulierten Kohlenstoffhaushalts auf das Wachstum von Pathogenen untersucht. Dafür wurden zuvor generierte transgenetische Tabakpflanzen, basierend auf ein regulierbarem Invertase Enzym verwendet. Es konnte gezeigt werden, dass induzierte und nicht-induzierte Expression von CIN1 unter der Kontrolle des Tet-Promotors das Wachstum von P. syringae pv. tabaci nicht beeinflusst. Darüber hinaus zeigten Linien, welche den Invertaseinhibitor NtCIF unter Kontrolle desselben Tet-Promotors exprimieren, keine differenzielle Veränderung des Wachstums von P. syringae pv. tabaci bei induziertem und nicht-induziertem Status der Pflanze. Ähnlich waren die Resultate in der transgenetischen Tomaten-Linie Lin6::NtCIF für P.syringae pv. tomato DC 3000. Interessanterweise zeigten die Blätter von Lin6::NtCIF Tomatenpflanzen starke Symptome nach Behandlung mit Botrytis cinerea verglichen zum Wildtyp. Eine mögliche Verbindung zwischen Cytokininen und Zuckermetabolismus im Bezug auf die Wirt-Pathogen Beziehung wurde ebenfalls untersucht. Die Expression des IPT-Gens unter der Kontrolle des pathogeninduzierbaren Promotors (4xJERE::IPT) im transgenetischen Hintergrund von Tet::CIN1 ergab lokale Unterschiede in der Entwicklung der Symptom von P. syringae pv. tabaci. Bei exogen appliziertem Kinetin an abgeschnittenen Tabakblättern von Tet::CIN1 verzögerte sich ebenfalls das Wachstum von P. syringae pv. Tabaci im Vergleich zu Tet-induzierten Blättern. Diese Ergebnisse führen zu der Schlussfolgerung, dass die extrazelluläre Invertase keine essentielle Rolle in der Cytokinin-vermittelten Resistenz gegen hemibiotrophe Pathogene spielt. / Phytohormones are known for their pivotal roles in promoting normal growth and development of the plants and contributing to the mechanism of defense. Although an over simplification, however, they may be categorized as stress specific and growth promoting. SA and JA/Ethylene are implicated in stress responses while auxins, cytokinins and gibberellins are involved in developmental processes. Phytohormones from the above perspective got much attention in the last few decades; however their reciprocal role is currently in focus. It is because of the reason that plant pathogens cause overall hormonal imbalance at host pathogen interface and alter host physiology for the sake of pathogenecity. Despite their importance in growth and development, cytokinins are among the most neglected phytohormones that are usually noticed as consequence rather than a cause of pathogen infection. Results presented in this thesis are based on the hypothesis that elevated levels of CKs embody plants with resistance against hemibiotrophic pathogens. To explore a connection between the spread of P. syringae and its tobacco host, CKs over producing transgenic plants were investigated whereby bacterial IPT gene was expressed under the control of pathogen inducible, tetracycline inducible and developmentally inducible promoters. To further validate the out-come of transgenic plants, various types of cytokinins were exogenously fed to detached tobacco leaves. Mentioned transgenics and exogenous CKs feeding approaches unanimously resulted in, “more cytokinins less disease symptoms” and vice versa. This state of cytokinins mediated resistance was further substantiated with various cellular, signaling, biochemical and microbial approaches wherein levels of SA and JA remained unaffected. Conversely, PR1 gene expression was strongly up-regulated in enhanced cytokinins accumulating samples. Moreover, less accumulation of ROS was observed in IPT expressing sites of the plants as compared to their corresponding controls. Additionally, we neither noticed any direct effect of cytokinins on the growth of P. syringae pv. tabaci nor found presence of anti-microbial peptides in cytokinins enriched extracts. Interestingly, enhanced accumulation of phtyoalexins in elevated CKs status of the plant proved to be a possible gesture in jeopardizing the spread of pathogen. Contrarily, no reduction was observed in the spread of fungal necrotrophic pathogen Sclerotinia sclerotiorum when leaves of elevated CKs were inoculated. Besides host-pathogen interaction in perspective of elevated cytokinins, impact of modulated sugar status of the plant on the spread of pathogen was also investigated. For this purpose, previously generated modulated invertase enzyme tobacco transgenic plants were analyzed. We showed that repression and de-repression of CIN1 gene under the control of tetracycline inducible-promoter did not affect the growth of P. syrinage pv. tabaci in Tet::CIN1 transgenic plants. Moreover, invertase inhibitor tobacco lines expressing NtCIF gene under the control of the same promoter failed to exhibit differential pathogenic responses in induced and non induced status of the plant. Similar was the case of tomato transgenic plants expressing NtCIF gene under the control of invertase gene Lin6 promoter in Lin6:: NtCIF plants for P.syringae pv. tomato DC 3000. Interestingly, when challenged Lin6:: NtCIF tomato plants with Botrytis cinerea, severe disease symptoms were observed on transgenic leaves as compared to control plants. To dissect a potential link between cytokinins and sugar metabolism with its effect on the growth of pathogen, invertase transgenic plants with elevated CKs were probed. When expressed exogenous IPT gene under the control of pathogen inducible promoter (4xJERE::IPT) in transgenic background of Tet::CIN1, we observed localized differences in symptom development for P.syringae pv. tabaci. Similarly, when exogenously fed with kinetin, detached leaves of Tet::CIN1 exhibited retarded growth of P.syringae pv. tabaci as compared to the tetracycline induced leaves. These results led to the conclusion that extracellular invertase may not play an essential role in cytokinins mediated disease resistance against hemibiotrophic pathogens.

1. OH-FS wurden in vitro hergestellt, um als Standardsubstanzen zur gaschromato-graphischen Identifizierung von OH-FS in Pflanzenmaterial eingesetzt zu werden. 2. Für die Untersuchung der Oxylipin-Gehalte in A. thaliana wurden der virulente Pst-Stamm DC3000 sowie der avirulente Stamm avrRPM1 verwendet, um die kompatible Interaktion mit der inkompatiblen Interaktion vergleichen zu können. Die Konzentrationen der Oxylipine sowie SA wurden innerhalb einer Versuchsdauer von 60 h verfolgt. Dabei wurden PPF1 sowie 12- und 16-OH-FS, als Vertreter der nicht-enzymatisch entstandenen Oxylipine, 9- und 13-OH-FS, sowohl als enzymatisch als auch nicht-enzymatisch entstandene Oxylipine, sowie JA und deren Vorstufe OPDA als enzymatisch gebildete Phytohormone untersucht. Es wurden monophasische Konzentrationsanstiege, bei allen untersuchten Substanzen, in der kompatiblen Interaktion ermittelt, wohingegen die Konzentrationsanstiege in der inkompatiblen Interaktion biphasisch waren. In beiden Interaktionen wurden nach 48 bis 60 h Konzentrationsmaxima der freien sowie der veresterten OH-FS und PPF1 nachgewiesen, ein früher Konzentrationsanstieg nach 5 bis 10 h konnte ausschließlich in der inkompatiblen Interaktion ermittelt werden. Die gleichzeitige Akkumulation von 9-, 10-, 12-, 13, 15- und 16-OH-FS und PPF1 deutet auf eine parallel ablaufende Oxylipin-Synthese durch enzymatische, Photo-oxidative und über freie Radikale vermittelte Prozesse hin. Die Akkumulation veresterter OH-FS und PPF1 erfolgte in beiden Interaktionen 5 bis 12 h früher als die Konzentrationsanstiege der freien OH-FS und PPF1. Die Ergebnisse bestätigen die Hypothese, dass nicht-enzymatische Oxylipine in Membranen gebildet werden können und anschließend vermutlich durch eine Lipase frei gesetzt werden. In der inkompatiblen Interaktion konnte ein erstes frühes Konzentrationsmaximum von JA und OPDA nach 5 h beobachtet werden, während späte Maxima in beiden Interaktionen nach 24 bis 36 h erfolgten. Somit akkumulierten die OH-FS und PPF1 in der inkompatiblen Interaktion zeitgleich mit den Jasmonaten nach 5 h. 3. Bei einer Kälteexposition von A. thaliana bei 4°C über 2 h wurde jeweils ein 3,3-facher Konzentrationsanstieg der freien und der veresterten enzymatisch gebildeten 13-OH-FS nachgewiesen. Darüberhinaus wurde ein 4,6-facher Anstieg der enzymatisch entstandenen 9-OH-FS ermittelt. Die nicht-enzymatisch gebildeten 12- und 16-OH-FS zeigten dagegen keine signifikanten Konzentrationsanstiege über die basalen Konzentrationen hinaus. Die angewendeten Stressbedingungen bewirken demnach ausschließlich eine enzymatische Bildung von OH-FS in A. thaliana. 4. Zur Untersuchung der OH-FS-Synthese in der inkompatiblen Interaktion in Abhängigkeit von der bei der Pflanzenanzucht eingesetzten Lichtstärke wurden A. thaliana bei Licht und in Dunkelheit mit Pst avrRPM1 infiziert. Nach 10 h wurde eine 1,1- bis 3,7-fach stärkere Bildung der freien sowie eine 2,0- bis 3,4-fach stärkere Akkumulation der veresterten 9-, 10-, 12-, 13, 15- und 16-OH-FS bei den Pflanzen ermittelt, die bei Licht angezogen wurden. Die Lichtintensität, der Pflanzen während der Infektion mit Pst ausgesetzt sind, hat demnach große Bedeutung für die Entstehung enzymatisch und nicht-enzymatisch gebildeter OH-FS. Ein 4,9-facher Anstieg veresterter 15-OH-FS, ein Marker für eine photooxidative OH-FS-Entstehung, auch bei Dunkelheit widersprach der Hypothese, dass 15-OH-FS ohne Lichteinwirkung nicht gebildet werden können und deutet auf eine bisher unbekannte Licht-unabhängige Entstehung von 1O2 bzw. von 15-OH-FS hin. 5. Die Bestimmung von OH-FS in Blättern und Wurzeln von unbehandelten A. thaliana-Pflanzen ergab eine 13- bis 31-fach höhere Konzentration veresterter 9-, 10-, 12-, 13- und 16-OH-FS in den Blättern. Darüberhinaus wurde eine 111-fach höhere Konzentration von veresterten 15-OH-FS in Blättern im Vergleich zu Wurzeln nachgewiesen. 15-OH-FS wurden als selektiver Marker für eine Photo-oxidative OH-FS-Bildung durch 1O2 verwendet. Mit 0,57 µg/g TG kommt 15-OH-FS allerdings auch im Wurzelgewebe vor, was einen Hinweis darauf darstellt, dass neben einem Licht-abhängigen Hauptweg auch ein Licht-unabhängiger Entstehungsmechanismus von 15-OH-FS bzw. 1O2 existiert. Alternativ wäre es denkbar, dass ein Transport von 15-OH-FS von den Blättern in die Wurzeln stattfindet. 6. Eine Untersuchung der Gehalte an OH-FS und PPF1 in NahG-, lsd1-, atrbohD- und atrbohF-Mutanten ergab 48 h nach Infiltration von Pst avrRPM1 keine signifikanten Unterschiede im Vergleich zu den Pflanzen des jeweiligen Wildtyps Col-0 und WS. Unter den gewählten Versuchsbedingungen bewirken die genetischen Defekte der untersuchten Mutanten keine veränderte Akkumulation enzymatisch sowie nicht-enzymatisch gebildeter Oxylipine. / 1. To obtain reference-substances for the identification of OH-FA in plant material by GC-MS, OH-FA were made in vitro. 9- and 13-hydroxy-octadecadienoic acids were formed from linoleic acid, 9- and 13-hydroxy-octadecatrienoic acids from -linolenic acid by LOX-catalized reaction. 2. In order to compare the concentrations of oxylipins in A. thaliana during compatible and incompatible interaction, a virulent Pst-strain DC3000 and an avirulent strain avrRPM1 were utilized. The concentrations of oxylipins and salicylic acid were investigated within 60 h after inoculation. F1-phytoprostanes, 12- and 16-OH-FA were used as markers for non-enzymatically formed oxylipins. Concentrations of 9- and 13-OH-FA, either enzymatically or non-enzymatically formed, were measured as well as phytohormones, jasmonic acid and its precursor 12-oxo-phytodienoic acid. Within the compatible interaction an increase of the amount of all measured compounds was observed. In contrast, during incompatible interaction the increases of all measured substances was seperated into two phases. In both interactions, free and esterified OH-FA- and PPF1-concentrations exhibited maxima after 48 to 60 h, an early increase after 5 to 10 h was exclusively found in the incompatible interaction. The concurrent accumulation of 9-, 10-, 12-, 13, 15- und 16-OH-FA und PPF1 indicates that enzymatically, Photo-oxidative, and free-radical catalyzed synthesis of oxylipins takes place simultaneously. In both interactions the accumulation of esterified OH-FA and PPF1 occurred 5 - 12 h earlier than the increase of the concentrations of free OH-FA and PPF1. These results confirm the hypothesis that oxylipins are formed non-enzymatically from lipids inside cell membranes and are subsequently released by lipases. An early increase of JA- and OPDA-concentrations after 5 h was found in the incompatible interaction, while late maxima occurred in both interactions after 24 to 36 h. Therefore, OH-FA- and PPF1-accumulation took place at the same time as the increase of jasmonates after 5 h. 3. When A. thaliana plants were chilled for 2 h at 4°C an 3,3-fold incrceased formation of both, the enzymatically formed free and esterified 13-OH-FA was detected. The amount of enzymatically formed free 9-OH-FA increased 4,6-fold. In contrast, the amount of non-enzymatically formed 12- and 16-OH-FA did not increase significantly indicating that the applied mild stress conditions triggered exclusively enzymatical OH-FA-formation. 4. In order to get information about OH-FA-formation with regard to light intensity A. thaliana was infected by Pst avrRPM1 and cultivated either in the dark or in the light. When plants were cultivated in the light after 10 h an 1,1- to 3,7- fold increased formation of free and an 2,0- to 3,4-fold increased accumulation of esterified 9-, 10-, 12-, 13-, 15- und 16-OH-FA could be detected. Therefore, the light intensity during an infection with Pst avrRPM1 is an important factor for the formation of enzymatically as well as non-enzymatically formed OH-FA in planta. The 4,9-fold increase of esterified 15-OH-FA (a marker for photooxidative formation of OH-FA) in the dark contradicted the hypothesis that its formation is impossible without the impact of light. In contrast the accumulation of 15-OH-FA in the dark points to a light-independent 1O2- and subsequent 15-OH-FA-formation by a so far unknown mechanism. 5. Determination of the concentrations of OH-FA in leaves and roots of untreated plants of A. thaliana showed 13- to 31-fold higher amounts of esterified 9-,10-, 12-, 13 und 16-OH-FA in the leaves. Moreover, the amounts of esterified 15-OH-FA exceeded in leaves by the factor 111 in comparison to roots. 15-OH-FA was used as a selective marker for Photo-oxidative formation of OH-FA by 1O2. Though, 15-OH-FA occurred to an amount of 0,57 µg/g (dry weight) in roots as well. This indicates that, apart from an primarily used light depending mechanism, a second path for the formation of 15-OH-FA respectively 1O2 exists which does not depend on light strength. An alternative explanation could be the transport of 15-OH-FA from leaves to roots. 6. Compared to the wildtype plants no significant differences in the increase of OH-FA and PPF1 could be detected in NahG-, lsd1-, atrbohD and atrbohF-mutants 48 h after infiltration of Pst avrRPM1. Under the utilized conditions the genetic defects of the analyzed mutants did not cause a modified accumulation of both, enzymatically and not enzymatically formed oxylipins.

Publicación a texto completo no autorizada por el autor / Optimiza la producción de ramnolípidos a escala piloto con Pseudomonas aeruginosa 6K-11 en cultivos sumergidos por lotes. Con la finalidad de establecer los parámetros óptimos para la aireación, agitación y concentración de nitrógeno en la producción de ramnolipídos a escala piloto, se evaluaron dos niveles para cada factor: 0.25 vvm y 0.5 vvm (aireación), 50 RPM y 70 RPM (agitación) y 2.45 g/L y 4.89 g/L (concentración de NaNO3). Con estos tres factores y dos niveles para cada uno se evaluó la producción de ramnolípidos a escala piloto con el diseño experimental de Taguchi L4 [2]3 o arreglo L4. / Tesis

A dissertation submitted in partial fulfilment of the requirements of the degree of Master of Science in Environmental science School of Animal, Plant and Environmental Sciences University of Witwatersrand, Johannesburg. March 2017. / Plant growth promoting rhizobacteria (PGPR) promotes plant growth in a variety of modes of action and also suppresses several phytopathogens causing plant diseases. There is evidence that Pseudomonas strains are able to induce systemic resistance, thereby enhancing the defensive capacity of many plant species, and they do so without any negative impact on the environment. Currently, many agricultural systems rely more on the use of chemical pesticides to combat plants diseases. The chemicals have several negative impacts on both human health and the environment. Therefore, there is need to investigate the ability to fight plant pathogens of alternatives like the Pseudomonas spp that do not harm the environment. Several strains of this genus are yet to be tested to see if they induce systemic resistance. Previous studies showed that bio surfactants produced by Pseudomonas koreensis exhibited strong effect against oomycetes P. ultimum in tomato plants. Induced systemic resistance (ISR) potential of P. koreensis following exposure to viruses has not been fully demonstrated to date. This study sought to investigate whether this strain has an effect on viruses and if it is able to induce systemic resistance against viral pathogens. The study started by growing the model plant N. benthamiana. The second stage involved carrying out assays of tobacco mosaic virus (TMV) after inoculating this virus in three bio treatments: (i) seed treatment of N. benthamiana with P. koreensis (referred to as the early treatment), (ii) root treatment at the transplanting stage (late treatment) and (iii) the control. In bio treatments (i) seeds were first sterilized by dipping them into 70% alcohol for 3 minutes and 0.1 % HgCl2 for 1 minute and washing them with distilled water. Each seed was then soaked into 20ml of bacteria suspension for 30 minutes and in (ii) a litre of P. koreensis culture was then poured onto the roots of 36 N. benthamiana plants. The bacteria suspension was added at 107 colony forming units per gram of soil to each tray. It was observed that disease severity was lower in the P. koreensis plant treatments than for the control. Results of this investigation have shown that P. koreensis can induce systemic resistance in foliar parts when plant seeds or roots are inoculated with this strain. This was demonstrated by separation of plant growth promoting rhizobacteria (PGPR) bacteria and TMV. Seeds and roots were inoculated with bacteria while the leaves were inoculated with TMV. The early bio treatment had the lowest mean number of necrotic lesions, and exhibited the mildest effects from TMV compared to the late bio treatment and control. Plants in the late bio treatment were moderately affected while the control was severely affected (P˂0.0001) ˂0.05. The early and the late bio treatment both had higher leaf surface area than the control; (P˂0.0001) ˂0.05. The early bio treatment lost the fewest leaves, and the late bio treatment lost a moderate number while the control lost the highest number (P˂0.0001)˂0.05.The reduced symptoms exhibited by plants inoculated with P. koreensis is an indication that P. koreensis has anti-viral activity against TMV. It was concluded that P. koreensis can reduce plant‟s viral susceptibility and result in ISR. It is hence proposed that P. koreensis can be used as a biological control (bio control) agent against viruses. Key words: Tobacco Mosaic Virus (TMV), Pseudomonas koreensis (P. koreensis), induced systemic resistance (ISR) / LG2018

A unique undescribed gram-negative rod is extensively characterized in this study. The cells of this unusual water isolate measure 1.2 x 6.5 microns. The most distinguishing characteristic of the bacterium is a polar tuft of 35-40 flagella that aggregate to function as a single organelle which is visible under phase contrast.

Many strains of Gram-negative bacteria, such as Pseudomonas, are able to utilize a variety of unusual chemicals, including a wide range or aromatic hydrocarbons and their derivatives for growth. Bacteria with the potential to degrade sodium benzoate were isolated, identified and grown as biofilms on sodium benzoate in a laboratory-scale fluidized bed biofilm bioreactor. Four Pseudomonas strains identified as P. aeruginosa (BDS2) P. putida (BDS1 and GR1) and Burkholderia cepecia (GR3FAR) were used in a laboratory-scale FBBR together with two Bacillus strains - Bacillus macroides (SBSY4) and Bacillus simplex (MAR). Sodium benzoate biodegradation capacities of these species were compared under batch and continuous operations. Biofilm and planktonic bacterial growth dynamics were monitored by plate counts, and optical density measurements (230nm) determined benzoate biodegradation. Overall, higher attached and planktonic bacterial counts were determined under batch compared to continuous mode. In addition to this, the ability of attached cells to use sodium benzoate as their sole carbon source was compared to their suspended counterparts in a batch system. There were more attached counts compared to suspended cells and attached cells apparently degraded sodium benzoate better than planktonic cells. Similarly, higher rates of benzoate depletion were found to occur under batch compared to the continuous system. It thus appeared that more cell growth implied more substrate consumption. SEM showed attached cells and microcolonies of all the isolates on GAC, indicating their biofilmforming abilities.

Bacteriophages (phages) are obligate intracellular parasites of bacteria that usually kill the bacterial host. Bacteriophage therapy is a recently revived approach for treating bacterial infection that relies on the traits of the phage lytic cycle. A lot of attention has been given to phage therapy with new research being published weekly and international conferences organised every year, bringing together the academic and industrial phage communities. However, despite this huge effort and considerable scientific interest there is still a great lack of understanding on how to use phage effectively and overcome the many obstacles in the near future. One of the main triggers for such interest was the increasing evidence of antibiotic resistance among human bacterial pathogens, which were once efficiently eliminated by drugs but are now causing alarmingly high levels of morbidity and mortality. Also, bacteria when causing a disease are able to produce highly protective biofilm communities. Biofilms are major causes of impairment of wound healing and two of the most common and aggressive wound pathogens are Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative), both displaying a large repertoire of virulence factors and reduced susceptibility to antibiotics. This work reports and explores the use of phages to target both S. aureus and P. aeruginosa pathogen biofilm producers. Firstly, isolation of promising phage candidates was performed and cocktails were established. Two phages (DRA88 and phage K) formed the cocktail to target S. aureus and six phages (DL52, DL54, DL60, DL62, DL64 and DL68) formed a cocktail to target P. aeruginosa. A thorough characterisation of each of the selected phages was performed, including their range of host infectivity and their genome sequences were analysed. The phage’s ability to infect and kill planktonic cultures was successfully studied and afterwards such ability was assayed on biofilms using an in vitro static biofilm system (microtitre-plate), followed by an in vitro dynamic biofilm system (The Modified Robbins Device). Both cocktails were shown to be effective in reducing and dispersing biofilms formed by the clinical strains showing them to be promising not only to combat topical bacterial infections (related to biofilm production), but also to control biofilms produced on the surfaces of medical devices, such as catheters. Finally, the phage cocktail’s ability to treat systemic infections caused by the two pathogens was assessed in an in vivo G. mellonella infection model. In the case of the P. aeruginosa infection, although the phages were not able to fully treat the larvae, the cocktail allowed a delay of larval death, caused by the infection. For the S. aureus infection, the cocktail did not show the same trend, but most likely the high bacterial cell numbers involved in the experiment interfered with a successful study on the phage cocktail. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus and P. aeruginosa biofilms.

Many bacteria secrete secondary metabolites, whose production is decoupled from active growth in laboratory cultures. Historically, the advantages of secondary metabolite production have mostly been explored in the context of cellular interactions, such as antibiotic effects on competing organisms, damage caused to host tissues during infection, or cell density-dependent signaling. However, recent studies in the opportunistic pathogen Pseudomonas aeruginosa have brought into focus the physiological effects of secondary metabolites on their producer and their implications for multicellular behavior. P. aeruginosa produces antibiotics called phenazines, which can act as mediators to transfer reducing power to an extracellular oxidant and thereby support bacterial survival when oxygen is not accessible. In the crowded environments of biofilms, communities of bacteria surrounded by self-made matrices, this property of phenazines could support energy generation for cells in anoxic subzones. As biofilm formation is a hallmark of P. aeruginosa colonization at various infection sites within the body, I was motivated to investigate the regulation of phenazine production at the level of synthesis and transport, the distribution of phenazines in P. aeruginosa biofilms, and the effects of individual phenazines on P. aeruginosa gene expression and colony biofilm morphogenesis. As part of this work, a novel electrochemical device was developed that enables direct detection of phenazines released from intact colony biofilms. Application of this device and other electrochemical techniques enabled detection of the reactive phenazine intermediate 5-Me-PCA, which was found to be the primary phenazine affecting P. aeruginosa colony morphogenesis. The production of this phenazine was found to be sufficient for activation of the redox-active transcription factor SoxR and full induction of the RND efflux pump MexGHI-OpmD. Finally, results described in this thesis show that 5-Me-PCA is transported by MexGHI-OpmD, constituting a unique demonstration of the self-protective role of an efflux pump in a gram-negative antibiotic-producing bacterium. These findings raise broad questions about the effects of individual phenazines on biofilm cell physiology and have implications for the contributions of individual phenazines to virulence and survival during infection. The technology developed also has potential applications in novel diagnostic and therapeutic approaches. Chapters 1-3 introduce and highlight advances made in understanding secondary metabolite production, with a focus on P. aeruginosa. Chapter 1 provides an introduction to antibiotic production, the concept of self-resistance and other physiological effects of antibiotics in their producers, and infections caused by P. aeruginosa. Chapter 2 reviews recent studies that have brought into focus the physiological effects of secondary metabolites on their producers and their implications for multicellular behavior. Chapter 3 provides an overview of our current understanding of the regulation of phenazine production in pseudomonads and other bacterial species. Chapter 4 describes the development of an integrated circuit-based platform for detection of redox-active metabolites released from multicellular samples, and demonstrates its application to mapping phenazines released from P. aeruginosa biofilms. The study described in Chapter 5 investigates the role of the P. aeruginosa SoxR regulon, which is induced by phenazines, in phenazine transport and shows that the understudied reactive phenazine 5-methylphenazine-1-carboxylic acid (5-Me-PCA) is transported by the RND efflux pump MexGHI-OpmD and is required for wild-type biofilm formation. Chapter 6 describes the development of an assay for 5-Me-PCA production and studies exploring the role of the regulator PsrA in controlling phenazine biosynthesis. Chapter 7 provides an overview of the findings and open questions to be explored in future research. The P. aeruginosa genome contains two nearly identical operons that encode biosynthetic enzymes for the production of phenazine-1-carboxylic acid, the precursor to all of the other phenazines. The study described in Appendix A characterizes the respective contributions of these operons to phenazine production in shaken liquid cultures and biofilms. Appendix B presents evidence that electron acceptor availability influences, and is influenced by, the morphogenesis of P. aeruginosa colony biofilms. Finally, Appendix C describes a screen for commercially available compounds that inhibit production of the phenazine pyocyanin by P. aeruginosa. Together, these findings reveal the unique physiological roles of specific phenazine-related genetic loci and regulatory proteins and of 5-Me-PCA, a phenazine that was previously overlooked due to the technical challenges associated with its detection. They have also uncovered novel aspects of phenazine production in both shaken liquid cultures and biofilms relevant for the development of therapeutics.

A bacterial biofilm is a community of sessile cells encased in a matrix composed of polysaccharides, proteins, and extracellular DNA that develops according to a reproducible morphogenic program. This morphogenic program is deeply influenced by prevailing redox conditions within the biofilm, which are established by a gradient of terminal electron acceptor through the depth of the biofilm. Terminal electron acceptor limitation leads to redox stress, measured as an elevated ratio of reduced to oxidized forms of the metabolic cofactor nicotinamide adenine dinucleotide, NAD(H). In biofilms of the gram-negative bacterium Pseudomonas aeruginosa, redox stress is relieved by the presence of diffusible redox-cycling molecules, phenazines, that are able to act as an electrical conduit between intracellular NADH and oxygen in the aerobic zone of the biofilm. This is most apparent in the dramatically hyperspread and hyperwrinkled morphologies observed in colony biofilms unable to produce phenazines. However, the ability of phenazines to act as a biologically relevant redox couple between the reducing equivalents of metabolism and atmospheric oxygen also renders them toxic to producing cells. In order to avoid phenazine toxicity, P. aeruginosa encodes self-resistance mechanisms under the control of the redox-sensitive transcription factor SoxR. Two components of the SoxR regulon, the efflux pump MexGHI-OpmD and the monooxygenase PumA, are known to be major contributors to survival in the presence of toxic concentrations of phenazines. This work further details the role of the small protein MexG (Chapter 3) and PumA in phenazine resistance (Chapter 4), and presents an electrochemical platform for studying the effects of a phenazine redox gradient in biofilm morphogenesis (Chapter 5).