With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

Pseudomonas aeruginosa is an opportunistic pathogen that express a plethora of virulence components controlled through two-component regulatory systems that allow for sensing and responding to environmental stimuli. This study was aimed at identifying transcription regulators of algZ that encodes the histidine sensor kinase (AlgZ) of the AlgZR two-component regulatory system. To understand how the algZ gene is transcriptionally controlled, transposon mutagenesis was used to create a mutant library with varying algZ expression based on their b-Galactosidase activity. The gene PA3327 was identified as a potential regulator of algZ expression using arbitrary PCR. This gene encodes a probable non-ribosomal peptide synthetase responsible for the biosynthesis of secondary metabolites such as antibiotics. Further experiments are required to understand how PA3327 transcriptionally regulates algZ expression and its physiological role in the organism. Because the AlgZ/R system regulates virulence, it is possible to attenuate virulence by targeting the expression of algZ gene.

Please read the abstract in the front section of this document / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2005. / Medical Microbiology / unrestricted

Bacterial diversity has always been associated with micro-evolutionary events such as horizontal gene transfer and DNA mutations. Such events influence the rapid evolution of bacteria as a result of the environmental conditions which they encounter. They further establish beneficial phenotypic effects that allow bacteria to specialize in new habitats. Due to the increase in number of bacterial genomic sequences, studying microbial evolution has been made possible, and the impact of micro-evolution on bacterial diversity is becoming more apparent. To gain biological information from this ever increasing genomic data, a variety of computational tools are required. This thesis therefore, focuses on the development and application of computational approaches to identify genomic regions of divergence which have resulted from horizontal gene transfer or small mutational changes. The first and major part of the thesis describes the application of DNA patterns, termed oligonucleotide signatures to identify horizontally acquired genomic regions in prokaryotes. These DNA patterns are demonstrated to differentiate between signatures of the core genome and those which have been acquired through horizontal transfer events. DNA patterns are further demonstrated to: reveal the distribution patterns of horizontally acquired genomic elements, determine their acquisition periods, and predict their putative donor organisms. The second part of the thesis focuses on the evaluation of modern short read sequence data of geographically unrelated Pseudomonas aeruginosa to study their intraclonal genomic diversity. The work described in the thesis was purely in silico driven and performed at Hannover Medical School and the Bioinformatics and Computation Biology Unit at the University of Pretoria. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Biochemistry / unrestricted

Pseudomonas aeruginosa ist ein gramnegatives Bakterium, welches insbesondere bei abgeschwächter Immunabwehr schwere Infektionen auslösen kann. Es besitzt eine hohe intrinsische Resistenz gegenüber Antibiotika, so dass nur eine begrenzte Anzahl von Antimikrobiotika wie beispielsweise Aminoglykoside für die Behandlung zur Verfügung steht. Unter Antibiotikatherapie wird zudem eine schnelle Resistenzentwicklung beobachtet, daher ist die Weiterentwicklung und Optimierung der Therapieoptionen von großer Bedeutung. Die vorliegende Arbeit untersucht den Einfluss von Colistin auf die Aminoglykosidresistenz bei Pseudomonas aeruginosa. 25 Bakterienstämme, die zu Beginn gegenüber Amikacin, Tobramycin und Gentamicin resistent waren, wurden Colistin ausgesetzt. Mithilfe des Epsilometertests wurde die minimale Hemmkonzentration (MHK) der Antibiotika für die zu untersuchenden Bakterienstämme vor und nach Colistineinfluss bestimmt. Es konnte ein signifikanter Rückgang der minimalen Hemmkonzentration nach Colistineinwirkung dokumentiert werden. Zu den Hauptresistenzmechanismen von Pseudomonas aeruginosa gehören die Effluxpumpen, welche die Antibiotika aus dem Bakterium ausschleusen. Für den Transport von Aminoglykosiden ist die MexXY-Pumpe verantwortlich, die in dieser Arbeit weiteruntersucht wurde. Durch eine quantitative Echtzeit-PCR unter Nutzung des Fluoreszenz-Resonanz-Energie-Transfers (FRET) wurde die Expression der Pumpe vor und nach Colistin verglichen. Es konnte nachgewiesen werden, dass durch Colistin die Expression reduziert wurde. Ein linearer Zusammenhang zwischen MHK-Veränderung und mexXY-Expressionslevel wurde anhand der Untersuchungsergebnisse nicht ermittelt. Es ist dementsprechend davon auszugehen, dass andere Resistenzmechanismen ebenfalls durch Colistin beeinflusst werden und so die MHK-Reduktion erklären können.

Despite the significant progress in medicine, infectious diseases are life-threatening thanks to an increasing number of multiresistant strains of microorganisms and late detection of pathological agents. An opportunistic fungus Aspergillus fumigatus cause respiratory system diseases called aspergillosis. The invasive pulmonary aspergillosis affects immunocompromised patients after inhalation of ubiquitous conidia of A. fumigatus and results in 450,000 deaths per year. The biofilm formation in the infected tissue protects A. fumigatus against antimicrobial drugs. Late therapy may not be effective. Infection of immunocompromised patients and biofilm formation is characteristic also for gram negative bacteria Pseudomonas aeruginosa, which is due to the production of many factors of virulence and multiresistance a dreaded opportunistic pathogen. Scanning electron microscopy (SEM) provides detail information about morphology of microorganisms with the resolution in range of tens of nanometers that allows to observe microorganisms in the infected tissue and its pathological changes. Mass spectrometry allows to detect infection and its course based on identification of characteristic microbial molecules. The aim of this study was to optimize sample preparation of tissues infected with A. fumigatus or P....

No apparent repression of pyr gene expression in Pseudomonas aeruginosa is observed upon addition of exogenous pyrimidines to the growth medium. Upon introduction of the subcloned Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) into a P. aeruginosa pyrB mutant strain, repression was observed in response to exogenously fed pyrimidine compounds. The results proved that it is possible to bring about changes in pyrimidine nucleotide pool levels and changes in transcriptional regulation of gene expression as a result. Thus, the lack of regulatory control in P. aeruginosa pyr gene expression is not due to an inability to take up and incorporate pyrimidine compounds into metabolic pools, or to an inability of the RNA polymerase to respond to regulatory sequences in the DNA but is probably due to a lack of specific regulatory signals in the promoter of the genes themselves.

Pseudomonas aeruginosa and Burkholderia cepacia are opportunistic human pathogens. Hazards pose to immunocompromited patient groups, most of whom are patients suffering from cystic fibrosis, for which is the infection caused by Pseudomonas aeruginosa or Burkholderia cepacia, often lethal. Bacteria exhibit resistance to most antibiotics and the immunization of patients, paradoxically, worsens the patients' condition. Hen antibodies, unlike mammalian do not activate the complement cascade and do not cause the inflammatory response, therefore, seem to be a suitable tool to protect high risk populations as a means of passive immunization. In this work were prepared specific chicken antibodies against significant virulent factor - bacterial lectins PAIIL and BClA responsible for adhesion. Antibody specificity was demonstrated by ELISA and Western blot. Antibodies were affinity purified and cleaved to fragments. Pneumocytes type II from the lungs of patients with Cystic Fibrosis were isolated and cultured. Tests of the adhesion of bacteria cells were performed on them using ELISA. As an alternative model rat pneumocytes and tumor cell line A549 has been used. Prepared antibodies specifically detect bacterial lectins on the surface of bacteria. Antibody has been shown to reduce adhesion of bacteria to...

The primary clinical characteristics of cystic fibrosis (CF) are malnutrition caused by malabsorption secondary to pancreatic insufficiency, chronic pulmonary infections, and male infertility. The major cause of morbidity and mortality are bronchiectasis and obstructive pulmonary disease. Lung disease in CF is manifested by this chronic lung disease progression, with intermittent episodes of acute worsening of symptoms called pulmonary exacerbations. Once the patient has stabilized, and if suitable care can be arranged, these interventions are often transitioned to the home. This review summarizes important points pertinent to the use of intravenous and inhaled antimicrobials that may be encountered by prescribers, nurses, technicians, and case managers in the home health setting. Appropriate dosing, indications, adverse drug reactions, monitoring parameters, and practicality of both intravenous and inhaled antimicrobials are discussed.