Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 35-40). Also available on the World Wide Web.

Antibiotics have been extensively used to treat bacterial infections since Alexander Fleming’s discovery of penicillin 1928. Disease causing microbes that have become resistant to antibiotic drug therapy are an increasing public health problem. According to the world health organization (WHO) there are about 440 000 new cases of multidrug-resistant tuberculosis emerging annually, causing at least 150 000 deaths. Consequently there is an immense need to develop new types of compounds with new modes of action for the treatment of bacterial infections. Presented herein is a class of antibacterial ring-fused 2-pyridones, which exhibit inhibitory effects against both the pili assembly system in uropathogenic Escherichia coli (UPEC), named the chaperone usher pathway, as well as polymerization of the major curli subunit protein CsgA, into a functional amyloid fibre. A pilus is an organelle that is vital for the bacteria to adhere to and infect host cells, as well as establish biofilms. Inhibition of the chaperone usher pathway disables the pili assembly machinery, and consequently renders the bacteria avirulent. The focus of this work has been to develop synthetic strategies to more efficiently alter the substitution pattern of the aforementioned ring-fused 2-pyridones. In addition, asymmetric routes to enantiomerically enriched key compounds and routes to compounds containing BODIPY and coumarin fluorophores as tools to study bacterial virulence mechanisms have been developed. Several of the new compounds have successfully been evaluated as antibacterial agents. In parallel with this research, manipulations of the core structure to create new heterocycle based central fragments for applications in medicinal chemistry have also been performed.

The aim of the study is to investigate the causal relationship between membrane-damaging activity and antibacterial action of cardiotoxins from Naja naja atra (Taiwan cobra) cardiotoxin 3 (CTX3) and Naja nigricollis (Egyptian cobra) toxin gamma. Compared with that on Escherichia coli (E. coli, Gram-negative bacteria), CTX3 showed a greater growth inhibition on Staphylococcus aureus (S. aureus, Gram-positive bacteria). Antibacterial avtivity of toxin gamma toward E. coli and S. aureus was similar. Bactericidal action of cardiotoxins positively correlated with increase in membrane permeability of bacterial cells. Morphological examination showed that cardiotoxins disrupted the integrity of bacterial membrane. Cardiotoxins showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall promoted bactericidal effect of cardiotoxins on E. coli and S. aureus, respectively. CTX3 notably permeabilized model membrane of S. aureus and toxin gamma had similar activity on the permeabilization of bacterial model membrane used. Membrane-damaging activity of cardiotoxins was inhibited by either LPS or LTA, while increasing concentrations of cardiotoxins counteracted the inhibitory action of LPS and LTA. Oxidation of Met residues on loop II of cardiotoxins simultaneously attenuated membrane-permeabilizing activity and bactericidal effect of cardiotoxins. Taken together, our data indicate that antibacterial action of cardiotoxins depend on their ability to induce membrane permeability.

Soft corals of the genus Lobophytum (Alcyoniidae) have been well recognized as a rich source of various secondary metabolites that have attracted much interest for the natural products chemists due to their structural complexity and remarkable pharmacological activities such as cytotoxicity, antibacterial activities, anti-inflammatory properties, and antiviral activity. Twelve cembrane diterpenes including six new secondary metabolites 1−6 were isolated organic extracts of soft coral Lobophytum durum collected at Dongsha Atolls. The structures of these six new cembranolides were determined by 1H, 13C, DEPT, COSY, HMBC, HSQC, NOESY, IR and Mass spectra. Furthermore, these six new secondary metabolites 1−6 were evaluated in vitro for the cytotoxicity against A-459 (human lung carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cancer cell lines, and antiviral activity against HCMV (human cytomegalovirus) cells.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed Feb. 4, 2010). Additional advisors: Subramaniam Ananthan, David E. Graves, Craig D. Smith, Sadanandan E. Velu. Includes bibliographical references.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen often infecting the lungs of individuals with the heritable genetic disease cystic fibrosis and the peritoneum of those undergoing continuous peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this dissertation I used a rat dialysis membrane peritoneal model to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo. Included in this analysis are genes important for iron acquisition and growth in lowoxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in the environment. This lysis was shown to be dependent on antimicrobial quinolones produced by P. aeruginosa. I demonstrate that these quinolones are present in outer membrane vesicles (MVs). Not only were these quinolones present in MVs, but the quorum sensing molecule; 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal; PQS) was also packaged into MVs and was necessary for MV formation. These findings illustrate that a prokaryote possesses a signal trafficking system with features common to those used by higher organisms and outlines a novel mechanism for delivery of a signal critical for coordinating group behaviors in P. aeruginosa. Although MVs are involved in important processes besides signaling, the molecular mechanism is unknown. To provide insight into the molecular mechanism of MV formation, I examined the interaction of PQS with bacterial lipids. In this work, I demonstrated that PQS interacts strongly with the acyl chains and 4’-phosphate of bacterial lipopolysaccharide. The results of my studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signaling functions. Finally, I propose a model of PQSmediated MV formation where PQS interacts with specific outer membrane components to allow the necessary curvature for MV formation. / text

The global emergence of drug resistance is posing increasing difficulties in the public health control and treatment of tuberculosis (TB). Fluoroquinolones (FQs) are regarded as having a pivotal role among the antimicrobial agents in multidrug regimens against multidrug-resistant tuberculosis (MDR-TB). Thus, early diagnosis of fluoroquinolone-resistant (FQr) MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) by molecular tests has predictive value for the guidance of TB therapy. The pharmacokinetic (PK) and pharmacodynamic (PD) indices are valuable parameters to evaluate the activity and efficacy of fluoroquinolones (FQs) based upon the bactericidal effect and prevention of the emergence of resistance. In the first part of this study, the potencies of ofloxacin (OFX) and moxifloxacin (MXF) against clinical isolates of MDR-TB in terms of their PK/PD indices (Cmax/MIC90, AUC/MIC90, Cmax/MPC90 and AUC/MPC90) were investigated and compared. The results revealed that MXF displays higher ratios of PK/PD in vitro and could serve as a promising agent for the treatment of MDR-TB. Molecular tests on resistance genes are reliable and rapid technology for diagnosis of drug-resistant TB which facilitates timely patient management and public health control of TB. In the second part of the study, the feasibility of a PCRsequencing assay for the examination of mutations in the quinolone-resistance-determining- region (QRDR) of the gyrase A (gyrA) gene in FQ-resistant (FQr) Mycobacterium tuberculosis in direct clinical specimens was evaluated. As determined by gyrA QRDR DNA sequencing analysis, complete concordance of phenotypic and genotypic outcomes was demonstrated. The results indicate that the molecular assay is an accurate and effective method for the diagnosis of FQr TB and allows identification of mixed resistant variants in the same patient. GyrA mutations that associated with FQr in clinical isolates of M. tuberculosis were clustered in hotspot codons 88, 90, 91 and 94, corroborating other reports. We also detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from the respiratory specimens by using the PCR-DNA sequencing assay. In the third part of this study, the functional effect of the Ala74Ser mutant was verified through study of the DNA supercoiling inhibitory activities of OFX and MXF against the recombinant DNA gyrase. Fifty percent inhibitory concentrations (IC50) of FQs against the DNA supercoiling activities of the recombinant DNA gyrase complex reconstituted with gyrA Ala74Ser were eight-fold and 14-fold greater than the wild-type H37Rv reference strain, and results correlated well with their phenotypic drug susceptibilities. Besides, a combination of gyrA mutations (Glu21Gln, Ser95Thr and Gly668Asp) was also characterized to be non-functional polymorphisms. The impact of the gyrA Ala74Ser mutation on drug binding affinity was elucidated through a crystal structure model of the gyrA-MXF-DNA cleavage complex. Alanine at position 74 of gyrA in M. tuberculosis, which corresponds to the alanine at position 67 of gyrA in Escherichia coli, is an amino acid lying in the α3 helix domain which forms a hydrophobic interface between the gyrA-gyrA dimer. Perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative drug binding pocket, and leads to the reduction of the binding affinity of FQ due to the distance effect. This is the first report verifying that gyrA Ala74Ser mutation alone is responsible for FQr in M. tuberculosis. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

No description available.

Tetraciklinų nustatymas piene atliktas efektyviąja skysčių chromatografija ir įteisintas remiantis ES išleista direktyva 2002/657/EB. Metodas atitinka visus kriterijus aprašytus šioje direktyvoje. Antibakterinių medžiagų likučiai iš pieno ekstrahuojami Mcllvaine-EDTA buferiniu tirpalu, o po to valymas atliekamas naudojant OASIS HLB kietafazės ekstrakcijos kolonėles. Chromatografinėje kolonėlėje atskyrimas atliekamas naudojant 10 mM oksalo rūgšties vandeninio tirpalo ir ACN gradientinį režimą, tėkmės greitis 0,9 ml/min, 100 μl injekcija, bangos ilgis 355 nm ir optimali kolonėlės temperatūra (LiChroCART Superspher 60 RP C8 (250 x 4,6 mm, dalelių dydis 5 m)) – 30 oC. Visi rezultatai apskaičiuoti naudojant kompiuterinę duomenų apdorojimo programą “Interval”. Tetraciklinų išgavos nuo 86,20 % iki 107,19 %, o nustatymo ribos yra intervale nuo 26,7 μg/l iki 49,3 μg/l. / Analysis of tetracycline compounds was performed using high performance liquid chromatography and validated according to EU commition decision 2002/657/EC. Method is compliant at all criteria described in the decision. Antibacterial material residues were extracted from milk with Mcllvaine-EDTA buffer followed by clean-up on OASIS HLB cartridges. Separation was performed using 10 mM oxalic acid/ACN gradient with flow rate 0.9 ml/min, 100 μl injection, detection at 355 nm and the optimal temperature of column (LiChroCART Superspher 60 RP C8 (250 x 4.6 mm, size of particles 5 m)) – 30 oC. All the results were evaluated statistically with “Interval” data processing program. Recoveries are at the range from 86.20 % to 107.19 %. The determination limits for tetracyclines were carried out from 26.7 μg/L to 49.3 μg/L in this study.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: S. Ismat Shah, Dept. of Materials Science . Includes bibliographical references.