No description available.

Salmonella enterica serovar Typhi, a major public health concern in developing countries, continues to be a priority for the World Health Organization. S. Typhi possesses a viaB locus responsible for the biosynthesis of the Vi-capsular antigen, a significant virulence factor at the focus of developing improved prophylaxis for typhoid fever. Tissue culture experiments have demonstrated that S. Typhi wild-type capsule-expressing strain elicits less chemokine secretion than a viaB mutant. Calf experiments using the viaB mutant resulted in an increase inflammatory response. Osmolarity is one of the control signals that affect the biosynthesis of the Vi antigen. Under high osmolarity growth conditions of 300 mM and greater, Vi production is suppressed and S. Typhi is highly invasive. Studies reveal that the viaB mutant displays increased invasion towards intestinal epithelial cells. Our first objective was to implement direct and indirect methods to localize and detect Vi expression within intestinal epithelial cells and bovine Peyer's patch. The second objective was to compare the invasiveness between a viaB mutant, an ompR mutant, and S. Typhi grown under hyperosmolarity. We also measured the effects of these strains in eliciting inflammation in the calf model. We report that tviB was significantly up regulated intracellularly within T84 polarized cells. In the calf experiments, tviB was expressed at levels significantly higher in calf tissue following invasion compared to inoculum grown under Vi-suppressing conditions. Together, these results support the idea that the Vi capsular antigen is expressed after invasion of intestinal epithelial cells in vivo. We found that S. Typhi grown under high osmolarity, the viaB mutant, and the ompR mutant had increased invasion in polarized T84 cells and bovine ileal tissue. Fluid accumulation among Vi-deficient and Vi-suppressed strains was similar. The histopathology of the inflammatory lesions of the small intestine produced by the Vi-deficient and suppressed strains was quite comparable. Our data supports the notion that Vi-suppressed and Vi mutants of S. Typhi exhibit similar levels of increased invasion and inflammation, perhaps mechanistically through the inactivation of the Vi antigen.

The intracellular human pathogen Salmonella enterica serovar Typhi (S. typhi) causes the systemic disease known as typhoid fever. This disease afflicts approximately 17,000,000 people every year, of which over 600,000 cases are fatal. / Sequencing of the S. typhi genome has allowed a better understanding of the pathogenesis caused by this bacterium. In silico research on the genome sequence identified three open reading frames, termed prpZ gene cluster, present in the Ty2 and multi-drug resistant CT18 strains of S. typhi but absent in all other sequenced serovars of S. enterica. Further analysis of this gene cluster revealed that the three genes are transcribed as an operon that encodes two eukaryotic-like Ser/Thr kinases (PrkX and PrkY) and a protein phosphatase 2C (PP2C) (PrpZ). / A previous study has shown that the recombinant His-PrpZ protein has all the hallmarks of a PP2C. Typically, PP2Cs hydrolyze phosphoserine and phosphothreonine residues. In addition, His-PrpZ was found to hydrolyze phosphotyrosine residues, making it a dual specificity phosphatase. A subsequent investigation implicates the prpZ gene cluster in S. typhi virulence as the survival of a prpZ operon deletion mutant is compromised after 48 hours of macrophage infection when compared to wild type bacteria. / It is clear from these results that the prpZ operon plays a role in the pathogenesis of S. typhi. To determine the role of these three genes in virulence, an in vitro characterization of PrkY was carried out as well as an examination of the possible physiological roles of PrpZ. / We have demonstrated that PrkY is an active protein kinase capable of phosphorylating artificial substrates in the presence of Mg2+ and/or Mn2+. Optimal phosphorylation of substrates is achieved in the presence of 5mM Mg2+ at pH 8.0. In addition, we have identified a putative interaction between PrkY and PrpZ, leading to an inhibition of the kinase activity of PrkY. While exploring the possible physiological functions of PrpZ, we have found that this protein is secreted by Ty2 S. typhi in both LB and in the low pH, low phosphate and low Mg 2+ LPM medium. / These findings suggest that PrkY and PrpZ may have antagonistic effects in a S. typhi specific virulence pathway involved in the modulation of host cell signaling by secreted bacterial virulence factors.

No description available.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-10T13:46:45Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_CaracterizacaoMolecularFenotipica.pdf: 1040083 bytes, checksum: 48972a88ad6c49701dfcb31c811763bd (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-08T13:40:23Z (GMT) No. of bitstreams: 2 Dissertacao_CaracterizacaoMolecularFenotipica.pdf: 1040083 bytes, checksum: 48972a88ad6c49701dfcb31c811763bd (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-08T13:40:23Z (GMT). No. of bitstreams: 2 Dissertacao_CaracterizacaoMolecularFenotipica.pdf: 1040083 bytes, checksum: 48972a88ad6c49701dfcb31c811763bd (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2011 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Salmonella Typhi é o agente etiológico da febre tifóide, uma doença infecciosa, sistêmica, que constitui importante problema de saúde pública principalmente nos países em desenvolvimento da África, Ásia, América Central e do Sul. Amostras de Salmonella Typhi isoladas de casos clínicos no período de 1970 a 2009 no Estado do Pará foram caracterizadas por meio da técnica de Eletroforese em Gel de Campo Pulsado (PFGE). O perfil de suscetibilidade aos antimicrobianos foi realizado através dos métodos manual e automatizado. Foi empregada a técnica de Reação em Cadeia Mediada pela Polimerase (PCR) para a pesquisa das integrases de classe 1 e 2 e do fator de virulência Vi. Em 151 amostras pôde-se observar 68 diferentes pulsotipos, sendo 66 deles agrupados em 5 clusters. As amostras independente de sua procedência, fonte de isolamento ou ano, apresentaram elevada similaridade genética que variou de 80 a 100%. Verificou-se resistência (1,99%) e resistência intermediária (6,62%) somente à nitrofurontoína, em nenhuma amostra foi detectado integrase de classe 1 e 2, demonstrando, que, no Estado do Pará, as cepas circulantes não apresentam multi-resistência como observado em várias regiões do mundo. Foram encontradas 4 (2,65%) amostras Vi-negativo, que pode ser devido ao longo período de armazenamento, pois a ilha de patogenicidade SPI-7 é geneticamente instável e pode ser perdida após sucessivos repiques. / Salmonella Typhi is the causative agent of typhoid fever, a systemic infectious disease which is an important public health problem mainly in developing countries from Africa, Asia, America Central and South. Samples of Salmonella Typhi isolated from clinical cases between the period from 1970 to 2009 in the state of Pará were characterized by the Pulsed Field Gel Electrophoresis (PFGE) technique. The profile of antimicrobial susceptibility has been performed using manual and automated methods. Also, Polymerase Chain Reaction (PCR) has been used to the detection of class 1 and 2 integrons and the virulence factor Vi. In 151 samples, we could observe 68 different pulse types, with 66 pulse types grouped into five clusters. The samples, regardless their origin, source of isolation or year, have shown high genetic similarity ranging from 80 to 100%. Resistance (1.99%) and intermediate resistance (6.62%) only against nitrofurantoin has been noticed, integrons have not been found in class 1 and 2 related to resistance, demonstrating that, in the State of Para, the phenomenon of resistance and multidrug resistance found in several regions of the world has not occurred yet. We found four (2.65%) Vi-negative samples, which may be so due to the long storage period, as the pathogenicity island SPI-7 is genetically unstable and may be lost after repeated subcultures.

No description available.

The work in this thesis is concerned with the study of bacterial adaptation on short and long timescales. In the first section, consisting of three chapters, I describe a recently developed high-throughput technology for probing gene function, transposon-insertion sequencing, and its application to the study of functional differences between two important human pathogens, Salmonella enterica subspecies enterica serovars Typhi and Typhimurium. In a first study, I use transposon-insertion sequencing to probe differences in gene requirements during growth on rich laboratory media, revealing differences in serovar requirements for genes involved in iron-utilization and cell-surface structure biogenesis, as well as in requirements for non-coding RNA. In a second study I more directly probe the genomic features responsible for differences in serovar pathogenicity by analyzing transposon-insertion sequencing data produced following a two hour infection of human macrophage, revealing large differences in the selective pressures felt by these two closely related serovars in the same environment. The second section, consisting of two chapters, uses statistical models of sequence variation, i.e. covariance models, to examine the evolution of intrinsic termination across the bacterial kingdom. A first collaborative study provides background and motivation in the form of a method for identifying Rho-independent terminators using covariance models built from deep alignments of experimentally-verified terminators from Escherichia coli and Bacillus subtilis. In the course of the development of this method I discovered a novel putative intrinsic terminator in Mycobacterium tuberculosis. In the final chapter, I extend this approach to de novo discovery of intrinsic termination motifs across the bacterial phylogeny. I present evidence for lineage-specific variations in canonical Rho-independent terminator composition, as well as discover seven non-canonical putative termination motifs. Using a collection of publicly available RNA-seq datasets, I provide evidence for the function of some of these elements as bona fide transcriptional attenuators.

No description available.

M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology. / Water shortages, water pollution and climate changes are highly interrelated global issues. These have raised immense concerns about serious adverse effects on the quality, treatment and re-use of wastewater. A major role of water is for vitality of life on earth. Water is recognized as source of evolution from origin to degree of civilization, since it is an essential resource its treatment becomes a necessity for day to day for life. Nanoparticles and their application in treatment of wastewater is becoming a major area of research. It is mainly applicable to the removal of major contaminants like microorganisms. This study was carried out with an objective to investigate the antibacterial and antifungal potentials of nanoparticles. Cobalt and cobalt complexes of urea and thiourea were synthesized and characterized using UV-Vs, PL, FTIR, TEM, SEM, XRD and TGA techniques. The Co particles are in a mixture of rod, agglomerates with irregular shape around 50 – 100 nm in diameter. The Co/Thiourea particles appear to be around 10 – 30nm in size. The Co complexed with urea images showed spherical to hexagonal shape with 50 nm size in diameter. The antimicrobial activity was determined using Minimum Inhibitory and bactericidal concentration and the well diffusion method. The antibacterial and antifungal activities of ratios (1:1, 1:2, 1:3, 2:1 μg/mL) of doped cobalt nanoparticles were tested against a panel of five Gramnegative bacteria - (Escherichia coli, Pseudomonas aeruginosa, Shigella enterica, Salmonella typhi and Salmonella sonnei) human pathogenic bacteria; and two fungal strains - Aspergillus niger and Candida albicans. Zones of inhibition as a consequence of nanoparticles were compared with that of different standards like Neomycin for antibacterial activity and Amphotericin B for antifungal activity. The results showed a remarkable inhibition of the bacterial growth against the tested organisms. The most striking feature of this study is that Cobalt, Urea and Thiourea nanoparticles have antifungal activity comparable or more effective (as in case of Thiourea on A. niger) than Amphotericin B and nearly promising antibacterial activity although not comparable to Neomycin.

I performed two randomised controlled trials (RCTs) to determine the best treatments for enteric fever in Kathmandu, Nepal, an area with a high proportion of nalidixic acid resistant S. Typhi and S. Paratyphi A isolates. I recruited 844 patients with suspected enteric fever to compare chloramphenicol versus gatifloxacin. 352 patients were culture confirmed. 14/175 patients treated with chloramphenicol and 12/177 patients treated with gatifloxacin experienced treatment failure (HR=0.86 (95% CI 0.40 to 1.86), p=0.70). The median times to fever clearance were 3.95 and 3.90 days, respectively (HR=1.06 [CI 0.86 to 1.32], p=0.59). The second RCT compared ofloxacin versus gatifloxacin and recruited 627 patients. Of the 170 patients infected with nalidixic acid resistant strains, the number of patients with treatment failure was 6/83 in the ofloxacin group and 5/87 in the gatifloxacin group (Hazard Ratio, HR=0.81, 95% CI 0.25 to 2.65; p=0.73); the median times to fever clearance were 4.7 and 3.3 days respectively (HR=1.59 [CI 1.16 to 2.18], p=0.004). I compared conventional blood culture against an electricity free culture approach. 66 of 304 patients with suspected enteric fever were positive for S. Typhi or S. Paratyphi A, 55 (85%) isolates were identified by the conventional blood culture and 60 (92%) isolates were identified by the experimental method. The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% and 96.0%, respectively. This electricity free blood culture system may have utility in resource-limited settings or potentially in disaster relief and refugee camps. I performed a literature review of RCTs of enteric fever which showed that trial design varied greatly. I was interested in the perspective of patients and what they regarded as cure. 1,481 patients were interviewed at the start of treatment, 860 (58%) reported that the resolution of fever would mean cure to them. At the completion of treatment, 877/1,448 (60.6%) reported that they felt cured when fever was completely gone. We suggest that fever clearance time is the best surrogate for clinical cure in patients with enteric fever and should be used as the primary outcome in future RCTs for the treatment of enteric fever.