>Magister Scientiae - MSc / The aim of this study was to investigate the presence of Salmonella in the tea processing environment, to identify and detect potential virulence genes isolated from Salmonella in the tea, and to determine the antibiotic resistance levels of Salmonella isolated from fermented Rooibos. / National Research Foundation (NRF)

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

A HEp-2 cell culture model was used to investigate the invasive properties of Campylobacter species. Two of twenty-five Campylobacter isolates did not invade HEp-2 cells, and one of these isolates did not adhere to the epithelial cells. Penetration of HEp-2 epithelial cells by C. jejuni was significantly (P < 0.05) inhibited with C. jejuni lysates and a MAb (1B4) in competitive inhibition studies. Immunogold electron microscopic studies revealed that the 1B4 MAb bound to the flagella and cell surface of low passage (invasive) C. jejuni M 96, whereas only the flagella of high passage (non-invasive) C. jejuni were labelled. Western blot analysis revealed that the 1B4 MAb identified an epitope on antigens ranging in size from 66 to 44 kDa in invasive and non-invasive organisms. Antigens were also recognized in lysates prepared only from invasive strains from 42 to 38 kDa. Sodium meta-periodate chemical treatment of C. jejuni lysates significantly (P < 0.05) affected its inhibitory capacity. Additionally, proteinase K and sodium meta-periodate treatment of lysates changed the mobility of antigens recognized by the 1B4 MAb. This suggests that the antigens required for epithelial cell penetration by C. jejuni may be glycoprotein in nature and that the functional binding site is dependent upon an intact carbohydrate moiety. Co-infection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, polio virus (LSc type 1), porcine enterovirus and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of C. jejuni was significantly increased for HEp-2 cells pre-infected with echovirus 7, coxsackievirus B3, and UV-inactivated (non-infectious) coxsackievirus B3 particles. Polio and porcine enterovirus had no effect on C. jejuni adherence and invasiveness. C. hyointestinalis and C. mucosalis, two non-invasive isolates, did not invade virus-infected HEp-2 cells. The increase of invasiveness of C. jejuni appears to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter but are not sufficient to allow internalization by non-invasive bacteria.

Orientador: Jesus Aparecido Ferro / Banca: Henrique Ferreira / Banca: Rafael Marini Ferreira / Banca: Fabricio José Jaciani / Banca; Helen Alves Penha / Resumo: Diante da importância do cancro cítrico para a citricultura mundial, a bactéria Xanthomonas citri subsp. citri, o agente causal da doença, tem sido amplamente estudada. Pesquisadores brasileiros realizaram o sequenciamento completo do genoma da X. citri subsp. citri isolado 306 (Xac306), com o intuito de elucidar os genes e mecanismos envolvidos na patogenicidade e/ou virulência da bactéria. As ORFs XAC2361 e XAC3898 possuem tamanhos e locais diferentes no genoma da Xac306, mas têm em comum o domínio Peptidase_M23, cujo processo biológico predito é de hidrolase de parede celular, mas a real função destas proteínas em Xac ainda é desconhecida. Este trabalho teve como objetivo avaliar, por meio da mutação sítio-dirigida, se as ORFs XAC2361 e XAC3898 estão relacionadas com a virulência e/ou patogenicidade de X. citri subsp. citri 306 e com o desenvolvimento do cancro cítrico. Para isso, foram realizados testes in vitro e in planta dos mutantes obtidos, visando observar alterações fenotípicas. Devido a presença de peptídeo sinal, a ORF XAC2361 foi fundida à proteína fluorescente mCherry (XAC2361-mCherry) para expressão in vivo e avaliação por microscopia de fluorescência. As análises fenotípicas demonstraram que o mutante ΔXAC2361 não apresentou alterações nos sintomas quando avaliado em limoeiro cravo (Citrus limonia Osbeck), porém apresentou menor capacidade de crescimento in vitro, menor motilidade swimming e swarming e menor capacidade de sobrevivência em SDS. Por outro lado,... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In view of the importance of citrus canker in citrus culture worlwide, the bacterium Xanthomonas citri subsp. citri, the causal agent of this disease, has been extensively studied. Brazilian researchers performed the complete sequencing of the genome of X. citri subsp. citri isolate 306 (Xac306), in order to elucidate the genes and mechanisms involved in the pathogenicity and/or virulence of Xac. The ORFs XAC2361 and XAC3898 have different sizes and location in the genome, but have in common the M23 Peptidase domain, whose biological process is predicted to act as cell wall hydrolase, but the actual function of these proteins in Xac is still unknown. This work aimed to evaluate, through site-directed mutagenesis, whether XAC2361 and XAC3898 ORFs are related to the virulence and / or pathogenicity of Xac306 and the development of citrus canker. In order to observe phenotypic changes, in vitro and in planta tests of the mutants were carried out. Due to the presence of signal peptide, the ORF XAC2361 was fused to the mCherry fluorescent protein (XAC2361-mCherry) for in vivo expression and fluorescence microscopy evaluation. Phenotypic analyzes demonstrated that the ΔXAC2361 mutant showed no change in symptoms when evaluated in Mexican lime (Citrus limonia Osbeck), but presented lower in vitro growth capacity, lower swimming and swarming motility, and lower survival capacity in SDS. On the other hand, the ΔXAC3898 mutant showed a significant reduction in virulence, lower in plant... (Complete abstract click electronic access below) / Doutor

Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: Dr. Christian Baron. Includes bibliographical references (leaves 94-101).

The role of viral proteins in the pathogenesis of infectious hematopoietic necrosis virus (IHNV) was studied at the molecular level. The expression of the viral genes at the protein and RNA level, and their cellular localization, were characterized to further our understanding of viral pathogenesis. The pathogenic effect of individual viral proteins was also investigated and a method for detecting viral RNA in infected fish tissues was developed. The polarity of transcription was confirmed in terms of the relative amounts of each viral protein. Also, cells treated with glycosylation inhibitors did not exhibit cytopathic effect, demonstrating that a functioning host glycosylation system is necessary for viral replication. These studies also revealed a previously undescribed non-glycosylated protein, S, which appeared to be virus-encoded. The expression of the nonvirion protein (NV), was also detected in infected kidney tissues. The location of M2 and NV in the cell was found to be the nucleus and cytoplasm. The expression of the NV gene was further analyzed at the level of transcription and the regulation signals for IHNV transcription were investigated. Unique transcriptional initiation and terminational signals for the fish lyssa-like rhabdoviruses were identified. The transcriptional initiation signal, 3'-CGUG-5', was distinctly different from that of the other rhabdoviruses, 3'-UUGU-5'. The role of the M2 and NV proteins in viral pathogenesis was investigated by transient expression of these proteins individually in cultured fish cells. The M2 protein alone resulted in inhibition of host-directed gene expression at the level of transcription and induction of nuclear fragmentation. The NV protein was not involved in the regulation of the host gene expression, but was involved in another type of cytopathic effect characterized as cell rounding. This is the first biological function attributed to the NV protein. A PCR method was developed for detecting IHNV N-specific RNA in formalin-fixed, paraffin-embedded fish tissues. The method is sensitive and specific. The technique is capable of detecting viral RNA in samples that have been remained at room temperature in 10% buffered formalin for over 2 years. / Graduation date: 1997

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

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

Bacillus cereus causes emesis and/or diarrhea following ingestion of contaminated food due to the production of emetic toxins and enterotoxins. SYBR Green I is used as an intercalating dye and its florescence increases as a result of DNA amplification during real-time PCR. A second-derivative plot is obtained at the end of the PCR run, where amplicons are differentiated based on their DNA melting temperature (Tm). DNA was extracted from Tryptic Soy Broth (TSB) and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus at cell densities of 10',106,105,104,0,102, and 101 cfu/ml. In order to detect the multiple virulence determinants in pathogenic B. cereus, specific primers were used to target three enterotoxigenic genes (hblC, nheA, and hblA), followed by melt-curve analysis to confirm identity. Conditions used for this experiment allowed for the reproducible distinction of melt curves (characteristic Tm) for each amplicon (hblC = 74.5°C in TSB and 75°C in NFDM; nheA = 78°C; and hblA = 85.5°C in TSB and 84°C in NFDM) with an assay sensitivity of 106 CFU/ml in TSB and 10' CFU/ml in NFDM. B. cereus, nheA expression was examined in cells grown in TSB using transcript-specific, real-time nucleic acid sequence-based amplification (NASBA) with SYBR Green II. NASBA was applied to ascertain relative levels of nheA expression, when cells were subjected to subinhibitory levels of chloramphenicol as a stressor. B. cereus demonstrated consistently high levels of nheA expression at 15 hours when grown in TSB containing subinhibitory concentration (SIC) chloramphenicol (15.625 mg/ml). Relative levels of nheA expression differed in stressed B. cereus cells grown during the 30 hours incubation. / Department of Biology