Enterohaemorrhagic Escherichia coli (EHEC) have recently emerged as significant zoonotic pathogens. O157:H7 is one of the most common EHEC serotypes associated with human disease, which is transmitted faeco-orally from a bovine reservoir. EHEC O157:H7 preferentially colonises the bovine terminal rectum (BTR). Injection of virulence factors by type-III secretion is necessary for colonisation of cattle and results in re-modelling of the host cytoskeleton. Flagella machinery is evolutionarily related to the Type III secretion apparatus and O157 strains lacking H7 flagella show reduced adherence to the BTR. Vaccination with FliC, the main component of H7 flagella, has the potential to protect cattle against E. coli O157:H7 infection. The focus of this work was to investigate the molecular basis for H7 flagella binding to the BTR, in order to understand the basis for FliCH7 being an immuno-protective antigen. H7 flagella were shown to adhere across the surface and penetrate into BTR epithelial cells. Both the FliC shaft and the FliD cap components of flagella filaments showed the capacity to adhere to BTR epithelial cells. Preliminary studies indicate that the current FliCH7 vaccination of cattle results in FliD-specific antibodies where oral challenge with O157:H7 does not. FliD is more conserved than FliCH7, which contains a predicted 88aa structural insertion, but variation occurs along the full length of the FliD protein. There was no evidence for post-translational modification of FliCH7. A number of actin binding proteins were identified as potential FliC and FliD binding partners from BTR epithelial cell lysates. From this, a panel of purified galectin-4, cofilin-1 and βγ-actin was used to compare binding of flagella from different pathogens. H7 flagella bound more to cofilin-1 than βγ-actin, whereas phase-1 and phase-2 flagella from Salmonella Typhimurium bound more to βγ-actin, than to cofilin-1. Size-exclusion chromatography indicated that cofilin-1 alters H7 flagella filament polymerisation dynamics. αβ-ctin polymerisation and depolymerisation experiments indicate that H7, phase-1 and phase-2 flagella interactions with actin affect actin dynamics.

Antibiotic resistance is a serious global public health problem. ESBLs are enzymes that destroy expanded-spectrum beta-lactam antibiotics rendering these drugs ineffective. Infection with ESBL-producing K.pneumoniae are hard to treat and result in longer hospital stay and higher mortality rates. The Clinical Laboratory Standard Institute (CLSI) have standard methods for detection of ESBL producing strains of bacteria in infected patients to guide antibiotic therapy, reduce the risk of mortality and risk of transmission. The presence of K.pneumoniae and E.coli which produce ESBLs have been confirmed in natural environments such as soil and water but no standard methods exist to identify directly and quantify these bacteria to understand the risk of human exposure in these settings. The purpose of this research is to assess the ability of an agar dilution method, using a differential agar Bio-Rad Rapid E.coli 2 agar utilized in environmental water quality studies, to identify correctly ESBL-producing K.pneumoniae. The minimum inhibitory concentration (MIC) of ceftriaxone antibiotic for wild-type ESBL producing K.pneumoniae isolates were compared on Mueller-Hinton broth (MHB) and Bio-Rad Rapid E.coli 2 agar. Using the MIC values, the isolates were classified as susceptible, intermediate or resistant. The MIC of wild-type strains of K.pneumoniae were above 4μg/mL for both methods on all susceptibility tests performed. The results of this research suggest that Bio-Rad Agar dilution method performed well, correctly identifying these strains as resistant to ceftriaxone, an indication of ESBL production. The Bio-Rad agar dilution method can be considered as a viable standard method for direct identification of ESBL-producing K.pneumoniae in natural environments.

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Made available in DSpace on 2015-04-11T13:38:45Z (GMT). No. of bitstreams: 1 Bruna Lovizutto Protti Wohlke.pdf: 1421829 bytes, checksum: 1654b1beeb77d5566e82213f52b50a17 (MD5) Previous issue date: 2012-03-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A malária continua a ser a maior causa de morbidade e mortalidade mundial com até três milhões de mortes anuais. O tratamento da malária, causada por Plasmodium. falciparum, dependeu por décadas do uso da aminoquinolina cloroquina. Contudo a resistência à cloroquina em uma escala global expôs a capacidade com que o parasita pode desenvolver resistência a drogas. É, portanto, necessário o desenvolvimento de estudos que assegurem que drogas mais eficazes sejam descobertas de forma sustentável e que novos alvos moleculares para agentes antimalária sejam revelados. Através de análises de biologia celular e molecular foi possível sequenciar o genoma de P. falciparum e identificou-se novos alvos alvos terapêuticos antimalária. A proteína alvo estudada neste projeto foi a Hipoxantina guanina fosforribosiltransferase do P. falciparum, que está relacionada com a via de recuperação das purinas .O gene da enzima foi identificado no genoma do P. falciparum, e produzido por síntese química com códons preferenciais de Escherichia coli, clonado em um forte promotor de expressão para esta bactéria, expresso e a proteína recombinante purificada mostrou-se ativa. A enzima será utilizada futuramente em estudos de binding e inibição com novos compostos químicos e/ou componentes de extratos de microrganismos e plantas amazônicas

Sewage pollution from humans, animal and domestic sources (land and agricultural run-off) are recognized as a major cause of deteriorating water quality along Australia's coastline. Management of water quality has primarily relied on the use of bacterial indicator methods. However the validity and source-specificity of these methods have been met with increasing reservations for several years now. A relatively recent methodology uses a different chemical biomarker approach using 'sterols', a group of compounds related to the common bio-membrane lipid cholesterol and its derivatives. Sterols can offer an additional diagnostic tool to distinguish and discriminate between sources of faecal contamination in marine, freshwater and estuarine environments in both sediments and the water column. This study investigates for the first time, the degradation of coprostanol and selected faecal sterols in 'natural' sediments from a highly mixed (marine and estuarine) sub-tropical environment following a simulated pollution event (primary effluent); the use of faecal sterols as an additional indicator for determining non-point source sewage discharges at popular anchorages in the Moreton Bay and Gold Coast Broadwater system; and the use of sterol ratios in the determination of the fate and transportation of nutrients from a Sewage Treatment Plant (STP) point-source outlet pipe during plant malfunction. The microcosm degradation experiment revealed that faecal and selected sterols are continually synthesised and degraded over time by auto- and hetero trophic organisms within the sediment matrix. Coprostanol was the only sterol to degrade continually, with only minor fluctuations over a time period of two months. Results from this degradation experiment further revealed a sharp decline of coprostanol within the first week. From this it could be concluded that, without any further addition, external inputs of coprostanol are reduced to background levels within this time period. Therefore, removal of coprostanol after six days was 94% and 73% in mud and sand, respectively. The removal of coprostanol was much higher in mud than sand, reflecting a higher level of microbial activity in muddy sediments for assimilation of sterols. The field study undertaken at popular anchorages in Moreton Bay and the Gold Coast Broadwater revealed extremely low levels of sterols and bacterial indicators over both a spatial and temporal scale consistent with a shallow, oligotrophic, highly dynamic sand dominated system. Even though sterols analysed were found at extremely low levels (mostly in the nano-gram range), they were found to be highly correlated and were successful in identifying an unexpected once off pollution event from a point source at Moreton Bay Island. Other than this one incident, both sterol and bacterial levels were consistently low even when anchorages were at full capacity. Thus, sewage from recreational vessels was found to have very little, if any, effect on the water quality at anchorages in Moreton Bay and Gold Coast Broadwater. The point-source study conducted during a local sewage treatment plant malfunction revealed that even though absolute concentrations of sterols did not change during this event, the distribution of sterols within the samples changed, hence changing the sterol ratios. Further, nutrients (mainly nitrogen) can be transported several kilometres by currents, flocculate out of the water column and settle out into the sediment in areas with low tidal and hydrological flushing. There, the nutrients can cause in situ production of sterols in sediments changing sterol ratios. Overall, this study revealed that analyses of sterol biomarkers have the potential to indicate nutrient inputs (such as nitrogen) as well as sewage, post-hoc pollution events at extremely low levels/high dilutions in coastal sediments.

The fitness and macromolecular composition of the gram-negative bacterium E.coli are governed by a seemingly insurmountable level of complexity. However, simple phenomenological measures may be found that describe its systems-level response to a variety of inputs. This thesis explores phenomenological approaches providing accurate quantitative descriptions of complex systems in E.coli. Chapter 1 examines the relationship between unnecessary protein production and growth rate in E.coli. It was previously unknown whether the negative effects on growth rate due to multiple unnecessary protein fractions would add linearly or collectively to produce a nonlinear response. Within the regime of this thesis, it appears that the interplay between growth rate and protein is consistent with a non-interacting model. We do not need to account for complex interaction between system components. Appendix A describes a novel technique for real-time measurement of messenger RNA in single living E.coli cells. Using this technique, one may accurately describe the transcriptional response of gene networks in single cells. / Physics

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Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in travellers and young children in developing countries. We previously characterized three vaccine candidates (MipA, Skp, and ETEC_2479) which effectively protected mice in an intranasal ETEC challenge model after immunization. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. In this study, we examined the potential of these antigens to affect the host intestinal microbiota and subsequently found no significant impact on healthy of host after vaccination. In addition, we also optimized the types of adjuvants and forms of antigens and evaluated the efficacy in a mouse intranasal challenge model. Enterohemorrhagic Escherichia coli (EHEC) is an emerging zoonotic pathogen that cause global public health threads. EHEC possesses the potential to cause gastroenteritis, hemorrhagic colitis and hemolytic uremic syndrome (HUS), which may lead to renal failure. Type III secretion system (T3SS) is a hallmark of EHEC, characterized by the needle-like structure and a variety of effectors injected into host cells. NleB, one of T3SS effectors, is a glycosyltransferase with the ability to catalyze the transfer of N-acetyl-D-glucosamine (N-GlcNAc) to host proteins to suppress the activation of NF-kB signaling pathway. In this study, we employed luminescence-based glycosyltransferase assay and high-throughput screening using a chemical library of various compounds. A total of 128 chemicals was selected with significant inhibition on NleB glycosyltransferase activity for further pharmaceutical study as novel therapy against EHEC infection.

There is a need for a sensitive, specific, rapid and cost-effective assay that can be used as an early warning signal of contamination of aquatic ecosystems. The purpose of this work was to develop a sensitive stress-specific microbial bioreporter responsive to pro-oxidants. Furthermore, the bioreporter was designed to be applicable in environments possibly affected by metal processing activities. An E.coli bioreporter was developed containing a plasmid with the katG promoter sequence as the sensing sequence and with mCherry as the reporter protein. The bioreporter responded to metal pro-oxidants (Cd, As, Zn, Pb, Ag and Ag nanoparticles). A new assay growth-medium was developed and contributed to improve the sensitivity of our assay that has the best detection limit to inorganic pro-oxidants compared to other oxidative-stress sensitive bioreporters in the literature. The bioreporter detected pro-oxidants in environmental samples. The assay has a reasonable sensitivity, however, it still lacks sensitivity to detect pro-oxidants at concentrations lower than those shown to be toxic to many aquatic species. Within-lab reproducibility and robustness were determined to be acceptable. For stress-specific bioreporters to be incorporated in regulative legislations and industrial monitoring programs there is a need to improve the sensitivity of these assays, they need to be calibrated with other relevant pro-oxidants, inter-lab reproducibility needs to be established and robustness to environmental samples needs to be further tested. To further validate the sensitivity and ecotoxicological relevance of the bioreporter as a relevant predictive tool, stress-specific bioreporter assays need to be performed in parallel with traditional ecotoxicological assays using contaminated environmental samples.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20207号 / 理博第4292号 / 新制||理||1616(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 秋山 秋梅, 教授 沼田 英治, 教授 高橋 淑子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM