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Foo, Chuen-hing, and 符傳興. "Bacteremia due to Elizabethkingia and related species." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208519.
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Elizabethkingia spp. is a gram-negative, non-fermenting rod bacterium that is frequently implicated in hospital outbreaks. Elizabethkingia has a high rate of resistance to antibiotics and a shortage of effective parenteral antibiotics usually occurs in intensive care units. Infection includes neonatal sepsis and meningitis. Recently, a new species of Elizabethkingia, which is closely related to E. meningoseptica ATCC 13253 and E. miricola GTC862, was reported as a human pathogen in Central Africa and named E. anophelis. Our investigation involved 27 Elizabethkingia clinical isolates, which were fully identified through phenotypic and genotypic typing. The isolates were identified as E. meningoseptica by VITEK 2 (bioMereux) and Phoneix (Beckton Dickinson) automated bacterial identification systems. We then re-identified the isolates by 16S rRNA gene sequencing; 23 of the 27 strains were identified as E. anophelis and one was identified as E. miricola instead of E. meningoseptica. Subsequently, we evaluated the performance of the Bruker MALDI-TOF MS system for identification of the E. anophelis strains; many were misidentified as E. meningoseptica or were unidentified. All of the strains were correctly re-identified as E. anophelis when the original Bruker database was expanded with the inclusion of 10 E. anophelis clinical isolates and a standard 〖R26 〗^T strain. We also analysed 23 E. anophelis clinical isolates by biochemical tests, antimicrobial susceptibilities tests and pulsed-field gel electrophoresis. From the biochemical investigation of all isolates and type strain, showing that the conventional biochemical tests are not reliable to differentiate E. anophelis from other Elizabethkingia spp. More than 75% of the isolates tested were susceptible to cotrimoxazole, ciprofloxacin, and cefoperazone-sulbactam, however they were all resistant to aminoglycosides and beta-lactam drugs except one strain. At the PFGE investigation all the strains were not clonally related as shown by PFGE and displayed distinct PFGE fingerprints.<br>published_or_final_version<br>Medicine<br>Master<br>Master of Medical Sciences
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O'Hara, Heather Marie. "Comparison of the different spectra of some selected bacteria." Thesis, Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/27161.
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Kondacs, Laszlo. "Novel substrates for the improved detection and identification of pathogenic bacteria." Thesis, University of Sunderland, 2018. http://sure.sunderland.ac.uk/10222/.
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Many diseases are caused by pathogenic bacteria. A key example of this is sepsis, which is mostly caused by staphylococci and Gram-negative bacteria. In addition, the highly resistant ESKAPE pathogens are responsible for the majority of hospital acquired infections. In order to treat bacterial infections effectively, and to avoid promoting bacterial resistance against antibacterial drugs, the correct agents must be used, for which in turn the detection and identification of pathogenic strains is essential. This research aims to develop selective chromogenic culture media, by introducing new antibacterial agents for the improved selectivity and new chromogenic substrates for selective visualisation of certain bacterial strains. The intention of the major part of this work was to inhibit the growth of commensal bacteria in clinical samples, as they mask the growth of the infection-causing bacteria. New and known compounds were prepared for 3 evaluation as alanine racemase inhibitors. The compounds were tested on a range of clinical pathogenic and non-pathogenic bacterial strains. The molecules developed were based on the amino acid alanine and utilised bioisosteres and other replacements for the carboxylic acid moiety. Key compounds targeted included alanylmethanesulfonamide 27-L, 1-aminoethyl5-oxo-1,2,4-oxadiazole 33-L and 1-aminoethyltetrazole 32a-L. Each compound was tested initially as the alanyl-X dipeptide form. While most of the alanine bioisosteres were known structures, their novel peptide derivatives required synthetic development using both solution and solid phase techniques. The solid phase synthesis of several C-terminal 1aminoethyltetrazole peptides was successfully established by using 2-chlorotrityl chloride resin. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These compounds were shown to provide differentiation between Salmonella and Escherichia coli, or enterococci and streptococci. This work also gave a useful comparison between the different alanine bioisosteres, and showed the importance of di- and oligopeptide permease systems in order to reach sufficient bacterial activity. The microbiological activity of 1- aminoethyltetrazole peptide derivatives was studied in more detail, due to their potential in clinical applications for the diagnosis of food poisoning. In other work, also directed towards the rapid and selective detection and identification of pathogenic bacteria in a clinical environment, new chromogenic substrates were prepared. Each of these compounds contained a chromogen with a phenoxazin-3-one scaffold linked to an amino acid residue. The purpose of the amino acid is to act as a unit recognised and cleaved by specific hydrolytic bacterial enzymes. Upon liberation, electronic differences between the conjugated and free forms of the chromogen resulted in the development of distinct colour changes, which provide the basis of 4 bacterial detection and identification. Synthetic methods have been developed for the efficient and economical production of this series of substrates. After preparation, these compounds were tested against a panel of clinically relevant bacteria. The aim of these substrates was to present an alternative substrate for (N-β-alanyl)-7-amino-1-pentylphenoxazine-3-one 86a, which is applied commercially in chromID® Pseudomonas aeruginosa chromogenic medium designed for the clinical detection of P.aeruginosa. The new substrates are designed to fully explore the chemical space of phenoxazinonebased chromogenic substrates, and to decrease the colour, as substrate 86a causes significant background colour in culture media. The future application of these substrates in chromogenic media resides in their potential to advance the identification of specific pathogenic bacteria and to thus facilitate the treatment of bacterial infections.
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Li, Kwan-hing. "Identification of bacterial pathogens by 16S ribosomal RNA gene sequencing." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31971982.
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Li, Kwan-hing, and 李群卿. "Identification of bacterial pathogens by 16S ribosomal RNA gene sequencing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31971982.
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Yeung, Shiu-yan, and 楊兆恩. "Update and evaluation of 16SpathDB, an automated comprehensive database for identification of medically important bacteria by 16S rRNA gene sequencing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193552.
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Identification of pathogens is one of the important duties of clinical microbiology laboratory. Traditionally, phenotypic tests are used to identify the bacteria. However, due to some limitations of the phenotypic tests, the bacteria may not be identified sometimes and cannot be identified promptly. 16S rRNA gene sequencing is a rapid and accurate method to achieve this target. It is especially useful for identify rare or slow growing bacteria. However, the interpretation of the 16S rRNA gene sequencing result is one of the challenging duties to laboratory technicians and microbiologists. Apart from the well known 16S rRNA gene databases such as Genbank, The Ribosomal Database Project (RDP-II), MicroSeq databases, Ribosomal Differentiation of medical Microorganism database (RIDOM), SmartGene IDNS, 16SpathDB is an automated and comprehensive database for interpret the 16S rRNA gene result. The 16SpathDB first version was established in 2011. In this study, 16SpathDB was updated based on the all clinical important bacteria present in the 10th edition of the Manual of Clinical Microbiology (MCM)(Versalovic. et al., 2011) into this new version of database, 16SpathDB 2.0. The database was evaluated by using 689 16S rRNA gene sequences from 689 complete genomes of medically important bacteria. Among the 689 16S rRNA gene sequences, none was wrongly identified by 16SpathDB 2.0, with 247 (35.8%) 16S rRNA gene sequences reported in only one single bacterial species with more than 98% nucleotide identity with the query sequence (category 1), 440 (63.9%) reported as more than one bacterial species having more than 98% nucleotide identity with the query sequence (category 2), 2 (0.3%) reported to the genus level (category 3), and none reported as “no species in 16SpathDB 2.0 found to be sharing high nucleotide identity to your query sequence” (category 4). 16SpathDB 2.0 is an updated, automated, user-friendly, efficient and accurate database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories.<br>published_or_final_version<br>Microbiology<br>Master<br>Master of Medical Sciences
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Male, Abigail. "Identification of inhibitors of protein-protein interactions essential for virulence in pathogenic bacteria." Thesis, University of Southampton, 2014. https://eprints.soton.ac.uk/369351/.
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There is a continous requirement for broad-spectrum post-exposure antibiotic therapeutics. Meeting this challenge relies on the production of compounds that successfully disrupt bacterial systems identified as both conserved and essential. Here, inhibitors of protein-protein interactions involved in the Phage shock protein response and toxin internalisation, within Burkholderia pseudomallei and Bacillus anthracis, respectively have been identified. This was achieved using a high-throughput screen that combines a bacterial reverse two-hybrid system and an intein-mediated method for the generation of cyclic peptide libraries. A reverse two-hybrid system for Burkholderia pseudomallei PspA homodimerisation was constructed, alongside a heterodimeric system for the interaction between Bacillus anthracis protective antigen and the mammalian receptor, capillary morphogenesis gene-2. From both systems a series of peptide sequences were identified with potential inhibitory activity within the reverse two-hybrid system. These compounds were synthesised and their activity assessed using a selection of in vitro assays. This study identified two cyclic peptides sequences active in the reverse two-hybrid system against PspA oligomerisation; which were not active in vitro. In contrast, three linear peptides were isolated that demonstrated the ability to disrupt the interaction between protective antigen and the mammalian receptor, with one binding specifically to the receptor. This linear inhibitor provides the foundation for the development of a more potent antimicrobial for the arsenal against Bacillus anthracis.
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Kaittanis, Charalambos. "Magnetic nanosensors for multiplexed bacterial pathogenesis identification." Doctoral diss., University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4610.
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Developing diagnostic modalities that utilize nanomaterials and miniaturized detectors can have an impact in point-of-care diagnostics. Diagnostic systems that (i) are sensitive, robust, and portable, (ii) allow detection in clinical samples, (iii) require minimal sample preparation yielding results quickly, and (iv) can simultaneously quantify multiple targets, would have a great potential in biomedical research and public healthcare. Bacterial infections still cause pathogenesis throughout the world (Chapter I). The emergence of multi-drug resistant strains, the potential appearance of bacterial pandemics, the increased occurrence of bacterial nosocomial infections, the wide-scale food poisoning incidents and the use of bacteria in biowarfare highlight the need for designing novel bacterial-sensing modalities. Among the most prominent disease-causing bacteria are strains of Escherichia coli, like the E. coli O157:H7 that produces the Shiga-like toxin (Stx). Apart from diarrheagenic E. coli strains, others cause disease varying from hemolytic uremic syndrome and urinary tract infections to septicemia and meningitis. Therefore, the detection of E. coli needs to be performed fast and reliably in diverse environmental and clinical samples. Similarly, Mycobacterium avium spp. paratuberculosis (MAP), a fastidious microorganism that causes Johne's disease in cattle and has been implicated in Crohn's disease (CD) etiology, is found in products from infected animals and clinical samples from CD patients, making MAP an excellent proof-of-principle model. Recently, magnetic relaxation nanosensors (MRnS) provided the first applications of improved diagnostics with high sensitivity and specificity.; Furthermore, these MRnS achieved equally fast IS900 detection even in crude DNA extracts, outperforming the gold standard diagnostic method of nested Polymerase Chain Reaction (nPCR). Likewise, the MRnS detected IS900 with unprecedented sensitivity and specificity in clinical isolates obtained from blood and biopsies of CD patients, indicating the clinical utility of these nanosensors. Subsequently, we designed MRnS for the detection of MAP via surface-marker recognition in complex matrices (Chapter III). Milk and blood samples containing various concentrations of MAP were screened and quantified without any processing via MRnS, obtaining dynamic concentration-dependent curves within an hour. The MAP MRnS were able not only to identify their target in the presence of interferences from other Gram positive and Gram negative bacteria, but could differentiate MAP among other mycobacteria including Mycobacterium tuberculosis. In addition, detection of MAP was performed in clinical isolates from CD patients and homogenized tissues from Johne's disease cattle, demonstrating for the first time the rapid identification of bacteria in produce, as well as clinical and environmental samples. However, comparing the unique MAP quantification patterns with literature-available trends of other targets, we were prompted to elucidate the underlying mechanism of this novel behavior (Chapter IV). We hypothesized that the nanoparticle valency--the amount of probe on the surface of the MRnS --may have modulated the changes in the relaxation times (delta]T2) upon MRnS--target association. To address this, we prepared MAP MRnS with high and low anti-MAP antibody levels using the same nanoparticle formulation. Results corroborated our hypothesis, but to further bolster it we investigated if this behavior is target-size-independent.; Hence utilizing small-molecule- and antibody-carrying MRnS, we detected cancer cells in blood, observing similar detection patterns that resembled those of the bacterial studies. Notably, a single cancer cell was identified via high-valency small-molecule MRnS, having grave importance in cancer diagnostics because a single cancer cell progenitor in circulation can effectively initiate the metastatic process. Apart from cells, we also detected the Cholera Toxin B subunit with valencly-engineered MRnS, observing similar to the cellular targets' diagnostic profiling behavior. Finally, as bacterial drug resistance is of grave healthcare importance, we utilized MRnS for the assessment of bacterial metabolism and drug susceptibility (Chapter V). Contrary to spectophotometric and visual nanosensors, their magnetic counterparts were able to quickly assess bacterial carbohydrate uptake and sensitivity to antibiotics even in blood. Two MRnS-based assay formats were devised relying on either the Concanavalin A (Con A)-induced clustering of polysaccharide-coated nanoparticles or the association between free carbohydrates and Con A-carrying MRnS. Overall, taking together these results, as well as those on pathogen detection and the recent instrumentation advancements, the use of MRnS in the clinic, the lab and the field should be anticipated.; Nucleic acids, proteins, viruses and enzymatic activity were probed, yet neither large targets (for instance bacterial and mammalian cells) nor multiple bacterial disease parameters have been simultaneously monitored, in order to provide thorough information for clinical decision making. Therefore, the goal of this study was to utilize MRnS for the sensitive identification of multiple targets associated with bacterial pathogenesis, while monitoring virulence factors at the microorganism, nucleic acid and virulence factor levels, to facilitate improved diagnosis and optimal treatment regimes. To demonstrate the versatility of MRnS, we used MAP as our model system, as well as several other pathogens and eukaryotic cell lines. In initial studies, we developed MRnS suitable for biomedical applications (Chapter II). The resulting MRnS were composed of an iron oxide core, which was caged within a biodegradable polymeric coating that could be further functionalized for the attachment of molecular probes. We demonstrated that depending on the polymer used the physical and chemical properties of the MRnS can be tailored. Furthermore, we investigated the role of polymer in the enzyme-mimicking activity of MRnS, which may lead to the development of optimized colorimetric in vitro diagnostic systems such as immunoassays and small-molecule-based screening platforms. Additionally, via facile conjugation chemistries, we prepared bacterium-specific MRnS for the detection of nucleic acid signatures (Chapter III). Considering that MAP DNA can be detected in clinical samples and isolates from CD patients via laborious isolation and amplification procedures requiring several days, MRnS detected MAP's IS900 nucleic acid marker up to a single MAP genome copy detection within 30 minutes.<br>ID: 028916614; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (Ph.D.)--University of Central Florida, 2010.; Includes bibliographical references (p. 139-150).<br>Ph.D.<br>Doctorate<br>Burnett School of Biomedical Sciences<br>Medicine
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Pierce, Carrie. "High throughput mass spectrometry for microbial identification." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/43741.
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Bacteria cause significant morbidity and mortality throughout the world, including deadly diseases such as tuberculosis, meningitis, cholera, and pneumonia. Timely and accurate bacterial identification is critical in areas such as clinical diagnostics, environmental monitoring, food safety, water and air quality assessment, and identification of biological threat agents. At present, there is an established need for high throughput, sensitive, selective, and rapid methods for the detection of pathogenic bacteria, as existing methods, while nominally effective, have failed to sufficiently reduce the massive impact of bacterial contamination and infection. The work presented in this thesis focuses on addressing this need and augmenting conventional microorganism research through development of mass spectrometry (MS)-based proteomic applications. MS, a well established tool for addressing biological problems, offers a broad range of laboratory procedures that can be used for taxonomic classification and identification of microorganisms. These methods provide a powerful complement to many of the widely used molecular biology approaches and play critical functions in various fields of science. While implementation of modern biomolecule-identifying instrumentation, such as MS, has long been postulated to have a role in the microbiology laboratory, it has yet to be accepted on a large scale. Described in this document are MS methods that erect strong foundations on which new bacterial diagnostics may be based. A general introduction on key aspects of this work is presented in Chapter 1, where different approaches for detection of pathogenic bacteria are reviewed, and an overview regarding MS and microbial identification is provided. Chapter 2 presents the first implementation of microbial identification via rapid, open air Direct Analysis in Real Time MS (DART MS) to generate ions directly from microbial samples, including the disease-causing bacteria, Coxiella burnetii, Streptococcus pyogenes, and Escherichia coli. Chapter 3 expands on whole cell C. burnetii MS analysis and presents a rapid differentiation method to the strain-level for C. burnetii using mass profiling/fingerprinting matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and multivariate pattern recognition. Chapter 4 presents a unique "top-down" proteomics approach using 15N-labeled bacteriophage amplification coupled with MALDI-TOF MS as a detector for the rapid and selective identification of Staphylococcus aureus. Chapter 5 extends the idea of using isotopically labeled bacteriophage amplification by implementing a "bottom-up" proteomics approach that not only identifies S. aureus in a sample, but also quantifies the bacterial concentration in the sample using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) as a detector. In conclusion, Chapter 6, summarizes and contextualizes the work presented in this dissertation, and outlines how future research can build upon the experimentation detailed in this document.
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Gamieldien, Junaid. "Novel genomic approaches for the identification of virulence genes and drug targets in pathogenic bacteria." Thesis, University of the Western Cape, 2001. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_4400_1185438906.
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<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p>
<p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash<br>associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>
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Boucher, David. "Identification and characterisation of in vivo expressed genes of Pasteurella multocida." Monash University, Dept. of Microbiology, 2004. http://arrow.monash.edu.au/hdl/1959.1/9657.
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Pollard, Stephanie Kay. "Identification of food safety risks at Virginia farmers' markets and development of a food safety plan to help farmers market managers." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/78196.
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The growing popularity of farmers' markets coupled with a high percentage of produce-related foodborne outbreaks highlights the need for an emphasis on food safety within these markets to protect farmers, patrons and local economies. The number of farmers' markets registered in the United States has almost tripled in the last 15 years. Fresh produce constitutes the majority of food sold at farmers'markets. Between 1998 and 2008, raw produce accounted for almost half of the 4,589 foodborne illness outbreaks linked to a specific commodity. This research was conducted to identify practices at farmers' markets which may contribute to an increased risk of contamination, assess the microbial quality of produce sold at farmers' markets, as well as to develop a food safety management plan template for market managers to utilize to build their own food safety plan.
Using an observational data collection method, risky food safety practices were identified at Southwest Virginia farmers' markets. While market managers and vendors in three of the five markets observed had formal food safety training, numerous risky food safety behaviors were still observed including temperature abuse, cross contamination opportunities, and poor personal hygiene and sanitation. Additionally, the microbial quality of produce from Southwest Virginia farmers' markets was compared to produce sold at retail using culture based microbiological plating and molecular methods. Total aerobic bacteria and coliforms were enumerated, and the presence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and generic E. coli were determined. A significantly greater quantity of total aerobic bacteria was isolated from farmers' market leafy greens, onions and tomatoes when compared to a retail grocery store (P=0.0011, P=0.0395, and P<0.0001, respectively). Additionally, one or more target pathogen was isolated from 28 farmers' market samples and 16 retail grocery store samples. The observed risky food safety behaviors along with the bacterial data collected emphasize the need for a pathogen reduction focus on fresh produce not only at farmers' markets, but also with growers and other retail outlets.
To help promote proper food safety practices at farmers' markets, a farmers' market food safety management plan (FSMP) template was developed to address the top five risk factors contributing to foodborne illness as identified by the Centers for Disease Control and Prevention (CDC). The FSMP was evaluated for practicality and feasibility through interviews with market mangers in North Carolina and Virginia. Most market managers (66.7%) agreed that the FSMP was practical for their market while only 33.3% agreed that they could implement the plan immediately. Revisions suggested to the FSMP will be made and it will be made available in Virginia and North Carolina in spring 2016.<br>Ph. D.
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Nejad, Pajand. "Pathogenic and ice-nucleation active (INA) bacteria causing dieback of willows in short rotation forestry /." Uppsala : Dept. of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, 2005. http://epsilon.slu.se/200524.pdf.
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Woo, Chiu-yat Patrick, and 胡釗逸. "Defining novel clinical syndromes and emerging pathogens." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B27497343.
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Beshr, Ghamdan [Verfasser], and Alexander [Akademischer Betreuer] Titz. "Biochemical characterization and ligand identification of Pseudomonas aeruginosa lectins and their orthologs in other pathogenic bacteria / Ghamdan Beshr ; Betreuer: Alexander Titz." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2018. http://d-nb.info/1167770781/34.
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Golanowska, Malgorzata. "Characterization of Dickeya solani strains and identification of bacterial and plant signals involved in induction of virulence." Thesis, Lyon, INSA, 2015. http://www.theses.fr/2015ISAL0087/document.
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Les bactéries pectinolytiques des genres Pectobacterium (ancien nom Erwinia carotovora) et Dickeya (ancien nom Erwinia chrysanthemi) sont les agents des maladies de la jambe noire et de la pourriture molle. Ils provoquent des dommages aux cultures et des pertes économiques élevées. Les pertes causées par les bactéries pectinolytiques sont évaluées à environ 2 à 10% du rendement de pommes de terre, en fonction de l'année. En 2009, les pertes en pommes de terre en Europe ont été estimées à 250 millions d'euros. Au cours des dernières années, des souches de Dickeya ont été de plus en plus souvent isolées de plantes malades en Pologne, en France et d'autres pays européens. Le genre Dickeya est un groupe très diversifié, qui, selon la nomenclature actuelle contient sept espèces: D. aquatica, D. chrysanthemi, D. dadantii, D. dianthicola, D. paradisiaca, D. solani et D. zeae. Les résultats récents, obtenus dans différents pays européens, indiquent qu'un nouveau groupe de souches de Dickeya peut infecter efficacement les plantes de pomme de terre et causer des symptômes de la maladie en climat tempéré. Les souches de D. solani sont considérés comme plus agressives que les autres bactéries causant la jambe noire. Une analyse préliminaire a suggéré qu’elles ont besoin de plus faibles températures optimales pour le développement de la maladie ainsi que de niveaux d'inoculum inférieurs pour la propagation de l'infection. Elles semblent avoir une plus forte capacité à coloniser les racines de plantes de pomme de terre et à se propager à travers le système vasculaire de la plante. Les souches de D. solani produisent une large gamme d’enzymes dégradant de la paroi cellulaire végétale, qui sont les principaux facteurs de virulence. Les objectifs de l'étude étaient les suivants: 1) la caractérisation phénotypique et génotypique des souches de D. solani isolées dans des pays ayant des conditions climatiques différentes: Pologne, Finlande et Israël, 2) l'étude de l’influence d'extraits de pomme de terre sur l'expression de quelques gènes sélectionnés de D. solani: pelD, pelL, tssk, lfaA, 3) la génomique comparative de dix souches de D. solani, basée sur 4 génomes séquencés pour cette étude et 6 séquences génomiques disponibles dans la base de données GenBank. En conclusion, toutes les études génomiques ont montré que les souches de D. solani forment un groupe très homogène. Cependant, leur analyse phénotypique révèle une certaine variabilité entre les souches provenant de différentes conditions climatiques. La raison des variations observées dans les traits phénotypiques peut être liée à la régulation de l'expression des gènes codant les facteurs de virulence qui peuvent être influencés par la température, le pH, la carence en fer ou en oxygène et la disponibilité en azote, ainsi que par la présence de composés spécifiques des tissus végétaux<br>Dickeya solani is a species consisting of newly emerged plant pathogenic bacteria that cause blackleg and soft rot diseases. They are responsible for great damages to potato plantations in most of European countries. D. solani strains produce a wide range of plant cell-wall degrading enzymes which are the main virulence factors. The aims of the study were: 1) phenotypic and genotypic characterizations of the D. solani strains isolated in countries with different climatic conditions: Poland, Finland and Israel, 2) study of the potato tuber extract influence on the expression of a few selected D. solani genes : pelD, pelL, tssK, lfaA,3) comparative genomics of ten D. solani strains, performed on 4 genomes sequenced for this study and 6 genome sequences available in the GenBank databases. The results showed that the strains from different climatic conditions have identical profiles in rep-PCR (with three different primers) and in Restriction Fragments Lenght Polymorphism-Pulse Field Gel Electrophoresis. However, they do differ phenotypically, especially in the activity of plant cell-wall degrading enzymes. Polish strains have higher activities of pectinolytic, cellulolytic and proteolytic enzymes than Finnish and Israeli strains. D. solani mutants in the pelD, pelL, tssK, lfaA genes were constructed by site-specific mutagenesis. The highest induction by plant extracts was observed for the lfaA gene. The expression of pelL is also induced by plant derived signal(s), but not that of pelD and tssK. Comparative genomics helped to elucidate the D. solani pangenome. The 10 D. solani strains genomes are coding for a total of 41 947 proteins which were grouped into 5 045 Orthologous Groups, 3 809 belonging to the core genome, 413 to the accessory genome and 823 to the unique genome. Some pathogenicity-related genes as well as their regulators were selected on the basis of the knowledge available for D. dadantii 3937, the most studied Dickeya strain, which belongs to a closely related species. Analysis of their protein sequence showed no difference in the sequence of those genes within the 10 genomes. All the genetic studies proved that D. solani strains form a very homogenous group. On the other hand, the phenotypic analysis showed some variability among strains from different climatic conditions. The observed variations in the phenotypic traits can results from a different regulation of the expression of the genes encoding virulence factors which are influenced by temperature, pH, iron deprivation, oxygen and nitrogen availability, as well as by the presence of plant compounds
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Panji, Sumir. "Identification of bacterial pathogenic gene classes subject to diversifying selection." Thesis, University of the Western Cape, 2009. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_5842_1297942831.
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<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo<br>s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>
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Tam, Man-wah. "Identification of bacterial pathogens by 16S ribosomal RNA gene sequencing." Click to view the E-thesis via HKUTO, 2002. http://sunzi.lib.hku.hk/hkuto/record/B31970783.
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譚文華 and Man-wah Tam. "Identification of bacterial pathogens by 16S ribosomal RNA gene sequencing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31970783.
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Chambers, Jacob Richard. "Identification and Characterization of the Hfq protein and small RNAs in Francisella novicida." OpenSIUC, 2011. https://opensiuc.lib.siu.edu/dissertations/370.
Full textAbstract:
Francisella tularensis is the causative agent of the disease tularemia and a potential bioterrorism agent. Few regulators have been identified in this organism and little is known about its genetic regulatory networks. In this dissertation project, culture-based and molecular methods were used to both determine the role of the RNA chaperone protein Hfq and identify potential novel small RNAs in F. tularensis subsp. novicida strain U112. The Hfq protein is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis, and has been shown in several bacteria to interact with small RNAs as a post-transcriptional regulator of mRNA stability and translation. Molecular methods were employed to determine that hfq is potentially transcribed in an operon with both the immediate up- and downstream genes. Phenotypic analysis of two transposon insertions within the hfq ORF revealed that the N-terminal region of the Hfq protein is more important for stress tolerance than the C-terminal end. Complete deletion of hfq resulted in a variety of growth defects under certain stress conditions such as heat-shock, low pH, and oxidative stress. Gene expression of hfq under several of these conditions changed significantly, further suggesting a role for the protein during stress tolerance. Because Hfq likely functions as a global regulator, the expression of several genes in the hfq mutant strain were compared to wild-type and some were significantly altered in particular growth backgrounds. The hfq mutant also exhibited a delayed entry into stationary phase and increased biofilm formation under certain conditions. Shotgun cloning and high-throughput sequencing were used to generate a list of potential sRNAs, an important class of regulators that had yet to be studied in F. novicida. Three candidates were selected and their expression verified using Northern blot analysis and self-ligating RACE. The sRNA transcript designated CISC-1 appears important for certain aspects of cell growth and is differently expressed under several stress conditions. ISC-2 is a transcript that has a minor effect on cell growth during exponential phase, but is upregulated during stationary phase. The third sRNA, ISC-16, is highly conserved among Francisella species and is potentially important for the biosynthesis of bacterial fatty acids. These sRNAs represent an important group of regulators that, along with the Hfq protein, could be important for controlling global gene expression in Francisella.
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Wiltshire, Michael David. "The identification of genes important to the growth of Staphylococcus aureus in in vitro models mimicking infection." Thesis, University of Sheffield, 2001. http://etheses.whiterose.ac.uk/14812/.
Full textAbstract:
Staphylococcus aureus is a major pathogen, which causes a wide range of infections. Despite its obvious clinical importance, little is known about the mechanisms of pathogenesis. An in vitro model mimicking infection was developed in order to identify putative virulence determinants. The model involves the growth of S. aureus in serum under microaerobic conditions. All known virulence factors tested were shown not to be required for growth, or preferentially expressed, in serum. Tn917 transposon libraries of S. aureus were screened to identify genes preferentially expressed in serum, compared to a nutrient-rich growth medium. 73 clones were identified and the transposon insertion site was characterised for 23 of these clones. Analysis of sequence flanking the transposon insertion revealed the identity of the mutated loci. 10 out of 23 sequenced clones, contained transposons inserted within genes involved in the biosynthesis of the aspartate family of amino acids (lysine. threonine, methionine and isoleucine). These were: the two common pathway enzymes; aspartokinase (lysC) , and aspartate semi aldehyde dehydrogenase (asd) , along with; dihydrodipicolinate dehydrogenase (dapA), and cystathionine y-synthase (yjcf) , involved in the biosynthesis oflysine and methionine respectively. Analysis of methionine biosynthesis indicated that S. aureus possesses only a single pathway, which proceeds via cystathionine. Several genes encoding methionine biosynthetic enzymes were found clustered on the S. aureus chromosome. The genes lyse, asd and dapA were found to be encoded by the first three genes of an eight gene operon, which also contains three other genes involved in lysine biosynthesis. This operon named the dap operon, is the major lysine biosynthetic operon of S. aureus. lysC, asd and dapA were all found to be repressed at the transcriptional level primarily by lysine, although factors other than the availability of lysine may be responsible for the regulation of lysine biosynthetic gene expression in serum. lysC, asd and dapA were all found to be expressed in vivo, in a murine pyelonephritis model using both RT-PCR and TaqMan techniques. However, these genes were not found to be important in three murine pathogenicity models. Finally, in addition to the development of a model mimicking infection, and the identification of genes with a potentially important role in vivo, this thesis has enhanced our understanding of both methionine and lysine biosynthesis in S. aureus.
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Zhang, Xueying. "Identification, properties, and application of enterocins produced by enterococcal isolates from foods." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1206069582.
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Leung, Ka-ming, and 梁家銘. "Isolation, identification and establishment of bacterial culture collection of fish pathogens in Hong Kong." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/207649.
Full textAbstract:
The importance of fish culture has been increasing since 1990’s. The steady growth of fish culture helps to ensure a stable supply of fish for human consumption. However, when compared with capture fisheries, production from fish culture is greatly influenced by fish diseases. Outbreaks of fish diseases have caused great economic loss to fish culture. Research has been conducted to understand and reduce the occurrence of fish diseases in fish culture. In Hong Kong, bacterial infection is the most common cause of fish diseases. This project is therefore directed to isolate and identify the causative bacterial pathogen of some fish disease cases with the aim of setting up a local fish disease database for assisting the identification of diseases and improving the understanding of fish diseases in fish farms in Hong Kong. In this project, seven fish disease cases caused by bacteria were investigated with the AFCD officials in Hong Kong. Nine fish disease bacterial pathogens were isolated and identified using different methods (including commercial biochemical test kits, automated system and DNA sequencing). The bacteria identified included Aeromonas hydrophila, Lactococcus garvieae, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus iniae, Vibrio vulnificus and Aeromonas salmonicida. Sensitivity tests to 10 common antibiotics conducted for the identified bacteria showed that spectinomycin is the most broad spectrum antibiotics. In addition, a long-term physical storage of bacterial stock with glycerol and glass beads was established for further research of the identified bacteria. For efficient data analysis, an electronic database using Microsoft Access to hold the identification results and case history of each isolated bacteria was developed. Different data entry forms and reports were also constructed to facilitate easy data entry and data access for users. The three bacteria identification methods were compared for their efficiency and accuracy. Some limitations encountered in this project including time constraints and low accuracy of some biochemical identification tests were discussed and recommendations to overcome these limitations and improvements to the constructed database were made.<br>published_or_final_version<br>Environmental Management<br>Master<br>Master of Science in Environmental Management
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Wright, Lynda J. "Identification and characterisation of components expressed by gram-positive bacterial pathogens during human infection." Thesis, University of Sheffield, 2008. http://etheses.whiterose.ac.uk/10312/.
Full textAbstract:
Gram-positive pathogens are responsible for a wide range of global diseases, including nosocomial infections. The increasing incidence of antibiotic-resistant strains warrants the development of novel therapeutic strategies to combat these organisms.
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Lau, Ken Wan Keung. "The identification of novel marine bacteria, and the construction of single chain fragment variable antibodies for the control of a viral pathogen /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?AMCE%202006%20LAU.
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Richter, Lisa [Verfasser]. "Identification of immunological properties of natural products for the treatment of resistant tumors and bacterial pathogens / Lisa Richter." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2020. http://d-nb.info/1202603734/34.
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Zhou, Binbin. "Identification and characterization of target genes of RRS1-R, a protein conferring resistance in Arabidopsis thaliana to the pathogenic bacterium Ralstonia solanacearum." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2604/.
Full textAbstract:
Ralstonia solanacearum, agent du flétrissement bactérien, affecte près de 200 espèces végétales. Les gènes RRS1-R confèrent à l'écotype d'A. Thaliana Nd-1 une résistance à différentes souches de R. Solanacearum. RRS1-R code une protéine de structure modulaire associant les domaines typiques de nombreuses protéines de résistance TIR-NBS-LRR et un domaine signature de facteurs de transcription WRKY. Dans l'écotype sensible Col-0, le gène RRS1-S code pour une protéine qui présente une structure très semblable. Au cours de ce travail, nous avons montré que les gènes RRS1-R et RRS1-S s'expriment essentiellement dans les cellules du péricycle et les cellules de passage de l'endoderme des racines matures et de la base de l'hypocotyle, cellules qui correspondent aux sites de pénétration des bactéries dans le système vasculaire où elles se multiplient. Nous avons montré que les deux domaines WRKY des protéines codées par ces gènes se fixent spécifiquement aux boites W, reconnues par les facteurs de transcription de la famille WRKY. Nous avons par la suite développé une approche DamID (DNA adenine methyltransferase IDentification) visant à identifier les gènes cibles des protéines RRS1-R et RRS1-S in vivo. L'analyse a été focalisée sur l'identification des gènes cibles de RRS1-R, dans le fond génétique résistant Nd-1 exprimant, ou pas, la protéine d'avirulence PopP2 sous contrôle d'un promoteur inductible. Dans chacun des cas le séquençage d'une centaine de FARMs (Fragments Associated to RRS1-driven Methylation) a permis de proposer des cibles potentielles et un modèle de fonctionnement de RRS1-R comme régulateur transcriptionel. Ce travail se poursuit par une analyse globale au niveau du génome, grâce au séquençage haut débit des FARMS et par l'étude de la fonction dans la réponse de la plante et de la régulation transcriptionelle de quelques cibles d'intérêt. Les résultats de ce travail illustrent dans leur ensemble l'importance de RRS1-R pour réguler la réponse des plantes à R. Solanacearum<br>In nature, plants are constantly exposed to microbial pathogens and have evolved an effective and dynamic immune system in order to survive. R. Solanacearum, the causing agent of wilt disease, is a soil-borne bacteria pathogenic on more than 200 plant species. Bacteria enter roots, invade xylem vessels and then spread rapidly to aerial parts of the plant through the vasculature. In A. Thaliana Nd-1 plants, RRS1-R, with its partner RPS4 allows resistance to strains of R. Solanacearum that deliver PopP2, a type III effector, into the plant cells. Previous studies showed that RRS1 and RPS4 are two NBS-LRR receptor proteins involved in the perception of the effector. Interestingly, RRS1 also harbors a WRKY transcription factor domain in its C-terminal end. In a susceptible Arabidopsis ecotype Col 0, RRS1-S is an allelic gene of RRS1-R, which encodes a similar structure. The recognition of bacterial and plant proteins leads to RRS1 protein accumulation in the nucleus, triggering possibly transcriptional gene regulation. Important genomic reprogramming of the infected plant cells has indeed been shown. My work shows that the RRS1-S and RRS1-R genes are expressed mainly in mature roots and basal hypocotyls, in pericycle cells and passage cells from the endoderm. These cells correspond to entry sites of the invading R. Solanacearum bacteria within the vascular tissues. We also demonstrated the binding of WRKY domain of RRS1-R and RRS1-S, in vitro, to W boxes which are cis-regulatory elements recognized by WRKY transcription factors. In order to identify the in vivo target sequences of RRS1-R and RRS1-S, a DamID (DNA adenine methyltransferase IDentification) approach, detecting transitory DNA-protein associations was developed. DamID is based on the fusion of a protein of interest to a DNA Adenine Methyl-transferase from E. Coli, which will methylate DNA in the vicinity of the binding sites of this protein. The fingerprints can be further mapped by DNA restriction with methylation sensitive enzymes, and cloned or directly sequenced. Analysis was focused on RRS1-R, by cloning FARMs (Fragment Associated to RRS1 driven Methylation) from Nd-1 plants expressing or not an inducible PopP2 gene. This allowed the identification of several putative targets of RRS1-R and led us to propose a model for its function as a transcription factor. High throughput sequencing was then initiated at a whole genome scale analysis. The function and transcriptional regulation of a putative RRS1 target gene was evaluated. Taken together, the results of this study illustrate the important role of RRS1-R in the regulation of the plant response to R. Solanacearum
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Al_griw, Huda Hm. "Molecular detection of bloodstream pathogens in critical illness." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/molecular-detection-of-bloodstream-pathogens-in-critical-illness(5f143a31-3694-454c-8940-5ae434f1eb31).html.
Full textAbstract:
Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.
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Rezende, de Castro Moretti Fernanda. "Identification of candidate resistance metabolites to Leifsonia xyli subsp. xyli in sugarcane through metabolomic profiling." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512478087354334.
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Tallent, Sandra McKenzie. "Identification and Characterization of Helper Phage Gene Products Involved in Mobilization of Staphylococcal Pathogenicity Island SaPI1." VCU Scholars Compass, 2007. http://scholarscompass.vcu.edu/etd/1223.
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Staphylococcal pathogenicity island SaPI1 is excised from genomic DNA and extrachromosomal copies are amplified during the vegetative growth of staphylococcal phage 80α. The amplified genetic element is subsequently encapsidated and transduced at very high frequency. Previous studies have demonstrated that the transducing particles have virions with tails that appear identical to those of helper phage 80α but have smaller capsids, commensurate with the smaller genome of the SaPI (Lindsay et.al., 1998). The morphology of the transducing particles, coupled with the observation that the genomic sequence of SaPIl (GenBank U93688) does not reveal any obvious phage structural proteins, has led to the hypothesis that SaPIl is encapsidated in a virion comprised of 80α structural proteins. Analysis of SaPIl transducing particles supports this hypothesis. Further investigation of 80α genes involved in SaPI1 mobilization was accomplished by selection of phage mutants resistant to SaPI1 interference. Two classes of SaPI1 jnterference resistant (sir) mutants were obtained, and point mutations were identified in two adjacent genes. In order to confirm the roles of these genes, an in-frame deletion of each candidate gene was constructed in an 80α prophage. All mutant phage and deletion constructs were evaluated for phage replication, SaPI1 replication, SaPIl transduction and SaPI1 interference. One gene (ORF21) was required for 80α growth and replication, but was not required for SaPIl growth or replication. The second gene (ORF22) was not essential for phage replication, but was required for SaPIl replication and high frequency transduction. The product of this gene was subsequently shown to be required for SaPIl excision.
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Appel, Maryke. "Cloning and identification of genes involved in the interaction between the bacterial stone fruit pathogen Pseudomonas syringae pv. syringae strain NV and plum trees." Thesis, Stellenbosch : Stellenbosch University, 2001. http://hdl.handle.net/10019.1/52165.
Full textAbstract:
Thesis (PhD)--Stellenbosch University, 2001.<br>ENGLISH ABSTRACT: Bacterial canker of stone fruit, caused by Pseudomonas syringae pv. syringae, is one of
the most destructive crop diseases in South Africa. Chemical control has failed completely
and effective long-term management strategies will have to rely on the breeding of
resistant host trees. To assist in such breeding programmes, investigations into the
molecular basis of the interaction between P. s. pv. syringae and stone fruit trees have
been undertaken in collaboration with the ARC-Fruit, Wine and Vine Research Institute in
Stellenbosch.
The aim of this dissertation was to clone and identify genes that are involved in interaction
between the bacterial canker pathogen and stone fruit trees. In the first part of the study,
the harpin encoding gene of a local strain of the pathogen, P. s. pv. syringae NV, was
amplified in a polymerase chain reaction (PCR) strategy with primers based on the
hrpAZB sequences of the bean pathogen, P. s. pv. syringae 61. Sequencing of this
hrpZpssNvgene revealed a high degree of homology (96%) between the harpin encoding
genes and harpin proteins of the two strains. The hrpZpssNvgene was subsequently cloned
into the pMAL-c2 expression vector and expressed in Escherichia co/i. This system was
used for the production of purified, biologically active, recombinant HrpZpSSNV protein.
In the second part of the study, differential display (DD) technology was used to identify
genes that are induced in stone fruit trees in response to P. s. pv. syringae and/or its
harpin elicitor. For this purpose, actively growing shoots of two Prunus sa/icina cultivars,
the moderately resistant cv. 'Laetitia' and the highly susceptible cv. 'Songold' were treated
with recombinant harpinpssNvprotein or live P. s. pv. syringae NV bacteria. An untreated
control and wounding control was included in the experiment. Total RNA was isolated for
comparative mRNA analysis 24 hours after treatment. DD profiles were generated with
fifteen primer combinations. Eight candidate bands were re-amplified, cloned and
sequenced. Reverse transcription PCR was employed to verify the expression patterns of
the cloned bands in the original RNA sample set. Two bands, DDc and DD4 were shown
to be differentially expressed between treatments and/or cultivars, while no differences in
the expression levels of the remaining six bands (DDa, DDe, DD3, DD5, DD6 and DD7)
were observed. BLAST similarity searches yielded significant matches for DDe, DD4 and
DD7 with plant defense-related genes.<br>AFRIKAANSE OPSOMMING: Bakteriese kanker van steenvrugte, wat deur Pseudomonas syringae pv. syringae veroorsaak
word, is een van die mees verwoestende siektes van landbougewasse in Suid-Afrika.
Chemiese beheermaatreëls het geheel en al misluk en effektiewe langtermyn
beheerstrategieë sal op die teling van weerstandbiedende gasheerbome moet staatmaak.
Ondersoeke na die molekulêre basis van die interaksie tussen P. s. pv. syringae en
steenvrugbome is in samewerking met die LNR-Vrugte-, Wyn- en Wingerdnavorsingsinstituut
in Stellenbosch van stapel gestuur om tot sulke telingsprogramme by te dra.
Die doelwit van hierdie proefskrif was om gene wat betrokke is in die interaksie tussen die
bakteriese kanker patogeen en steenvrugbome te kloneer en te identifiseer. In die eerste
gedeelte van die studie is die harpien-koderende geen van 'n plaaslike ras van die patogeen,
P. s. pv. syringae NV, geamplifiseer in 'n polimerase kettingreaksie (PKR)-strategie met
peilers wat op die hrpAZB-geenopeenvolgings van die boontjiepatogeen, P. s. pv. syringae 61,
gebaseer is. Volgordebepaling van hierdie hrpZpssNv-geen het 'n hoë vlak van homologie (96%)
tussen die harpien-koderende gene en harpien proteïene van die twee rasse getoon. Die
hrpZpssNv-geen is vervolgens in die uitdrukkingsvektor pMAL-c2 gekloneer en uitgedruk in
Escherichia coli. Hierdie sisteem is vir die produksie van suiwer, biologies-aktiewe,
rekombinante HrpZpssNv-proteingebruik.
In die tweede gedeelte van die studie is die differensiaalvertoon (DD) tegniek gebruik om gene
te identifiseer wat deur P. s. pv. syringae en/of sy harpien elisitar in steenvrugbome
geïnduseer word. Vir hierdie doel is aktief-groeiende lote van twee Prunus sa/icina kultivars,
die matig weerstandbiedende kv. 'Laetitia' en die hoogs vatbare kv. 'Songold', met
rekombinante harpienpssNvproteïen of lewende P. s. pv. syringae NV bakterieë behandel. 'n
Onbehandelde- en verwondingskontrole is in die eksperiment ingesluit. Totale RNA is 24 uur
na behandeling vir vergelykende mRNA-analise geïsoleer. DD-profiele is met vyftien
peilerkombinasies gegenereer. Agt kandidaatbande is geheramplifiseer en gekloneer, waarna
hul DNA-opeenvolgings bepaal is. Trutranskriptase-PKR is gebruik om die ekspressiepatrone
van die gekloneerde bande in die oorspronklike RNA monsters na te gaan. Daar is vasgestel
dat twee van die bande, DDc en DD4, differensieel tussen kultivars en/of behandelings
uitgedruk is, terwyl geen verskille in die ekspressievlakke van die oorblywende ses bande
(DDa, DOe, 003, DOS, 006 en DO7) waargeneem is nie. BLAST-soektogte het betekenisvolle
ooreenkomste vir DDe, DD4 en DD7 met plant weerstandsgeassosieerde gene opgelewer.
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Cossé, Mathilde. "Identification et caractérisation d'un nouvel effecteur précoce de Chlamydia trachomatis." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066083/document.
Full textAbstract:
C. trachomatis est une bactérie Gram-négative intracellulaire obligatoire et un pathogène humain. Première cause de maladie sexuellement transmissible d'origine bactérienne, elle est également responsable, dans les pays en développement, d'infections oculaires pouvant conduire à la cécité (trachome). Son cycle de développement bi-phasique a lieu au sein d'un compartiment appelé inclusion. Grâce à un système de sécrétion de type 3 (SST3), Chlamydia sécrète des protéines dans le cytosol de la cellule afin de promouvoir sa survie et sa multiplication. Ces protéines sont désignées sous le terme d'effecteurs<br>C. trachomatis is an obligate intracellular Gram-negative bacteria and a human pathogen. It is the most prevalent cause of sexually transmitted diseases of bacterial origin and a leading cause of preventable blindness in the developing world. During their biphasic developmental cycle the bacteria remains in a membrane-bounded cellular compartment called an inclusion. Using a type 3 secretion system (T3SS) they translocate effector proteins inside the cytosol of the cell to promote its survival and multiplication.The aim of the PhD was to study the function of CT622, a hypothetic protein from C. trachomatis. We showed that CT622 is an effector protein from the T3SS and that it is secreted early during the infection. We identified a bacterial protein that binds to CT622, and we showed that it acts as a chaperone, stabilizing CT622 and enhancing its secretion. We obtained bacteria lacking CT622 expression, thus demonstrating that CT622 is not essential for bacterial growth in vitro. However, preliminary studies indicate that in the absence of CT622 bacterial development is delayed and T3SS is defective.We identified several molecules interacting with CT622: geranylgeranyl diphosphate, Rab39 and Atg16L1 proteins. Future work will aim at understanding how these identified interactions, or other bacterial or cellular partners still to be discovered, contribute to the establishment of a niche favorable to bacterial development
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Kühbacher, Andreas. "Identification of new molecular effectors involved in the internalization of the bacterial pathogen Listeria monocytogenes in host cells using high-throughput RNAi screening and targeted approaches." Paris 7, 2013. http://www.theses.fr/2013PA077193.
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La bactérie pathogène Listeria monocytogenes infecte les cellules non-phagocytaires, s'échappe de la vacuole et se réplique dans le cytoplasme de la cellule-hôte. L'internalisation implique l'interaction de récepteurs de la cellule avec des protéines de surface de la bactérie de la famille des internalines. Notamment, InlA interagit avec E-cadherin et In1B interagit avec le récepteur de facteur de croissance hépatocytaire Met, ce qui provoque l'activation de la cascade de signalisation, qui déclenche l'internalisation de la bactérie, dépendant de l'actine et de la clathrine. Afin de mieux comprendre les mécanismes cellulaires qui régulent l'invasion par L. Monocytogenes, nous avons établi une procédure de screening à haut débit avec des ARNs d'interférence et des inhibiteurs. Ce projet a été accompli dans le cadre du consortium suisse InfectX. En combinant ces screens avec des approches de microbiologie, biochimie et biologie cellulaire, nous avons mis en évidence de nouveaux aspects de l'infection par L. Monocytogenes. Des nouveaux facteurs cellulaires impliqués directement ou indirectement ont pu être identifiés. Nous avons caractérisé en détail l'implication d'un de ces facteurs, Lmodl, De plus, l'utilisation de nouvelles approches de protéomique, en collaboration avec l'équipe de Bernd Wollscheid à l'ETH de Zurich, ont permis d'identifier de nouveaux facteurs interagissant avec InIB, ainsi que le rôle des compartiments de l'endocytose tardive pendant l'entrée de L. Monocytogenes et son échappement de la vacuole. Enfin, nous avons pu montrer via des approches ciblées que le réseau d'actine au site d'entrée de L. Monocytogenes est modulé par la lipide phosphatase OCRL<br>The gram-positive bacterium Listeria monocytogenes is able to enter non-phagocytic cells, to disrupt the phagocytic vacuole and to replicate in the host cell cytoplasm. Invasion of epithelial cells requires the interaction of L. Monocytogene surface proteins internalin (InIA) or In1B with cellular receptors which are the adherens junctions molecule E-cadherin or the hepatocyte growth factor receptor Met respectively. These ligand/receptor-interactions lead to the activation of signaling cascades which result in actin- and clathrin-dependent uptake of the bacterium. In order to obtain a better understanding of the cellular fonctions regulating the L. Monocytogenes invasion process, a high-content screening procedure was established and applied to siRNA, miRNA mimic, miRNA inhibitor and drug libraries. This project was performed in the framework of the Swiss consortium InfectX (www. Infectx. Ch). In combination with standard microbiology, biochemistry and cell biology approaches, we shed light on several new aspects of L. Monocytogenes invasion. New host factors that are directly or indirectly involved in the invasion process could be identified. One of these factors, Lmodl, has been further characterized. In addition, novel proteomics approaches were applied in collaboration with the group of Bernd Wollscheid at ETH Zurich leading to the identification of novel interactors of the bacterial In1B and to the description of a rote of late endocytic compartments during L. Monocytogenes entry and vacuolar escape. Finally, by targeted approaches we could show that the lipid phosphatase OCRL modulates actin dynamics at the L. Monocytogenes entry site
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Goad, David Wesley. "Methods for detection and identification of pathogenic bacteria." 2008. http://digital.library.okstate.edu/etd/umi-okstate-2826.pdf.
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Zaraee, Negin. "Interferometric imaging for pathogenic bacteria identification and antibiotic susceptibility testing." Thesis, 2021. https://hdl.handle.net/2144/42614.
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Pathogenic bacterial infections are a serious threat to public health, claiming millions of lives every year. In order to contain the spread of infectious diseases sensitive and timely diagnosis of pathogenic bacteria is of significant importance. The rapid detection of low abundance analytes is still challenging in the most common bacteria detection techniques including, culture and colony counting, Enzyme-linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR). Conventional bacteria detection techniques suffer from limitations such as low sensitivity, cost, long procedural time and requiring complex lab equipment. Thus, there is a critical need for rapid, sensitive and low-cost bacterial detection platform in various applications ranging from water and food safety to medical diagnosis. The quest to overcome these limitations have sparked significant interest in innovative biosensor development, with considerable emphasis on optical techniques. Among optical biosensors, label-free methods are highly desirable over label-based alternatives for eliminating the additional cost and sample processing required for labeling. Also, techniques for whole-cell bacteria detection are preferred to detection of pathogenic molecular components detection due to the requirement for extracting and isolating the desired bacterial components such as nucleic acids or proteins. Overall, label-free whole-cell detection of pathogenic bacteria has a significant advantage of simplicity in sample preparation that translates to time and cost reduction.
An additional benefit of detecting whole-cell bacteria without labels, thus in their natural environment, is the ability of monitoring the growth and replication of individual pathogens with a potential application in antimicrobial susceptibility determination. Despite the significant advantages of antibiotics as one of our most powerful tools for fighting infections, their extensive misuse and overuse over the years, have resulted in the emergence of antibiotic resistant bacteria as the global health crisis of our time. The current gold-standard technique for antibiotic susceptibility testing (AST) used in clinics, is culture-based disk diffusion assays. The time-consuming diagnosis method of the common clinical susceptibility testing, which is an inherent limitation of culture-based techniques, have necessitated the need for an alternative AST analysis platform. A clinical diagnosis test that could perform rapid pathogenic bacteria identification and determine its susceptibility to a panel of selected antibiotics, would greatly reduce the hospital stay time for patients with bacterial infection, therefore decreasing mortality and morbidity rate. In addition, it will have a great economic impact on the global healthcare system by advising optimal antibiotic use and maintaining the value of existing drugs.
In this dissertation, we describe the design and development of a rapid, sensitive, and multiplexed biosensor platform that can both identify pathogenic bacteria and perform image-based AST on a single reader instrument. The simple and low-cost design of our biosensing platform makes it a perfect candidate as a point-of-care (POC) diagnostic tool in clinical setting. The biosensor presented in this dissertation is based on interferometric enhancement of the visibility of individual biological particles, such as viruses and bacteria, afforded by Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS), previously developed in our group. The integration of SP-IRIS with microfluidic flow cells provides kinetic measurements capability, by enabling in-liquid imaging of the sensor surface in real-time, therefore making it a promising diagnostic platform. Here, we build upon the SP-IRIS platform and utilize it for pathogenic bacteria identification and image-based AST analysis. To validate our biosensor's functionality, we demonstrate E. coli detection and characterization in end-point and real-time measurement modality through particle detection and tracking analysis of the acquired images from sensor surface. In addition, we perform rapid image-based AST analysis for E. coli bacteria against two antibiotics, ampicillin and gentamicin, by monitoring single cell morphological variations and tracking their growth rate under various antibiotic challenges.
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Hsieh, Pei-Chi, and 謝沛旂. "Identification of pathogenic bacteria in body fluids of canine and feline by 16S rRNA." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/us58bv.
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碩士<br>國立臺灣大學<br>臨床動物醫學研究所<br>105<br>Regarding to urinary tract and bloodstream infections in dogs and cats, bacterial culture is the gold standard to confirm the diagnosis. As the duration of culture and strain identification need 72 hours at least, the clinicians often use antibiotics before the cultural results; consequently the problems of antibiotic abuse and resistance increase. Thus the less time and higher sensitivity diagnostic methods were indeed needed. The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria. In addition to highly conserved sites, 16S rRNA gene sequences also contain hyper-variable regions that can provide species-specific signature sequences useful for identification of bacteria, which can be divided into v1 to v9 regions. Those species-specific sequences within a given hyper-variable region constitute useful targets for diagnostic assays and other scientific investigations. In this study, universal PCR primers that can amplify 16S rRNA hyper-variable regions from a large number of different bacterial species were designed. Also, minimum detectable concentration with serial dilution method to determine PCR sensitivity and specificity was determined and applied for the cases with bacterial culture of urine, blood and other body fluids such as ascites and pleural effusions at the same time. We aimed to identify infectious pathogens quickly to achieve the goal of early diagnosis and proper treatment without antibiotic abuse in the near future.
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Lall, Namrita. "Isolation and identification of Naphthoquinones from Euclea natalensis with activity against Mycobacterium tubercolosis, other pathogenic bacteria and Herpes simplex virus." Thesis, 2001. http://hdl.handle.net/2263/25097.
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The antimycobacterial activity of twenty South African medicinal plants were investigated using two methods commonly used; the conventional agar plate method and the BACTEC radiometric method. Fourteen of the twenty acetone extracts of medicinal plants used to treat pulmonary diseases showed inhibitory activity at a concentration of 0.5 mg/ml against a sensitive strain of Mycobacterium tuberculosis using the conventional agar plate method. These fourteen extracts were also tested against M tuberculosis by the BACTEC radiometric method against a sensitive as well as a strain resistant to the drugs isoniazid and rifampin. Eight plants showed activity against both the strains at a concentration of 1.0 mg/ml. Susceptibility testing of M tuberculosis by the agar plate method is reliable, economical, and reproducible whereas the BACTEC radiometric method is much faster and probably more accurate than the agar plate method. A cytotoxicity assay of the fourteen plants on primary vervet monkey kidney cells showed that the crude acetone extracts of E. natalensis was the least cytotoxic extract with significant antimycobacterial properties. It was therefore, chosen for the isolation of active compound(s). An antibacterial assay of the water and acetone extracts of the roots of E. natalensis showed that they inhibited the growth of Gram-positive bacteria at concentrations ranging between 0.1 and 6.0 mg/ml. The water extract did not exert any inhibitory action on Gram-negative bacteria while the acetone extract showed inhibitory activity at a concentration of 5.0 mg/ml. The MIC of diospyrin, isolated from E. natalensis, was found to be 100 µg/ml for a drug-sensitive and a number of drug-resistant strains of M. tuberculosis and Gram-positive bacterial species. An antiviral investigation of the crude extracts of E. natalensis showed that the water extract of the roots of the plant inhibited the replication of herpes simplex virus type 1 moderately at a concentration of 0.2 mg/ml whereas, acetone extract at concentrations ranging from 0.1 to 0.02 mg/ml. Diospyrin exhibited no inhibitory effect against the virus. The MIC of 7-methyljuglone, isolated from E. natalensis, was found to be 50 µg/ml for both drug-sensitive and drug-resistant strains of M. tuberculosis. The compound inhibited the growth of Gram-positive bacterial species at concentrations ranging from 50 to 100 µg/ml. No inhibitory effect of the compound was observed on any Gram-negative bacteria at the highest concentration tested. A significant synergistic effect of the two naphthoquinones was observed against M tuberculosis and some of the bacterial species. MICs obtained were 10 µg/ml and 50 µg/ml for M tuberculosis and the bacterial species respectively. No synergistic effect was observed on any Gram-negative bacterial species investigated. In view of the encouraging results obtained from this study on the biological activity of the two naphthoquinones; diospyrin and 7 -methyljuglone, it appears that the compounds deserve further investigation in order to explore its potential as antimycobacterial agents.<br>Thesis (DPhil (Plant Physiology))--University of Pretoria, 2007.<br>Plant Science<br>unrestricted
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HsiYang and 楊晞. "Identification and characterization of mobilizable plasmids that benefit bacterial pathogens during infections." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/853445.
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Almeida, Maria Teresa da Conceição Malheiro Pinto de. "Identification of new host signaling pathways hijacked by bacterial pathogens during infection." Tese, 2015. https://repositorio-aberto.up.pt/handle/10216/79924.
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Almeida, Maria Teresa da Conceição Malheiro Pinto de. "Identification of new host signaling pathways hijacked by bacterial pathogens during infection." Doctoral thesis, 2015. https://repositorio-aberto.up.pt/handle/10216/79924.
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Yang, Wen-Shin, and 楊雯馨. "Identification and detection of bacterial pathogens infecting papaya in central and southern Taiwan." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/63216427490919062936.
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碩士<br>中興大學<br>植物病理學系所<br>95<br>A bacterial disease with symptoms as that of black rot on papaya, seriously occurred in central and southern Taiwan in recent years. Based on physiological, biochemical and pathogenicity tests, there were 36 strains of plant pathogenic bacteria isolated from diseased papayas from Pingtung and Changhua, including 21 strains of Erwinia sp., 5 strains of Pectobacterium carotovorum subsp. carotovorum and 10 strains of Pseudomonas aeruginosa. Physiological and biochemical assays revealed that the characteristics of the Erwinia sp. strains were similar to that of Pectobacterium cypripedii (Erwinia cypripedii) strain from papaya reported in 1980. Phylogenetic trees based on the sequences of 16S rDNA indicated that the Erwinia sp. strains isolated from papaya were more closely related to the Erwinia papayae than Pectobacterium cypripedii. Sixty different random primers were tested to amplify specific DNA fragments from strains of Erwinia sp. using random amplified polymorphic DNA (RAPD) analysis. A specific DNA fragment (about 950 bp in size) form Erwinia sp. strains was amplified by the random primer OPC 5. This specific DNA fragment was cloned and sequenced to design specific primer pair C5-1L/C5-1R. This primer pair could amplify a distinct band of 300 bp that was specific to strains of Erwinia sp. isolated from papaya by polymerase chain reaction (PCR). The minimum amount of DNA from Erwinia sp. that could be amplified by PCR was 10 pg. The results indicated that the primer pair C5-1L/C5-1R could be a useful tool for identification and detection of Erwinia sp. strains from black rot of papaya.
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Chu, Chun-Yu, and 朱俊育. "Development of an Oligonucleotide Array for Identification of Bacterial Pathogens of Bovine Mastitis." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/57444483394659710774.
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碩士<br>國立屏東科技大學<br>食品科學系所<br>98<br>Cow mastitis is caused by a wide variety of microbial pathogens. In this study, an oligonucleotide array was developed to identify the pathogens causing mastitis. These pathogens include Corynebacterium bovis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Mycoplasma bovis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus simulans, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis. Specific oligonucleotide probes were designed from the 16S-23S rRNA gene intergenic spacer (ITS) region and immobilized on Nylon membrane. The identification method consisted of PCR amplification of the ITS region using bacterial universal primers, followed by hybridization of the digoxigenin-labeled PCR product with oligonucleotide probes on the array. A total of 35 species-specific oligonucleotide probes were immobilized on the array (0.6 cm × 0.5 cm) to identify 18 species of bacteria. After testing a collection of 322 target strains and 154 nontarget strains, the sensitivity and specificity of the array was 100 % and 96.2 %, respectively. The current array has a great potential to rapidly and correctly identify bacteria causing bovine mastitis.
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Lin, Jiunn-Yow, and 林俊佑. "Rapid Identification of Bacterial Food Poisoning Pathogens by High-Resolution Melting Curve Analysis." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/24203145926023478068.
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碩士<br>輔仁大學<br>生命科學系碩士班<br>101<br>Foodborne illness is one of the most common infectious diseases worldwide. Among them, food poisoning syndromes caused by bacterial pathogens are known to be very diversified and complicated. Therefore, how to clarify food poisoning pathogens as soon as possible is an important issue. The conventional methods used for the diagnosis of bacterial food poisoning pathogens are very time-consuming. However, with the advance of cross-cutting diagnostic technology, many new tools are available for an accurate and rapid bacterial detection and diagnosis. Among them, nucleic acid-based detection and diagnosis is one of the most promising sectors in in vitro diagnostics. In this study, a high-resolution melting curve method (HRM) was employed for developing the detection and identification methods of the above- mentioned pathogens. Totally, 7 genera of DNA samples, including Bacillus spp., Staphylococcus spp., Escherichia spp., Salmonella spp., Shigella spp., Pseudomonas spp. and Vibrio spp. were used. The ribosomal DNA sequences of each species were retrieved from the GenBank and aligned, the primers for polymerase chain reaction (PCR) was subsequently designed. The HRM profiles of the 16S rDNA and ITS (internal transcribed spacer) amplicons were examined to characterize inter-species and inter-subspecies difference. The amplicons of the 16S rDNA variable regions were obtained lastly with the hn-PCR(hemi-nested PCR) protocol, and resolved then by the HRM analyses. The results have clearly demonstrated that the HRM profiles revealed by the aforementioned approach could differentiate at least 7 species of the 5 different genera. Moreover, it was capable of resolving the difference at the subspecies level. Additionally, the phylogenetic relationship reconstructed using the neighbor-joining method, based on the 16S rDNA sequences of the examined species, was also shown that the closer the phylogenetic relationship, the similar the HRM profile. The potential application and prospect of this study as well as the plausible approach to build an identification and diagnostic platform were addressed finally.
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Πρωτόπαπα, Μαρία. "Μοριακή ανίχνευση παθογόνων βακτηρίων που ενέχονται στην περιοδοντίτιδα". Thesis, 2010. http://nemertes.lis.upatras.gr/jspui/handle/10889/3683.
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Τα Gram αρνητικά βακτήρια είναι οι κύριοι αιτιολογικοί παράγοντες της περιοδοντικής νόσου, όπως τα βακτήρια Porphyromonas gingivalis, Actinοbacillus actinomycetemcomitans και Bacteroides forsythus. Σκοπός της εργασίας είναι η μοριακή ανίχνευση και ταυτοποίηση των βακτηρίων αυτών, σε δείγματα ασθενών με περιοδοντίτιδα, μέσω εντοπισμού ειδικών αλληλουχιών του DNA του γονότυπου κάθε παθογόνου. Τα δείγματα συλλέγονται σε PBS και DNA παρασκευάζεται με το QIAmp Tissue kit (Qiagen). Η μοριακή ανάλυση γίνεται με PCR και multiplex PCR, με ένα εκκινητή ειδικό για κάθε βακτήριο και ένα κοινό για τα τρία βακτήρια, για ειδική αλληλουχία του γονιδίου 16S rRNA.Το αποτέλεσμα της PCR ελέγχεται με ηλεκτροφόρηση αγαρόζης 2%.Η ταυτοποίηση του προϊόντος της PCR γίνεται με κατάτμηση του γενετικού υλικού με ειδικά ένζυμα περιορισμού. Ο προσδιορισμός της ευαισθησίας της μεθόδου γίνεται με συνεχείς αραιώσεις του DNA για κάθε βακτήριο και PCR.Εξετάστηκαν 42 δείγματα οδοντικής πλάκας. Τα εξεταζόμενα παθογόνα βακτήρια ανιχνεύτηκαν σε 40 δείγματα, εκ των οποίων: 15 περιέχουν τη Porphyromonas gingivalis, 6 τον Actinοbacillus actinomycetemcomitans και 19 το Bacteroides forsythus. Σε 5 δείγματα συνυπάρχουν τα βακτήρια Porphyromonas gingivalis και Bacteroides forsythus, ενώ σε 4 η Porphyromonas gingivalis και ο Actinοbacillus actinomycetemcomitans. Η μεθοδολογία αυτή παρουσιάζει υψηλή ειδικότητα καθότι δεν ανιχνεύεται μη ειδικό προϊόν PCR και υψηλή ευαισθησία, με το χαμηλότερο όριο ανίχνευσης για το Porphyromonas gingivalis είναι 280 pgr/αντίδραση και για το Actinοbacillus actinomycetemcomitans, 120 pgr/αντίδραση.
Η υψηλή αποτελεσματικότητα, ειδικότητα και ευαισθησία μεθόδου, σε συνδυασμό με τον μικρό χρόνο ανάλυσης, καθιστούν τη μοριακή ανίχνευση και ταυτοποίηση των περιοδοντικά παθογόνων βακτηρίων αποτελεσματικότερη, σε σχέση με τις συμβατικές μεθόδους.<br>Gram- bacteria are the main aetiological factors for periodontal disease(i.e.Porphyromonas gingivalis, Actinοbacillus actinomycetemcomitans και Bacteroides forsythus). The aim of this study is the molecular identification of these bacterias in samples of patients with periodontal disease. The molecular analysis is performed with PCR and multiplex PCR.42 samples with dental plague were evaluated. The pathogenic bacterias were found in 40 of these samples. The great efficacy, sensitivity and speciality of the method make the molecular identification of the periodontal pathogenic bacteria more useful than the conventional methods.
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Wen, Bing-Cheng, and 溫秉丞. "Detection and identification of the pathogen of bacterial meningitis by Surface Enhanced Raman Scattering (SERS)." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/69350776873346999616.
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碩士<br>國立陽明大學<br>微生物及免疫學研究所<br>98<br>Before the discovery of antibiotic, meningitis was a fatal disease. At the beginning of the infection, the symptoms of meningitis are hard to differentiate from other illnesses. Besides, it takes about 2~3 days to culture and diagnosis the pathogen after acquisition of the Cerebrospinal fluid(CSF) from patient. It is fatal for the patients if appropriate antibiotics are not prescribes soon after infection, therefore, it is of utmost important to identify the pathogen as quickly as possible.
Raman Scattering was discovered in 1928, it has been used to analyze pure chemical elements. The spectrum can represent the specific chemical bond. Since the signal of Raman Scattering is quite weak, and is not appropriate to apply to biological samples. After the discovery of the technique of Surface Enhanced Raman Scattering(SERS). The Raman Scattering signal has now been applied to biological samples.
This research focus on the 10 pathogens of bacterial meningitis, and two methods including Flow Cytometry and Filter are used to differentiate the bacteria in the CSF from patients. The result shows the spectrum of different bacteria is specific, and after filtration we are able to isolate the bacteria from CSF, which will be detected by SERS spectrometer.
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Chen, Yi-Jeng, and 陳以錚. "Bacterial wilt of vegetable sweet potato in Taiwan - pathogen identification, inoculum sources and management strategy." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/60053076402405567788.
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博士<br>國立中興大學<br>植物病理學系所<br>103<br>Abstract
Sweet potato [Ipomoea batatas L. (Lam)], the dicotyledonous plant of the family Convolvulaceae, is the seventh most important food crop in the world after wheat, rice, maize, potato, barley and cassava. Besides food supply, sweet potato has diverse uses in green source, ornamental, feed, starch and liquor manufacture, human consumption, biofuel and bioplastic production, etc. Many cultivars have been currently developed for different uses through the world. Among these cultivars, vegetable sweet potato (VSP) has been bred for edible leafy vegetable and can produce a lot of leaves, tender shoots and small/non tubers constantly throughout the growth period in whole year period, especially, summer and rainy season. The VSP is considered as an important green source in summer or rainy season in Taiwan, and similar cultivars of VSP have been bred and grew in Japan. A new disease, Bacterial wilt (BW) caused by Ralstonia solanacearum (RS) broke out during 2000’s and reduced 30 to 80% yield of VSP last decade in Taiwan. The R. solanacearum isolates obtained from diseased VSP were identified as Ralstonia solanacearum phylotype I race 1 biovar 4 (R1bv4) based on physical and molecular analyses. Moreover, these isolates also caused wilting in convolvulaceous, solanaceaous and cruciferous plants. Field investigation indicated that R1bv4 was generally distributed in soil of VSP fields with 1.3×102 to 9.5×105 cfu/g soil. Further detection showed R1bv4 could latently infect healthy VSP cuttings with 2 to 98% isolation frequency. The severity of BW was closely related to R1bv4-carried VSP cuttings (R=0.913); however, the severity of BW did not show significant correlation with the R1bv4 density in soil (R=0.086). Similar phenomenon was observed in greenhouse test. Thus, the cuttings carried R1bv4 were more important inocula source than the R1bv4 residing in soil. The distribution of R1bv4 in VSP indicated that the terminal shoots or erect stems had low R1bv4 containation perecentage (<31%) and creeping stem had high R1bv4 containation percentage (45 to 100%) 8 wks after the VSP planted in infested soil (106 cfu/g soil). Results demonstrated that R1bv4 did not consistently move to the part of erect stem cuttings. For confirming the efficacy of the erect stem cutting on control BW of VSP in the fields, the erect stem cuttings were collected from VSP field. The results revealed that the erect stem cuttings used as new plants could decrease the BW in field. Moreover, companied with early R1bv4 detection in erect stems could increase the control efficient of BW in VSP production area. In this study, a bacterial endophyte, Bacillus amyloliquefaciens SPX1 from healthy VSP in Dali, had good ability to inhibit the growth of R1bv4 on TTC medium. The further experiment showed that the SPX1 isolate could decrease the wilting development of VSP in greenhouse and field conditions by soaking treatment. In addition, a resistance line of VSP, VSPSL-1, showed high reistance to bacterial wilt in field. Thus, the VSPSL-1 line is a promising cultivar resistant to R1bv4 in the future.
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Hwang, Chung-Hsing, and 黃中興. "Rapid Detection and Identification of Foodd-Borne Bacterial Pathogens by Multiplex PCR and Restriction Endonuclease Digestion." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/16304518326196407602.
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碩士<br>國立中山大學<br>生物科學系研究所<br>89<br>英文摘要
Multiplex PCR amplification of 16S rRNA gene、virA、tpl、and H1d genes was developed enabling simultaneous detection in Escherichia coli，an indicator of fecal contamination and food-borne microbial pathogens，Shigella flexneri、Citrobacter freundii、Salmonella typhi、Vibrio cholerae、Vibrio parahaemolyticus、and Staphylococcus aureus。Each of the nine pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene。The optimized multiplex PCR reaction utilized a primer annealing temperature of 59 ℃and used agarose gel electrophoresis for detection of the PCR-amplified products。Selection of appropriate target genes、oligonucleotide primers 、PCR reaction、and cycling parameters resulted in the amplification of four target genes simultaneously in a single PCR reaction with the sensitivity of detection was 102 CFU after 32 cycles。Multiplex PCR amplification followed by differential PCR for E. coli / Shigella， and Citrobacter / Salmonella，sequenced for the PCR-amplified products of 16S rRNA gene of the seven pathogens in this study，and used restriction endonuclease AfaI to confirm the PCR-amplified products of V. cholerae，V. parahaemolyticus and Staphylococcus aureus，has been shown to be an sensitive，specific，and rapid method to detect food-borne bacterial pathogens。