Rozprawy doktorskie na temat „RRNA gene analysis”

Nucleic acid sequencing can provide a detailed overview of microbial communities in comparison with standard plate-culture methods. Expansion of high-throughput sequencing (HTS) technologies and reduction in analysis costs has allowed for detailed exploration of various habitats with use of amplicon, metagenomics, and metatranscriptomics approaches. However, due to a capital cost of HTS platforms and requirements for batch analysis, genomics-based studies are still not being used as a standard method for the comprehensive examination of environmental or clinical samples for microbial characterization. This research project investigated the potential of a novel nanopore-based sequencing platform from Oxford Nanopore Technologies (ONT) for rapid and accurate analysis of various environmentally complex samples. ONT is an emerging company that developed the first-ever portable nanopore-based sequencing platform called MinIONTM. Portability and miniaturised size of the device gives an immense opportunity for de-centralised, in-field, and real-time analysis of environmental and clinical samples. Nonetheless, benchmarking of this new technology against the current gold-standard platform (i.e., Illumina sequencers) is necessary to evaluate nanopore data and understand its benefits and limitations. The focus of this study is on the evaluation of nanopore sequencing data: read quality, sequencing errors, alignment quality but also bacterial community structure. For this reason, mock bacterial community samples were generated, sequenced and analysed with use of multiple bioinformatics approaches. Furthermore, this study developed sophisticated library preparation and data analyses methods to enable high-accuracy analysis of amplicon libraries from complex microbial communities for sequencing on the nanopore platform. Besides, the best performing library preparation and data analyses methods were used for analysis of environmental samples and compared to high-quality Illumina metagenomics data. This work opens a new possibility for accurate, in-field amplicon analysis of complex samples with the use of MinIONTM and for the development of autonomous biosensing technology for culture-free detection of pathogenic and non-pathogenic microorganisms in water, soil, food, drinks or blood.

Les abcès cérébraux sont des infections potentiellement mortelles, entraînant souvent des séquelles graves. La prise en charge médicale en reste empirique en raison d’un manque de connaissance approfondie des microorganismes responsables de cette condition. Dans la plupart des laboratoires microbiologiques, le diagnostic d’abcès cérébral est basé sur la culture du pus recueilli chirurgicalement. Malheureusement, cette procédure a de nombreuses limites et ne permet l’identification que d’une petite partie de la population microbienne en cause. L’amplification par PCR et le séquençage du gène codant la fraction 16S de l’ADN ribosomal ont récemment été utilisées pour surmonter les limites de la culture, et ont été démontré leur efficacité dans la documentation des infections bactériennes. Malheureusement, cette procédure présente un degré de discrimination limité en cas d’infection polymicrobienne. Des études métagénomiques de flores complexes de l’homme, basées sur une combinaison de PCR, clonage et séquençage des produits de PCR se sont avérées utiles pour évaluer la diversité bactérienne des flores dentaires, vaginales et intestinales. Nous avons appliqué cette technique à des échantillons d’abcès cérébral pour étudier la flore associée à cette maladie. Dans une première étape, nous avons réalisé une enquête en utilisant la culture et les techniques moléculaires. Le but de cette étude était d’analyser et d’évaluer les bactéries de la flore responsable des abcès cérébraux, en comparant la culture à trois techniques moléculaires basées sur le gène 16S rDNA, incluant le séquençage direct, le clonage suivi de séquençage par méthode de Sanger, et le séquençage direct des produits de PCR par pyroséquençage. Cette enquête a déterminé que la variété des espèces bactériennes associée aux abcès cérébraux est beaucoup plus grande que précédemment décrite, et inclut de nombreuses bactéries anaérobies et des bactéries incultivables de la flore buccale. Cette étude préliminaire a identifié 49 agents bactériens différents, et a permis l’identification de 27 bactéries jamais détectées auparavant dans des abcès du cérébraux, dont 15 n’avaient jamais été cultivées. Un tel nombre d’espèces bactériennes impliquées dans les abcès cérébraux a motivé l’étude de 51 nouveaux spécimens dans le but de décrire plus en détail la flore associée aux abcès cérébraux en fonction de leurs étiologies. Ainsi, nous avons effectué une analyse métagénomique, basé sur le gène 16S rDNA, de 51 patients ayant développé un abcès cérébral. Notre stratégie a été beaucoup plus discriminatoire et a permis à l’identification d’un plus grand nombre de bactéries que la culture et l’amplification et le séquençage direct de l’ANRr 16S. La combinaison des données de 71 patients (20 de la première étude et 51 de la deuxième étude) a permis l’identification de plusieurs associations à l’aide de la méthode de data mining.En outre, notre étude a permis l’identification de deux nouvelles bactéries, la première étant une nouvelle espèce de genre Staphylococcus (Staphylococcus massiliensis) et la seconde étant une bactérie anaérobie qui représente une nouvelle espèce dans un nouveau genre au sein du phylum des Bacteroidetes (Phocaeicola abscesses). En outre, nous avons décrit deux cas inhabituels d’abcès du cerveau, à Mycoplasma hominis après curetage utérin, et à Nocardia carnea chez un greffé rénal. Malgré les limites inhérentes à la procédure de clonage, nos résultats suggèrent que le clonage et le séquençage de gène DNAr 16S est une méthode très performante pour identifier les agents bactériens associés aux abcès cérébraux
Brain abscess is a life-threatening infection with frequent serious sequelae. The medical management remains empirical due to a lack of comprehensive knowledge of the microorganisms responsible for this condition. In most microbiology laboratories the diagnosis of brain abscess is based on culture from pus collected surgically. Unfortunately, this procedure has many limitations and reveals only a small portion of the true microbial population. PCR-amplified 16S rDNA sequencing has recently been used to overcome the limitations of culture-based bacterial detection in brain abscess pus, and it was demonstrated to be effective in the documentation of monomicrobial infections. Unfortunately, this procedure failed to discriminate among polymicrobial floras.Metagenomic studies of complex human floras using a combination of 16S rDNA PCR and cloning-sequencing of PCR products proved useful to evaluate the bacterial diversity of dental, vaginal and intestinal floras. Thus, we applied this technique to brain abscess samples to study the flora associated with this condition. In a first step, we performed an investigation using culture and molecular techniques. The purpose of this investigation was to analyze and evaluate the bacterial flora responsible for brain abscess by comparing standard culture technique to three techniques using 16S rDNA amplification, that is, direct sequencing, multiple sequencing following cloning, and multiple sequencing via high throughput pyrosequencing. This investigation has determined that the variety of brain abscess-associated bacterial species is much larger than previously reported, and it includes many anaerobes and uncultured bacteria from the oral cavity flora. This preliminary study identified 49 distinct brain abscess bacterial agents, and enabled the identification of 27 bacteria never detected before in brain abscess, 15 of which were uncultured.Such a high number of bacterial species involved in brain abscess prompted the study of 51 new specimens in an effort to describe further the flora associated with brain abscesses and their etiologies. Thus, we performed a 16S rDNA-based metagenomic analysis of cerebral abscesses from 51 patients. Our strategy was significantly more discriminatory and enabled the identification of greater number of bacterial taxa, than culture and conventional 16S rDNA PCR/sequencing, respectively. The combination of data from 71 patients (20 from the first study and 51 from the second study) enabled the identification of several associations using the data mining analysis. Also, these studies permitted the identification of two novel bacteria, the first being a novel Staphylococcus species (Staphylococcus massiliensis) and the second being a novel anaerobic bacterium that represents a novel species in a new genus within the phylum Bacteroidetes (Phocaeicola abscesses). In addition, we reported tow unusual cases of brain abscess, the first case was a Mycoplasma hominis brain abscess following uterus curettage and the second case was a Nocardia carnea infection in a kidney transplant recipient patient.Despite limitations inherent to the cloning procedure, our results suggest that cloning and sequencing of PCR-amplified 16S rDNA is a highly valuable method to identify bacterial agents of brain abscesses

In order to analyze the bacterial community of river otter scat (fecal material) at the class level, river otter scat samples were collected from Grizzly Island Wildlife Area (Solano County, CA) and the Cosumnes River Preserve (Sacramento County, CA). DNA was isolated from each sample with the MOBIO PowerSoil™ DNA Isolation Kit and 16S rRNA gene sequences were amplified from each sample. After digestion with Mspl, TRFLPs were analyzed in an ABI Prism™ 310 Genetic Analyzetin triplicate and data peak information from each electropherogram was uploaded into the Phylogenetic Assignment Tool (PAT). Species belonging to the Class Bacilli were the most abundant followed by unclassified species. Two road killed river otters were necropsied to recover brain and blood tissue. DNA was isolated using the Qiagen Tissue DNeasy Kit. Samples from both otters were amplified with a singe tube nested PCR primer set for the detection of the ITS 1 region of Toxoplasma gondii. Scat samples used in the T-RFLP analysis were also tested for the presence ofT. gondii using the same nested primer set. Neither the river otter tissue samples nor any of the scat samples used in this analysis showed evidence of infeGtion with T. gondii.

16S rRNA gene analysis has shown that bacterial diversity in the GAB bores studied was limited to the genera Hydrogenobacter in the phylum Aquificae, Thermus in the phylum Deinococcus-Thermus, Desulfotomaculum in the phylum Firmicutes, the alpha-, beta- and gamma-classes of the phylum Proteobacteria and the phylum Nitrospirae. There was no clone closely related to members of the delta-proteobacteria and epsilon-proteobacteria classes detected. The number of bacterial strains directly isolated from the Fairlea and the Cooinda bores were far less than the numbers of distinctive phylotypes detected by the 16S rRNA gene characterisation. In addition none of the bacterial strains directly isolated from the water samples were represented in the 16S rRNA gene clone libraries. Similar discrepancies between the bacterial populations obtained from the 16S rRNA gene analysis and those obtained from direct isolation have been reported in the literature (Dunbar et al., 1999; Kampfer et al., 1996; Suzuki et al., 1997; Ward et al., 1998; Ward et al., 1997). However, in general, the phyla with which the isolates were affiliated were the same as those phyla to which the clones belonged. The environmental changes introduced (by bringing the artesian water up to the surface and exposing it to four types of metal coupons made of carbon steels identified by codes ASTM-A53B, ASTM-A53, AS-1074 and AS-1396 and commonly used in bore casings) led to changes in the bacterial community structures. In general, the species which proliferated in the communities before and after the changes were different. The diversity of the bacterial species in the community decreased following the environmental changes. Clones dominating the clone libraries constructed from newly established bacterial communities also differed from the clones dominating the libraries constructed from the bacterial communities which had existed naturally in the bores. These trends toward change in the bacterial communities were observed at both the Fairlea and the Cooinda bore sites. All four metal types incubated in the Fairlea bore water lost between 3.4 and 4.7% of their original weight. In contrast none of the metals incubated in Cooinda bore water lost weight. Clone library A1 showed that the natural population of the Fairlea bore was dominated by clone A1-3, which represented a novel species related to the isolate boom-7m-04. But after metal incubation (and recording of the metal weight loss), the bacterial community was dominated by clone PKA34B, which has a 95% similarity in its 16S rRNA gene sequence with Desulfotomaculum putei. Desulfotomaculum species are known to cause metal corrosion due to their byproduct H2S. But the low level of phylogenetic relatedness found does not provide enough information to speculate on whether the species represented by clone PKA34B is a member of the genus Desulfotomaculum or not. However, the fact that clone PKA34B dominated the PKA clone library by 50% makes the species it represents a suspected candidate likely to be involved with the metal weight loss at the Fairlea bore. In contrast, clone library 4381 showed that the natural population of the Cooinda bore was dominated by clone 4381-15 representing a species distantly related to a hydrogen oxidiser Hydrogenophaga flava (95% similarity). The dominating clone of the new community formed after metal incubation was clone COO25, which has 99% similarity with Thermus species that have not been reported to be involved with metal corrosion to my knowledge. In this project detection, identification and comparative quantification by 16S rRNA gene-targeted PCR probing with probes 23B and 34B were successfully developed for a Leptothrix-like species and for a Desulfotomaculum-like species represented by clones PKA23B and PKA34B respectively. This method of probing permits a fast, sensitive and reproducible detection, identification and at least a comparative quantification of the bacteria in the environment without the need for culturing. Therefore it is extremely suitable for use in bacterial population monitoring. PCR probing with the 34B probe has a potential commercial use as a means of screening for bores with a potential high risk of corrosion due to this Desulfotomaculum-like species. Direct isolation of bacteria from the GAB water has resulted in the isolation of seven strains from the Fairlea bore and eight from the Cooinda bore. Among these isolates, three novel strains were studied in detail. Reports on the characterisation of strain FaiI4T (T=Type strain) from the Fairlea bore (Kanso & Patel, 2003) and strain CooI3BT from the Cooinda bore have been published (Kanso et al., 2002). The data generated during this project add to our current information and extend our knowledge about the bacterial communities of the GAB's sub-surface environment. This information will provide a basis for further ecological studies of the GAB. Studies on involvement of certain groups of bacteria with the corrosion of metals used in bore casings could provide a foundation for further studies to develop maintenance and managing strategies for the GAB bores.

16S rRNA gene analysis has shown that bacterial diversity in the GAB bores studied was limited to the genera Hydrogenobacter in the phylum Aquificae, Thermus in the phylum Deinococcus-Thermus, Desulfotomaculum in the phylum Firmicutes, the alpha-, beta- and gamma-classes of the phylum Proteobacteria and the phylum Nitrospirae. There was no clone closely related to members of the delta-proteobacteria and epsilon-proteobacteria classes detected. The number of bacterial strains directly isolated from the Fairlea and the Cooinda bores were far less than the numbers of distinctive phylotypes detected by the 16S rRNA gene characterisation. In addition none of the bacterial strains directly isolated from the water samples were represented in the 16S rRNA gene clone libraries. Similar discrepancies between the bacterial populations obtained from the 16S rRNA gene analysis and those obtained from direct isolation have been reported in the literature (Dunbar et al., 1999; Kampfer et al., 1996; Suzuki et al., 1997; Ward et al., 1998; Ward et al., 1997). However, in general, the phyla with which the isolates were affiliated were the same as those phyla to which the clones belonged. The environmental changes introduced (by bringing the artesian water up to the surface and exposing it to four types of metal coupons made of carbon steels identified by codes ASTM-A53B, ASTM-A53, AS-1074 and AS-1396 and commonly used in bore casings) led to changes in the bacterial community structures. In general, the species which proliferated in the communities before and after the changes were different. The diversity of the bacterial species in the community decreased following the environmental changes. Clones dominating the clone libraries constructed from newly established bacterial communities also differed from the clones dominating the libraries constructed from the bacterial communities which had existed naturally in the bores. These trends toward change in the bacterial communities were observed at both the Fairlea and the Cooinda bore sites. All four metal types incubated in the Fairlea bore water lost between 3.4 and 4.7% of their original weight. In contrast none of the metals incubated in Cooinda bore water lost weight. Clone library A1 showed that the natural population of the Fairlea bore was dominated by clone A1-3, which represented a novel species related to the isolate boom-7m-04. But after metal incubation (and recording of the metal weight loss), the bacterial community was dominated by clone PKA34B, which has a 95% similarity in its 16S rRNA gene sequence with Desulfotomaculum putei. Desulfotomaculum species are known to cause metal corrosion due to their byproduct H2S. But the low level of phylogenetic relatedness found does not provide enough information to speculate on whether the species represented by clone PKA34B is a member of the genus Desulfotomaculum or not. However, the fact that clone PKA34B dominated the PKA clone library by 50% makes the species it represents a suspected candidate likely to be involved with the metal weight loss at the Fairlea bore. In contrast, clone library 4381 showed that the natural population of the Cooinda bore was dominated by clone 4381-15 representing a species distantly related to a hydrogen oxidiser Hydrogenophaga flava (95% similarity). The dominating clone of the new community formed after metal incubation was clone COO25, which has 99% similarity with Thermus species that have not been reported to be involved with metal corrosion to my knowledge. In this project detection, identification and comparative quantification by 16S rRNA gene-targeted PCR probing with probes 23B and 34B were successfully developed for a Leptothrix-like species and for a Desulfotomaculum-like species represented by clones PKA23B and PKA34B respectively. This method of probing permits a fast, sensitive and reproducible detection, identification and at least a comparative quantification of the bacteria in the environment without the need for culturing. Therefore it is extremely suitable for use in bacterial population monitoring. PCR probing with the 34B probe has a potential commercial use as a means of screening for bores with a potential high risk of corrosion due to this Desulfotomaculum-like species. Direct isolation of bacteria from the GAB water has resulted in the isolation of seven strains from the Fairlea bore and eight from the Cooinda bore. Among these isolates, three novel strains were studied in detail. Reports on the characterisation of strain FaiI4T (T=Type strain) from the Fairlea bore (Kanso & Patel, 2003) and strain CooI3BT from the Cooinda bore have been published (Kanso et al., 2002). The data generated during this project add to our current information and extend our knowledge about the bacterial communities of the GAB's sub-surface environment. This information will provide a basis for further ecological studies of the GAB. Studies on involvement of certain groups of bacteria with the corrosion of metals used in bore casings could provide a foundation for further studies to develop maintenance and managing strategies for the GAB bores.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

A cultivation-independent approach, sequence analysis of 18S rRNA genes PCR-amplified from environmental DNA, was used to explore the diversity and distribution of eukaryotic microbes inhabiting algal mats in two acidic geothermal streams in Yellowstone National Park. The objectives were to: (1) clarify the identity of mat forming algae in Nymph Creek (2) survey microbial species in the Nymph Creek mat over seasonal intervals along a thermal gradient (3) compare microbial species in the Nymph Creek mat with those in Alluvium Creek mats (4) evaluate microbial species in algal mats formed on different substrates in Alluvium Creek. The results show that a novel red alga dominates high temperature regions (~50ºC) of Nymph Creek and two "Chlorella-like" algae predominate the cooler regions (<38ºC). The predominant algae in Alluvium Creek were distinctly different from those in Nymph Creek. Several stramenophiles and fungi were detected in each algal mat.

Les hydrocarbures aromatiques polycycliques (HAP) sont des composés ubiquitaires issus de la combustion incomplète de matières organiques. Ils sont à l'origine de pollutions de l'environnement, surtout liées à l'exploitation des produits pétroliers, car ce sont des composés toxiques pour les êtres vivants et pour l'homme en particulier. De nombreuses bactéries capables de dégrader les HAP ont été isolées et étudiées, mais celles qui les dégradent in situ sont mal connues, car moins de 5% des bactéries du sol sont cultivables en laboratoire. Le premier objectif de cette étude était d'identifier les bactéries qui dégradent les HAP dans le sol par des méthodes moléculaires indépendantes de la culture. A cette fin, une stratégie de marquage isotopique in situ a été mise en œuvre qui repose sur l'utilisation du phénanthrène, un HAP à trois cycles, dans lequel l'isotope naturel du carbone a été remplacé par le 13C. Cette molécule a été introduite comme traceur dans des microcosmes contenant du sol provenant d'un bassin de rétention des eaux de ruissellement d'autoroute. Les bactéries ayant incorporé le 13C ont ensuite été identifiées par séquençage des gènes d'ARNr 16S amplifiés à partir de l'ADN marqué extrait du sol. Les résultats montrent que des Betaprotéobactéries peu étudiées à ce jour, appartenant aux genres Acidovorax, Rhodoferax, Hydrogenophaga et Thiobacillus, ainsi que des Rhodocyclaceae, étaient les principaux acteurs de la dégradation du phénanthrène. La prépondérance des Betaprotéobactéries a été établie par des mesures de PCR quantitative. Une analyse dynamique de la diversité bactérienne a montré que celle-ci changeait en fonction de la biodisponibilité du phénanthrène. En outre, la diversité d'arène-dioxygénases impliquées dans la dégradation des HAP a été explorée sur le plan phylogénétique et fonctionnel. Nous avons ainsi détecté des séquences nouvelles, pour la plupart apparentées à des dioxygénases de Sphingomonadales et de Burkholderiales. Grâce à la construction et l'expression d'enzymes hybrides, il a été possible, pour la première fois, d'associer une activité catalytique d'oxydation des HAP à des séquences partielles de gènes, amplifiées à partir de l'ADN du sol. Les résultats obtenus et les outils mis au point dans cette étude pourront servir à développer des méthodes de diagnostic et de suivi de biodégradation de polluants, par exemple dans le cadre d'opérations de bioremédiation de sites pollués par les HAP.

碩士
國立陽明大學
生物醫學資訊研究所
103
Asthma is a chronic inflammatory disease of the airways and the etiology of asthma is a complicated mechanism containing unknown genetic and environmental factors. Microbiota in airway is hypothesized to play an important role in the development of asthma. The aim of this study is to explore the changes of the microbiota between children with atopic diseases and controls using 16S rRNA gene sequencing data. We have applied the QIIME (the Quantitative Insights Into Microbial Ecology) unsupervised analytic method to 16S rRNA sequencing data in order to understand the composition of microbiota in the airways of children with asthma and healthy controls.

The Rhodophyta (red algae) are an ancient crown group of the Eukarya (ca. 1400-1500 million years), comprised of 5000 - 6000 species. Gametophytes of taxa excluding the speciose Class Florideophyceae are typically of very simple unicellular, filamentous or foliose morphologies. These simple morphologies are often homoplasious (resulting from convergent or parallel evolution) and can be indistinguishable among distinct taxa, leading to cryptic species. As a result, historical morphology-based taxonomy is often not congruent with evolutionary history. Intraspecific genetic variation is not yet characterized for non-Florideophyceae taxa. Here the intraspecific genetic variation was characterized for a locally endemic, morphologically distinct bangiophyte red alga, Bangia maxima Gardner using inter simple sequence repeat (ISSR) patterns from 91 individual filaments across seven local populations. A high degree of genetic variation was observed over very small distances (< 25 cm) and very little genetic exchange was observed between populations. It is possible that B. maxima is a true endemic species and its population dynamics may differ from other Bangia species. Metrics of sequence-based identification rely on genetic divergence among isolates to distinguish taxonomic units independent of morphology. Such metrics are especially useful for morphologically simple or cryptic species. The mitochondrial cytochrome oxidase c subunit 1 gene has been proposed for the Florideophyceae. An evaluation of this gene as a metric for non-Florideophyceae taxa was undertaken and limited utility was demonstrated in most lineages of Rhodophyta due to poor or inconsistent amplification and conflicts with nuclear and plastid phylogenies. Patterns of genetic divergence among taxa are used to infer evolutionary relationships. The nuclear ribosomal small subunit (nSSU rRNA) is the taxonomically broadest pool of gene sequence data for the Rhodophyta. The use of stochastic models of nucleotide evolution is the most common approach to inferring phylogenies using this gene, ignoring much of its evolutionary information as different characters that contribute to secondary structure (e.g. paired nucleotides) are treated independently. The incorporation of structural information leads to more biologically realistic evolutionary models increasing phylogenetic resolution. Parametric models incorporating structural information were used here to more fully resolve phylogenies for all known Rhodophyta lineages. Novel phylogenetic topologies were observed and well supported for each Class within the Rhodophyta resulting in a number of formally proposed or suggested taxonomic revisions. These include phylogenetic resolution of Rhodophyta Classes, support for the introduction of 11 genera within the Bangiales and support for various taxonomic revisions within the Florideophyceae previously proposed but not yet fully adopted. As structure evolves more slowly than its constituent sequence, secondary structure elements can further resolve evolutionary relationships, especially in lineages as old as the Rhodophyta. A novel encoding of secondary structure elements and subsequent multivariate analysis was performed for all known Rhodophyta nSSU rRNA gene sequences, reinforcing phylogenetic results. Computer programs developed for these analyses are publicly available. The analyses presented here significantly advanced understanding of the evolutionary distribution of cryptic species within the Rhodophyta. Furthermore, useful methods for the characterization of such species are presented, as is a demonstration of the utility of biologically realistic sequence models parameterizing nSSU rRNA structure in resolving ambiguous phylogenetic relationships. Most importantly, this work also represents a significant improvement toward taxonomy congruent with evolutionary history for the Rhodophyta.

Histologically, colorectal cancer (CRC) resides in the abnormal proliferation of epithelial cells of the colon mucosa, progressing from adenoma to adenocarcinoma. This cancer continues to be the third with the highest incidence and mortality worldwide. It is caused by an accumulation of genetic mutations and epigenetic silencing, in addition to other intrinsic and extrinsic risk factors. Due to the high rates of incidence and mortality, diagnostic and prevention tools have been created, implemented, and optimized. However, the need to develop tools that provide an increasingly early, rigorous and sensitive diagnosis remains a pressing need. In this sense, the objectives of this dissertation were: (1) to develop the state of the art about CRC diagnostic tools, (2) to summarize "omic" applications in order to identify microbial biomarkers related to CRC and (3) to compare the microbiome of Portuguese patients with CRC and healthy individuals. Currently, the diagnosis of CRC has been driven by more (e.g., colonoscopy) or less (e.g., imaging techniques, molecular biomarkers) invasive procedures. Recently, the search for microbial biomarkers through "omic" tools has been an alternative, mainly due to the relevance of the microbiome in the metabolic and physiological homeostasis, as well as in the functioning of the host immune system. Thus, the intestinal microbiome has been assigned an active role in the evolution of CRC, being able to influence or be influenced by the disease. In particular, the metagenomic and metabolomic analysis of the CRC-associated microbiome in stool samples has stimulated the scientific community in the search for sensitive, reliable, differential, stable, and early biomarkers in the non-invasive detection of the disease. However, these advances lack representativeness for several geographic areas, given the cultural, genetic, and environmental impact on the incidence of this disease. In this sense, the microbiome (with a focus on Bacteria) was analyzed in feces of two clinical groups constituted by Portuguese individuals (patients with CRC and healthy individuals), through the sequencing of the 16S rRNA gene using Ilumina MiSeq. This study is a contribution to fill the gap of existing knowledge about the microbiome associated with CRC in the Portuguese population. Although the structure of the fecal microbiome assumes homogeneous patterns among individuals of the same clinical group, there was some variability in the abundance of taxa between these groups and at different stages of CRC. For example, Prevotella, Alloprevotella, Sutterella, Desulfovibrio and Olsenella observed in CRC samples can serve as microbial biomarkers. In the future, the study will be extended to larger population samples, as well as to other types of human samples and clinical groups, in order to identify sensitive and specific microbial signatures that can translate the development of CRC, thus reducing incidence rates and mortality.
Do ponto de vista histológico, o cancro coloretal (CCR) reside na proliferação anormal de células epiteliais da mucosa do cólon, progredindo de adenoma a adenocarcinoma. Este cancro continua a ser o terceiro com maior incidência e mortalidade mundialmente. É causado por um acúmulo de mutações genéticas e silenciamento epigenético, para além de outros fatores de risco intrínsecos e extrínsecos. Devido às altas taxas de incidência e mortalidade, têm vindo a ser criadas, implementadas e otimizadas ferramentas de diagnóstico e prevenção. No entanto, continua premente a necessidade de desenvolver ferramentas que forneçam um diagnóstico cada vez mais precoce, rigoroso e sensível. Neste sentido, os objetivos desta dissertação consistiram em (1) desenvolver o estado da arte acerca das ferramentas de diagnóstico de CCR, (2) resumir as aplicações “ômicas” para indentificar biomarcadores microbianos relacionados com CCR e (3) comparar o microbioma de pacientes portugueses com CCR e indivíduos saudáveis. Atualmente, o diagnóstico de CCR tem vindo a ser conduzido por procedimentos mais (e.g., colonoscopia) ou menos (e.g., técnicas de imagem, biomarcadores moleculares) invasivos. Muito recentemente, a procura de biomarcadores microbianos através de ferramentas “ómicas” tem sido uma alternativa, principalmente devido à relevância do microbioma nas homeostase metabólica e fisiológica, assim como no funcionamento do sistema imunitário do hospedeiro. Assim, ao microbioma intestinal tem sido atribuído um papel ativo na evolução do CCR, podendo influenciar ou ser influenciado pela doença. Em particular, a análise metagenómica e metabolómica do microbioma associado a CCR em amostras de fezes tem estimulado a comunidade científica na procura de biomarcadores sensíveis, fidedignos, diferenciais, estáveis e precoces na deteção não invasiva da doença. Contudo, estes avanços carecem de uma representatividade para diversas áreas geográficas, dada o impacto cultural, genético e ambiental na incidência desta doença. Neste sentido, realizou-se a análise do microbioma (com enfoque em Bacteria) em fezes de dois grupos clínicos constituídos por indivíduos Portugueses (pacientes com CCR e indivíduos saudáveis), através da sequenciação do gene 16S rRNA usando Ilumina MiSeq. Este estudo é um contributo para colmatar a lacuna de conhecimento existente sobre o microbioma associado a CCR na população Portuguesa. Apesar da estrutura do microbioma de fezes assumir padrões homogéneos entre indivíduos do mesmo grupo clínico, houve alguma variabilidade na abundância de taxa entre esses grupos e em diferentes estádios do CCR. Por exemplo, maiores abundâncias de Prevotella, Alloprevotella, Sutterella, Desulfovibrio e Olsenella observadas em amostras de CCR podem servir como biomarcadores microbianos. No futuro, o estudo será alargado a amostras populacionais maiores, assim como a outro tipo de amostras humanas e grupos clínicos, no sentido de identificar assinaturas microbianas sensíveis e específicas, que possam traduzir o desenvolvimento de CCR, reduzindo, assim, as taxas de incidência e mortalidade.
Mestrado em Biologia Molecular e Celular

The main objective of this study was the characterization of surface associated microbial communities in breweries. In addition, the beer-spoiling potential of isolated strains and biofilm samples was investigated. Some studies reported the identity of cultivatable organisms from industrial plants. However, there were no data available about the composition of biofilm communities from these habitats for cultivation-independent techniques. Consequently, the fatty acid methyl esters (FAMEs) analysis, the fluorescence in situ hybridization (FISH) and the construction and investigation of 16S rRNA gene clone libraries were applied to reveal the structure of these communities. All of these methods have different advantages and therefore, they complement each other to get a more reliable picture of the biofilm communities. The cultivation method was included in this study because it enables a verification of results from other studies. Furthermore, the obtained strains are genuine brewery isolates and can be used for physiological tests. Isolates were obtained from seven different sample sites (Chapter 1 and 5). They were identified and affiliated to 25 different genera. Some of these strains were inoculated in beer but none of them was able to grow in it (Chapter 1 and 5). However, these strains can still be harmful for the industry, e.g. if they are able to form biofilms. This aspect was investigated by analyzing the potential of the isolates to produce acyl-homoserine lactones (AHLs) (Chapter 6). These quorum sensing mediating molecules are involved in the maturation process of biofilms. Indeed, some strains were found to secrete these autoinducer molecules, they mainly belonged to the genus Pseudomonas. An abundant proportion among the isolates was constituted by members of the Enterobacteriaceae (Chapter 7). In the beginning of this study, there was a minor suspicion concerning their beer-spoiling potential. Indeed, all isolated Enterobacteriaceae were found to be able to multiply in non-alcoholic beer under access of oxygen but they represented no risk for filled beer. The beer-spoiling potential of biofilm communities was investigated by inoculating them in beer (Chapter 3). These enrichments allowed the detection of minor proportions of beer-spoiling organisms. About 25% of the biofilms contained microorganisms which were able to multiply in beer with 4.8% of ethanol (v/v). The absence of anaerobic beer-spoiling bacteria in most of the biofilms was confirmed by using specific FISH probes for Pectinatus and Megasphaera cells (Chapter 9). However, Pectinatus cells constituted one of the most abundant groups in two biofilm communities. These samples clearly demonstrated that brewery biofilms can become hazardous for the quality of the product. The acetic acid bacteria were supposed to be abundant brewery biofilm organisms. This was not confirmed by any method used (Chapter 8). Instead, FISH signals were found for many other taxa in considerable proportions, e.g. communities from the conveyors consisted of members of the Eukarya, Archaea, Alpha-, Beta-, Gammaproteobacteria, Cytophaga-Flavobacteria, Planctomycetales, Actinobacteria and Firmicutes (Chapter 1). Such diverse communities were also evidenced for three other biofilms analyzed by FISH (Chapter 2 and 9). Whereas the FISH technique allows the specific detection of single cells, the FAME analysis targets all organisms present, except the Archaea. The fatty acid profiles of 78 biofilms indicated significant differences between the communities, even between those which were exposed to similar conditions. In addition, repeated sampling of identical sites revealed a temporal variability of the microbial communities (Chapter 3). Characteristical fatty acids of beer-spoiling bacteria were almost absent. Typical fatty acids of Eukarya dominated nearly half of all biofilms. The high proportions of Eukarya in some biofilms was not confirmed, as these samples were also investigated by FISH. This divergence was found to be due to the higher biomass of eukaryotic cells compared to bacterial cells (Chapter 3). As some wild yeast strains were isolated and characterized, they are a potential source of these fatty acids. In contrast to the revealed bacterial diversity, most of the isolated yeasts were assigned to Saccharomyces or Candida spp. (Chapter 4). The Saccharomyces spp. showed a high beer-spoiling potential and many Candida species were able to form biofilms. The construction of 16S rRNA gene clone libraries and the analysis of the clones with amplified ribosomal DNA restriction analysis (ARDRA) was performed with two biofilm communities (Chapter 2). Clones with identical ARDRA patterns were grouped and some representatives were identified by sequencing. These clone sequences were affiliated to 30 different genera, most of which were members of the Alpha- and Gammaproteobacteria and the Bacteroidetes. In addition, some clone sequences were assigned to uncultured organisms. Despite of the presence of 53 and 59 different ARDRA patterns in the two clone libraries, respectively, they had only four patterns in common. This result underlined the differences in the microbial composition of these communities. In conclusion, breweries represent a habitat with high cleaning and disinfecting pressure, which might have selected for a limited number of more resistant or adopted species. Instead, the community structures of biofilms in industrial environments were found to be diverse and variable in their compositions.

This thesis attempts to broaden what is known about bacterial symbionts within amoebae by the use of a number of different molecular methods. Initially a number of different amoeba strains were screened for bacterial symbionts by 16S rRNA gene PCR, then the symbionts were identified by comparative sequence analysis and phylogenetic analysis. The amoeba strains containing bacterial symbionts were characterised by cell morphology, 18S rRNA gene sequencing, internal transcribed spacer sequencing and allozyme electrophoresis. Amoebae belonging to the genera Acanthamoeba, Naegleria, Ripidomyxa and Saccamoeba were identified as containing symbionts that belonged to a wide range of different bacterial genera. Relationships between bacterial symbionts and their host amoebae were analysed by the use of transmission electron microscopy and fluorescent in situ hybridisation using symbiont specific probes. Also described are attempts that were made to isolate and grow the bacterial symbionts outside of their host amoebae as well as experiments to try to transfer bacterial symbionts from one amoeba strain to another. Lastly the results from this study are discussed as a whole to put into perspective how they contribute to the body of knowledge of symbionts within protozoa.

碩士
中山醫學大學
醫學檢驗暨生物技術學系碩士班
103
Background and aim: Periodontitis has a high prevalent rate in Taiwan and Porphyromonas gingivalis is a major pathogen in destructive periodontal disease in humans. Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain A (RgpA). Gingipains have been implicated in the pathogenicity of Porphyromonas gingivalis, a major etiologic agent of chronic periodontitis. The purpose of our studies is to determine the phylogenies of 16S RNA gene and Arg-gingipain (RgpA) gene of clinical P. gingivalis strains among different years. Methods: Samples were collected from gingival pockets of periodontitis patients. All samples were treated with genomic DNA extraction, and PCR was used to amplify 16S RNA and RgpA genes for DNA sequencing. DNA sequences of all amplicons were subjected to phylogenetic analysis by using MEGA 6.01. In addition, the DNA sequences of 16S RNA genes were also deposited and submitted to National Center for Biotechnology Information Genbank database to acquire accession numbers. Results: Fifty four samples were collected from gingival pockets of periodontitis patients. The positive rates of P. gingivalis were 38.9% and 18.5% by PCR and by culture, respectively. 16S RNA and RgpA gene fragments from ten samples were amplified and sequencing. Phylogenetic analysis of 16S RNA genes indicated that P. gingivalis strains were divided into two clusters and our isolates from each other were similar to two clusters. Similarly, phylogenetic analysis of RgpA genes showed that compared with the other strains, the P.g_CSMU24、P.g_CSMU33、P.g_CSMU38、P.g_CSMU41 and P.g_CSMU51 of our clinical isolates were significantly different. Conclusion: P. gingivalis was steadily evolved on both 16S RNA gene and RgpA gene. The correlation between gene variations, fitness on human being and disease severity is requires further studies.

碩士
臺灣大學
植物病理與微生物學研究所
98
A new disease named as periwinkle leaf yellowing (PLY) was first observed in a flower production farm in Dayuan Township (Taoyuan county, Taiwan) in August 2005. Sequence analysis of 16S rDNA, 16S-23S rDNA ISR, and partial 23S rDNA sequence revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows (AY) group (16SrI group) which cause diseases in many horticultural and vegetable crops worldwide, and can be delineated into 10 subgroups. Six cultivated plants including periwinkle plant, chrysanthemum, cosmos, torenia, Persian violet, goosegrass and cucumber, were determined to be the host plants of PLY phytolasma. Monthly PCR detection indicated that PLY phytoplasma was detected in host plants from June to October in 2007, and from July to October in 2007. However, it can be detected earlier since April in 2009. To further clarify the phylogenetic relationship of strain PLY among 16SrI phytoplasmas, six phylogenetic trees were constructed in this study. Beside the phylogenetic trees based on the independent analysis of 16S rRNA, rplV-rpsC, secY and tuf gene sequences, two other trees based on the analysis of the comprising gene sequence of 16S rRNA, rplV-rpsC and secY, and the comprising gene sequence of rplV-rpsC and secY were also constructed. The results indicate that the strain PLY was closely related to 16SrI-B and 16SrI-D subgroup, and could be a new subgroup based on the phylogenetic tree constructed by rplV-rpsC gene sequences. In addition, the phylogenetic analysis of the comprised gene sequences showed similar tree topology when compared with sequence analysis of the rplV/ rpsC gene or of the secY gene alone. The main difference is that the branch lengths were elongated in the comprised gene tree. To further confirm the subgroup affiliation of PLY phytoplasma, the 16S rRNA gene sequences of 10 closely related phytoplasma strains were digested in silico, and the similarity coefficients were then calculated. The results also support the conclusion that PLY phytoplasma might belongs to a new 16SrI subgroup. The putative restriction site analysis can also distinguish PLY phytoplasma from other close related phytoplasma strains in phylogenetic analysis.

Many studies of hyper saline environments have been performed, mainly on aquatic systems. However, the microbial community in terrestrial thallasohaline environments has not been studied extensively. To our knowledge, this is the first study of a natural terrestrial thallasohaline environment using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). We studied the great salt plains (GSP) for this purpose. The GSP is a perfect example of an extreme environment. The environment of the GSP changes frequently due to rain events that can change the salinity of the soil. Salt gradient samples and core samples were collected from the GSP in different years and DNA extractions were performed. 16S rRNA genes were amplified using PCR and examined on DGGE gels. Banding patterns of the DGGE gels were analyzed using Quantity One-Versa Doc software. Based on the banding patterns after DGGE, it was shown that the low- and high-salt soil samples had greater band richness than medium-salt soil samples. A dendrogram of relatedness was made and the samples were placed in different phenons using NTSYS. Core samples collected from the same locations exhibited similar microbial communities, and samples collected in different years from same location exhibited different microbial communities. Environmental factors such as soil salinity, water flow, and temperature, vary from year to year and from place to place on the GSP, which can select for different microbial communities in soil samples collected from the same place in different years and soil samples collected from different places on the GSP.
Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Biological Sciences.
"May 2006."

碩士
國立成功大學
醫學檢驗生物技術學系
93
Many species belonging to Enterococcus, Streptococcus, and nutritionally variant streptococci (NVS) including Abiotrophia and Granulicatella, are human pathogens. The feasibility of sequence analysis of the 16S-23S ribosomal DNA intergenic spacer (ITS) for identification of strains belonging to the four genera was evaluated. In this study, ITS sequences of 62 type strains (55 species) and 12 ITS sequences obtained from the GenBank database were used to construct an ITS database for identicication of strains belonging to Enterococcus, Streptococcus, and NVS. The ITS lengths of different species varied from 189 to 602 bp, with interspecies sequence similarities ranging from 0.31 to 0.996. ITS sequences were highly conserved among strains within the same species, with intraspecies similarity ranged from 0.98 to 1.0, except for strains of Granulicatella elegans. A total of 227 strains, including 89 reference strains and 138 clinical isolates, were identified by ITS sequencing. Cinical isolates producing discrepant species names by ITS sequencingwere reconfirmed by the Rapid ID 32 STREP system (bioMe’rieux Vitek, Marcy l’Etoile, France). A total of 27 strains (11.9%, 27/227), including reference strains and clinical isolates, were found to produce discrepant identification results between by ITS sequencing and. The sequences of 16S rRNA genes were further determined for species confirmation of strains producing discrepant identification. The sensitivity of ITS sequence analysis for species identification was 98.2% (226/230). Four strains were not identified by ITS sequencing because their corresponding ITS sequences were not available in the ITS sequence database. Based on ITS sequence, an oligonucleotide array was designed to identify these bacteria. A total of 391 strains, including 336 target strains (160 reference strains and 176 clinical isolates) and 55 nontarget strains were identified by the array. The sensitivity and specificity of the array were 100% (336/336) and 96.4% (53/55), respectively. In conclusion, both ITS sequence analysis and the array hybridization method could be used as accurate alternatives for species identification of Abiotrophia, Enterococcus, Granulicatella, and Streptococcus.

碩士
國立清華大學
生命科學系
89
Helicobacter pylori is a Gram-negative microaerophilic bacterium which colonizes the gastric mucosa of the human stomach. Infection with H. pylori is associated with gastritis, gastric ulcer, duodenal ulcer. The common treatment of H. pylori infection includes antibiotics and a combination of a proton pump inhibitor. Because of an increased use of antibiotic agents, resistance to antibiotics is the major cause of the therapy failure. The aim of this study is to investigate the efficacy of the triple therapy and the prevalence of the resistance of the three antibiotics used in eradication therapy, which are metronidazole, clarithromycin, and amoxicillin. 151 patients who took the triple therapies containing lansoprazole, clarithromycin, and metronidazole/amoxicillin in Taichung Veterans General Hospital were enrolled in this study and 137 patients completed the course of treatment. The resistance of fifty-one strains collected from patients in 2000 were determined by its minimal inhibitory concentration (MIC) values of the three antibiotics, which were determined by E-test. Two regimens were used in this study: the eradication rate of the LAC regimen total is 88.41% and it is 85.29% in LMC regimen. In LAC regimen, the prevalence of resistance of metronidazole and clarithromycin is 39.29% and 7.14% respectively. In LMC regimen, the prevalence of these two antibiotics are 43.48% and 4.35%. No amoxicillin-resistant strains are found in this study. Eighteen different strains from fifteen patients were analyzed for the reason of therapy failure. The strains were examined by polymerase chain reaction (PCR) and PCR-based restriction fragment length polymorphism (RFLP) of the vacA and ureA-ureB genes to examine whether the strains obtained before and after treatment are the same. Their MIC values were also determined by E-test. The rdxA and 23s rRNA genes from both antibiotic resistant and sensitive strains were sequenced to investigate the difference between those strains. We find that there are two re-infection cases in LAC regimen and six different strains six patients are resistant to metronidazole or clarithromycin and four different strains from four patients are resistant to both metronidazole and clarithromycin. In the gene analysis, most our data correspond with published data. We also find one interesting strain has the three amino acids deletion in its rdxA gene, which is not seen in other data.