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Members of the Legionellaceae cause respiratory infections in man; the most severe, pneumonic form is known as Legionnaires' disease. Of the 39 species described to date 16 have been associated with human disease, however the majority of reported cases of legionellosis are caused by Legionella pneumophila serogroup 1. A number of pathogenic bacteria regulate their virulence gene expression in response to environmental stimuli. Temperature and the availability of iron are considered to be stimuli which signal entry to a host environment. The first part of this study utilised chemostat culture to investigate the influence of growth temperature and the availability of iron on the physiology, morphology and virulence of L. pneumophila serogroup 1. This study demonstrated, for the first time, that the virulence of L. pneumophila was significantly reduced (P < 0.05) when the culture temperature was lowered from 37 to 24°C and this modulation was reversed by returning the temperature to 37°C which resulted in a statistically significant (P < 0.05) increase in virulence. Further experiments demonstrated that the concentration of iron in the growth medium also had an effect on virulence. Contrary to expectations iron-limited cultures were less virulent than those grown iron-replete. This modulation was also reversible with a return to virulence when iron-replete conditions were restored. The physiology and morphology of L. pneumophila were also influenced by both growth temperature and iron-limitation. At 24°C cultures consisted of flagellated short rods, whereas cultures grown at 37°C were pleomorphic and flagella were not evident. It was demonstrated that L. pneumophila accumulates the intracellular carbon storage compound, polyhydroxybutyrate, and that the proportion of the cell dry weight which it comprised varied with growth temperature, being maximal at 24°C. The ratio of saturated to unsaturated fatty acids in L. pneumophila decreased as the temperature was reduced to 24QC; this is a common strategy designed to maintain membrane fluidity. Siderophore production was detected in iron-limited cultures but not in iron replete cultures. Protease production was also affected by both growth temperature and iron-limitation. The BIOLOG bacterial identification system was modified for use with legionellae and this was used to investigate the metabolic versatility of these bacteria. A database containing substrate utilisation profiles of Legionella species was constructed using the modified system; this was then used to identify legionella isolates to species level. Evaporative cooling towers are a significant source of Legionnaires' disease accounting for the majority of outbreak cases in the United Kingdom. In the second part of this study a microbiologically-contained, fully-functional evaporative cooling tower was constructed and used to investigate factors that could influence the growth of L. pneumophila in such systems. The mode of operation of the cooling tower was found to influence the multiplication of legionellae in the system. Low-usage situations resulted in enhanced growth of L. pneumophila. Growth of L. pneumophila demonstrated a significant positive correlation with water temperature but its concentration decreased with increased conductivity. The concentrations of calcium, magnesium, potassium and zinc and the total hardness of the water all exhibited inverse relationships with legionella population size. The protocol for the emergency disinfection of cooling systems recommended in the Report of the Department of Health Expert Advisory Committee on Biocides did not eradicate L. pneumophila from the experimental cooling tower.

The objective was to compare pentachlorophenol (PCP) degradation in contaminated groundwater by indigenous and bio-augmented (Sphingobium chlorophenolicum and Burkholderia cepacia) PCP degrading bacteria. Indigenous bacteria were identified by cloning and sequencing of 16S rDNA fragments while PCP concentrations were determined by GC-ECD. Gene expression for PCP degrading enzymes: chlorophenol 4-monooxygenase (TftD, B. cepacia) and pentachlorophenol-4-monooxygenase (pcpB, S. chlorophenolicum), was determined by RT-PCR. B. cepacia, a PCP degrading bacteria was identified as dominant indigenous bacteria. PCP concentrations correlated negatively with PCP tolerant bacteria and relative fold gene expression in treatments with air-sparging (phase2) compared to without air-sparging (phase1). PCP concentrations decreased and TftD or pcpB expressions were higher in treatments inoculated with B. cepacia (49%, 10.7 fold) or S. chlorophenolicum (32%, 7 fold), respectively, than un-inoculated (indigenous) or mixed culture inoculated treatments. Thus bio-augmentation of indigenous bacteria with B. cepacia or S. chlorophenolicum resulted in more PCP degradation than indigenous bacteria.

No sentido de colaborar com o estudo da redução de microrganismos em Endodontia, foi realizada uma análise crítica dos modelos metodológicos in vivo sobre as técnicas moleculares utilizadas na avaliação da microbiota endodôntica e na capacidade de medir a antissepsia que o tratamento endodôntico proporciona. Desta maneira, os trabalhos foram agrupados de acordo com a proposta do presente estudo. Na análise critica e comparativa, pode-se observar uma grande variedade de métodos moleculares aplicados nos estudos, sendo que o mais utilizado nos ensaios para avaliação da microbiota foi o PCR. Havia estudos que utilizavam uma combinação de vários métodos onde foi possível identificar microrganismos ainda não conhecidos. Nos ensaios que utilizam iniciadores universais e que se valem da identificação de microrganismos através do DNA, foram listados de maneira a poder observar que o iniciador universal formado pela seguinte cadeia de oligonucleotídeos: F: 5- AGA GTT TGA TCC TGG CTC AG-3/R: 5-ACG GCT ACC TTG TTA CGA CTT-3 foi o mais descrito pela literatura. / In order to collaborate with the study of disinfection in Endodontics, was performed a study of critical methodological models in vivo on the molecular techniques used in the evaluation of endodontic microbiota and the ability to measure the disinfection capacity of endodontic treatment. Thus, the studies were grouped according to the purpose of this study. Under in critical and comparative analysis its can be seen a wide variety of molecular techniques used in the studies, and as used in the tests was to evaluate the microbial PCR studies using a combination of various methods which could be identified microorganisms has not known. In assays using universal primers which rely on the identification of microorganisms using the DNA, were listed as to be able to observe that the universal primer chain formed by the following primers: F: 5\'-AGA GTT TGA TCC TGG CTC AG-3 \'/ R: 5\'-ACG GCT ACC TTG TTA CGA CTT-3\' was as described in the literature.

No sentido de colaborar com o estudo da redução de microrganismos em Endodontia, foi realizada uma análise crítica dos modelos metodológicos in vivo sobre as técnicas moleculares utilizadas na avaliação da microbiota endodôntica e na capacidade de medir a antissepsia que o tratamento endodôntico proporciona. Desta maneira, os trabalhos foram agrupados de acordo com a proposta do presente estudo. Na análise critica e comparativa, pode-se observar uma grande variedade de métodos moleculares aplicados nos estudos, sendo que o mais utilizado nos ensaios para avaliação da microbiota foi o PCR. Havia estudos que utilizavam uma combinação de vários métodos onde foi possível identificar microrganismos ainda não conhecidos. Nos ensaios que utilizam iniciadores universais e que se valem da identificação de microrganismos através do DNA, foram listados de maneira a poder observar que o iniciador universal formado pela seguinte cadeia de oligonucleotídeos: F: 5- AGA GTT TGA TCC TGG CTC AG-3/R: 5-ACG GCT ACC TTG TTA CGA CTT-3 foi o mais descrito pela literatura. / In order to collaborate with the study of disinfection in Endodontics, was performed a study of critical methodological models in vivo on the molecular techniques used in the evaluation of endodontic microbiota and the ability to measure the disinfection capacity of endodontic treatment. Thus, the studies were grouped according to the purpose of this study. Under in critical and comparative analysis its can be seen a wide variety of molecular techniques used in the studies, and as used in the tests was to evaluate the microbial PCR studies using a combination of various methods which could be identified microorganisms has not known. In assays using universal primers which rely on the identification of microorganisms using the DNA, were listed as to be able to observe that the universal primer chain formed by the following primers: F: 5\'-AGA GTT TGA TCC TGG CTC AG-3 \'/ R: 5\'-ACG GCT ACC TTG TTA CGA CTT-3\' was as described in the literature.

L'exploration complète de la diversité bactérienne reste incomplète, due à des limitations techniques (extraction d'ADN incomplète, limites des techniques de caractérisation…), à la complexité et l'hétérogénéité de la matrice sol (présence de microenvironnements…) et à l'existence de populations numériquement faibles. Bien que ces populations minoritaires jouent probablement un rôle important dans le fonctionnement de la communauté, elles sont rarement détectées par les techniques conventionnelles. Nos objectifs dans le cadre de ce travail de thèse ont été de développer des approches tant conceptuelles que techniques qui permettent d‟accéder à un niveau plus important de la diversité bactérienne présente dans les environnements complexes comme les sols. La première approche a pour but de favoriser le développement de bactéries minoritaires grâce à l'inoculation de communautés bactériennes dans des sols stérilisés avec des propriétés physico-chimiques différentes, afin de confirmer ou d‟infirmer le concept de Baas Becking « tout est partout et l‟environnement sélectionne ». Les changements de structures des communautés bactériennes inoculées, sont analysés à l'aide de puces à ADN taxonomique nouvellement développées. Les résultats confirment clairement l'impact de l'environnement sur la structure des populations bactériennes. De plus l'analyse des puces à ADN révèle des bactéries précédemment non détectées, confirmant la présence de populations minoritaires et la possibilité d‟augmenter leur abondance relative sous différentes conditions. La deuxième approche consiste au développement d'une souche bactérienne capable de capturer in situ des fragments d‟ADN. Elle a pour but d'éviter l'étape d'extraction d‟ADN. Cet outil est basé sur la construction d'un système de contre sélection positive permettant de sélectionner les clones ayant intégré des gènes d'intérêt. Nos travaux montrent clairement qu'il est possible d'améliorer l'accès à cette « rare biosphere » sans toutefois pouvoir répondre entièrement à la question : est-ce que tout est partout? / No abstract

Typical bacterial analysis involves culturing and visualizing colonies on an array of agar plates. The growth patterns and colors among the array are used to identify the bacteria. For fast growing bacteria such as Escherichia coli, analysis will take one to two days. However, slow growing bacteria such as mycobacteria can take weeks to identify. In addition, there are some species of bacteria that are viable but nonculturable. This lengthy analysis time is unacceptable for life-threatening infections and emergency situations. It is clear that to decrease the analysis of the bacteria, the culturing and growth steps must be avoided. The goal of this research is to design, build, and test a device that could decrease the analysis time of bacteria. Device design accommodates for the varied growth and environmental conditions of expected samples for bacterial analysis. Clinical samples containing bacteria come in a wide variety of forms including urine, saliva, sputum, blood, etc. Each medium will have associated debris and other contaminants that must be isolated from bacteria before identification. This process can be challenging as bacteria and debris can range in size from a fraction of a micrometer to tens of micrometers. In addition, a device must be equipped to accurately identify bacteria regardless of growth conditions. Thus, to decrease the analysis time of bacteria, a device must be capable of isolation, concentration, and identification at a micron level. In this dissertation, a device was designed, built, and tested that incorporates dielectrophoresis for cell sorting and Raman spectroscopy for identification. Using the device, bacteria (1 μm in length) were successfully isolated away from 5 μm polystyrene spheres and Raman spectra of the trapped bacteria were collected. The simultaneous isolation and identification of bacteria from a mixed sample indicates the capability for the cDEP-Raman device to decrease the analysis time of bacteria from clinical samples.

<p>  </p> <p>An optical technique that discriminates microbial organisms using an elastic light scatter (ELS) pattern, known as BActeria Rapid Detection using Optical Technology (BARDOT), has shown excellence in pathogen screening which effectively saves time and cost during the identification. Owing to the successful implementation of the light-scattering technique in microbiology, a series of studies on the light scatter pattern have been conducted to improve the technology. As an extended study of the multispectral application in BARDOT, a hyperspectral elastic light-scatter phenotyping instrument (HESPI) was developed to increase the ability to discriminate and detect foodborne pathogens. The newly designed instrument integrated a supercontinuum (SC) laser into the traditional BARDOT system to provide a broad spectrum of the light source. An acousto-optic tunable filter (AOTF) was utilized to select the wavelength of interest, allowing multiple spectral patterns in a single measurement. Owing to the filtering mechanism of AOTF, the wavelength of the laser was shifted rapidly so the overall acquisition time of 80 hyperspectral patterns was less than30 seconds. A pair of optical lenses were used to compensate for the beam spot movement caused by the wavelength-dependent separation angle at the exit of AOTF. To capture the transmitted scattering patterns, a complementary metal-oxide-semiconductor (CMOS) sensor was placed under the bacteria sample plate.</p> <p>For a comprehensive understanding of the ELS patterns, at first, the diverse nature of bacterial colony morphology was explored. Using the optical scatter model based on the scalar diffraction theory, the forward light-scatter patterns were simulated with respect to various colony shapes. The numerical predictions were then compared to the scattering patterns that were experimentally obtained from the colonies with various elevation profiles. The experimental verification proved a strong correlation between the colony morphology and the ELS pattern, as an excellent agreement between the simulation and the experiment observed. Second, the wavelength-dependent characteristics of the ELS patterns were investigated. Based on the theoretical and experimental interrogation, the wavelength of the incident laser beam affected the shape of ELS patterns by the overall size, the number of diffraction rings, and the gap distance between the rings. </p> <p>The performance of HESPI was validated by differentiating green leafy microflora using the hyperspectral ELS patterns. A group of bacteria that were poorly classified with the traditional single-wavelength method was selected to prove the improvement of the classification by the hyperspectral application. HESPI was utilized to measure the hyperspectral ELS patterns of their colonies, and for the classification, the descriptive features were extracted from the patterns at 70 selected wavelengths within the 473 – 709 nm region. A classification model was constructed for every wavelength, and the classification accuracy of the individual model ranged from 88.7% to 93.2%. The classification result also showed that colonies of varied species produced distinctive scatter features at a particular spectral band. When employing the entire wavelengths for the classification, the more number of wavelengths consequently led to an increase in the number of scatter-pattern features. This could cause the classifier's overfitting and negatively affect the classification. Therefore, the presented work incorporated various feature reduction and selection procedures to enhance the robustness and ultimately lessen the complexity of data collection. A classification model with feature reduction improved the overall classification rate to 95.9% after selecting meaningful predictors.</p>

Natural products chemistry is the pursuit of bioactive small molecules from living organisms. These can be classified as primary metabolites if they are essential to survival, and secondary metabolites if they are accessory, playing a role in communication, defense, recruitment, etc.. Natural products have made a significant contribution to society – of 1,881 FDA-approved drugs from 1981 to 2019, 4% were pure natural products, 19% were natural products derived, and 3% were synthetic drugs with a natural products pharmacophore targeting a wide range of diseases and infections (Newman & Cragg, 2020). Pharmacophores are structural components of drugs that are responsible for the observed biological activity. Natural products often contain unique pharmacophores that exhibit potent bioactivity, thereby serving as inspiration for synthetic chemists to manufacture exciting new drug leads. Bacteria are ubiquitous in the environment. Marine bacteria are a prolific source of chemically diverse natural products due to the high biodiversity and competition in the marine environment. In 2018, 240 new marine natural products were reported in the literature from bacteria (Carroll et al., 2020). It is hypothesized that secondary metabolites offer an advantage to the producer, however, the roles that natural products play in their environment are not as well characterized. These pursuits are classified as chemical ecology. Throughout my thesis, I aim to identify the bacteria present from these environments and begin to understand the ecological role small molecules play in their environment. Staphylococcus aureus is notorious for causing chronic infections and resisting therapeutic treatment by forming biofilms. Biofilms are extracellular polymeric substance (EPS) matrices containing bacteria that attach to biotic and abiotic surfaces. The EPS matrix provides a refuge and anchorage to a surface, allowing biofilm inhabitants to be shielded from full strength of therapeutic treatments leading to resistance. Variovorax paradoxus is a gram-negative bacteria that also produces biofilms. It has been previously reported that V. paradoxus inhibits S. aureus biofilm formation. Preliminary data suggests V. paradoxus produces a small molecule that has biofilm inhibition activity. My work focuses on characterizing a GLP and another secondary metabolite produced by V. paradoxus that inhibits S. aureus biofilms through both molecular biology and natural products chemistry. Caulerpa spp. is a macroalgae native to tropical and subtropical oceans. Due to global warming, the temperature of oceans continues to rise, allowing Caulerpa spp. to inhabit higher latitudes. It has been hypothesized that successful invasion occurs by outcompeting native organisms via exerting adverse effects on the surrounding environment. The secondary metabolites of this algae are well characterized however their ecological role is hardly characterized. We hypothesize that Caulerpa spp. could be chemically mediating its surface microbiome by recruiting a higher percentage of Vibrio spp.. Vibrio spp. are known pathogens to humans and marine organisms by causing infections and forming biofilms. My goal was to identify a panel of culturable Caulerpa spp. surface-associated bacteria through molecular and microbiology methods. Microalgae are an exciting alternative source of biofuels. However, microalgae are grown in open algal ponds which are susceptible to crashing causing the total loss of an algal crop. Pond crashes are caused by a number of factors, one of which is contamination by unwanted pests such as protozoans and fungi. Previous studies focused on the use of bacterial communities as a built-in biocontrol to inhibit pests from causing algal pond crashes. Preliminary data demonstrated the addition of a bacterial community protected the microalgae Microchloropsis salina from grazing by the marine rotifer Brachionus plicatilis (Fisher et al., 2019). My work focuses on analyzing the composition of the protective bacterial community added to the microalgae that have been size filtered to observe bacterial association with algae, rotifers, or free-floating. M. salina cultures in the presence and absence of B. plicatilis were analyzed for the identification of protective bacterial species that were algae-, rotifer-associated, or free-floating. This work has been submitted to the journal Algal Research and is under review (Fisher et al., 2022). Bacteria play a significant role in their environment. The identification of bacterial species and the role their suite of small molecules play is crucial to fully characterizing the observed interactions. My thesis surveys several means of bacterial community analysis through identification and small molecule characterization.

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis