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Extensive use of next-generation sequencing has the potential to transform our knowledge on how genomic variation within bacterial species impacts phenotypic versatility. Because different environments have unique selection pressures, they drive divergent evolution. However, there is also parallel or convergent evolution of traits in independent bacterial isolates inhabiting similar environments. The application of tools to describe population-wide genomic diversity provides an opportunity to measure the predictability of genetic changes underlying adaptation. Here, we describe patterns of sequence variations in the core genome among 99 individual Pseudomonas aeruginosa clinical isolates and identified single-nucleotide polymorphisms that are the basis for branching of the phylogenetic tree. We also identified single-nucleotide polymorphisms that were acquired independently, in separate lineages, and not through inheritance from a common ancestor. Although our results demonstrate that the Pseudomonas aeruginosa core genome is highly conserved and in general, not subject to adaptive evolution, instances of parallel evolution will provide an opportunity to uncover genetic changes that underlie phenotypic diversity.

Background The prevalence of peri-implantitis ranges between 7 and 38.4% depending on risk indicators such as smoking, diabetes mellitus, lack of periodontal maintenance program, and history or presence of periodontitis. Currently, the possible effect of the type of superstructure on peri-implant health is unclear. This cross-sectional study aims to investigate the influence of the superstructure on the prevalence of peri-implant mucositis, peri-implantitis and peri-implant dysbiosis. Methods During a 32-month recruitment period dental implants were assessed to diagnose healthy peri-implant tissues, mucositis or peri-implantitis. The study included 1097 implants in 196 patients. Out of all peri-implantitis cases 20 randomly chosen submucosal biofilms from implants with fixed denture (FD) originating from 13 patients and 11 biofilms from implants with removable dentures (RD) originating from 3 patients were studied for microbiome analysis. Composition of transcriptionally active biofilms was revealed by RNAseq. Metatranscriptomic profiles were created for thirty-one peri-implant biofilms suffering from peri-implantitis and microbiome changes associated with superstructure types were identified. Results 16.41% of the implants were diagnosed with peri-implantitis, 25.00% of implants with RD and 12.68% of implants with FD, respectively. Multivariate analysis showed a significant positive association on patient (p = p = 0.03) between the prevalence of peri-implantitis and RD. Eight bacterial species were associated either with FD or RD by linear discriminant analysis effect size method. However, significant intergroup confounders (e.g. smoking) were present. Conclusions Within the limitations of the present work, RDs appear to be a risk indicator for peri-implantitis and seem to facilitate expansion of specific periodontopathogens. Potential ecological and pathological consequences of shift in microbiome from RDs towards higher activity of Fusobacterium nucleatum subspecies animalis and Prevotella intermedia require further investigation.

Summary Gut colonization with multidrug-resistant (MDR) bacteria enhances the risk of bloodstream infections in susceptible individuals. We demonstrate highly variable degrees of ex vivo colonization resistance against a carbapenem-resistant Klebsiella pneumoniae strain in human feces samples and subsequently isolate diverse K. oxytoca strains from protected donors. Several of these K. oxytoca strains reduce gut colonization of MDR K. pneumoniae strains in antibiotic-treated and gnotobiotic mouse models. Comparative analysis of K. oxytoca strains coupled with CRISPR-Cas9-mediated deletion of casA, a protein essential for utilization of selected beta-glucosides, identified competition for specific carbohydrates as key in promoting colonization resistance. In addition to direct competition between K. oxytoca and K. pneumoniae, cooperation with additional commensals is required to reestablish full colonization resistance and gut decolonization. Finally, humanized microbiota mice generated from K. pneumoniae-susceptible donors are protected by K. oxytoca administration, demonstrating the potential of commensal K. oxytoca strains as next-generation probiotics.

Health-care-associated infections by multi-drug-resistant bacteria constitute one of the greatest challenges to modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds, among which the acquisition of carbapenemases is one of the most concerning. In Klebsiella pneumoniae, the dissemination of the K. pneumoniae carbapenemase is tightly connected to the global spread of certain clonal lineages. Although antibiotic resistance is a key driver for the global distribution of epidemic high-risk clones, there seem to be other adaptive traits that may explain their success. Here, we exploited the power of deep transcriptome profiling (RNA-seq) to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origins. We identified a large set of 3346 genes which was expressed in all isolates. While the core-transcriptome profiles varied substantially between groups of different sequence types, they were more homogenous among isolates of the same sequence type. We furthermore linked the detailed information on differentially expressed genes with the clinically relevant phenotypes of biofilm formation and bacterial virulence. This allowed for the identification of a diminished expression of biofilm-specific genes within the low biofilm producing ST258 isolates as a sequence type-specific trait.

Health-care-associated infections by multi-drug-resistant bacteria constitute one of the greatest challenges to modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds, among which the acquisition of carbapenemases is one of the most concerning. In Klebsiella pneumoniae, the dissemination of the K. pneumoniae carbapenemase is tightly connected to the global spread of certain clonal lineages. Although antibiotic resistance is a key driver for the global distribution of epidemic high-risk clones, there seem to be other adaptive traits that may explain their success. Here, we exploited the power of deep transcriptome profiling (RNA-seq) to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origins. We identified a large set of 3346 genes which was expressed in all isolates. While the core-transcriptome profiles varied substantially between groups of different sequence types, they were more homogenous among isolates of the same sequence type. We furthermore linked the detailed information on differentially expressed genes with the clinically relevant phenotypes of biofilm formation and bacterial virulence. This allowed for the identification of a diminished expression of biofilm-specific genes within the low biofilm producing ST258 isolates as a sequence type-specific trait.

A fundamental study of the undrained behaviour of sands under multi-axial loading is presented. The study was performed by using the hollow cylinder torsional (HCT) device. The HCT is the only device that permits a soil specimen to be subjected to multi-axial loading with controlled variations in the magnitudes of the three principal stresses and the direction of the major principal stress with the vertical deposition direction. The main objective of the study was to assess the effects of principal stress magnitude, directions and their rotation on sand liquefaction. This is achieved by a systematic study of static and cyclic undrained behaviour of reconstituted loose sand. Shear loading is carried out under strain control. Only such loading permits the needed capture of post peak strain softening characteristics of loose sands. Undesirable runaway strains are inevitable in stress controlled loading modes. In addition to the investigations in the hollow cylinder torsional device, sand behaviour in simple shear as well as under the triaxial conditions was also assessed as reference for comparisons with that under multi-axial stresses. The investigations were carried out using two sands - Fraser River sand and Syncrude sand. Sand specimens were reconstituted by water pluviation, which is considered to duplicate the fabric ofin-situ fluvial and hydraulic fill deposits. Independence of the effective stress path and stress-strain characteristics from the total stress path under fixed principal stress directions and constant value of intermediate principal stress parameter is illustrated. The undrained response of loose sand is highly dependent on the loading direction, implying inherent anisotropy. The friction angle mobilized at phase transformation or steady state is a unique material property, independent of the mode of loading static or cyclic, direction of principal stresses, intermediate principal stress level, consolidation history and the stress and void ratio state prior to undrained shear. There is no unique relationship between steady state or phase transformation strength and void ratio that is independent of the stress path, implying that a unique steady state line does not exist for a sand. The influence of intermediate principal stress, on undrained response is small when the intermediate principal stress parameter, that reflects value of this stress relative to the major and the minor values, is less than about 0.5. At constant values of other parameters increasing confining stress and decreasing relative density under multi-axial loading promote a higher degree of contractive response. The history of principal stress directions during principal stress rotation does not seem to have any appreciable effect on the peak and steady state or phase transformation strength. These strengths are apparently controlled by the peak value of major principal stress inclination experienced during shearing with respect to vertical direction. Principal stresses undergo continuous rotation from 0 to about ±45° in simple shear deformation. A simultaneous change in intermediate principal stress occurs as the major principal stress rotates. The maximum shear stress and maximum shear strain in conventional simple shear deformation approximately equals the horizontal shear stress and shear strain respectively. For a given initial stress and void ratio state, the number of cycles to liquefaction is smaller under cyclic triaxial than under similar 90° jump rotation that do not invoke the weakest triaxial extension loading mode during shear. For a given direction of principal stresses, if the sand is contractive under static loading, it would also be contractive under cyclic loading, provided that the cyclic deviator stress amplitude is higher than the steady state or phase transformation strength in static loading.

Dynamic properties of soils have to be well understood in order to assure stability and acceptable performance of soil structures under seismic and wave loadings. It has been found the two important dynamic properties - shear modulus and damping factor are complex functions of many variables. In order to study the influence of various factors on shear modulus and damping factor, drained cyclic torsion shear tests were carried out in the hollow cylinder torsion device using medium Ottawa A S T M C-109 sand. Effects of shear strain amplitude, stress history, effective mean normal stress (σ'm = l/3(σ-'1 +σ'2 + σ'3)), principal effective stress ratio (R = σ1/σ3), intermediate principal stress parameter (b = σ2 —σ3)/(σ1 — σ-3)), void ratio, number of cycles of loading are some of the factors studied in this thesis. During the application of cyclic shear stress σ'm ,R and b were kept constant at pre-selected values for each test. This technique allows to study the effect cr'm, R and b independently. For example, the effect of R on dynamic properties can be isolated by a series of tests on specimens that have identical σ'm and b but different levels of R and all parameters σ'm, R and b are held constant during cyclic shear application. It is shown that shear modulus increases with number of cycles of a constant amplitude cyclic shear stress when the induced shear strain is higher than a certain threshold value. The damping, however, decreases with number of cycles even at strain amplitudes less than this threshold value. There is also a threshold value of shear strain below which zero volumetric strain occurs due to cyclic shear loading, and hence no pore pressure would develop if cyclic loading was undrained. Effects of stage testing and small strain history on dynamic properties is shown to be insignificant. With decrease of void ratio, shear modulus increases and damping factor decreases. It is shown that for a given b, the void ratio factor F(e) = (2.17 —e)² ( l + e), collapses the modulus degradation curves obtained at different void ratios in to a single curve. For a given initial stress state and shear strain amplitude, shear modulus obtained at different R levels do not show any significant difference when R < 3. Damping factors, however, seems to be unaffected by the change in R at all R levels. When R < 3, shear moduli in triaxial extension condition (b = 1) are found to be less than those in triaxial compression condition (b = 0) and damping factors for b = 1 are higher than those for b = 0. Both triaxial compression and extension state of loadings yielded same values shear modulus and damping factors at large amplitude of shear strain at R = 3. Test results indicate that when b < 1, the dynamic properties are independent of intermediate principal stress. Effects of stress history due to decrease in R from 3 to 2, is significant only in the small strain range, and as the strain level increase, the effects of stress history diminishes.