In healthcare settings and beyond, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) among other pathogens exchange antibiotic resistance and virulence factors and emerge as new infectious clones. According to the Saudi General Authority for Statistics (stats.gov.sa), Saudi Arabia is a country where more than 27 million pilgrims meet in annual continual mass-gathering events. This massive influx of people could introduce novel pathogens to the community that could not necessarily be detected with traditional culture-dependent clinical microbiological tests. Conventional clinical microbiology and environmental pathogen detection methods have had many limitations and narrow search scope. These methods can only target known and culturable pathogens. Over the past decade, applications of next-generation sequencing (NGS) and bioinformatics tools have revolutionized the way pathogens are detected and their relevant phenotypes such as clonal types, antibiotic resistance are predicted to aid in clinical decision making as additional practice to traditional clinical microbiology-based testing protocols. The aim of this study was to apply whole-genome sequencing (WGS) and bioinformatic analysis tools on clinical samples and bacterial isolates in order to pave the way for transforming current clinical microbiology practices in a tertiary referral hospital in Jeddah, Saudi Arabia. My attempt to utilize WGS as a tool on pathogenic strains in this study combined with the clinical data has resulted in discovering a silent outbreak of an emerging hypervirulent strain of Klebsiella pneumoniae (Chapter 2). Analysis of the strains antimicrobial profiles genetically has yielded the first characterization of a misidentified Klebsiella quasipneumoniae harboring plasmid-mediated carbapenemases of Klebsiella pneumoniae carbapenemases (KPC) (Chapter 3). Similarly, I was able to study mobile colistin resistance genes in the isolates and identify a novel occurrence of mcr-1 and mcr-8 (Chapter 4). I applied clinical metagenomic protocol on an intestinal biopsy of an inflammatory bowel disease patient with Crohn’s disease, where I identified an association of three co-occurring and an actively replicating non-tuberculosis mycobacteria (Chapter 5). The deployment of whole-genome sequencing and metagenomic in infectious disease surveillance and diagnostics could prove beneficial in limiting epidemics and detect transmission patterns of antimicrobial-resistant genes.

<p>Mycobacterium abscessus (Mab) is a non-tuberculous mycobacterium that is highly resistant to many antibiotics. Mab causes pulmonary infections in immunocompromised individuals. The presence of efflux pumps to pump out antibiotics and its ability to form biofilms makes Mab a virulent pathogen. Studies have been done on Mab antibiotic tolerance but there are still a lot of gaps in knowledge about the effects of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis in this bacterium during biofilm formation. In this study, we investigated the effects of the EPIs chlorpromazine (CPZ), 1-(1-naphthylmethyl)-piperazine (NMP), thioridazine (TRZ), Phenylalanine-arginine β-naphthylamide (PBN) and plumbagin (PLU) on antibiotic resistance, efflux, biofilm formation and lipid biosynthesis associated with log-phase growth and biofilm formation in Mab. We used the resazurin assay to determine the minimum inhibitory concentration (MIC) of the EPIs. We investigated the effects of the EPIs during biofilm-forming growth conditions on the MICs of antibiotics such as clarithromycin, amikacin, cefoxitin, ciprofloxacin which are the frontline antibiotics used to treat non-tuberculous mycobacterial infections. We also assessed the effects of the EPIs on the accumulation and efflux activities of the Mab cells through ethidium bromide (EtBr) assay. Furthermore, we evaluated the effects of the EPIs at sub-MIC concentrations on Mab biofilm formation under normoxic and hypoxic conditions. We utilized metabolic radiolabeling methods using 14C-palmitic acid and 14C- acetic acid which are precursors of lipid biosynthesis and analyzed lipids by silica-thin layer chromatography and autoradiography. We observed that Mab cells developed higher tolerance to the EPIs in a biofilm-stimulating medium. Furthermore, a decrease in the MICs of antibiotics was observed in the presence of the EPIs. Also, in the presence of the EPIs, there was less efflux activity within the Mab cells. In addition, EPIs inhibited biofilm formation significantly. We also noticed that NMP and PBN inhibited 14C-palmitic acid and 14C- acetic acid incorporation into polar lipids such as glycopeptidolipids, trehalose monomycolate, phosphatidylethanolamine, phosphatidylglycerol/cardiolipin, phosphatidylinositol mannosides at specific tested conditions. Our findings suggest that the EPIs inhibited the activities of the efflux pumps associated with the efflux of the antibiotics and lipid biosynthesis involved in biofilm formation. In conclusion, the results from this study gives insights on possible therapeutic opportunities.</p>

<div>As aging proceeds, the occurrence of cardiovascular diseases increases independent of other risk factors. At atherosclerotic sites, the rise in the senescent cell population was also observed. Patients with Hutchinson Gilford Progeria Syndrome (HGPS) also showed accelerated aging syndromes and extensive atherosclerosis progression, which was due to missense mutations on the LMNA gene that led to the production of progerin, an aberrant lamin A isoform instead of regular lamin A protein. Lamins act as structural and functional components in nuclear lamina, and recent findings suggested that the ectopic expression of mutant lamin A or lamin A precursor (prelamin A) not only caused defects in cell mechanics but also disturbed mechanotransduction pathways involving lamin A, both of which may contribute to vascular dysregulation. Moreover, the observation of the accumulation of prelamin A in normal aged vascular cells further suggests shared dysregulations involving lamin A in the vascular system between aged people and HGPS patients.</div><div>In the vascular system, endothelial cells were well regulated by hemodynamic forces in vivo to maintain vascular homeostasis. Endothelial dysfunction, including impaired vasodilation and increased permeability, was regarded as the initial marker of atherosclerosis. Despite recent advancements and discussions about the potential mechanisms of progerin-induced vascular disorders, how progerin triggers endothelial dysfunction in a mechanical environment as an early event during atherosclerotic lesion formation has not been studied intensively.</div><div>To help answer the gap question, we first set our goal to understand the effect of laminar flow at arterial levels on endothelial lamins as part of the aging process. Spatial and temporal changes in lamin A/C expression were observed as cell passage went up without flow present. As shear stress was applied, lamin A/C expressions were modulated on both transcriptional and translational levels, which were also dependent on PDL. To further examine how progerin was involved in EC functions with a particular focus on the flow effects, we next generated a stable endothelial cell line that expressed progerin as our EC aging model. Endothelial wound repair under laminar flow at different rates was characterized, and differential cell proliferation activities, as well as migration deficiencies in progerin-expressing ECs during the process, were also recognized. Furthermore, we also showed the overactivated mTORC2 pathway and unusual actin polymerization activities in these cells after flow application. Our results reported changes in cell migration by progerin with flow application for the first time and provided potential candidate pathways that were disturbed by progerin under arterial flow, which may help explain the high occurrence of atherosclerotic lesions in HGPS vasculature, even at straight portion. The reported progerin-induced wound recovery defects in endothelial cells in the presence of physiological flow may also suggest a mechanism of how progerin disturbs endothelial integrity and functions under mechanical stimuli in the development of vascular pathologies.</div><div>Further extended studies may help to understand the roles of progerin in initiating atherosclerosis, which will aid in the development of potential therapies for those suffering from prelamin A-associated accelerated aging syndromes.</div>

Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete. This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification. Four main areas are examined: (i) The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples. (ii) The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus. (iii) Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study. (iv) Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.

Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A are a major public health concern in Kathmandu. The aim of this thesis was to identify and assess the population most at risk by investigating epidemiologic trends of enteric fever within a subset population of Kathmandu. Therefore,the burden and incidence of enteric fever within the study population and the seasonal and gender distribution of enteric fever was assessed. Considerable burden of enteric fever, unrelated to population density, correlating with the seasonal fluctuations in rainfall was observed. This thesis also aimed to improve the understanding of enteric fever transmission by identifying probable transmission routes,hence various water and food samples were analysed and the extent of faecal contamination in them was determined. S. Typhi isolates were sequenced and genotyped and combined with GPS data to longitudinally study the local distribution and infer transmission of this human restricted bacterial pathogen. Extensive clustering of typhoid independent of population size and density and existence of an extensive range of genotypes within typhoid clusters including individual households with multiple cases was observed. These observations predict that indirect transmission had an overwhelming contribution for disease persistence, potentially through contaminated water. Consistent with this hypothesis, S. Typhi and S. Paratyphi A were detected in water supplies and it was observed that typhoid was spatially associated with public water sources and low elevation. A concurrent case-control study was also conducted which allowed for the determination of risk factors in the population at risk. These studies imply that resources should be allocated toward controlling the most important vectors of enteric fever, including food sold by vendors, chlorination of drinking water, construction of proper water distribution and sewage networks,vaccination campaigns and hygiene education.

The poor stability of most therapeutic viruses in the human bloodstream is a major obstacle in the field of cancer virotherapy, preventing systemic intravenous delivery to treat tumour metastases. Delivery is typically limited by inactivation of virus particles by blood components and rapid scavenging by hepatic phagocytes. Members of the Alphavirus family are exposed to blood during natural infections; as such, we hypothesised that evolutionary pressure may have led to blood stability and clearance kinetics superior to those of other viruses currently in development for use as oncolytic agents. Sindbis virus is a member of the Alphavirus family that has shown promising anti-cancer activity in pre-clinical models. A concern for the clinical use of Sindbis virus as an anticancer agent is its pathology in humans, known as Pogosta disease. The symptoms of Pogosta disease may be a result of Sindbis virus replication in neuronal, muscle or haematopoietic tissues. Inhibiting virus replication in these tissues could, therefore, alleviate such potential side effects of virotherapy treatment. Introduction of microRNA response elements, perfectly complementary to microRNAs specifically expressed in liver (miR122), neuronal (miR124), muscle (miR133 and miR206) and haematopoietic (miR142-3p) cells, successfully attenuated SV replication in these tissues. In contrast to all other viruses studied, data presented in this thesis show that Sindbis virus infectivity in vitro is not significantly inhibited by incubation with neat, whole naïve human blood. Despite full infectivity in naïve mouse blood, virus particles were rapidly cleared from the circulation of mice in vivo by the liver. An attempt to decrease the clearance rate by depletion of Kupffer cells through pre-treatment of mice with clodronate liposomes was ineffective. We also explored the use of Sindbis virus packaged in mosquito cells to more closely mimic virus particles exposed to blood in the wild during mosquito mediated transmission, but this also failed to improve virus circulation kinetics in mice. Despite rapid clearance from the circulation, intravenous administration of Sindbis virus had significant anti-cancer efficacy in C57BL/6 mice bearing syngeneic B16F10 metastatic melanomas. Overall, data presented support our proposed use of Sindbis virus as a systemically delivered oncolytic agent and suggest decreasing the rate of clearance by the liver could dramatically enhance therapeutic outcomes. In addition it is shown that microRNA targeting of Sindbis virus provides a means of alleviating potential side effects of the administration of large virus doses.

Bien que considérées comme étant sous contrôle durant la seconde partie du 20ième siècle avec la découverte des antimicrobiens, les maladies infectieuses demeurent une menace bien réelle pour l’humanité. Quelque soit l’état de connaissance que nous avons sur ces maladies, toutes demeurent imprédictibles. Afin de lutter contre ce phénomène, de nombreuses stratégies de surveillance ont été développées amenant à la mise en place de divers outils informatiques de surveillance épidémiologique visant à détecter et identifier, le plus précocement possible, des événements anormaux. L’objectif initial de notre travail a consisté à mettre en place, au sein de l’Institut Hospitalo-Universitaire Méditerranée Infection et à partir du logiciel Microsoft Excel, deux nouveaux outils informatiques de surveillance épidémiologique visant à identifier, de façon hebdomadaire et automatisée, des événements anormaux sur la base des données de microbiologie clinique issues du laboratoire du Centre Hospitalo-Universitaire Timone à l’Assistance Publique- Hôpitaux de Marseille (AP-HM). Une fois cette étape achevée, nous avons par la suite travaillé au développement d’une structure de surveillance complète intégrant l’investigation et la validation des alarmes émises par les systèmes de surveillance créés, l’émission d’alertes à l’Agence Régionale de Santé (ARS) de la région Provence-Alpes Côte d’Azur (PACA), la valorisation des cas d’événements anormaux confirmés par des publications scientifiques, ainsi que la mise en place de rétro-informations et de bulletins épidémiologiques hebdomadaires visant à informer les acteurs locaux de la surveillance épidémiologique des maladies infectieuses. / Although considered under control in the second half of the 20th century with the discovery of antimicrobials, infectious diseases remain a serious threat to humanity. Regardless of the state of knowledge we possess on these diseases, all remained unpredictable. To fight this phenomenon, many monitoring strategies have been developed leading to the implementation of various epidemiological surveillance computer programs to detect and identify, as soon as possible, abnormal events including epidemic phenomena. The initial objective of our work was to implement, within the Hospitalo-Universitaire Méditerranée Infection and based on the Microsoft Excel software, two new automated computer-based programs for the weekly automated epidemiological surveillance of abnormal epidemic events using clinical microbiological data from the Timone teaching hospital of of Assistance Publique- Hôpitaux de Marseille (AP-HM). Once completed, we then worked to develop a comprehensive monitoring structure incorporating the investigation and the validation of alarms emitted by the established surveillance systems, the transmission of alerts to the Regional Health Agency (ARS) of the Provence-Alpes Côte d'Azur (PACA), the public dissemination of confirmed abnormal events by publishing scientific articles, and the implementation of feedback and weekly epidemiological bulletins to inform local infectious diseases epidemiological surveillance actors.

Le répertoire des bactéries est mal connu en Afrique du fait des méthodes d’étude essentiellement basées sur des techniques de culture sur milieux simples associées à des tests biochimiques, ce qui ne permet pas son exploration. Il a néanmoins été récemment bouleversé par l’usage systématique de la spectrométrie de masse de type MALDI-TOF MS.Au cours de nos travaux de thèse de doctorat, nous avons utilisé deux types de spectromètres de masse: le MALDI-TOF Vitek MS, nouvellement installé à Dakar; le MALDI-TOF Microflex LT, installé à Marseille. Les résultats que nous avons obtenus ont montré, pour la première fois, que la technique du MALDI-TOF est efficace et tout à fait adaptée en Afrique pour le diagnostic spécifique de routine. Cette performance a conduit le laboratoire clinique de l’HPD à opter pour son utilisation à la place des traditionnelles techniques d’identification phénotypique telles que les galeries API. Nous avons également confirmé que le MALDI-TOF est un puissant outil d’identification des espèces bactériennes rarement impliquées dans les maladies infectieuses humaines. De plus, cet outil nous a permis de détecter sept nouvelles espèces de bactéries isolées pour la première fois chez l’homme. En Afrique, il faudrait donc multiplier l’installation de spectromètre de masse MALDI-TOF, ou mettre en place des réseaux autour de plateformes MALDI-TOF sous-régionales partagées entre plusieurs structures sanitaires et/ou de recherche. / The Africa bacteria repertory is unfamiliar because the available tools in this region are not allowed its best knowledge. In fact, bacteria are most often identified using culture techniques on simple media and biochemical tests which enable the identification of some common characters. These methods do not facilitate an exhaustive knowledge of the bacterial repertory; consequently they have recently been revolutionized by the systematic use of MALDI-TOF mass spectrometry (MS).In our thesis we used two mass spectrometers, respectively, MALDI-TOF Vitek MS currently installed at Dakar (Senegal) and MALDI-TOF Microflex LT installed in Marseille (France). In addition we have also confirmed that MALDI-TOF is a powerful tool for identifying bacterial species rarely involved in human infectious diseases. Thus in adopting the MALDI-TOF as a first-line tool in bacterial identification before Gram staining or other techniques of phenotypic identifications based on chemical characteristics, we discovered seven new species of bacteria isolated for first time in humans. Microbial identification using MALDI-TOF MS is currently feasible in Africa. Its performance and effectiveness in routine diagnosis of clinical microbiology laboratories have been proven. It is necessary either to increase the installation of MALDI-TOF, or establishing a network around a shared MALDI-TOF platform between several structures located in the same area, especially in the underdeveloped countries of Africa amortization of investment costs of the device, because it allowed reducing the time of reporting results and indirectly facilitating better care for patients.

RNA-dependent RNA polymerases (RdRps) play a vital role in the life cycle of RNA viruses, being responsible for genome replication and mRNA transcription. In this thesis viral RdRps (vRdRps) of dsRNA bacteriophage phi6 (phi6 RdRp) and Severe Acute Respiratory Syndrome (SARS) coronavirus [non structural protein 12 (NSP-12)] are studied. For SARS polymerase NSP-12, a library-based screening method known as ESPRIT (Expression of Soluble Protein by Random Incremental Truncation) was employed in an attempt to isolate domains of NSP-12 that express solubly in Escherichia coli (E. coli) and are thereby suitable for structural studies. This experiment identified for the first time in a systematic fashion, conditions under which the SARS polymerase could be solubly expressed at small scale and allowed mapping of domain boundaries. Further experiments explored different approaches for increasing expression levels of tractable fragments at large scale. Bacteriophage phi6 RdRp is one of the best studied vRdRps. It initiates RNA synthesis using a de novo mechanism without the need for a primer. Although formation of the de novo initiation complex has been well studied, little is known about the mechanism for the transition from initiation to elongation (i.e. extension of an initiated dinucleotide daughter strand). In the phi6 RdRp initiation complex the C-terminal domain (CTD) blocks the exit path of the newly synthesised dsRNA which must be displaced for the addition of the third nucleotide. The crystal structure of a C-terminally truncated phi6 RdRp (P2T1) reveals the strong non-covalent interactions between the CTD and the main body of the polymerase that must be overcome for the elongation reaction to proceed. Comparing new crystal structures of complexes of both wild-type (WT) and a mutant RdRp (E634 to Q, which removes a salt-bridge between the CTD and main body of the polymerase) with various oligonucleotides (linear and hairpin), nucleoside triphosphates (NTPs) and divalent cations, alongside their biophysical and biochemical properties, provides an insight into the precise molecular details of the transition reaction. Thermal denaturation experiments reveal that Mn2+ acquired from the cell and bound at the phi6 RdRp non-catalytic ion site sufficiently weakens the polymerase structure to facilitate the displacement of the CTD. Our crystallographic and biochemical data also indicate that Mn2+ is released during this displacement and must be replaced for the elongation to proceed. Our data explain the role of the non-catalytic divalent cation in vRdRps and pinpoint the Mn2+-dependent step in viral replication. In addition, by inserting a dysfunctional Mg2+ at the non-catalytic ion site for both WT and E634Q RdRps we captured structures with two NTPs bound within the active site in the absence of Watson-Crick base pairing with template and could map movements of divalent cations during preinitiation through to initiation. Oligonucleotides present on the surface of phi6 RdRp allowed mapping of key residues involved in template entry and unwinding of dsRNA; these preinitiation stages have not been observed previously. Considering the high structural homology of phi6 RdRp with other vRdRps, particularly from (+)ssRNA hepatitis C virus (HCV), insights into the mechanistic and structural details of phi6 RdRp are thought to be relevant to the general understanding of vRdRps.

The efficacy of Bacille-Calmette Guerin (BCG) vaccination in protection against pulmonary tuberculosis (TB) is highly variable between populations. One possible explanation is increased exposure of certain populations to non-tuberculous mycobacteria (NTM). Given the variable efficacy of BCG an improved vaccine against TB is required. The novel TB vaccine MVA85A has shown promising results, however, the immunogenicity of the vaccine is reduced when it is administered in the Expanded Programme on Immunisation (EPI) schedule. This thesis aims to explore: (A) the effect of exposure to NTM on the level of protection afforded by BCG vaccination against Mycobacterium tuberculosis (M. tb) and (B) the immunological mechanisms behind EPI interference with MVA85A. The effect of M. avium (MA) exposure via systemic and oral routes on the efficacy of BCG was tested using M. tb aerosol infection in a mouse model. The adaptive immune response was profiled in BCG vaccinated mice with and without exposure to MA pre- and post- M. tb infection. The results showed BCG efficacy could be enhanced by exposure to dead MA by a systemic route; T helper 1 and T helper 17 responses were associated with increased protection. In contrast, BCG efficacy may have been reduced by exposure to live MA by the oral route; T helper 2 and regulatory T cells were associated with reduced protection. To answer the second aim MVA85A was co-administered to mice with aluminium adjuvants or aluminium-containing vaccines to replicate the effect of co-administration in the EPI schedule; the adaptive immune response was profiled. T helper 2 and regulatory T cell responses induced by aluminium-containing vaccines were associated with a reduction in the immunogenicity of MVA85A.