Klebsiella pneumoniae is an opportunistic pathogen responsible for lobar pneumoniae, liver abscess, and septicemia. Clinical isolates are found to be extended spectrum beta lactamase positive with differential expression of the two classical porins, OmpK35 and OmpK36. Porin loss is associated with increased minimum inhibitory concentrations of beta lactam, cephalosporin, and carbapenem antibiotics that target the peptidoglycan. However, little is known about how porin loss affects other aspects of the cell envelope. The focus of this study was to characterize clinical isolates exhibiting differential porin expression and determine if the cumulative changes altered the resistance to phagocytosis by macrophages. The results support the hypothesis that porin loss significantly impacts the overall cell envelope composition, which in turn alters interactions with macrophages.

<p>  </p> <p>Microbiome studies are varied and involve the examination of microorganisms at different levels: individual cells to determine individual functions, populations of specific microorganisms to determine interactions between organisms, and/or communities of microorganisms for a broader investigation of interactions between organism and environment. These studies are typically done within the context of a particular niche or environment. There are two parts to this dissertation, separated by the types of research involved. First, the analysis of bacterial communities using 16S rRNA sequencing and analysis. In this first part the bacterial communities of the reproductive tract of bulls and the gastrointestinal tract of weanling pigs were studied. The reproductive organs of the male, domestic species had not been studied from an ecological perspective prior to the study. As such, the research was mainly focused on characterizing the bacterial communities found within the prepuce of bulls that were considered to be healthy, or that the breeding soundness exam was satisfactory and the bulls had no clinical disease in the urogenital tract. Through this study two distinct types of bacterial communities were found based on the diversity of the observed taxa; the groups were split into a low diversity group identified by the presence of <em>Bradyrhizobium</em> and a high diversity group distinguished by the abundance of mucosal-associated bacteria found in oral, respiratory, and vaginal communities of cattle. Second, the effects of supplementary, soluble fiber on the intestinal bacterial communities of piglets pre- and/or post-weaning were studied. The rationale behind this study was to determine if pre-weaning fiber could alter the microbiome prior to weaning and the change of diet from liquid to solid. Pre-weaning, supplementary, soluble fiber was found to increase short-chain fatty acid concentrations and bacterial taxa potentially involved in their production. Additionally, bacterial taxa implicated in an increased inflammatory response were reduced in groups fed supplementary fiber. Taken together, the two bacterial community studies highlight the gaps in knowledge for reproductive communities in male animals as well as the potential for reducing weaning stress in pigs. Part two of this dissertation focuses on whole genome sequence analysis as a way to study bacterial populations associated with bovine respiratory disease (BRD), a common and potentially fatal disease in cattle. Identification of BRD has low accuracy and the presence of antibiotic resistant bacteria increases the chance of treatment failure. Using machine learning, the prediction of antibiotic resistance in bacterial isolates from animals with BRD was performed to find potential sequences for use in future molecular assays. While using known resistance genes was helpful for some antibiotics, several of the antibiotics used in treating BRD were better predicted using the machine learning models. Model output sequences should be further tested using molecular methods to determine function and importance before using as an assay target. Put together, the contents of this dissertation should serve as an introduction to bacterial ecology as well as how the concepts can be applied to food animal production systems.</p>

<p>Bacteria employ virulence mechanisms to promote fitness that are generally detrimental to a host organism. The Gram-negative pathogen <em>Salmonella enterica </em>utilizes type three secretion systems (T3SS) to inject proteins termed effectors into the host cell cytoplasm where normal cellular function is modified. The coordinated T3SS assembly, and delivery of effectors to the cytoplasmic face of the T3SS is aided by virulence chaperones. The interaction of effector-chaperone complex with the T3SS occurs via an ATPase protein, where the complex is dissociated and the effector is unfolded, presumably for passage through the T3SS. The virulence chaperone network associated with the <em>Salmonella </em>pathogenicity island two (SPI-2) encoded T3SS has not been fully characterized. Additionally, the T3SS ATPase protein encoded within SPI-2, SsaN, has yet to be examined for functional motifs or a precise role in effector secretion. The contents of this thesis describe the characterization of two novel virulence chaperones, SrcA and SscA, and the T3SS ATPase SsaN. SrcA is a virulence chaperone for the effector substrates SseL and PipB2, and adopts the characteristic horseshoe-like structure common amongst effector chaperones. SscA is a chaperone for the translocon component SseC of the T3SS structure, and both proteins impact the regulation of SPI-2 promoters. The structure of SsaN resembles other T3SS ATPases, although different conformations exist between the structures, potentially highlighting regions with T3SS function. Additionally, an N-terminal domain was found to be dispensable for membrane localization, and residues within the predicted hexamer model impact effector secretion. These results identify novel virulence chaperones essential for T3SS function, and characterize the T3SS ATPase protein encoded within SPI-2. These findings greatly expand our knowledge of the virulence mechanisms utilized by <em>S. enterica</em>.</p> / Doctor of Philosophy (PhD)

Ce manuscrit décrit la distribution géographique des génotypes de Mycobacterium tuberculosis dans une large partie de la Chine, et l'étude d'une éventuelle corrélation avec la vaccination BCG et la résistance aux antibiotiques. La prévalence de la résistance a été analysée dans 10 provinces, et les mutations responsables de la résistance à la rifampicin et à l'INH ont été recherchées. La distribution des différents génotypes a été analysée par spoligotypage, par l'identification de délétions génomiques et par l'analyse du polymorphisme de répétitions en tandem (MLVA). Le génotype Beijing représente 55 à 93% des souches dans les provinces étudiées, avec un gradient allant du Sud vers le Nord de la Chine. Plusieurs autres familles sont identifiées, toutes appartenant à la clade dite " Moderne ". Les familles " Chine 2 " et " Chine 3 " qui représentent l'essentiel des autres souches sont rares en dehors de la Chine. Quelques souches de la famille CAS (Central Asia) sont également rencontrées dans une province et un ancêtre possible de la lignée Beijing dans une autre. La prévalence de la résistance aux antibiotiques a été mesurée parmi plus de 2000 isolats de 10 provinces. Le pourcentage de souches résistantes à au moins une drogue est de 45% et celui des souches multirésistantes est d'environ 29%. En Chine, à la différence d'autres pays, aucune corrélation n'est observée entre le génotype Beijing et la vaccination BCG. La distribution des mutations du gène rpoB a été déterminée ainsi que les mutations responsables de la résistance à l'INH. Aucune mutation n'est observée dans les gènes oxyR et ahpC. Cette étude est la première étude de grande envergure effectuée en Chine sur la diversité génétique de M. tuberculosis et sur les mécanismes de résistance aux antibiotiques.

Après trois pandémies majeures responsables de millions de morts, la peste n'a pas encore disparu. Cette maladie est causée par la bactérie Yersinia pestis, dont les mécanismes de virulence sont encore mal compris. Le suivi d'infection de la peste bubonique chez la souris, méthode classique pour étudier le processus infectieux, requiert beaucoup d'animaux et de temps pour obtenir des résultats significatifs. L'imagerie in vivo et en temps réel par bioluminescence permet de suivre la progression du pathogène au cours du processus infectieux en observant les animaux de façon non invasive. Nous avons transformé la souche virulente CO92 avec le plasmide pEm7-luxCDABE et confirmé la production de bioluminescence in vitro et in vivo. Nous avons pu quantifier la charge bactérienne dans plusieurs organes colonisés sans sacrifier l'animal et établir le schéma de progression de la bactérie au cours de la maladie. Après formation d'un foyer infectieux au site d'injection, la colonisation du ganglion lymphatique inguinal drainant ce site a été observée. Nous avons démontré que la bactérie suit un trajet direct du ganglion lymphatique inguinal au ganglion lymphatique axillaire. L'étape suivante est la colonisation des organes filtrant le sang, puis survient la septicémie dans les phases terminales de la mort. Nous avons établi que la forte variabilité dans le processus infectieux était due au temps pendant lequel la bactérie était contenue au site d'injection. À partir du moment où les ganglions lymphatiques sont colonisés, la cinétique de progression est à la fois régulière et rapide ; la septicémie survient dans les deux jours, suivie de près par la mort.

Before the consolidation of the germ theory of human diseases at the end of the nineteenth century, medical explanations about disease causation were dominated by the environmental notions of medical geography. This dissertation explores how nineteenth-century Colombian physicians transformed the medical geographical approach using the early concepts and technologies of the emerging Pasteurian germ theory. I follow this transformation in the cases of periodic fevers (yellow fever and malaria), continuous fevers (typhoid fever and typhus) and leprosy. The analysis reveals that by mid century physicians had incorporated neo-Hippocratic versions of disease causation and French medical geographical ideas in order to make sense of disease of the warm, temperate and cold lands. Their conceptual network revolved around the specific, predisposing and occasional causes in which climate and geography played a determinant role. Evidence indicates that this was the case of periodic fevers of the warm lands (yellow fever and malaria). I argue that the “parasitic” hypothesis of yellow fever was accepted during the controversy around the prophylactic inoculations inspired by Pasteurism that were applied in Colombia in 1887. However, doctors struggled to reconcile the medical geographical and the bacteriological perspective of both yellow fever and malaria. Continuous fevers, on the other hand, were also framed within the medical geography scheme of disease causation. I show how during the debates about typhoid fever and typhus happening in the Colombian highlands during the 70s, 80s and 90s, doctors used medical geographical notions and developed anti-pasteurian arguments, while the international scientific community had identified the specific bacilli for typhoid fever. Finally, I argue that the strong interest of Colombian doctors on leprosy –also understood in neo-Hippocratic terms- that foster the search for local treatments based on Pasteurism (antiseptics in the 1880s and serotherapy in the 1890s) also prompted the extension of the bacteriological model and techniques to other diseases in those decades.

A mechanistic model was developed, which described the growth of a bacterial colony on an agar medium. Diffusion of substrate (nutrients/oxygen) through the colony are considered. Rate of substrate use by the organisms is assumed to follow Michaelis-Menton kinetics for substrate concentrations between prescribed limits. Above and below the prescribed concentration limits, the substrate use rate is assumed constant and zero, respectively. Supply of substrate to the colony was assumed to be non-limiting. Under these conditions, the model predicted that diffusion of substrate through the colony will eventually control colony growth. It also described a slower eponential growth rate of the colony when the organisms utilized an alternate substrate for one that became deficient throughout a portion of the colony, and a constant linear growth rate when an alternate substrate was not utilized. Consistent with published literature, a mathematical description of substrate supply through the agar indicated that, under normal conditions, glucose supply through the agar to the colony would not be expected to limit colony growth before oxygen diffusion through the colony limited growth.

Obligate intracellular pathogens must acquire host cell-derived nutrients to facilitate their survival. One such bacterial pathogen, Anaplasma phagocytophilum, replicates within neutrophils and non-phagocytic cells in a bacterial-modified, host cell-derived vacuole. The bacterium exploits host cell vesicular trafficking pathways to route nutrients to its vacuole and utilizes Rab GTPases, guanine nucleotide-dependent, vesicular trafficking regulators, to do so. We previously discovered that the A. phagocytophilum vacuolar membrane is decorated with a specific subset of Rab GTPases - Rab1, Rab4A, Rab10, Rab11, Rab14, Rab22A and Rab35. Rab1 is exclusively found on the endoplasmic reticulum (ER) and thus its localization suggests that the bacterium intercepts the ER. Rab10, which is found on the ER, trans-Golgi and recycling endosomes, localizes to the vacuolar membrane in a guanine nucleotide-independent and bacterial protein synthesis-dependent manner. This suggests that a bacterial-encoded protein is binding to and recruiting Rab10. In this study, we determined that A. phagocytophilum hijacks two very nutrient-rich sources in the secretory pathway - trans-Golgi- and endoplasmic reticulum-derived vesicles. A. phagocytophilum localizes perinuclearly adjacent to the Golgi apparatus during infection. A. phagocytophilum and Anaplasma marginale, an intravacuolar bovine pathogen, also localize near the smooth ER and rough ER in both mammalian and tick host cells. These results are supported by transmission electron microscopy analyses of infected cells. Membrane markers for the rough ER label the peripheries of A. marginale and A. phagocytophilum organisms in both mammalian and tick host cells, which suggests that they are translocated into the pathogen vacuole. Furthermore, membrane markers for trans-Golgi-derived vesicles, including endogenous Rab10, label the periphery of intravacuolar A. phagocytophilum organisms. Markers for the trans-Golgi and the ER co-fractionate with A. phagocytophilum in density gradient centrifugation studies. siRNA knockdown of Rab10 pronouncedly reduces delivery of trans-Golgi markers into the pathogen-occupied vacuole, significantly reduces infection, and impedes bacterial conversion to the bacterium’s dense-cored form. These results suggest that trans-Golgi recruitment is Rab10 dependent and is critical for bacterial development. We identified an outer membrane A. phagocytophilum moonlighting protein, uridine monophosphate kinase that specifically binds GST-Rab10 in affinity chromatography assays and interacts with Rab10 in vivo. We hypothesize that this surface protein is mediating the interaction of the bacteria with intravacuolar trans-Golgi derived vesicles. This interaction could be critical for the delivery of essential nutrients. Taken together, these data suggest that nutritional virulence mechanisms of A. phagocytophilum and A. marginale target the host secretory pathway. Additionally, they suggest a novel mechanism whereby pathogens translocate nutrient rich vesicles into the pathogen vacuole, thus delivering essential nutrients right to their front door.

The gene encoding methicillin resistance in Staphylococcus aureus (MRSA) is carried in the chromosome on a large genomic island called SCCmec and is always inserted at the att site within orfX. SCCmec has been designated a mobile genetic element but a mechanism by which it moves among different strains and species of staphylococci has never been demonstrated. This work shows that bacteriophage 80α is capable of transducing SCCmec into a recipient cell, after which it can integrate into the bacterial chromosome via homologous recombination. More importantly, this work characterizes a conjugative mechanism of SCCmec transfer. Results demonstrate the capture of a 30.8 kb SCCmec element on a conjugative plasmid for the first time, its transfer into both S. aureus and S. epidermidis recipients, and its excision from the plasmid with insertion in the orfX att site in recipients. The element was integrated into the plasmid by recombination between IS elements invariably present on all SCCmec types and pGO1/pSK41-like conjugative plasmids. These data explain the movement of SCCmec from reservoirs in commensal coagulase-negative staphylococci into different Staphylococcus aureus lineages using a ubiquitous conjugative plasmid that can transfer among staphylococci of different species and, thus, describes a mechanism for the environmental dissemination of methicillin resistance in nature.

Preterm birth, defined at birth before 37 weeks gestation, affects millions of newborns worldwide every year. Preterm birth is a leading cause of infant morbidity and mortality. One major cause of preterm birth is preterm premature rupture of membranes (PPROM), which can be triggered by bacterial infection and inflammation. A bacterial species that has been implicated in preterm birth and other obstetric complications is Sneathia amnii. The goals of this study were to observe cytopathogenic effects caused by S. amnii strain Sn35 and identify putative virulence factors causing those effects. Sn35 was able to adhere to, invade, and damage/kill various host cell lines. We characterized these virulence attributes. A putative virulence determinant was identified, and a fragment of the protein was expressed for polyclonal antiserum production. Antiserum was used to characterize the expression and subcellular localization of the protein in Sn35. However, antiserum was unable to prevent cytopathogenic effects.