The purpose of this study was to investigate the prevalence and mechanisms of extended-spectrum cephalosporin- (ESC) and carbapenem-resistance in Escherichia coli from the GTA. A total of 526 non-duplicate E. coli clinical isolates were collected during March 1-5, 2010 from 13 participant hospitals. Among these, 71 isolates showed reduced susceptibility (rS, intermediate, and/or resistant phenotype) to cefoxitin (FOX) and/or ESC. No carbapenem resistance was detected. Extended-spectrum ß-lactamase genes detected (n=37; 52.1%) belong to the CTX-M-family, including blaCTX-M-15 (78.4%), blaCTX-M-23 (2.7%), blaCTX-M-14 (18.9%). The only plasmid-mediated ampC gene identified among FOXrS isolates (n=49; 69%) was blaCMY-2 (n=7; 14.3%). Seventeen strains (24%) were negative for all ß-lactamase genes tested. Analysis of the chromosomal ampC promoter revealed mutations associated with AmpC hyperproduction. Other mechanisms of resistance (e.g. impermeability and/or unidentified ß-lactamases) cannot be discarded. The most prevalent clone detected was ST131. IncFIA, FIB and Frep were the most common plasmid replicon types detected.

The enteropathogenic bacteria Salmonella are the main cause of food borne gastroenteritis worldwide. The activation of Toll-like receptor 4 (TLR4) by LPS triggers an immune response to counter infection. Signaling by TLR4 requires the adaptor proteins, TIRAP and TRAM. Recruitment and activation of these molecules is dependent on the membrane lipid, PIP2. The Salmonella effector, SigD, is a 4-phosphatase that depletes PIP2 from the host plasma membrane during invasion. Thus, we investigated if SigD could lead to the interruption of the TLR4 pathway. We observed that SigD expression caused the disappearance of TIRAP from the Salmonella containing vacuoles (SCVs) in HeLa cells. Furthermore, we demonstrated that SigD attenuates NF-κB activation, implicating SigD in the disruption of the MyD88 dependent pathway. In addition, the observed inhibition of PKCε phosphorylation suggests SigD may also block the other branch of the TLR4 signaling cascade, the MyD88 independent pathway.

Bisphosphonate-induced osteonecrosis of the jaw (BPONJ) has been identified as a severe complication of dental treatment in 1-10% of patients previously treated with intravenous bisphosphonates. The mechanism by which bisphosphonates induce BPONJ is uncertain. It has been noted that necrotic bone from BPONJ sites display signs of bacterial infection that suggests that an immune defect may play a role in the pathophysiology of BPONJ. The purpose of this thesis examined the effect of a potent bisphosphonate, zoledronate, on the innate immune system, specifically, neutrophil function, differentiation and survival with in vitro and in vivo murine models. Zoledronate exposure leads to decreased neutrophil migration, neutrophil NADPH oxidase activity, circulating neutrophil counts, as well as neutrophil survival, however does not appear to affect neutrophil differentiation. We present evidence that bisphosphonates have the potential to depress the immune system in mice and a subset of patients, possibly contributing to the pathogenesis of BPONJ.

Legionella pneumophila (Lp) is the pathogen responsible for Legionnaires disease. Lp invades and survives in human macrophages to form an intracellular compartment, called the Legionella containing vacuole (LCV). Within the LCV, Lp avoids degradation and replicates, before killing its host and releasing its virulent progeny. Lp is pleomorphic, exhibiting a combination of short rod phenotypes and filaments. Filamentous Lp has been reported in patient samples, but current studies of Lp virulence are exclusively focused on rods. Our results show that filamentous Lp can invade and replicate in human and murine macrophages. Filaments are phagocytosed gradually into tubular phagocytic cups. Interestingly, the formation of the LCV starts at this stage of phagocytosis, and LCV markers were detected before the sealing of the phagosome occurred. We present evidence that the filamentous morphology acts cooperatively with Lp effectors to subvert the microbicidal activities of the macrophage, contributing to the survival of Lp.

The purpose of this study was to investigate the prevalence and mechanisms of extended-spectrum cephalosporin- (ESC) and carbapenem-resistance in Escherichia coli from the GTA. A total of 526 non-duplicate E. coli clinical isolates were collected during March 1-5, 2010 from 13 participant hospitals. Among these, 71 isolates showed reduced susceptibility (rS, intermediate, and/or resistant phenotype) to cefoxitin (FOX) and/or ESC. No carbapenem resistance was detected. Extended-spectrum ß-lactamase genes detected (n=37; 52.1%) belong to the CTX-M-family, including blaCTX-M-15 (78.4%), blaCTX-M-23 (2.7%), blaCTX-M-14 (18.9%). The only plasmid-mediated ampC gene identified among FOXrS isolates (n=49; 69%) was blaCMY-2 (n=7; 14.3%). Seventeen strains (24%) were negative for all ß-lactamase genes tested. Analysis of the chromosomal ampC promoter revealed mutations associated with AmpC hyperproduction. Other mechanisms of resistance (e.g. impermeability and/or unidentified ß-lactamases) cannot be discarded. The most prevalent clone detected was ST131. IncFIA, FIB and Frep were the most common plasmid replicon types detected.

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.

Bacterial species of the genus Neisseria include pathogens that are responsible for diseases of humans including bacterial meningitis (Neisseria meningitidis) and the sexually transmitted disease gonorrhea (Neisseria gonorrhoeae). These diseases are often characterized by a massive influx and activation of neutrophils, white blood cells involved in the early/innate immune response to pathogens, at the infection site. Neisseria spp. bind to and activate neutrophils via their Opacity-associated (Opa) outer membrane proteins, which interact with some members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. One of these CEACAMs, CEACAM3 (CD66d), is unique in its restriction to neutrophils and its expression of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-8YxxL/I). In the course of my thesis work, I have shown that this motif is critically dependent for the activation of the neutrophil response to Neisseria, through the coupling of neisserial binding to activation of the tyrosine kinase, Syk, which initiates downstream signaling responsible for the antimicrobial responses of neutrophils. These data contribute to the knowledge of how seemingly unrelated receptors of neutrophils (such as the IgG-binding Fcγ receptors, the fungal receptor Dectin-1, and the bacterial-binding CEACAM3) converge functionally on the presence of the ITAM.

Bacterial species of the genus Neisseria include pathogens that are responsible for diseases of humans including bacterial meningitis (Neisseria meningitidis) and the sexually transmitted disease gonorrhea (Neisseria gonorrhoeae). These diseases are often characterized by a massive influx and activation of neutrophils, white blood cells involved in the early/innate immune response to pathogens, at the infection site. Neisseria spp. bind to and activate neutrophils via their Opacity-associated (Opa) outer membrane proteins, which interact with some members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. One of these CEACAMs, CEACAM3 (CD66d), is unique in its restriction to neutrophils and its expression of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-8YxxL/I). In the course of my thesis work, I have shown that this motif is critically dependent for the activation of the neutrophil response to Neisseria, through the coupling of neisserial binding to activation of the tyrosine kinase, Syk, which initiates downstream signaling responsible for the antimicrobial responses of neutrophils. These data contribute to the knowledge of how seemingly unrelated receptors of neutrophils (such as the IgG-binding Fcγ receptors, the fungal receptor Dectin-1, and the bacterial-binding CEACAM3) converge functionally on the presence of the ITAM.

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), continues to be one of the leading global health challenges causing ~2 million deaths annually. In the majority of infected individuals, the bacteria establish a latent, asymptomatic infection capable of persisting for decades with 5-10% of infected individuals developing active disease in their lifetime. Currently it is estimated that one-third of the world’s population is latently infected, representing a large reservoir for disease reactivation and subsequent spread. Latent TB infection is a paucibacillary disease in which a small heterogeneous population of bacilli is present in the body. M. tb persisters, which are characterized by reduced or altered metabolic activity and enhanced drug tolerance, are thought to be the major contributor towards latent infection and disease relapse following chemotherapy; however, the molecular mechanisms governing persisters formation remain poorly understood. My thesis concerns the characterization of the highly conserved DNA binding protein Lsr2 of mycobacteria. Previous biochemical study of Lsr2 revealed it exhibits DNA-bridging activity analogous to H-NS, an important nucleoid associated protein found in the proteobacteria. iii Here I show using in vivo complementation assays that Lsr2 is functionally equivalent to H-NS, even though these proteins share no sequence similarity. I also present genetic evidence that Lsr2 is a global regulator of M. tb that acts primarily as a transcriptional repressor. Notably, I found that Lsr2 represses a large cohort of genes induced in M.tb during in vitro models of latency including genes implicated in persister formation. I also present evidence that lsr2 is selectively inactivated during long-term hypoxia, a condition thought to be important for persister formation during latency. Lastly, I tested the lsr2deletion mutant in a mouse model of infection and found it had reduced growth relative to the WT but was still able to persist. Taken together my work implicates Lsr2 as a central regulator of persister formation and opens up exciting future research avenues on latent TB infection.

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), continues to be one of the leading global health challenges causing ~2 million deaths annually. In the majority of infected individuals, the bacteria establish a latent, asymptomatic infection capable of persisting for decades with 5-10% of infected individuals developing active disease in their lifetime. Currently it is estimated that one-third of the world’s population is latently infected, representing a large reservoir for disease reactivation and subsequent spread. Latent TB infection is a paucibacillary disease in which a small heterogeneous population of bacilli is present in the body. M. tb persisters, which are characterized by reduced or altered metabolic activity and enhanced drug tolerance, are thought to be the major contributor towards latent infection and disease relapse following chemotherapy; however, the molecular mechanisms governing persisters formation remain poorly understood. My thesis concerns the characterization of the highly conserved DNA binding protein Lsr2 of mycobacteria. Previous biochemical study of Lsr2 revealed it exhibits DNA-bridging activity analogous to H-NS, an important nucleoid associated protein found in the proteobacteria. iii Here I show using in vivo complementation assays that Lsr2 is functionally equivalent to H-NS, even though these proteins share no sequence similarity. I also present genetic evidence that Lsr2 is a global regulator of M. tb that acts primarily as a transcriptional repressor. Notably, I found that Lsr2 represses a large cohort of genes induced in M.tb during in vitro models of latency including genes implicated in persister formation. I also present evidence that lsr2 is selectively inactivated during long-term hypoxia, a condition thought to be important for persister formation during latency. Lastly, I tested the lsr2deletion mutant in a mouse model of infection and found it had reduced growth relative to the WT but was still able to persist. Taken together my work implicates Lsr2 as a central regulator of persister formation and opens up exciting future research avenues on latent TB infection.