Identification of bacterial factors associated with the survival of Burkholderia cenocepacia in a murine host

The Burkholderia cepacia complex (BCC) is a family of Gram-negative bacteria of evolving importance as opportunistic pathogens, particularly in patients with cystic fibrosis (CF). BCC infections are difficult to treat and control due to the BCC's intrinsic resistance to antibiotics and ability to spread among patients. Moreover BCC colonization can develop into septicemia. Although recognized as a serious respiratory pathogen, little is known about the pathogenesis of the BCC. The purpose of these studies was to identify bacterial factors involved in BCC infection and colonization using animal models that show differential persistence and virulence among BCC strains in a murine host. Serial mouse passage of non-persistent BCC strain C1394 in a pulmonary infection model resulted in its adaptation to the murine host and produced a variant, C1394mp2 that persisted in the murine lung. The parent strain and variant were indistinguishable by genetic typing, but differed in colonial morphology and were compared to identify the bacterial determinants required for long-term infection in a murine host. The parent strain, C1394 had a matte colonial phenotype, made scant exopolysaccharide (EPS), and was lightly piliated. The variant, C1394mp2 had a shiny colonial phenotype, produced abundant EPS, and was heavily piliated. Matte to shiny colonial transformation was induced by growth at 42°C. Proteomic analysis of C1394 and C1394mp2 protein profiles at 37°C revealed increased flagellin production in C1394mp2 whereas C1394 had increased production of metabolizing enzymes of the tricarboxylic acid cycle. Differential expression of the stress-induced protein peroxiredoxin was observed in the proteomes of C1394 and C1394mp2 at 37°C and 42°C respectively. Further comparisons included in vitro assays examining host cell interactions with C1394 and C1394mp2. Though both isolates had poor nonopsonic association with primary human phagocytic cells, C1394mp2 had even less association than C1394. Binding assays with A549 epithelial cells also revealed lower association with C1394mp2 than C1394. The results in this thesis identified BCC surface determinants that were associated with a colonial morphology change, which could be induced under stress, and correlated with long-term infection in a murine host and decreased association with human host cells. Taken together, these results suggest putative bacterial factors that are necessary for BCC survival and chronic infections susceptible hosts.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat