Date on Master's Thesis/Doctoral Dissertation

5-2013

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Committee Chair

Warawa, Jonathan Mark

Author's Keywords

Klebsiella pneumoniae; ATCC 43816; Capsule polysaccharide; Genetic characterization; Bioluminescent

Subject

Bacterial genetics

Abstract

OBJECTIVE: Klebsiella pneumoniae is a Gram-negative enterobacterium that is a major cause of community-acquired and nosocomial infections. We performed a genetic characterization of K. pneumoniae strain ATCC 43816, which is a well-studied strain with a K2 serotype capsular polysaccharide. To provide the ability to monitor bacteria within a host, we will develop a bioluminescence strain of K. pneumoniae by integrating the lux operon into the chromosome. METHODS: The ATCC 43816 capsule cluster sequencing was performed by closing gapE, in the Next Generation Sequencing by long range peR for template and primer walking. We constructed a bioluminescent K. pneumoniae by using allelic exchange strategies and subsequently characterized the bioluminescent strain JSKP001 in a macrophage infection model. RESULTS: We have sequenced and annotated the CPS cluster as a 23,804-base pair sequence and identified eight homologous genes conserved between all serotypes of Klebsiella. Using our bioluminescent strain JSKP001, we demonstrate that K. pneumoniae may enter a reduced metabolic state within macrophages. CONCLUSION: We have successfully sequenced and annotated a K2 serotype CPS cluster with high homology to other K2 serotypes K. pneumoniae strains. We observed that K. pneumoniae is internalized by macrophages, but proliferates at a slow rate. We tested our bioluminescent strain in numerous growth conditions and found that rich growth media supports higher metabolic activity while the lowest activity was detected in K. pneumoniae internalized within rnacrophages. This suggests that macrophages may only provide a minor role as an intracellular replicative niche in vivo, but might be a potential niche for chronic persistence in sublethal infections.

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