Genetic characterization of the K2 serotype capsule of Klebsiella pneumoniae ATCC 43816 and the development of a bioluminescent strain.

Abstract

Scott, Jacob Brandon 1987-, "Genetic characterization of the K2 serotype capsule of Klebsiella pneumoniae ATCC 43816 and the development of a bioluminescent strain." (2013 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 wellstudied strain with a K2 serotype capsular polysaccharide. To provide thl~ ability to monitor bacteria within a host, we will develop a bioluminescence stra n 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 allelit:: exchange strategies and subsequently characterized the bioluminescent strain JSKP001 in a macrophage infection model. RESULTS: We have sequerced and annotated the CPS cluster as a 23,804-base pair sequence and identified eight homologous genes conserved between all serotypes of Klebsiella. 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 suggeBts 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.vi

In vivo monitoring of therapeutic efficacy and virulence profile by bioluminescent Klebsiella pneumoniae.

“…The capsular polysaccharide genetic cluster was manually sequenced to close contig gaps between five contigs, as described elsewhere [181], and the complete sequence for the ATCC 43816 capsular polysaccharide locus have been deposited with DDBJ/EMBL/GenBank with the accession number KJ541664.…”