Klebsiella pneumoniae is a bacterial pathogen of worldwide importance and a significant contributor to multiple disease presentations associated with both nosocomial and community acquired disease. ATCC 43816 is a well-studied K. pneumoniae strain which is capable of causing an acute respiratory disease in surrogate animal models. In this study, we performed sequencing of the ATCC 43816 genome to support future efforts characterizing genetic elements required for disease. Furthermore, we performed comparative genetic analyses to the previously sequenced genomes from NTUH-K2044 and MGH 78578 to gain an understanding of the conservation of known virulence determinants amongst the three strains. We found that ATCC 43816 and NTUH-K2044 both possess the known virulence determinant for yersiniabactin, as well as a Type 4 secretion system (T4SS), CRISPR system, and an acetonin catabolism locus, all absent from MGH 78578. While both NTUH-K2044 and MGH 78578 are clinical isolates, little is known about the disease potential of these strains in cell culture and animal models. Thus, we also performed functional analyses in the murine macrophage cell lines RAW264.7 and J774A.1 and found that MGH 78578 (K52 serotype) was internalized at higher levels than ATCC 43816 (K2) and NTUH-K2044 (K1), consistent with previous characterization of the antiphagocytic properties of K1 and K2 serotype capsules. We also examined the three K. pneumoniae strains in a novel BALB/c respiratory disease model and found that ATCC 43816 and NTUH-K2044 are highly virulent (LD50<100 CFU) while MGH 78578 is relatively avirulent.

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

Introduction. Klebsiella pneumoniae is a major threat to public health worldwide. It is the causative agent for multiple disease presentations including urinary tract infection, septicemia, liver abscess, wound infection and respiratory tract infection. K. pneumoniae causes community- and hospital-acquired pneumonia, which is a devastating disease associated with high mortality rates. Hypothesis. There is a growing concern about the emergence of multidrug-resistant K. pneumoniae strains complicating the treatment with the current available therapeutics; therefore, there is an urgent need for the development of new antimicrobial agents. Aim. K. pneumoniae causes an acute respiratory disease in mice and in the current work we investigated the capability to perform non-invasive monitoring of bioluminescent Klebsiella to monitor therapeutic efficacy. Methodology. We engineered a bioluminescence reporter strain of K. pneumoniae to monitor the impact of antibiotics in a murine respiratory disease model. Results. We demonstrate that bioluminescence correlates with bacterial numbers in host tissues allowing for a non-invasive enumeration of bacterial replication in vivo. Light production is directly linked to bacterial viability, and this novel bioluminescent K. pneumoniae strain enabled monitoring of the efficacy of meropenem therapy in arresting bacterial proliferation in the lung. Conclusion. The use of non-invasive bioluminescent imaging improves preclinical animal model testing to detect study outcome earlier and with higher sensitivity.