| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 98 === The nosocomial infection of Klebsiella pneumoniae (KP) had increased gradually in Taiwan in the past few years. KP is an opportunistic pathogen that infects immunocompromised patients, especially who are suffering from diabetes mellitus and the infection leads to many other complications, such as liver abscess or urinary tract infection (UTI). Treatment is difficult due to multiple-drug resistance. Many studies revealed that the thick capsular polysaccharide (CPS) on this bacterial surface is closely related to its virulence. The liver abscess strain is more virulent than the UTI strain, and we have found that there is an obvious difference in the amount of fucose on their capsule. We further confirmed that most liver abscess strains contain genes coding for two enzymes GDP-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxymannos-3,5-epimerase-4-reductase (WcaG), that are necessary for fucose biosynthesis from mannose, whereas UTI strains do not. In this study, we constructed a gmd-mutant of KP5, a highly virulent strain in mice and demonstrated that the downstream wcaG and wcaH genes were expressed by RT-PCR. According to the phenotype on the agar plate, the colony of mutant strain is smaller and non-mucoid as compared to the wild-type strain. In addition, mutant strain displayed much lower virulence than the wild-type strain in mice survival experiment. Examination of the internal organs in abdomem, ulceration and discoloration with pus formation were found in mice infected with wild-type KP5, while only minor inflammation was observed in mice infected with mutant KP5 strain. Both wild-type and mutant KP5 were used to infect HepG2 cells to investigate cell viability. At 6-hour post infection, we observed 68.55% of cells infected with mutant KP5 and 22.91% of cells infected with wild-type KP5 were viable. Moreover, the bacterial growth curves showed that the growth of the mutant KP5 was much slower than the wild type either with or without HepG2 cells. The bacterial interaction with mice peritoneal phagocytes was readily visibly for mutant KP5, but not apparent for the wild-type KP5 by Giemsa stain. The result showed that mutant-type strain was more easily recognized by phagocytes and then adhered to their surface than wild-type strain. The ability of biofilm formation was found to be the strongest for KP3, a UTI KP strain, followed by the mutant KP5, while the wild-type KP5 showed the least ability to form biofilm.
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