| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 89 === Chlorhexidine ( CHX ) is one of the most widely used antiseptic product today, in particular in handwashing and oral products. According to the experimental data, Klebsiella pneumoniae could be a highly CHX-resistant bacterium but the mechanism is still unclear. Thus, the aim of this study is to find CHX-resistance related genes in K. pneumoniae. We contructed an expression library with chromosomal DNA from CHX-resistant K. pneumoniae and screened for E. coli XLOLR carrying phagemid library which can tolerate 16 mg/ml CHX. The phenotype remained the same after retransformation. There are 20 mutants with 3 kind of inserts and all contain a same open reading frame and we designated it as cation efflux pump A, cepA. The sequences of cepA show 90 % similarity with yiip, a gene of putative transporter function in E.coli. In TIGR database, it was predicted as an cation efflux pump. Because CHX is a cation biquanide, the cepA may act as a cation efflux pump to export CHX out. We try to knock out cepA by inserting an antibiotic cassette into cepA, and transform the construct into K. pneumoniae by electroporation or conjugation. We didn’t get the knock-out mutant except the integration-forms. Therefore, this gene is possiblely vital. Then we delivered cep::pCR-TOPOII into the CHX-sensitive K. pneumoniae, we found it can elevate Minimal inhibitory concentration of CHX. Base on the above results, cepA is a cation efflux pump in K. pneumoniae, and associated for CHX resistance.
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