| Summary: | 碩士 === 國立中正大學 === 分子生物研究所 === 103 === Antibiotic resistance among infectious microorganisms is a serious and growing crisis worldwide. The carbapenem antibiotics are drugs of last resort, however, their misuse has already led to carbapenem-resistant bacteria. In Taiwan carbapenemase genes were detected in only 6.0% of Klebsiella pneumoniae isolates but no Klebsiella pneumoniae carbapenemase (KPC)-producing isolates detected in 2010. However, in 2012 the carbapenemase genes were detected in 22.3% of K. pneumoniae isolates and 75% of them are KPC-2. The KPC carbapenemase gene has been identified within Tn3-type transposon, Tn4401, and it can insert into diverse plasmids of Gram-negative bacteria and functions as origin of blaKPC-like gene dissemination. Identification of isolates harboring KPCs has proved to be especially challenging in clinical microbiology laboratories. The aim of this study is to establish a quick antibody-based detecting platform to screen KPC-2 harboring clinical isolates. The outcome can serve as a reference for clinical antibiotic administration.
The KPC-2 gene from a carbapenem-resistant K. pneumonia (CRKP) isolate was obtained by colony PCR, subcloned into pQE30 expression vector and KPC-2 protein was overproduced as insoluble inclusion bodies upon IPTG induction in E. coli M15. The urea-solubilized KPC-2 proteins were purified with Ni-column and further resolved by SDS-PAGE to exclude trace contaminates and used to immunize rabbit to produce antiserum against KPC-2 protein by a commercial biotech company.
The KPC-2 antiserum showed high sensitivity toward purified KPC-2 protein by Western blot and Dot blot. Screening 50 clinical CRKP isolates showed that 26 (52%) of them expressing KPCs proteins by Western blot using this KPC-2 antiserum. Colony PCR also confirmed presence of KPCs genes in these 26 corresponding immunoblot-positive isolates, but not in other immunoblot-negative isolates. The mechanisms of carbapenem resistance of those KPC-negative isolates are being under investigation. Nevertheless, our data suggested that this antibody-based method can serve as a quick and efficient screening for KPC-harboring CRKP.
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