Plasmid isolation and curing in gram-positive Enterococcus faecalis

摘要

This research reports a novel and efficient method for rapid plasmid isolation from a clinical isolate of Enterococcus faecalis exhibiting resistance to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50μg/ml). Results showed that the method which involves the use of sodium N-lauroylsarcosinate allowed for the reproducible isolation of two plasmids of molecular weight ~7 kb and 5.7 kb, respectively, as determined by gel electrophoresis in 1% agarose. The plasmid DNA thus prepared would transform E. coli and render it resistant to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50 μg/ml). Comparatively, the routine alkaline lysis procedure produced only one plasmid, barely detectable by agarose gel electrophoresis in one out of three preparations; the boiling procedure, although more efficient than the alkaline lysis procedure, also led to the detection of one plasmid only. Thus the method gave results superior to those from the conventional procedures used for gram-negative bacteria.