1991 Volume 39 Issue 11 Pages 1001-1013
A 2.2-kb Hind III DNA fragment containing the norfioxacin (NFLX) resistance gene was cloned from chromosomal DNA of new quinolone-resistant Staphylococcus epidermidis. The cloned DNA fragment was introduced into quinolone-susceptible Staphylococcus aurcus SA 113, for which MICs of NFLX, enoxacin, ciprofloxacin, ofloxacin, tosufloxacin and sparfloxacin were 0.39, 0.39, 0.1, 0.2, 0.01 and 0.02μg/ml, respectively. The resultant transformant was resistant to the above new quinolones with MICs of 25, 12.5, 3.13, 1.56, 0.2 and 0.1μg/ml, respectively. The cloned DNA fragment has an open reading frame of 1, 161 base pairs that encoded a polypeptide of 387 amino acid residues with the molecular weight of 42, 118. This polypeptide was rich in hydrophobic amino acids and was estimated to be membrane-associated. The amino acid sequence of the polypeptide was one amino acid fewer than that of the norA polypeptide of S. aureus in numbers, and showed 79% amino acid identity with the latter polypeptide. The polypeptide produced by DNA-directed translation using the cloned DNA fragment as a template showed the molecular weight of about 42, 000 which was consistent with the estimated value. Therefore, the open reading frame was considered to be the norA gene of S. epidermidis, a homologue of the S. aureus norA gene cloned previously. The cells of S. aureus SA 113 having the S. epidermidis norA gene uptook less enoxacin (about 50% of S. aureus SA 113 lacking the norA gene) and the uptake was recovered to about 70% when carbonyl cyanide m-chlorophenyl hydrazone was added. These results suggest that the NorA polypeptide encoded by the norA gene of S. epidermidis is located in the cell membrane and concerns energy-dependent active efflux of hydrophilic new quinolones.
