Tankovic J, Perichon B, Duval J, Courvalin P
Unité des Agents Antibactériens, Centre National de la Recherche Scientifique EP J0058, Institut Pasteur, Paris, France.
Antimicrob Agents Chemother. 1996 Nov;40(11):2505-10. doi: 10.1128/AAC.40.11.2505.
We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84-->His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), and in two mutants obtained in vitro (Ser-80-->Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.
我们通过基因扩增和测序，分析了在治疗期间或体外获得的耐氟喹诺酮肺炎链球菌突变体的gyrA、gyrB和parC基因喹诺酮耐药决定区的突变情况。在体内获得的两个突变体BM4203-R（第84位天冬氨酸被组氨酸取代[Asp-84→His]；以金黄色葡萄球菌的编号为准）和BM4204-R（Ser-80→Phe）以及体外获得的两个突变体（Ser-80→Tyr）中，检测到导致ParC发生替换的突变。体外获得的另一个突变体BM4205-R3表现出更高水平的氟喹诺酮耐药性，其gyrA基因发生突变，导致Ser-84→Phe的变化。我们在体外获得的其余三个突变体中未检测到任何突变。用来自BM4203-R、BM4204-R和BM4205-R3的总DNA通过氟喹诺酮选择来转化肺炎链球菌CP1000。对于parC突变体，获得了表型与供体无法区分的转化体，其频率（5×10⁻³至8×10⁻³）与单基因转化相符。相比之下，对于gyrA突变体，转化体的获得频率较低（4×10⁻⁵），与两个独立基因的转化相符。用来自BM4203-R和BM4204-R的parC扩增片段也获得了CP1000的耐药转化体，但用来自BM4205-R3的gyrA片段未获得。所有转化体的突变都与供体中的相同。这些数据有力地表明，ParC是肺炎链球菌中氟喹诺酮的主要靶点，并且BM4205-R3在获得两个突变（包括gyrA中的一个突变）后对更高水平的药物具有耐药性。
