Yasuda M, Fukuda H, Yokoi S, Ishihara S, Kawada Y, Deguchi T
Department of Urology, Gifu University School of Medicine, Gifu, Japan.
J Urol. 2000 Sep;164(3 Pt 1):847-51. doi: 10.1097/00005392-200009010-00060.
We attempted to select increasingly fluoroquinolone-resistant strains of Neisseria gonorrhoeae in vitro and to assess whether selected mutants harbored alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV, which were analogous to those in fluoroquinolone-resistant clinical isolates.
A fluoroquinolone-susceptible strain was exposed to norfloxacin in vitro. Selected mutants were sequentially exposed to norfloxacin, and this procedure was repeated. For 11 mutants, minimum inhibitory concentrations (MICs) of antimicrobial agents were determined, and mutations in the region corresponding to the quinolone resistance-determining region (QRDR) of the Escherichia coli gyrA gene and the analogous region of the parC gene were analyzed.
Mutants obtained in one step exhibited significantly increased MICs of norfloxacin, ofloxacin and ciprofloxacin and had a single amino acid change in GyrA. Two-step mutants exhibited significantly higher norfloxacin MICs. Three of four two-step selected strains had single amino acid changes in both GyrA and ParC. Three-step mutants exhibited further increases in fluoroquinolone MICs and were assigned to the ciprofloxacin-resistant category. Two had a double amino acid change in GyrA, and one had a double GyrA change and a single amino acid change in ParC.
We selected fluoroquinolone-resistant strains that carried GyrA and ParC alterations analogous to those in clinical isolates. The serial accumulation of changes in the QRDR of GyrA and the analogous region of ParC was associated with a stepwise increase in fluoroquinolone resistance, although the development of additional alterations in other regions of GyrA and ParC or other mechanisms of fluoroquinolone resistance also might contribute to the enhancement in fluoroquinolone resistance. The clinical emergence of fluoroquinolone-resistant strains may be due to in-vivo stepwise selection of strains with genetic alterations in GyrA and ParC, as observed here in the in-vitro selection of fluoroquinolone-resistant mutants.
我们试图在体外筛选对氟喹诺酮类药物耐药性不断增加的淋病奈瑟菌菌株，并评估所选突变体的DNA回旋酶GyrA亚基和DNA拓扑异构酶IV的ParC亚基是否发生了改变，这些改变与氟喹诺酮类耐药临床分离株中的改变类似。
将一株对氟喹诺酮类药物敏感的菌株在体外暴露于诺氟沙星。将所选突变体依次暴露于诺氟沙星，并重复此过程。对11个突变体测定了抗菌药物的最低抑菌浓度（MIC），并分析了与大肠杆菌gyrA基因喹诺酮耐药决定区（QRDR）相对应区域以及parC基因类似区域的突变情况。
一步筛选获得的突变体对诺氟沙星、氧氟沙星和环丙沙星的MIC显著增加，且GyrA中有一个氨基酸发生改变。两步突变体的诺氟沙星MIC显著更高。四个两步筛选菌株中有三个在GyrA和ParC中均有一个氨基酸发生改变。三步突变体的氟喹诺酮类MIC进一步增加，并被归类为对环丙沙星耐药。两个在GyrA中有双氨基酸改变，一个在GyrA中有双氨基酸改变且在ParC中有一个氨基酸改变。
我们筛选出了携带与临床分离株中类似的GyrA和ParC改变的氟喹诺酮类耐药菌株。GyrA的QRDR和ParC类似区域变化的连续积累与氟喹诺酮类耐药性的逐步增加相关，尽管GyrA和ParC其他区域额外改变的出现或氟喹诺酮类耐药的其他机制也可能导致氟喹诺酮类耐药性增强。氟喹诺酮类耐药菌株在临床上的出现可能是由于体内对GyrA和ParC发生基因改变的菌株进行逐步选择，正如我们在此体外筛选氟喹诺酮类耐药突变体时所观察到的那样。
