【摘要】 目的　研究伤寒杆菌DNA旋转酶A亚单位基因 (gyrA)变异与其耐喹诺酮类的关系。方法　应用聚合酶链反应 (PCR)检测、限制性片段长度多态性 (RFLP)、单链构象多态性分析 (SSCP)及序列测定 ,对伤寒杆菌S2 75 (临床分离敏感菌株 )及其自发耐药突变株RG1,DNAgyrA喹诺酮类耐药决定区进行了研究。结果　伤寒杆菌S2 75 gyrA第 12 8～ 42 6位碱基与大肠杆菌KL 16高度同源 ,仅 7.49%有差异 ,且大部分为静变异 ,最终仅使DNA旋转酶A亚单位产生Thr 45→His、Arg 49→Leu及Val 5 6→Gly ,均位于喹诺酮耐药决定区 (第 6 7～ 10 6位氨基酸 )外。RG1仅有第 2 47位碱基T→G变异 ,相应Ser 83→Ala ,使萘啶酸、氧氟沙星及环丙沙星对伤寒杆菌的MIC由 2、0 .0 6、<0 .0 3mg/L上升为 5 12、2、1mg/L。Ala替换与文献报道沙门杆菌该位以苯丙氨酸、酪氨酸替换为主不同。PCR RFLP及SSCP分析结果与上述情况类似。结论　gryA第 83位变异为其耐药主要原因更多还原

【Abstract】 Objective To study the relationship between the gene mutations of DNA gyrase subunit A (gyrA) and quinolone resistance in Salmonella typhi. Methods The genes of gyrA DNA of Salmonella typhi S275 (a clinically isolated quinolone susceptible strain) and its spontaneous quinolone resistant mutant RG 1 were examined in this study with polymerase chain reaction (PCR), restrictive fragments length polymorphism (RFLP), single strand conformational polymorphism (SSCP) and nucleotide sequencing. Results Nudeotide sequencing of gyrA in Salmonella typhi S275 revealed that the bases of 128～426 kept highly conservative as compared with those of Escherichia coli KL 16, with only 7.49% difference in the gyrA nucleotides 128～426 between the two strains. Most of the mutations were silent mutations,which contributed to 3 amino acid substitutions in gyrase (including Thr 45→His,Arg 49→Leu and Val 56→Gly), and all these substitutions were located outside the quinolone resistance determining region (amino acids 67 106 of subunit A of gyrase). In comparison with Salmonella typhi S275, a single mutation was found at base 247 of gyrA of Salmonella typhi RG 1, with change transferred from T to G and led to a substitution of Ser 83→Ala. The mutation might be responsible for the increase of MICs of nalidixic acid, ofloxacin and ciprofloxacin against Salmonella typhi from 2,0.06 and <0.03 to 512, 2, and 1 mg/L respectively. Ser 83→Ala was also a newly discovered substitution in gyrA of Salmonella spp. The results of PCR RFLP and SSCP were in concordance with results of nucleotide sequencing. Conclusions The mutation of gyrase at the 83rd amino acid maybe play a principal role in the resistance of Salmonella typhi to quinolone.更多还原