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伤寒杆菌耐喹诺酮类机制分子生物学基础研究 |
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肖永红 王其南
【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S275(临床分离敏感菌株)及其自发耐药突变株RG1，DNA gyrA喹诺酮类耐药决定区进行了研究。结果 伤寒杆菌S275 gyrA第128～426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-56→Gly，均位于喹诺酮耐药决定区(第67～106位氨基酸)外。RG1仅有第247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、0.06、<0.03 mg/L上升为512、2、1 mg/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。
【关键词】沙门菌，伤寒； 旋转酶A；聚合酶链反应-限制性片段长度多态性、单链构象多态性分析；脱氧核糖核酸序列
A study on the molecular basis of quinolone resistance mechanism in salmonella typhi
XIAO Yonghong, WANG Qinan.
（The First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China）
【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 RG1 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[1] [2] [3] 下一页
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