伤寒杆菌耐喹诺酮类机制分子生物学基础研究

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％

A study on the molecular basis of quinolone resistance mechanism in salmonella typhi

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-re-sistant mutant RGl were examined in this study with polymerase chain reaction(PCR),restrictive frag-ments 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 re-gion(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 RG1,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→A1a 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. Conclu.sions The mutation of gyrase at the 83rd amino acid maybe play a principal role in the resistance of Salmonella typhi to quinolone.