伤寒沙门菌DNA旋转酶gyrA及拓扑异构酶Ⅳ parC基因与耐喹诺酮类关系研究

【 摘要 】 目的 研究DNA旋转酶、拓扑异构酶Ⅳ在伤寒沙门菌耐喹诺酮类药物的作用。 方法对临床分离喹诺酮类敏感伤寒沙门菌275及其自发耐药变异菌RG₁的gyrA、parC基因喹诺酮类耐性决定区进行PCR DNA直接测序分析。 结果 表明伤寒沙门菌275 gyrA、parC与大肠埃希菌相应DNA序列分别有92.51％与97.01％同源性，推定氨基酸序列仅有3与6个的差异。伤寒沙门菌gyrA及parC也有较好同源性，相应区段氨基酸有60.92 ％相同。伤寒沙门菌RG₁与275相比，gyrA第247位T→G变异，致Ser 83 Ala氨基酸替换，两者parC完全相同。喹诺酮类药物对RG₁ MIC较对伤寒沙门菌275上升32倍或以上。 结论 DNA旋转酶、拓扑异构酶Ⅳ为喹诺酮类药物作用靶位，但以DNA旋转酶为主，该酶变异为伤寒沙门菌耐药的主要原因。 A study on the correlation of quinolone resistance with mutationsin DNA gyrase gyrA and topoisomerase Ⅳ parC genes in Salmonella typhi

XIAO Yonghong, WANG Qi′nan. The First Affiliated Hospital, Chongqing

University of Medical Sciences, Chongqing 400016, P.R.China

【 Abstract 】 Objective To study the correlation of quinolone resistance with mutations in DNA gyrase gyrA and topoisomerase Ⅳ parC genes in Salmonella typhi. Methods The quinolone-resistance determining regions of gyrA and parC in Salmonella typhi 275(a clinically isolated strain, S275) and its spontaneous resistant mutant RG₁ were sequenced. Results The results showed that the sequenced regions of gyrA and parC in S275 had 92.51% and 97.01% homology with those of Escherichia coli respectively, which led to 3 and 6 substitutions in deduced amino acids in gyrA and parC. The gyrA and parC of S275 also had high homology in amino acid sequence as much as 60.92%. In comparison with S275, gyrA of RG₁ had a mutation of T247 G which contributed to the substitution of Ser 83 to Ala in gyrA. No difference was found in parC of S275 and RG₁. The MICs of quinolones on RG₁ were higher than those on S275 by 32 folds or more. Conclusion These results indicated that both DNA gyrase and topoisomerase Ⅳ of S.typhi are targets of quinolones while DNA gyrase is more important as the target molecules. Its mutation seems to be the major mechanism to explain quinolone-resistance in S.typhi.

【 Subject words 】 Salmonella typhi; Quinolones; DNA gyrase; Topoisomerase Ⅳ; Sequence analysis

伤寒为我国常见传染病，氟喹诺酮类作为主要治疗药物受到广泛认可。喹诺酮类主要通过抑制细菌DNA复制、转录起到杀菌作用，其作用位点为DNA旋转酶。近年发现细菌DNA拓扑异构酶Ⅳ活性也受喹诺酮类抑制，与DNA旋转酶共同构成喹诺酮类作用靶位，对不同细菌两者所起作用有所差异，在革兰氏阳性菌拓扑异构酶Ⅳ为第一靶位^〔1,2〕。为研究在伤寒沙门菌中两者所起作用，我们用临床分离喹诺酮敏感伤寒沙门菌275(简称S275)及其体外筛选的耐药菌RG₁(简称RG₁)对DNA旋转酶gyrA及拓扑异构酶ⅣparC基因之耐喹诺酮类决定区(quinolone-resistant determining region, QRDR)进行PCR扩增及核酸序列分析，结果如下。

菌株：伤寒沙门菌S275为我院1996年临床病人血培养分离所得对喹诺酮类敏感的菌株，用平板法筛选其耐药变异株RG₁。挑取单个伤寒沙门菌S275菌落接种于10ml Mueller-Hinton肉汤(MHB，1000ml含牛肉浸膏5g，水解酪蛋白17.5g，可溶性淀粉1.5g)，过夜培养后，以少许菌液均匀涂布于含4μg/ml(2×MIC)萘啶酸的Mueller-Hinton琼脂(MHA)，37℃孵育48h后，得耐药变异株RG₁，与S275共同用于下列研究。

抗菌药物及主要试剂：氧氟沙星(OFLX)由日本第一制药株式会社提供，环丙沙星(CPFX)为太原制药厂提供，萘啶酸购自Sigma公司。多聚酶链反应扩增DNA旋转酶A亚单位基因(gyrA)上游引物为：5′-TGTCCGAGATGGCCTGAAGC-3′，位于大肠埃希菌gyrA 108～127位碱基；下游引物为5′-TGCCGTCATAGTTATCAACG-3′，与大肠埃希菌gyrA 435～454位碱基互补。拓扑异构酶Ⅳ亚单位C基因(parC)上游引物为5′-GTATGCGATG TCTGAACTGG-3′，位于大肠埃希菌parC基因第163～182位碱基；下游引物5′-GTGGTGCCGTTAAGCAAA-3′与大肠埃希菌parC基因第517～534位碱基互补。上述引物均委托中国科学院微生物研究所基因工程中心合成。合成仪为Beckman Oligo 1000型。dNTP、Taq酶、限制性内切酶HinfⅠ、Wizard PCR产物纯化试剂盒、DNA银染试剂盒均购自Promega公司；DNA测序试剂盒为Perkin-Elmer之末端标记试剂盒。