摘要
目的 :研究大肠埃希菌对氟喹诺酮类高水平耐药的机制。方法 :用Etest法筛选出 30株环丙沙星高水平耐药 (MIC>32 μg/ml)的临床大肠埃希菌株 ;用梯度平皿法测定诺氟沙星、四环素、氯霉素和氨苄西林的MIC ;己烷和环己烷用来研究菌株对有机溶剂的耐受性 ;聚合酶链反应扩增 gyrA、parC、gyrB 3种基因的喹诺酮类耐药决定区域 (QRDR)并测序。利用Westernblot和Northernblot检测通用调节子 (marA、soxS)和主动外排蛋白AcrA的表达。结果 :30株菌中 ,2 9株多重耐药 ,19株耐受环己烷。所有菌株的 gyrA有双突变 ,即第 83位的丝氨酸变为亮氨酸 ,87位的天冬氨酸替换为天冬酰胺或酪氨酸 ;6株还有第 3个突变 ,即 93位的丙氨酸变为苏氨酸或丝氨酸。在 parC基因上 ,2 4株菌有单突变 ,或是 80位的丝氨酸变为异亮氨酸 (n =2 1) ,或是 84位的谷氨酸变为赖氨酸 (n =3) ;其他 6株菌除了 80位的突变外 ,合并有另一突变 (3株 84位谷氨酸变为甘氨酸 ,3株 10 8位的丙氨酸变为缬氨酸 )。gyrB基因未发现氨基酸的改变。在所有的菌株中未发现marA和soxS表达增加。 19株高产AcrA ,这些菌同时耐受环己烷 ;当诺氟沙星MIC >32 μg/ml时 ,更多的菌高产AcrA(分别为 17/2 4和 2 /6 )。结论 :靶位点 gyrA和 parC的改变 。
Objective: To investigate the mechanism of high-level fluoroquinolone resistance in nosocomial Escherichia coli strains. Methods: Thirty strains of E.coli highly resistant to ciprofloxacin (MIC>32 μg/ml) from Peking Union Medical College Hospital were selected by E test. Gradient plates method was used to determine the MIC of norfloxacin, tetracycline, chloramphenicol and ampicillin. Hexane and cyclohexane were used to study the organic solvent tolerance of these strains. The quinolone resistant-determining region of gyrA, parC and gyrB were amplified and sequenced. Western blot and Northern blot were used to study the overexpression of the regulators (marA and soxS) and the efflux pump protein (AcrA). Results: Among 30 strains, 29 were multiple drug resistant and 19 strains were tolerant to cyclohexane. All isolates contained double mutations at amino acid codon 83 and 87 of gyr A. Ser-83 was replaced by leucine; Asp-87 was substituted by asparagines (n=27) or tyrosine (n=3). Six isolates had an additional mutation (Ala-93→Thr or Ser) in gyrA. Twenty-four isolates had a single mutation in parC, either a Ser-80 to IIe (n=21) or Glu84 to Lys (n=3); the other 6 isolates had an additional mutation besides Ser-80→IIe, of either Glu84 to Gly (n=3) or Ala108 to Val (n=3). No amino acid change was found in gryB. The overexpression of marA and soxS were not found in all the isolates. Nineteen strains overproduced AcrA; all these strains were tolerant to cyclohexane. Among strains with norfloxacin MIC greater than 32?μg/ml (17/24 vs. 2/6), there were more strains overproducing AcrA. Conclusions: Mutations in gyrA and parC are the most important factors leading to fluoroquinolone resistance. The regulators other than marA or soxS contribute to the high-level fluoroquinolone resistance through up-regulating acrAB efflux pump.
出处
《中国抗感染化疗杂志》
2002年第2期87-91,共5页
Chinese Journal of Infection and Chemotherapy
基金
1999年联合国教科文组织-美国微生物协会旅行奖学金的资助
