肺炎克雷伯菌对喹诺酮类药物的耐药机制研究

A study on quinolone resistant mechanisms in klebsiella pneumoniae

为了探讨肺炎克雷伯菌对喹诺酮类药物的耐药机制，将肺炎克雷伯菌９００１２９（临床分离的敏感菌）及其诱导耐药株Ｇ＿１（第一步变异株）与Ｇ＿２（第二步变异株）用于本研究。细菌ＤＮＡ旋转酶以新生霉素亲合层析分离，细菌外膜蛋白质及细菌对环丙沙星聚积分别以ＳＤＳ－ＰＡＧＥ及荧光法分析。结果发现：与９００１２９相比，Ｇ＿１和Ｇ＿２对喹诺酮类、四环素、氯霉素及β－内酰胺抗生素敏感性明显降低；喹诺酮类对Ｇ＿１与Ｇ＿２ＤＮＡ旋转酶活性５０％抑制剂量增加３～１１倍；Ｇ＿１与Ｇ＿２外膜４２ｋＤ及Ｇ＿２外膜３４、２４．７、２１ｋＤ蛋白消失；Ｇ＿１与Ｇ＿２对环丙沙星的聚积仅分别为９００１２９的１／３与１／１０，经氰氯苯踪处理后，分别上升达９００１２９与９００１２９的１／３水平。本文结果提示：ＤＮＡ旋转酶变异、外膜蛋白质改变及药物主动外流均为肺炎克雷伯菌耐喹诺酮类原因。

To study the quinolone resistant mechanisms in Klebsiella pneumoniae,we usedKlebsiella pneumoniae 900129(a quinolone sensitive clinical isolate)and its spontaneous quinoloneresistant mutants G1(single-step mutant)and G2(double-step mutant)in our experiment.DNAgyrases of the bacteria were purified by novobiocin affinity chromatography and the inhibitory effectof quinolone on its supercoiling activity was examined.Bacterial outer membrane protein profileswere analysed by SDS-PAGE.Accumulation of ciprofloxacin by the bacterial cell was determined byusing fluorometric assay.The results demonstrated that compared to 900129, the susceptibilities ofG1 and G2 to quinolones,tetracycline,chloramphenicol andβ-lactams decreased.ID50 s ofquinolones on DNA gyrases from mutants increased by 3-11 times,In both G1 and G2 a protein of42kD and in G2 proteins、of 34,24.7 and 21kD disappeared from their outer membranes.The accu-mulation of ciprofloxacin in G1 and G2 decreased to 1/3and 1/10 of that in 900129 respectively.Af-ter treatment with carbonylcyanid m-chloro-phenylhydra-zone,the drug uptake in G1 and G2 in-creased to the same or 1/3 level of that in 900129.The results indicated that DNA gyrase mutation,outer membrane permeability declining and active drug efflux were the major causes of quinolone re-sistance in K.pneumoniae.