两种氟喹诺酮类药物对肺炎克雷伯菌防耐药突变浓度及耐药突变体耐药性的研究论文.docVIP

　　两种氟喹诺酮类药物对肺炎克雷伯菌防耐药突变浓度及耐药突变体耐药性的研究论文 邓晓慧 郑风劲 何文富 孙爱慧 刘小康 【摘要】 目的 测定两种氟喹诺酮类药物(FQNs)对肺炎克雷伯菌(KP)的防耐药突变浓度(MPC)，比较其防耐药突变能力，了解突变耐药菌对FQNs的耐药性。方法 肉汤法富集1010CFU/ml的菌液接种于不同浓度环丙沙星及加替沙星琼脂平皿上，采用琼脂二倍稀释法测定环丙沙星、加替沙星对临床分离肺炎克雷伯菌ATCC700603及其耐药突变体的最低抑菌浓度(MIC)、防耐药突变浓度(MPC)。对不同药物浓度筛选出的耐药突变株进行编码拓扑异构酶VIC亚单位基因parC和回旋酶A亚单位基因gyrA喹诺酮耐药决定区的PCR扩增和测序，并测定外排泵抑制剂羟基氰氯苯腙(Carbonyl Cyanide mchlorophenylhydrazone，CCCP)对环丙沙星和加替沙星MIC的影响。结果 环丙沙星、加替沙星对ATCC700603的MPC分别为4、1.6mg/L，细菌耐药选择指数分别为16、4。两种药物的耐药突变体均出现了gyrA的第83位(TCC→ATC/TTG)均出现了突变，引起了相应氨基酸的改变(Ser83→Ile/Leu)。其中有一株同时也出现了第87位点(GAC→AAC)的改变.freeline mutant prevention concentration (MPC) of toniae (KP) and pare the capacity of fluoroquinolones to prevent the resistant mutant strains. Methods The KP strain ATCC700603 ing units per milliliter. The minimal inhibitory concentration (MIC) and MPC of gatifloxacin and ciprofloxacin for KP ined by agar plates dilution method. Quinolone resistancedeternining region (QRDR) of parC and gyrA genes plification and gene sequencing. The effect of effluxpump inhibitor CCCP on MICs of gatifloxacin and ciprofloxacin for resistant mutants ided by agar dilution method. Results The MPC of gatifloxacin and ciprofloxacin for KP strain ATCC700603 g/L, and the MPC/MIC utation in codon 83, leading to amino acid change: Ser→Leu or Ser→Ile. At the same time, a GAC→AAC mutation ino acid change: Asp→Asn. Except for the secondstep mutant, DNA sequencing analysis of parC did not reveal point mutation (AGC→ATC) leading the substitution of a Ile for Ser in 4 strains. As for the MICs, of e as the MICs utants is stronger than that of ciprofloxacin. The mutation of gyrA and parC genes are the primary mechanisms responsing for resistance of Klebsiela pneumoniae to FQNs. KEY utant prevention concentration 肺炎克雷伯菌(Klebsiella pneunoniae，KP)是兼性厌氧的革兰阴性菌，通常存在于人类肠道及呼吸道，是目前重要的条件致病菌，其发病率［1］仅次于大肠埃希菌和铜绿假单胞菌。氟喹诺酮类药物(FQNs)是治疗KP感染的常用药，由于其广泛应用，KP对其耐药性逐年递增。目前，就新一代FQNs的耐药性及延长其临床使用寿命，国外学者Drlica提出了防耐药突变浓度(mutant prevention concentration，MPC)这一概念［2］，该理论在关注抗菌