重症监护病房铜绿假单胞菌的耐药性分析及其耐氟喹诺酮的分子机制研究

Drug Resistance Analysis of Pseudomonas aeruginosa in Intensive Care Unit and Molecular Mechanism of Its Resistance to Fluoroquinolones

目的了解重症监护病房铜绿假单胞菌的耐药情况以及对氟喹诺酮类耐药的分子机制,为临床合理用药提供依据。方法用E试验法测定83株铜绿假单胞菌对13种抗菌药物的最低抑菌浓度(MIC),从中筛选28株氟喹诺酮类耐药菌,以标准敏感菌株ATCC27853作为质控菌株,PCR扩增gyrA及parC基因的喹诺酮耐药决定区(QRDR),扩增产物片段进行直接测序分析。结果83株铜绿假单胞菌在痰标本的阳性率最高71.08%;28株氟喹诺酮耐药菌株的gyrA基因在83位(ACC-ATC)有突变,导致氨基酸Thr-Ile的改变;有11株高耐药菌gyrA基因同时在87位(GAC-GGC)有突变,导致氨基酸Asp-Gly的改变;有14株耐药菌株的parC基因在87位有TCG-TTG突变,导致氨基酸由Ser-Leu的改变,未发现parC突变单独存在。结论重症监护病房铜绿假单胞菌对美罗培南保持较高的抑菌活性;而亚胺培南和氟喹诺酮类药物的滥用,已造成细菌对其耐药率急剧升高,gyrA和parC基因突变与铜绿假单胞菌耐喹诺酮类药物密切相关。

OBJECTIVE To study the Pseudomonas aeruginosas drug resistance in intensive care unit and its fluoroquinolone-resistant molecular mechanism, and provide scientific basis for rational employment of antibiotics in clinic. METHODS E test was used to determine the minimal inhibitory concentrations（MIC） of 13 antibiotics against 83 P. aeruginosa strains. Twenty-eight fluoroquinolone-resistant strains were selected with standard sensitive strain-ATCC27853 as control. The quinolone resistance-determining region（QRDR）of the gyrA and parC genes was amplified by PCR and sequenced. RESULTS The positive rate of P. aeruginosa in sputum specimen was the highest from 83 strains （71.08%）. gyrA Genes of all resistant strains had an ACC to ATC mutation in codon 83, leading to the amino acid substitution of threonine for a an isoleucine and 11 high level resistant strains also showed a GAC to GGC mutation in codon 87, leading to the substitution of an aspartic acid a glycine for. In addition, 14 resistant strains also had an TCG to TTG mutation in codon 87 of parC gene, leading to the amino acid substitution of a serine for a leucine. We didn＇t find parC gene mutation existing independent in fluoroquinolone-resistant P. aeruginosa. CONCLUSIONS Meropenem remains highly active against P. aeruginosa. But the abuse of imipenem and other fluoroquinolones leads to rise in their drug-resistance rate. Fluoroquinolone-resistance has increased rapidly, the mechanism of resistance is gene mutant. It displayed that gyrA and parC gene mutation is associated with fluoroquinolone resistance.