铜绿假单胞菌临床分离株主动外排泵的检测

目的了解铜绿假单胞菌临床分离株的耐药特点及其外排泵存在的状况。方法采用K-B法检测铜绿假单胞菌临床分离株对9种常用抗生素（庆大霉素、氨曲南、头孢他啶、亚胺培南、阿米卡星、哌拉西林/他唑巴坦、环丙沙星、左氧氟沙星、多粘菌素B）的耐药情况;通过外排泵抑制剂氰氯苯腙（CCCP）对四种药物（羧苄青霉素、红霉素、亚胺培南、庆大霉素）的琼脂稀释法抑制试验分别检测铜绿假单胞菌的四种外排泵（MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM）的表型;应用PCR检测MexAB-OprM外排泵调节基因mexR的存在情况。结果铜绿假单胞菌对9种常用抗生素的耐药率依次为：庆大霉素36.67%（22/60）,氨曲南33.33%（20/60）,头孢他啶31.67%（19/60）,亚胺培南28.33%（17/60）,阿米卡星26.67%（16/60）,哌拉西林/他唑巴坦23.33%（14/60）,环丙沙星21.67%（13/60）,左氧氟沙星11.67%（7/60）,多粘菌素B为0。外排泵表型阳性株依次为：MexAB-OprM35株,阳性率58.33%（35/60）;MexXY-OprM13株,阳性率21.67%（13/60）;MexCD-OprJ11株,阳性率18.33%（11/60）;MexEF-OprN10株,阳性率16.67%（10/60）。35株MexAB-OprM外排泵表型阳性株中有28株扩增出mexR基因片段,检出率为80.0%（28/35）。结论铜绿假单胞菌临床分离株对庆大霉素耐药率最高,对多粘菌素B耐药率最低;MexAB-OprM外排泵的存在是铜绿假单胞菌临床分离株的重要耐药机制。

Detection of active efflux pumps in clinical isolates of Pseudomonas aeruginosa

Objective To investigate the features of drug resistance and the effux pump expressed in clinical isolated strains of Pseudomonas aeruginosa.Methods K-B test was used to detect the rate of drug resistance.9 antibiotics,namely Gentamicin,Aztreonam,Ceftazidime,Imipenem,Amikacin,Piperacllin/tazobactam,Ciprofloxacin,Levofloxacin and Polymyxin B,were studied.Phenotypes of the four effux pumps（MexAB-OprM,MexCD-OprJ,MexEF-OprN,and MexXY-OprM） were studied using the efflux pump inhibitor cyanide chorophenyl hydrazone（CCCP）.This inhibitor is known to inhibit carbenicillin,erythromycin,imipenem,and gentamicin.PCR was used to detect the regulatory gene（mexR） of MexAB-OprM.Results The rates of drug resistance in Pseudomonas aeruginosa to 9 antibiotics were：gentamicin 36.67%（22/60）,aztreonam 33.33%（20/60）,ceftazidime 31.67%（19/60）,imipenem 28.33%（17/60）,amikacin 26.67%（16/60）,piperacillin/tazobactam 23.33%（14/60）,ciprofloxacin 21.67%（13/60）,levofloxacin 11.67%（7/60） and polymyxin B（0%）.The positive rates of efflux pump phenotype were：MexAB-OprM 58.33%（35/60）,MexXY-OprM 21.67%（13/60）,MexCD-OprJ 18.33%（11/60）,and MexEF-OprN 16.67%（10/60）.28 out of 35 MexAB-OprM positive strains had amplified MexR gene and its detection rate was 80.0%（28/35）.Conclusion The rate of gentamycin resistance in the clinical isolates of Pseudomonas aeruginosa was the high and the rate of polymyxin B resistance was the low.MexAB-OprM efflux pump is responsible for the drug resistance of Pseudomonas aeruginosa.