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目的探讨铜绿假单胞菌对碳青霉烯类抗生素的耐药性和耐药机制。方法选取温州医学院附属第一医院162株铜绿假单胞菌临床分离株,采用琼脂稀释法检测抗生素亚胺培南和美罗培南对铜绿假单胞菌的最低抑菌浓度(M IC);PCR扩增分离株外膜蛋白基因OprD2和碳青霉烯酶基因VIM、IMP、SPM、KPC,对阳性产物测序确定基因亚型;羰基氰氯苯腙(CCCP)协同抑制试验检测膜外排机制。结果 162株分离菌株中,亚胺培南和美罗培南的耐药率分别为37.0%和30.9%;60株耐亚胺培南和/或美罗培南菌株(简称耐药株)中,50株对2种抗生素均耐药,另10株仅对亚胺培南耐药;耐药株中有18株OprD2基因缺失,102株不耐亚胺培南或美罗培南菌株(简称敏感株)中有20株OprD2基因缺失;耐药株中检出13株碳青霉烯酶阳性,其中5株产VIM型,8株产IMP型,敏感株中未检出VIM、IMP基因,所有菌株中未检出SPM-1、KPC基因;耐药株有48.3%外排泵表型试验阳性,以亚胺培南和美罗培南作为底物的分别有19株和24株,敏感株有10.8%外排试验阳性。结论铜绿假单胞菌对碳青霉烯类抗生素耐药严重,与外膜蛋白OprD2缺失、产碳青霉烯酶和主动外排共同作用有关。
Objective To investigate the resistance and resistance mechanisms of Pseudomonas aeruginosa to carbapenem antibiotics. Methods 162 clinical isolates of Pseudomonas aeruginosa from the First Affiliated Hospital of Wenzhou Medical College were selected. The minimal inhibitory concentration (MIC) of antibiotics imipenem and meropenem against Pseudomonas aeruginosa was determined by agar dilution method. The outer membrane protein gene OprD2 and carbapenemase genes VIM, IMP, SPM and KPC were isolated and sequenced to determine the gene subtype. The CCCP synergistic inhibition assay was used to detect the membrane efflux mechanism. Results The resistance rates of imipenem and meropenem were 37.0% and 30.9% respectively in 162 isolates. Among the 60 imipenem-resistant and meropenem-resistant strains (resistant strains), 50 isolates The antibiotics were all resistant, and the other 10 strains were resistant to imipenem only. 18 strains of resistant OprD2 gene were deleted in drug-resistant strains, 20 of which were immature insensitive to imipenem or 20 strains of meropenem (referred to as sensitive strains) OprD2 gene was deleted. Among the drug-resistant strains, 13 strains were positive for carbapenema. Among them, 5 strains were VIM-type and 8 strains were IMP-type. VIM and IMP genes were not found in the susceptible strains and SPM was not found in all the strains -1 and KPC genes. The drug-resistant strains were positive for 48.3% of the efflux pump phenotype test. Imipenem and meropenem as substrate were 19 and 24, respectively, and 10.8% of the susceptible strains were positive for efflux assay. Conclusion Pseudomonas aeruginosa is highly resistant to carbapenem antibiotics and is associated with the loss of OprD2 and the production of carbapenemase and active efflux.