Yi Meiying, Wang Pengyuan, Liu Yucun
Department of Clinical Laboratory, China-Japan Friendship Hospital, Ministry of Health, Beijing 100029, China.
Department of General Surgery, Peking University First Hospital, Beijing 100034, China.
Chin Med J (Engl). 2014;127(6):1071-6.
Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa (P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanisms of P. aeruginosa isolated from a surgical intensive care unit (SICU) in China.
The molecular typing was analyzed by REP-PCR. Enzyme activity was measured with a 260 nm wavelength spectrophotometer. The levels of outer membrane proteins OprD and OprN were measured by Western blotting. The levels of mexA gene transcriptional expression were measured by quantitative real-time PCR. The metallo-beta-lactamase genes IMP, VIM, SPM, GES, and GIM were amplified by PCR. DNA fragments were sequenced by an automated ABI PRISM 3700.
Forty-two strains resistant to carbapenems isolated from a SICU were analyzed. REP-PCR revealed 34 belonging to type A, a predominant strain in this SICU. But we did not find metallo-beta-lactamases IMP, VIM, SPM, GES, or GIM genes by PCR. With a three-dimensional extract test, we found 34 strains producing high levels of AmpC enzymes. We also observed the activity of beta-lactamases enzymes in the imipenem resistant group, which was statistically different from the sensitive group. Western blotting revealed that 23 strains showed loss of OprD, 18 strains had decreased OprD expression, and 14 strains expressed OprN. We discovered 27 strains that overexpressed mexA by quantitative real-time PCR, and the resistance rate to meropenem was statistically different between the overexpressing group and the low-expressing group. Nucleotide sequences and deduced amino acid sequence analysis revealed that eight strains carried mutations in the mexR gene operon down regulating MexAB-OprM. The nucleotide sequences of mexR genes from PA36, PA41 and PA48 were submitted to the Genebank with accession numbers of AY899299, AY899300, and AY899301.
There was a predominant strain in the SICU of our hospital. Imipenem resistance is mainly mediated by OprD deficiency or loss, and high activity AmpC enzymes. Overexpression of MexAB-OprM is one of the mechanisms of meropenem resistance, which are partly upregulated by mutations in the mexR gene. The expression of MexEF-OprN also plays an important role in the carbapenem resistance.
碳青霉烯类是治疗铜绿假单胞菌感染的一类重要药物。然而，在非发酵菌中碳青霉烯耐药现象普遍存在。本研究旨在调查从中国一家外科重症监护病房（SICU）分离出的铜绿假单胞菌的分子流行病学及碳青霉烯耐药机制。
通过重复序列聚合酶链反应（REP-PCR）分析分子分型。用波长260nm的分光光度计测量酶活性。通过蛋白质免疫印迹法检测外膜蛋白OprD和OprN的水平。通过定量实时聚合酶链反应测量mexA基因转录表达水平。通过聚合酶链反应扩增金属β-内酰胺酶基因IMP、VIM、SPM、GES和GIM。DNA片段由自动ABI PRISM 3700测序。
分析了从一个SICU分离出的42株对碳青霉烯耐药的菌株。REP-PCR显示34株属于A类型，是该SICU中的优势菌株。但通过聚合酶链反应未发现金属β-内酰胺酶IMP、VIM、SPM、GES或GIM基因。通过三维提取试验，发现34株产生高水平的AmpC酶。我们还观察到亚胺培南耐药组中β-内酰胺酶的活性，与敏感组相比有统计学差异。蛋白质免疫印迹法显示23株OprD缺失，18株OprD表达降低，14株表达OprN。通过定量实时聚合酶链反应发现27株mexA过表达，过表达组和美罗培南低表达组之间对美罗培南的耐药率有统计学差异。核苷酸序列和推导的氨基酸序列分析显示8株在下调MexAB-OprM的mexR基因操纵子中携带突变。来自PA36、PA41和PA48的mexR基因的核苷酸序列已提交至Genebank，登录号分别为AY899299、AY899300和AY899301。
我院SICU存在优势菌株。亚胺培南耐药主要由OprD缺陷或缺失以及高活性的AmpC酶介导。MexAB-OprM的过表达是美罗培南耐药的机制之一，部分是由mexR基因的突变上调。MexEF-OprN的表达在碳青霉烯耐药中也起重要作用。
