CAJ | 학술논문

目的研究一株临床分离的碳青霉烯类药物耐药产气肠杆菌的耐药机制和耐药基因传播机制。方法采用琼脂稀释法检测菌株对抗菌药物的最低抑菌浓度（MIC），采用质粒接合试验、质粒提取、DNA分子杂交、等电聚焦电泳（IEF）、聚合酶链反应（PCR）、DNA测序和外膜蛋白分析研究菌株的耐药基因及其传递机制。结果IEF显示临床分离的产气肠杆菌株含有3条β内酰胺酶条带，等电点（pI）分别为5．4、6．7和7．8。PCR扩增及测序结果表明它们分别为TEM1（pI5.4）、KPC2（pI6．7）、DHA1（pI7．8）β内酰胺酶。接合菌含有2条β内酰胺酶条带，pI为6．7和7．8。接合试验、质粒提取和分子杂交试验结果显示KPC2和DHA1基因定位于同一个约56kb大小的质粒上。与临床野生产气肠杆菌株相比，外膜蛋白电泳分析显示耐药分离株缺失41000外膜蛋白。结论产气肠杆菌分离株对碳青霉烯类抗菌药物耐药可能由A类2f组KPC2酶介导，DHA1酶合并外膜蛋白缺失也可能与碳青霉烯类药物耐药机制形成有关。
목적연구일주림상분리적탄청매희류약물내약산기장간균적내약궤제화내약기인전파궤제。방법채용경지희석법검측균주대항균약물적최저억균농도（MIC），채용질립접합시험、질립제취、DNA분자잡교、등전취초전영（IEF）、취합매련반응（PCR）、DNA측서화외막단백분석연구균주적내약기인급기전체궤제。결과IEF현시림상분리적산기장간균주함유3조β내선알매조대，등전점（pI）분별위5．4、6．7화7．8。PCR확증급측서결과표명타문분별위TEM1（pI5.4）、KPC2（pI6．7）、DHA1（pI7．8）β내선알매。접합균함유2조β내선알매조대，pI위6．7화7．8。접합시험、질립제취화분자잡교시험결과현시KPC2화DHA1기인정위우동일개약56kb대소적질립상。여림상야생산기장간균주상비，외막단백전영분석현시내약분리주결실41000외막단백。결론산기장간균분리주대탄청매희류항균약물내약가능유A류2f조KPC2매개도，DHA1매합병외막단백결실야가능여탄청매희류약물내약궤제형성유관。
Objective To investigate the resistance and transmission mechanisms of a clinical isolate of carbapenem-resistant Enterobacter aerogen.Methods The minimal inhibition concentrations (MIC)of antimicrobial agents were determined by agar dilution method,and plasmid conjugation experiment,plasmid extraction and DNA molecular hybridization, isoelectric focusing electrophoresis (IEF ), polymerase chain reaction (PCR ), DNA sequencing and outer-membrane protein analysis were used for analyzing the resistant gene and transmission mechanism. Results IEF showed that there were 3 bands of beta-lactamases with isoelectric point (pI)of 5.4,6.7 and 7.8 in the clinical isolate of carbapenem-resistant Enterobacter aerogen.These 3 bands of beta-lactamases were confirmed to be TEM-1 (pI 5.4),KPC-2(pI 6.7)and DHA-1 (pI 7.8)by PCR amplification and DNA sequencing,and 2 of them (pIs of 6.7 and 7.8)were found that they can be transferred by plasmid conjugantion experiment.KPC-2 and DHA-1 genes were located on an about 56 kb plasmid by plasmid conjugation experiment,plasmid extraction and DNA molecular hybridization.Outer-membrane protein electrophoresis analysis revealed that a 41 000 outer-membrane protein was absent in the clinical isolate of carbapenem-resistant Enterobacter aerogen comparing with clinical wild-type Enterobacter aerogen.Conclusions The clinical isolate of Enterobacter aerogen resistant to carbapenem produces a plasmid-mediaed carbapemase KPC-2 which belongs to Group 2f,Class A beta-lactamase.DHA-1 enzyme with outer-membrane protein absence may be related with the resistant mechanism of carbapenem-resistance in the isolate of Enterobacter aerogen.