[摘要] 目的 探讨鲍曼不动杆菌对氨基糖苷类抗菌药物的双圈耐药现象。 方法 用琼脂稀释法检测庆大霉素、阿米卡星、妥布霉素、依替米星等4种氨基糖苷类药物对56株鲍曼不动杆菌的最低抑菌浓度（MIC）。用阿米卡星诱导试验检测介导该表型的基因是否存在诱导现象。用PCR法检测该类菌是否携带整合子。 结果 庆大霉素、依替米星对56株鲍曼不动杆菌的MIC均＞1024 mg/L；71.4%（40/56）的鲍曼不动杆菌妥布霉素的MIC≥1024 mg/L；阿米卡星的MIC呈抑制后再生长现象，大多在512 mg/L出现抑制，而到4096mg/L又开始生长。 用K-B法进行诱导试验，传至15代双圈耐药现象转为完全耐药。56株菌中53株检测到Ⅰ类整合子。 结论 MIC试验证实了K-B法中的双圈现象，介导这一现象的基因呈诱导型表达，这类菌大多携带Ⅰ类整合子。
[关键词] 鲍曼不动杆菌；氨基糖苷类；整合子
An initial exploration of Acinetobacter baumannii double-circle resistance to aminoglycoside antibiotic
AHAO Hai-feng，DENG Jinjin?WANG Wei-ping，Gao Fei, WANG Jiejie，SHI Lin-ing， FAN Ming.
Department of Central Laboratory Medicine, Institute of Clinical Laboratory Medicine, Nanjing General Hospital of Nanjing Command of?PLA, Nanjing 210002, China
[Abstract] Objective To explore the phenomenon of Acinetobacter baumannii double-circle resistance to aminoglycoside antibiotic. Methods The minimal inhibitory concentrations (MICs) of gentamicin,amikacin,tobramycin and etimicin against 56 strains of acinetobacter baumannii were examined by agar two-fold dilution. The amikacin induction experiment was used to test whether the gene which mediates this phenotype had induction phenomenon or not. The PCR was performed to determine if this baumannii carried integrons. Results Gentamicin and etimicin in 56 strains of resistant Acinetobacter baumannii were all more than 1024 ug/ml;tobramycin in 40 strains of resistant Acinetobacter baumannii were all more than 1024 ug/ml;the MICs of amikacin exhibited regrowth after inhibition, inhibition mostly at 512 mg/L and regrowth at 4096 mg/L. When the induction was made with the K-B method, the double-circle resistance was change to complete resistance at the fifteen generation. Class 1 integrons were found in the 53 strains. Conclusions The MICs confirm the double-circle
resistance phenomenon in K-B test; the gene mediating this phenomenon can be induced expression; the most of these baumannii carry class 1 integrons.
[Keyords]: Acinetobacter baumannii; aminoglycoside antibiotic; integrons?
鲍曼不动杆菌(Acinetobacter baumannii)是仅次于铜绿假单胞菌的引起医院内感染（如肺炎、败血症、尿路感染、伤口感染及脑膜炎等）的重要病原菌之一。由于广谱抗菌药物的广泛应用和不合理使用，近年来鲍曼不动杆菌的耐药现象越来越严重，对包括?-内酰胺类、氨基糖苷类、喹诺酮类等临床使用的各类抗菌药物均出现耐药。我们对本院分离的鲍曼不动杆菌采用K-B法进行氨基糖苷类抗菌药物的药敏试验中发现阿米卡星纸片周围出现了双圈耐药现象，即在抑菌圈与纸片之间出现了一个生长圈。本文就这一现象展开研究，探讨导致这一现象的机理。
１ 材料与方法
1.1 菌株来源?2005年1月到2006年11月本室从各类临床细菌学检验标本中分离的出现对阿米卡星双圈耐药的56株鲍曼不动杆菌，收集后用10%无菌脱脂奶粉液-70冰箱保存。
1.2 药物敏感试验?应用K-B法和琼脂稀释法进行阿米卡星,庆大霉素,妥布霉素和依替米星等4种药物的药敏试验[1、2]，稀释法的药物浓度从2048 mg/L倍比稀释至0.5mg/L,阿米卡星增加了3000、4000、5000、6000、7000、8000、9000 mg/L等7个高浓度。药敏纸片、MH琼脂及MH肉汤均为Oxiod公司产品。用大肠埃希菌ATCC 25922、铜绿假单胞菌ATCC 27853作为质控菌株，判断标准按CLISA-2006执行。
1.3 诱导试验?取1株对阿米卡星及高浓度庆大霉素呈双圈耐药现象的鲍曼不动杆菌,按K-B法操作，取抑菌圈内生长圈上的菌落调0.5麦氏浊度涂布后贴药敏纸片，35℃孵育18h，如此重复，每重复一次即为一代，直至出现表型改变为止。
1.4 PCR扩增编码Ⅰ类、Ⅱ类整合子酶基因
1.4.1 模板DNA的提取?挑取单个菌落接种于3ml LB肉汤管中，37℃摇床过夜，4000r/min离心10min取沉淀，再4000r/min、10min离心洗涤，留沉淀加灭菌蒸溜水500μL，100℃加热15-20min，10000r/min 离心5min，上清即模板DNA，分装-20℃保存。
