鲍曼不动杆菌临床分离株表型、基因型和耐药基因的对比研究

目的 对比研究鲍曼不动杆菌临床分离株基因型和编码耐药基因的差异,并分析其与临床多重耐药性的关系.方法 随机收集中南大学湘雅二医院2008年9月至2009年9月分离的77株鲍曼不动杆菌,采用WHO推荐的K-B法对鲍曼不动杆菌进行临床常见15种抗生素药物敏感试验,并对药敏谱进行分析.用随机扩增多态性DNA法(RAPD)技术进行基因分型.并利用PCR对β-内酰胺酶基因TEM-1、IMP、OXA-23、OXA-24、AmpC和氨基糖苷类修饰酶基因aac(3)-Ⅰ、aac(6')-Ⅰ、ant(3")-Ⅰ和16S rRNA甲基化基因armA、rmtA、rmtB进行扩增及序列分析.对比分析鲍曼不动杆菌耐药基因的携带情况,以及与基因型和耐药性的关系.结果 77株鲍曼不动杆菌中敏感菌株有31株,对5种或5种以上抗生素耐药的多重耐药菌株46株,内含全耐药菌株10株.RAPD技术将其分为17型,为A-G型,多重耐药株中E型为优势克隆株(17株),在重症监护病房(ICU)中流行最广,占47.1%(8/17).敏感株中各型散在分布.PCR扩增结果显示,多重耐药株和敏感株携带TEM-1、IMP、OXA-23、OXA-24、AmpC、aac(3)-Ⅰ、aac(6')-Ⅰ、ant(3")-Ⅰ和armA耐药基因的比率分别为95.7%、39.1%、84.8%、54.3%、87.0%、89.1%、84.8%、45.7%、63.0%和58.1%、9.7%、32.3%、48.4%、48.4%、29.0%、45.2%、12.9%、9.7%,未发现rmtA和rmtB基因阳性菌株.经x2检验,除OXA-24外,其余各耐药基因携带率比较差异有统计学意义(P＜0.05).药敏分析提示携带以上耐药基因的鲍曼不动杆菌菌株的耐药率明显高于未携带该基因的菌株,其中对阿米卡星和庆大霉素耐药的菌株,其氨基糖苷类酶基因均阳性(34.8%),含所测的所有β-内酰胺酶基因的菌株均为全耐药株.结论 与临床分离的敏感鲍曼不动杆菌相比,多重耐药株耐药谱广,耐药率高,其携带β-内酰胺酶基因和氨基糖苷类酶基因种类多,分离率高,且同一克隆的多重耐药株可在病室内和病室间传播.

Comparison of phenotype,genotype and drug resistance genes of Acinetobacter baumannii isolated from clinic

Objective To investigate the genotypes and encoding resistance genes differences of Acinetobacter baumannii and analyze their interrelations with multi-drug resistance.Methods A total of 77strains Acinetobacter baumannii were collected random from the second Xiangya Hospital during September 2008 to September 2009.The K-B method which was WHO recommended was adopted to Acinetobacter baumannii drug sensitivity test to 15 kinds of antibiotics to establish susceptibility spectrum.At the same time,random amplified polymorphic DNA(RAPD)technique was used to establish DNA fingerprinting.The genes of β-lactamase(TEM-1,IMP,OXA-23,OXA-24,AmpC),aminoglycoside-modifying enzymesaac(3')-Ⅰ ,aac(6')-Ⅰ ,ant(3")-Ⅰ]and 16S rRNA methylase(armA,rmtA,rmtB)were detected by PCR and sequenced,and find the relationship between the gene encoding and multi-drug resistance.In addition,we compared the rates of resistance genes of Acinetobacter baumannii and the relations with the genotype and the multi-resistance.Results Thirty-one sensitive strains and 46 multi-drug resistance strains(10 Pan-drug resistances)were isolated.Seventeen types from A to Q were separated using RAPD technique.E genotype widely popular in the ICU was the advantage type in multi-drug resistance strains,and the rate was 47.1%.While the various types scattered in sensitive strains.The positive rates of TEM-1,IMP,OXA-23,OXA-24,AmpC,aac(3')-Ⅰ ,aac(6')-Ⅰ ,ant(3")-Ⅰ ,armA in the multi-drug resistance strains and the sensitive strains were 95.7%,39.1%,84.8% ,54.3%,87.0%,89.1%,84.8%,45.7%,63.0% and 58.1%,9.7%,32.3%,48.4%,48.4%,29.0%,45.2%,12.9%,9.7%,respectively,and there was significant difference except for OXA-24 using the X2 test(P ＜ 0.05).All isolates were negative for rmtA gene and rmtB gene.Drug susceptibility analysis showed that the resistant rate was significantly higher of the strains carrying resistant genes than that of the resistance negative strains.When the strains were resistant to gentamicin and amikacin,the rate of three aminoglycoside genes positive was 34.8%.The trains containing all the measured β-lactamase genes were all resistant strains.Conclusion Compared with the sensitive Acinetobacter baumannii strains,a broad resistance spectrum and a high drug resistance rate were showed in multidrug resistance strains isolated from clinic,which harboring many kinds of β-lactamase genes and aminoglycosides genes with a high separation rate,and the same clone of multiple drug-resistant strains may be transmitted in and among wards.