目的 研究TEM-1型β-内酰胺酶及CarO蛋白介导的鲍曼不动杆菌临床株对舒巴坦的耐药机制.方法 将24株非重复鲍曼不动杆菌临床株通过琼脂稀释法分为舒巴坦非敏感组(18株)和敏感组(6株),用纸片扩散法(K-B)行药敏试验,PCR扩增blaTEM-1和carO基因,选取4株blaTEM-1阳性菌株(A3、A5、1327、C1),对其扩增产物测序,BLAST软件分析;应用实时荧光定量RT-PCR技术检测2组菌株blaTEM-1和carO基因mRNA转录情况.结果 敏感组临床株对美罗培南、亚胺培南、头孢哌酮、环丙沙星、庆大霉素、氨苄西林耐药率分别为1/6、2/6、3/6、1/6、5/6、5/6;非敏感组分别为6/18、10/18、18/18、18/18、18/18.敏感组未检出blaTEM-1,非敏感组中16株临床株扩增出blaTEM-1;24株临床菌株全部扩增出carO基因.测序显示4株临床株blaTEM-1基因均未出现有义突变;A3、A5、1327启动序列为P4,C1为P3.BlaTEM-1基因阳性菌株mRNA相对表达量与其对舒巴坦MIC值呈正相关(rs=0.551,P=0.027);carO基因mRNA相对表达在敏感组和非敏感组中无差异.结论 临床分离鲍曼不动杆菌受试菌株耐药严重,其对舒巴坦的耐药机制与TEM-1型β-内酰胺酶高表达有关,而blaTEM-1基因启动子调控可能是TEM-1型β-内酰胺酶高表达的原因之一.%Objective To investigate the mechanism of drug resistance of sulbactam mediated by TEM-1 beta-lactamases and porin CarO in clinical strains of acinetobacter baumannii. Methods Twenty-four unrepeated clinical acinetobacter baumannii strains were divided into sensitive strain group (n=6) and insensitive strain group (n=18) by susceptibility testing to sulbactam.Antibiotics susceptibility test was carried out using the Kirby-Bauer method.BlaTEM-1and carO genes were amplified by PCR.Four blaTEM-1positive strains(A3,A5,1327 and C1)were selected,and their amplified products were sequenced.The quantitative real-time RT-PCR was used to analyze mRNA transcriptional levels of blaTEM-1 and carO genes. Results Resistant rates of the sensitive strain group for meropenem, imipenem, cefoperazone, ciprofloxacin,gentamicin and ampicillin were 1/6,2/6,3/6,1/6,5/6 and 5/6,and resistant rates of the insensitive strain group were 6/18,10/18,18/18,18/18 and 18/18.BlaTEM-1genes were amplified in 16 insensitive strains,and blaTEM-1was negative in sensitive strains. The carO genes were amplified in all 24 strains.There was no significative mutation in the 4 strains of blaTEM-1genes. The promoters of the strains A3, A5 and 1327 were P4, and C1 was P3. There was a positive correlation between the mRNA expression of blaTEM-1and the MIC value of sulbactam (rs=0.551, P=0.027). There was no difference in the mRNA expression of carO between the two groups.Conclusion The clinical strains are seriously resistant to antibiotics.The main resistance mechanism of clinical strains to sulbactam is the high mRNA expression of blaTEM-1,and the promoter may be one of the reasons of high expression of TEM-1.
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