鲍曼不动杆菌多黏菌素E耐药机制研究

Mechanism study of polymyxin E resistance in Acinetobacter baumannii

为探讨鲍曼不动杆菌新的多黏菌素E耐药机制,采用体外诱导法将临床菌株和参考菌株诱导成多黏菌素E耐药株。利用生长曲线、生物膜形成、体外竞争和抗血清试验检测多黏菌素E耐药前后菌株生物学性状变化。PCR和qRT-PCR试验检测多黏菌素E耐药后相关基因序列及表达量的变化。结果表明:多黏菌素E耐药前后菌株生长曲线未发现显著差异。AB13-R耐药株生物膜形成能力显著增强,另外2菌株生物膜形成能力在耐药前后无显著差异。鲍曼不动杆菌获得多黏菌素E耐药后均表现出体外竞争缺陷。AB156-R与其敏感菌相比抗血清增强,而ATCC19606-R和AB13-R与其敏感菌相比抗血清降低。AB13-R中pmrB基因谷氨酸缺失,脂多糖(LPS)合成及脂质A合成基因下调,药物外排泵基因上调,达到LPS合成减少并降低菌体内药物浓度,使AB13-R对多黏菌素E耐药。ATCC19606-R及AB156-R中均无基因突变,可能由其他机制介导菌株对多黏菌素E耐药。综上,鲍曼不动杆菌获得多黏菌素E耐药后会出现适应性改变,pmrB基因中谷氨酸的缺失可能是新的多黏菌素E耐药机制。

To investigate the polymyxin E resistant mechanisms of Acinetobacter baumannii, strains were induced to polymyxin E resistance in vitro. Growth curve, biofilm formation, competition and antiserum test were used to detect the changes of biological characters of strains before and after polymyxin E resistance. PCR and qRT-PCR were used to detect the changes of gene sequences and expression. Results showed that there was no significant difference in strain growth before and after polymyxin E resistance. Biofilm formation of AB13-R was significantly enhanced, while the other two strains had no significant difference before and after polymyxin E resistance. Competitive defects were shown after A.baumannii acquired polymyxin E resistance. Antiserum of AB156-R was increased compared with that of sensitive bacteria, while ATCC19606-R and AB13-R were decreased. Deletion of glutamate in pmrB gene was detected in AB13-R. Down-regulated lipopolysaccharide and lipid A synthesis genes and up-regulated drug efflux pump genes leading to reduced LPS synthesis and drug concentration, thus making AB13-R resistant to polymyxin E. There was no gene mutation in ATCC19606-R and AB156-R. This study suggests that there are changes in fitness after A.baumannii acquired polymyxin E resistance, and deletion of glutamate in pmrB gene may be a new mechanism of polymyxin E resistance.