Ruzin Alexey, Visalli Melissa A, Keeney David, Bradford Patricia A
Wyeth Research, Department of Infectious Disease, 401 North Middletown Rd., Bldg. 200, Rm. 3219, Pearl River, NY 10965, USA.
Antimicrob Agents Chemother. 2005 Mar;49(3):1017-22. doi: 10.1128/AAC.49.3.1017-1022.2005.
Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 microg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903phikan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 microg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 x 10(-8). These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.
替加环素是一种广谱抗菌剂，对包括肺炎克雷伯菌在内的许多临床相关细菌病原体具有活性。大多数肺炎克雷伯菌分离株对替加环素完全敏感；然而，已分离出一些敏感性降低的菌株。选择了一株分离株G340（其替加环素MIC为4μg/ml，表现出多重耐药[MDR]表型），通过使用IS903phikan转座子诱变分析这种敏感性降低的机制。获得了G340的替加环素敏感突变体GC7535（替加环素MIC为0.25μg/ml）。转座子插入分析将其定位到ramA基因，该基因先前被确定与肺炎克雷伯菌的多重耐药有关。对于GC7535，ramA的破坏导致替加环素MIC降低16倍，同时也抑制了多重耐药。用质粒携带的ramA进行反式互补恢复了对替加环素敏感性降低的原始亲本表型。Northern印迹分析显示，与替加环素敏感菌株相比，G340中ramA的组成型过表达与AcrAB转运蛋白的表达增加相关。肺炎克雷伯菌对替加环素敏感性降低的实验室突变体可以以约4×10⁻⁸的频率被选择。这些结果表明，ramA由于其在AcrAB多药外排泵表达中的作用，与肺炎克雷伯菌对替加环素敏感性降低有关。
