A putative RND-type efflux pump, H239_3064, contributes to colistin resistance through CrrB in Klebsiella pneumoniae.
A putative RND-type efflux pump, H239_3064, contributes to colistin resistance through CrrB in Klebsiella pneumoniae.
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DOI：
10.1093/jac/dky054
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发表时间：
2018-06-01
期刊：
影响因子：
--
通讯作者：
Wang JT
中科院分区：
文献类型：
--
作者：
Cheng YH;Lin TL;Lin YT;Wang JT
粘菌素是用于治疗耐碳青霉烯肺炎克雷伯菌感染的最后手段抗生素之一。我们之前的研究表明，编码氨基酸取代的CrrB临床菌株比编码突变体MgrB、PmrB或PhoQ的粘菌素耐药菌株具有更高的粘菌素耐药性（mic≥512 mg/L）。除了用4-氨基-4-脱氧-l-阿拉伯糖和磷酸乙醇胺修饰LPS外，CrrAB可能调节另一个未知的机制，导致粘菌素耐药性。为了确定这些潜在的未知机制，我们构建了A4528 crrB（N141I）的转座子突变文库。通过缺失和互补实验证实了可能参与粘菌素耐药并受crrB调控的位点。在2976个转座子突变体的筛选中，有47个突变体的黏菌素mic较亲本显著降低。除了crrAB、crrC和pmrHFIJKLM操作子外，这47个转座子插入突变体还包括另外13个基因座。值得注意的是，与A4528亲本菌株相比，A4528 crrB（N141I）中其中一个插入靶点H239_3064（编码一个假定的rnd型外排泵）的转录物水平显著增加。在A4528 crrB（N141I）背景中缺失H239_3064导致粘菌素MIC降低8倍；缺失突变体与H239_3064的互补恢复了对粘菌素的抗性。同时检测了a4528衍生菌株对其他抗生素的敏感性。crrB突变导致对四环素和替加环素的敏感性降低，A4528 crrB（N141I）中H239_3064的缺失减弱了这一现象。本研究表明，肺炎克雷伯菌crrB错义突变导致H239_3064表达增加，进而导致对粘菌素、四环素和替加环素的敏感性降低。
Colistin is one of the last-resort antibiotics used to treat carbapenem-resistant Klebsiella pneumoniae infection. Our previous studies indicated that clinical strains encoding CrrB with amino acid substitutions exhibited higher colistin resistance (MICs ≥512 mg/L) than did colistin-resistant strains encoding mutant MgrB, PmrB or PhoQ. CrrAB may regulate another unknown mechanism(s) contributing to colistin resistance, besides modifications of LPS with 4-amino-4-deoxy-l-arabinose and phosphoethanolamine. To identify these potential unknown mechanism(s), a transposon mutant library of A4528 crrB(N141I) was constructed. Loci that might contribute to colistin resistance and were regulated by crrB were confirmed by deletion and complementation experiments. Screening of 2976 transposon mutants identified 47 mutants in which the MICs of colistin were significantly decreased compared with that for the parent. Besides crrAB, crrC and pmrHFIJKLM operons, these 47 transposon insertion mutants included another 13 loci. Notably, transcript levels of one of these insertion targets, H239_3064 (encoding a putative RND-type efflux pump), were significantly increased in A4528 crrB(N141I) compared with the A4528 parent strain. Deletion of H239_3064 in the A4528 crrB(N141I) background resulted in an 8-fold decrease in the MIC of colistin; complementation of the deletion mutant with H239_3064 restored resistance to colistin. Susceptibilities of A4528-derived strains to other antibiotics were also tested. Mutations of crrB resulted in decreased susceptibility to tetracycline and tigecycline, and deletion of H239_3064 in A4528 crrB(N141I) attenuated this phenomenon. This study demonstrated that missense mutations of K. pneumoniae crrB lead to increased expression of H239_3064, leading in turn to decreased susceptibility to colistin, tetracycline and tigecycline.
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