Tigecycline resistance in clinical isolates of Enterococcus faecium is mediated by an upregulation of plasmid-encoded tetracycline determinants tet(L) and tet(M)
Tigecycline resistance in clinical isolates of Enterococcus faecium is mediated by an upregulation of plasmid-encoded tetracycline determinants tet(L) and tet(M)
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DOI：
10.1093/jac/dkv420
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发表时间：
2016-04-01
影响因子：
5.2
通讯作者：
Werner, G.
中科院分区：
文献类型：
--
作者：
Fiedler, S.;Bender, J. K.;Werner, G.
替加环素是对抗多重耐药细菌病原体（包括 VRE 和 MRSA）的最后一线疗法之一。德国国家葡萄球菌和肠球菌参考中心近年来收到了 73 株耐替加环素的粪肠球菌和粪肠球菌分离株。肠球菌如何对替加环素产生耐药性的确切机制仍不清楚。本研究记录了肠球菌临床分离株中外排泵在替加环素耐药性中的作用的分析。对于所分析的不同分离株，发现了各种替加环素 MIC。通过全基因组测序和 RT-qPCR 分析替加环素耐药菌株的基因组和转录组差异。在单核细胞增多性李斯特菌中克隆并表达感兴趣的基因，以验证其功能。对三种同基因菌株进行详细的全基因组比较分析，显示出不同水平的替加环素耐药性，揭示了主要促进子超家族（MFS）外排泵TetL和核糖体保护蛋白TetM可能是耐药蛋白。随后的 RT-qPCR 证实了各个基因的上调。从进一步的 qPCR 分析推断出基因拷贝数和 MIC 水平的相关性。单增李斯特菌中 tet(L) 和 tet(M) 的表达明确证明了获得任一基因座后增加替加环素 MIC 的潜力。我们的结果表明，两个四环素抗性决定簇（tet(L) 编码的 MFS 泵和 tet(M) 编码的核糖体保护蛋白）的表达增加能够赋予替加环素抗性 在肠球菌临床分离株中。
Tigecycline represents one of the last-line therapeutics to combat multidrug-resistant bacterial pathogens, including VRE and MRSA. The German National Reference Centre for Staphylococci and Enterococci has received 73 tigecycline-resistant Enterococcus faecium and Enterococcus faecalis isolates in recent years. The precise mechanism of how enterococci become resistant to tigecycline remains undetermined. This study documents an analysis of the role of efflux pumps in tigecycline resistance in clinical isolates of Enterococcus spp.Various tigecycline MICs were found for the different isolates analysed. Tigecycline-resistant strains were analysed with respect to genome and transcriptome differences by means of WGS and RT-qPCR. Genes of interest were cloned and expressed in Listeria monocytogenes for verification of their functionality.Detailed comparative whole-genome analyses of three isogenic strains, showing different levels of tigecycline resistance, revealed the major facilitator superfamily (MFS) efflux pump TetL and the ribosomal protection protein TetM as possible drug resistance proteins. Subsequent RT-qPCR confirmed up-regulation of the respective genes. A correlation of gene copy number and level of MIC was inferred from further qPCR analyses. Expression of both tet(L) and tet(M) in L. monocytogenes unequivocally demonstrated the potential to increase tigecycline MICs upon acquisition of either locus.Our results indicate that increased expression of two tetracycline resistance determinants, a tet(L)-encoded MFS pump and a tet(M)-encoded ribosomal protection protein, is capable of conferring tigecycline resistance in enterococcal clinical isolates.