Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex
Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex
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DOI：
10.1128/aac.02840-15
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发表时间：
2016-04-01
影响因子：
4.9
通讯作者：
Giard, Jean-Christophe
中科院分区：
文献类型：
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作者：
Guerin, Francois;Lallement, Claire;Giard, Jean-Christophe
在革兰氏阴性菌中，主动外排是抗菌素耐药性的重要机制，但人们对阴沟肠杆菌复合体（ECC）知之甚少。它主要由属于 RND（抵抗-结节-细胞分裂）家族的泵介导，并且只有 AcrB（AcrAB-TolC 三方系统的一部分）在 ECC 中得到了表征。然而，阴沟肠杆菌亚种的详细基因组序列分析。泄殖腔 ATCC 13047 向我们揭示了其他 10 个基因可能编码 RND 型转运蛋白。然后，我们通过构建相应的缺失突变体来表征所有这些候选者的作用，并测试了它们对 36 种化合物的抗菌敏感性、它们在无脊椎动物大蜡螟感染模型中的毒力以及它们形成生物膜的能力。与野生型菌株相比，只有 Delta acrB 突变体表现出显着不同的表型：几种抗生素、防腐剂和染料的 MIC 降低 4 至 32 倍，生物膜的产生增加，并减弱大蜡螟的毒力。为了鉴定每个泵的特定底物，我们将含有 RND 泵编码基因的所有操纵子分别反式表达到 Delta acrB 高敏感菌株中。我们表明，其他三种 RND 型外排系统（ECL_00053-00055、ECL_01758-01759 和 ECL_02124-02125）能够部分恢复野生型表型，并超级增加甚至扩大广泛的抗菌药物耐药性。这是第一项评估 ECC 染色体编码的所有 RND 外排泵的作用的全球研究，证实了 AcrB 在致病性和耐药性中的主要作用，以及其他 RND 型成员在获得性耐药中的潜在参与。
In Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about the Enterobacter cloacae complex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strain E. cloacae subsp. cloacae ATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrate Galleria mellonella model of infection, and their ability to form biofilm. Only the Delta acrB mutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence in G. mellonella. In order to identify specific substrates of each pump, we individually expressed in trans all operons containing an RND pump-encoding gene into the Delta acrB hypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance.