Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity
Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity
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DOI：
10.1128/aac.01817-18
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发表时间：
2019-01-01
影响因子：
4.9
通讯作者：
Larsson, D. G. Joakim
中科院分区：
文献类型：
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作者：
Kotsakis, Stathis D.;Flach, Carl-Fredrik;Larsson, D. G. Joakim
虽然革兰氏阴性菌对碳青霉烯类抗生素的耐药性主要是由于产生高效的碳青霉烯酶，但谱较窄的β-内酰胺酶与其他机制相结合也可能导致耐药性。OXA-10型D类β-内酰胺酶，以前被证明是弱碳青霉烯酶，可以代表这种情况。在这项研究中，两个新的OXA-10变异体被鉴定为从医院废水中分离的耐美罗培南肠杆菌中唯一的碳青霉烯类水解酶，并通过下一代测序发现表达了额外的V-内酰胺耐药机制。新的变异体OXA-655和OXA-656由两个相关的IncQ1广寄主质粒携带。与OXA-10的序列相比，它们都含有Thr26Met取代，OXA-655在SAV催化基序的第117位也含有亮氨酸而不是缬氨酸。对复制天然blaOXA质粒的实验室菌株和表达OXA-10及其新变异体的重组克隆在等基因背景下的敏感性分析表明，OXA-655是一种更有效的碳青霉烯酶。稳态动力学实验表明，OXA-655的碳青霉烯酶活力源于Val117Leu取代，其中美罗培南的kcat提高了4倍。相反，OXA-655对氧亚氨基-β-内酰胺类抗生素没有活性，而对苯唑西林的催化效率显著降低。此外，Val117Leu变异体对替莫西林和头孢西丁更有效。分子动力学表明，Val117Leu影响117-Leu155位置的相互作用，导致活性中心的结构移动，可能改变碳青霉烯的排列。OXA-10酶对碳青霉烯类抗生素水解酶的进化潜力，以及它们通过混杂质粒传播的可能性，表明它们可能会构成未来的临床威胁。
While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, beta-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D beta-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional V-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural blaOXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the kcat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-beta-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.