Department of Biomedical and Forensic Science, Anglia Ruskin University, Cambridge, UK.
School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK.
J Antimicrob Chemother. 2021 Feb 11;76(3):587-595. doi: 10.1093/jac/dkaa502.
To measure the variability in carbapenem susceptibility conferred by different OxaAb variants, characterize the molecular evolution of oxaAb and elucidate the contribution of OxaAb and other possible carbapenem resistance factors in the clinical isolates using WGS and LC-MS/MS.
Antimicrobial susceptibility tests were performed on 10 clinical Acinetobacter baumannii isolates. Carbapenem MICs were evaluated for all oxaAb variants cloned into A. baumannii CIP70.10 and BM4547, with and without their natural promoters. Molecular evolution analysis of the oxaAb variants was performed using FastTree and SplitsTree4. Resistance determinants were studied in the clinical isolates using WGS and LC-MS/MS.
Only the OxaAb variants with I129L and L167V substitutions, OxaAb(82), OxaAb(83), OxaAb(107) and OxaAb(110) increased carbapenem MICs when expressed in susceptible A. baumannii backgrounds without an upstream IS element. Carbapenem resistance was conferred with the addition of their natural upstream ISAba1 promoter. LC-MS/MS analysis on the original clinical isolates confirmed overexpression of the four I129L and L167V variants. No other differences in expression levels of proteins commonly associated with carbapenem resistance were detected.
Elevated carbapenem MICs were observed by expression of OxaAb variants carrying clinically prevalent substitutions I129L and L167V. To drive carbapenem resistance, these variants required overexpression by their upstream ISAba1 promoter. This study clearly demonstrates that a combination of IS-driven overexpression of oxaAb and the presence of particular amino acid substitutions in the active site to improve carbapenem capture is key in conferring carbapenem resistance in A. baumannii and other mechanisms are not required.
测量不同 OxaAb 变体赋予碳青霉烯类药物敏感性的可变性，对 oxaAb 的分子进化进行特征描述，并使用 WGS 和 LC-MS/MS 阐明临床分离株中 OxaAb 和其他可能的碳青霉烯类药物耐药因素的贡献。
对 10 株临床鲍曼不动杆菌分离株进行了抗菌药物敏感性试验。评估了所有克隆到 A.baumannii CIP70.10 和 BM4547 中的 oxaAb 变体的碳青霉烯类药物 MIC，同时评估了有无其天然启动子。使用 FastTree 和 SplitsTree4 对 oxaAb 变体的分子进化进行分析。使用 WGS 和 LC-MS/MS 研究临床分离株中的耐药决定因素。
只有具有 I129L 和 L167V 取代的 OxaAb 变体，即 OxaAb(82)、OxaAb(83)、OxaAb(107)和 OxaAb(110)，在没有上游 IS 元件的敏感 A.baumannii 背景下表达时，才会增加碳青霉烯类药物的 MIC。在添加其天然上游 ISAba1 启动子时，会赋予碳青霉烯类药物耐药性。对原始临床分离株的 LC-MS/MS 分析证实了四种 I129L 和 L167V 变体的过度表达。未检测到与碳青霉烯类药物耐药性相关的蛋白质表达水平的其他差异。
通过表达携带临床常见取代 I129L 和 L167V 的 OxaAb 变体，观察到碳青霉烯类药物 MIC 升高。为了驱动碳青霉烯类药物耐药性，这些变体需要其上游 ISAba1 启动子的过度表达。本研究清楚地表明，oxaAb 的 IS 驱动过度表达与活性位点中特定氨基酸取代以提高碳青霉烯类药物捕获相结合是导致鲍曼不动杆菌和其他机制产生碳青霉烯类药物耐药性的关键。
