Supplementary Material for: Biofilm Formation Restrained by Subinhibitory Concentrations of Tigecyclin in Acinetobacter baumannii Is Associated with Downregulation of Efflux Pumps

Background: Tigecycline, one of the few therapeutic options against multidrug-resistant Acinetobacter baumannii, reaches subinhibitory serum concentrations only with cautious clinical dosing and pharmacokinetics. Subinhibitory concentrations of tigecycline might induce an A. baumannii biofilm. Methods: Biofilm formation was assessed via the crystal violet staining method. We further analyzed the main biofilm components with NaIO4, proteinase K, and DNase. Real-time RT-PCR was applied for quantitative detection of biofilm potential-associated genes. Results: In this study, A. baumannii proved to be a strong biofilm producer, and we found that proteins and extracellular DNA are crucial components of the A. baumannii biofilm. Quantitative real-time RT-PCR revealed positive correlations between biofilm formation restrained by subinhibitory concentrations of tigecycline and the expression of biofilm potential-associated genes, especially the AdeFGH efflux pump gene. Conclusion: Our results suggest that downregulation of efflux pumps, especially the AdeFGH efflux pump, is probably responsible for the decline in biofilm formation in A. baumannii treated with subinhibitory concentrations of tigecyclin.背景：替加环素（Tigecycline）是对抗多重耐药鲍曼不动杆菌（multidrug-resistant Acinetobacter baumannii）为数不多的治疗选择之一，仅在谨慎制定临床给药方案并结合药代动力学特征的前提下，方能达到亚抑菌血清浓度。亚抑菌浓度的替加环素可能诱导鲍曼不动杆菌形成生物被膜。 方法：采用结晶紫染色法评估生物被膜形成能力。本研究进一步通过高碘酸钠（NaIO4）、蛋白酶K与脱氧核糖核酸酶（DNase）分析生物被膜的主要组成成分。应用实时逆转录聚合酶链反应（Real-time RT-PCR）定量检测生物被膜形成相关潜能基因的表达水平。 结果：本研究证实鲍曼不动杆菌为强生物被膜形成菌株，且发现蛋白质与细胞外DNA是鲍曼不动杆菌生物被膜的关键组成成分。实时定量RT-PCR结果显示，亚抑菌浓度替加环素所抑制的生物被膜形成过程，与生物被膜潜能相关基因的表达水平呈正相关，其中尤以AdeFGH外排泵基因的相关性最为显著。 结论：本研究结果表明，外排泵（尤其是AdeFGH外排泵）的表达下调，可能是亚抑菌浓度替加环素处理后鲍曼不动杆菌生物被膜形成能力下降的核心原因。