Santiago Ariel J, Ahmed Marwa N A, Wang Shu-Lin, Damera Krishna, Wang Binghe, Tai Phang C, Gilbert Eric S, Derby Charles D
Department of Biology, Georgia State University, Atlanta, Georgia, USA
Department of Biology, Georgia State University, Atlanta, Georgia, USA.
Antimicrob Agents Chemother. 2016 Aug 22;60(9):5554-62. doi: 10.1128/AAC.02984-15. Print 2016 Sep.
Escapin is an l-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. We initiated the present study to investigate how the combination of EIP and H2O2 affected bacterial biofilms, using Pseudomonas aeruginosa as a model. Specifically, we examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to biofilm formation in untreated controls or with EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to that of the controls. Area layer analysis of biofilms posttreatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, and these concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to the effect of either EIP or H2O2 alone. Collectively, our results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix.
逃逸素是一种L-氨基酸氧化酶，作用于赖氨酸以产生过氧化氢（H2O2）、氨以及几种有机酸的平衡混合物，这些有机酸统称为逃逸素中间产物（EIP）。先前的研究表明，合成的EIP和H2O2组合作为一种抗菌剂，对多种浮游细菌具有协同作用。我们开展本研究，以铜绿假单胞菌为模型，探究EIP和H2O2组合如何影响细菌生物膜。具体而言，我们检测了抑制生物膜形成或促进已形成生物膜破坏的EIP和H2O2浓度。使用微量滴定板和结晶紫染色对生物膜形成进行的高通量检测表明，EIP和H2O2配对产生了显著效果，相对于未处理对照或单独使用EIP或H2O2时的生物膜形成，抑制了生物膜的形成。同样，流动池分析和共聚焦激光扫描显微镜显示，与对照相比，EIP和H2O2组合降低了已形成生物膜的生物量。生物膜处理后的面积层分析表明，生物量的破坏一直延伸到底层。仅需纳摩尔至微摩尔浓度的EIP和H2O2就能影响生物膜的形成或破坏，而且这些浓度显著低于对浮游细菌产生杀菌作用的浓度。与单独使用EIP或H2O2的效果相比，微摩尔浓度的EIP和H2O2组合增强了铜绿假单胞菌的游动能力。总体而言，我们的结果表明，EIP和H2O2组合可能通过干扰细菌附着和破坏生物膜基质来影响生物膜。
