Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India.
Center of Excellence: Tropical and Infectious Diseases, Gangwal School of Medical Sciences and Technology, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India.
ACS Infect Dis. 2024 Sep 13;10(9):3233-3244. doi: 10.1021/acsinfecdis.4c00252. Epub 2024 Aug 23.
Depending upon nutrient availability, bacteria transit to multiple growth phases. The transition from the active to nongrowing phase results in reduced drug efficacy and, in some cases, even multidrug resistance. However, due to multiple alterations in the cell envelope, probing the drug permeation kinetics during growth phases becomes perplexing, especially across the Gram-negative bacteria's complex dual membrane envelope. To advance the understanding of drug permeation during the life cycle of Gram-negative bacteria, we sought to address two underlying objectives: (a) how changes are occurring inside the bacterial envelope during growth and (b) how the drug permeation and accumulation vary across both the membranes and in subcellular compartments during growth. Both objectives are met with the help of nonlinear optical technique second-harmonic generation spectroscopy (SHG). Specifically, using SHG, we probed the transport kinetics and accumulation of a quaternary ammonium compound (QAC), malachite green, inside in various growth phases. Further insight about another QAC molecule, propidium iodide, is accomplished using fluorescence microscopy. Results indicate that actively growing cells have faster drug transport and higher cytoplasmic accumulation than slow- or nongrowing cells. In this regard, the gene plays a crucial role in limiting drug transport across the saturation phase cultures. Moreover, within a particular growth phase, membrane permeability undergoes gradual changes much before the subsequent growth phase commences. These outcomes signify the importance of reporting the growth phase and rate in drug efficacy studies.
根据营养物质的可用性，细菌会经历多个生长阶段。从活跃生长到非生长阶段的转变会降低药物的疗效，在某些情况下甚至会导致多药耐药性。然而，由于细胞包膜的多种改变，在生长阶段探测药物渗透动力学变得复杂，特别是在革兰氏阴性细菌复杂的双层膜包膜中。为了深入了解革兰氏阴性细菌生命周期中的药物渗透，我们试图解决两个基本目标：（a）在生长过程中细菌包膜内部发生了哪些变化；（b）在生长过程中，药物渗透和积累如何在两个膜和亚细胞隔室中变化。这两个目标都可以通过非线性光学技术二次谐波产生光谱（SHG）来实现。具体来说，我们使用 SHG 探测了季铵化合物（QAC）孔雀石绿在各种生长阶段在细菌包膜内的运输动力学和积累情况。使用荧光显微镜可以进一步了解另一种 QAC 分子碘化丙啶。结果表明，与缓慢生长或非生长细胞相比，活跃生长的细胞具有更快的药物运输和更高的细胞质积累。在这方面，基因在限制药物在饱和相培养物中的运输方面起着至关重要的作用。此外，在特定的生长阶段，膜通透性在随后的生长阶段开始之前就会发生逐渐变化。这些结果表明，在药物疗效研究中报告生长阶段和生长速度非常重要。
