Growth Phase Contribution in Dictating Drug Transport and Subcellular Accumulation inside <i>Escherichia coli</i>

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 <i>Escherichia coli</i> 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 <i>rpoS</i> 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.