Membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, including multidrug resistance (MDR), cellular signaling, and cell-cell communication. Pseudomonas aeruginosa is renowned for its intriguing membrane transport mechanisms, such as the interplay of membrane permeability and extrusion machinery, leading to selective accumulation of specific intracellular substances and MDR. Despite extensive studies, the mechanisms of membrane transport in living microbial cells remain incompletely understood. In this study, we directly measure real-time change of membrane permeability and pore sizes of P. aeruginosa at the nanometer scale using the intrinsic color index (surface plasmon resonance spectra) of silver (Ag) nanoparticles as the nanometer size index probes. The results show that Ag nanoparticles with sizes ranging up to 80 nm are accumulated in living microbial cells, demonstrating that these Ag nanoparticles transport through the inner and outer membrane of the cells. In addition, a greater number of larger intracellular Ag nanoparticles are observed in the cells as chloramphenicol concentration increases, suggesting that chloramphenicol increases membrane permeability and porosity. Furthermore, studies of mutants (nalB-1 and DeltaABM) show that the accumulation rate of intracellular Ag nanoparticles depends on the expression level of the extrusion pump (MexAB-OprM), suggesting that the extrusion pump plays an important role in controlling the accumulation of Ag nanoparticles in living cells. Moreover, the accumulation kinetics measured by Ag nanoparticles are similar to those measured using a small fluorescent molecule (EtBr), eliminating the possibility of steric and size effects of Ag nanoparticle probes. Susceptibility measurements also suggest that a low concentration of Ag nanoparticles (1.3 pM) does not create significant toxicity for the cells, further validating that single Ag nanoparticles (1.3 pM) can be used as biocompatible nanoprobes for the study of membrane transport kinetics in living microbial cells.

Multidrug resistance (MDR) has been reported in both prokaryotes and eukaryotes, underscoring the challenge of design and screening of more efficacious new drugs. For instance, the efflux pump of Pseudomonas aeruginosa (gram-negative bacteria) can extrude a variety of structurally and functionally diverse substrates, which leads to MDR. In this study, we present a new platform that studies modes of action of antibiotics in living bacterial cells (P. aeruginosa), in real-time, at nanometer scale and single-cell resolution using nanoparticle optics and single living cell imaging. The color index of silver (Ag) nanoparticles (violet, blue, green, and red) is used as the sized index (30 +/- 10, 50 +/- 10, 70 +/- 10, and 90 +/- 10 nm) for real-time measurement of sized transformation of the cell wall and membrane permeability at the nanometer scale. We have demonstrated that the number of Ag nanoparticles accumulated in cells increases as the aztreonam (AZT) concentration increases and as incubation time increases, showing that AZT induces the sized transformation of membrane permeability and the disruption of the cell wall. The results demonstrate that nanoparticle optics assay can be used as a new powerful tool for real-time characterization of modes of action of antimicrobial agents in living cells at the nanometer scale. Furthermore, studies of mutants of WT bacteria (nalB-1 and DeltaABM), suggest that an efflux pump (MexA-MexB-OprM) effectively extrudes substrates (nanoparticles) out of the cells, indicating that the MDR mechanism involves the induction of changes in membrane permeability and the intrinsic pump machinery.

We have developed and applied single live cell imaging for real-time monitoring of resistance kinetics of Pseudomonas aeruginosa. Real-time images of live cells in the presence of a particular substrate (EtBr) provided the first direct insights of resistance mechanism with both spatial and temporal information and showed that the substrate appeared to be accumulated in cytoplasmic space, but not periplasmic space. Three mutants of P. aeruginosa, PAO4290 (a wild-type expression level of MexAB-OprM), TNP030#1 (nalB-1, MexAB-OprM over expression mutant), and TNP076 (DeltaABM, MexAB-OprM deficient mutant), were used to investigate the roles of these three membrane proteins (MexAB-OprM) in the resistance mechanism. Ethidium bromide (EtBr) was chosen as a fluorescence probe for spectroscopic measurement of bulk cell solution and single cell imaging of bulk cells. Bulk measurement indicated, among three mutants, that nalB-1 accumulated the least EtBr and showed the highest resistance to EtBr, whereas DeltaABM accumulated the most EtBr and showed the lowest resistance to EtBr. This result demonstrated the MexAB-OprM proteins played the roles in resistance mechanism by extruding EtBr out of cells. Unlike the bulk measurement, imaging and analysis of bulk cells at single cell resolution demonstrated individual cell had its distinguished resistance kinetics and offered the direct observation of the regulation of influx and efflux of EtBr with both spatial and temporal resolution. Unlike fluorescent staining assays, live cell imaging provided the real-time kinetic information of transformation of membrane permeability and efflux pump machinery of three mutants. This research constitutes the first direct imaging of resistance mechanism of live bacterial cells at single cell resolution and opens up the new possibility of advancing the understanding of bacteria resistance mechanism.

It is well known that surface plasmon absorption (color) of Au nanoparticles highly depends on its size, shape, and surrounding surface environment, providing a great opportunity for the development of Au nanoparticles as a probe for chemical and biochemical sensing and as a building block for nano-optical devices. Currently, it remains incompletely understood how the surface properties of Au nanoparticles ultimately define its optical properties, hindering the progress of rational design of Au nanoparticles for biochemical sensing and assembly of nano-optical devices. Ru(bpy)3 2+ possesses rich photochemical and electrochemical properties, offering a unique probe to study the surface environment of Au nanoparticles. In this study, we investigate the kinetics of surface plasmon absorption of Au nanoparticles (6.5, 19, 48, 97 nm) in the presence of Ru(bpy)3 2+ at 24.0, 33.0, and 51.0 °C. We find that the color-change rate of Au nanoparticles is highly sensitive to the amount of Ru(bpy)3 2+. In addition, the minimum (critical) concentration of Ru(bpy)3 2+ needed to change the color of Au nanoparticles is highly dependent upon temperature and nanoparticle size. TEM images illustrate that the color-change mechanism of Au nanoparticles induced by Ru(bpy)3 2+ highly depends on the size of nanoparticles. Control experiments using NaCl and MgCl2 to replace Ru(bpy)3 2+ show no color change, suggesting that the color change of Au nanoparticles induced by Ru(bpy)3 2+ is not solely attributable to the salt effect. Other factors, such as adsorption of Ru(bpy)3 2+ on the Au nanoparticle surface, may be involved. Moreover, in the presence of the critical concentration of Ru(bpy)3 2+, as temperature increases from 24.0 °C to 51.0 °C, the color-change rate of 19 nm Au nanoparticles increases 8-fold, whereas the color-change rate of 48 nm Au nanoparticles decreases 3-fold, showing the temperature dependence of surface plasmon absorption of Au nanoparticles in the presence of surface adsorbates, Ru(bpy)3 2+.