Nonlethal Molecular Nanomachines Potentiate Antibiotic Activity Against Gram-Negative Bacteria by Increasing Cell Permeability and Attenuating Efflux

Antibiotic resistance is a pressing public health threat. Despite rising resistance, antibiotic development, especially for Gram-negative bacteria, has stagnated. As the traditional antibiotic research and development pipeline struggles to address this growing concern, alternative solutions become imperative. Synthetic molecular nanomachines (MNMs) are molecular structures that rotate unidirectionally in a controlled manner in response to a stimulus, such as light, resulting in a mechanical action that can propel molecules to drill into cell membranes, causing rapid cell death. Due to their broad destructive capabilities, clinical translation of MNMs remains challenging. Hence, here, we explore the ability of nonlethal visible-light-activated MNMs to potentiate conventional antibiotics against Gram-negative bacteria. Nonlethal MNMs enhanced the antibacterial activity of various classes of conventional antibiotics against Gram-negative bacteria, including those typically effective only against Gram-positive strains, reducing the antibiotic concentration required for bactericidal action. Our study also revealed that MNMs bind to the negatively charged phospholipids of the bacterial inner membrane, leading to permeabilization of the cell envelope and impairment of efflux pump activity following light activation of MNMs. The combined effects of MNMs on membrane permeability and efflux pumps resulted in increased antibiotic accumulation inside the cell, reversing antibiotic resistance and attenuating its development. These results identify nonlethal MNMs as pleiotropic antibiotic enhancers or adjuvants. The combination of MNMs with traditional antibiotics is a promising strategy against multidrug-resistant Gram-negative infections. This approach can reduce the amount of antibiotics needed and slow down antibiotic resistance development, thereby preserving the effectiveness of our current antibiotics

Attaching the NorA Efflux Pump Inhibitor INF55 to Methylene Blue Enhances Antimicrobial Photodynamic Inactivation of Methicillin-Resistant <i>Staphylococcus aureus in Vitro</i> and <i>in</i> <i>Vivo</i>

Antimicrobial photodynamic inactivation (aPDI) uses photosensitizers (PSs) and harmless visible light to generate reactive oxygen species (ROS) and kill microbes. Multidrug efflux systems can moderate the phototoxic effects of PSs by expelling the compounds from cells. We hypothesized that increasing intracellular concentrations of PSs by inhibiting efflux with a covalently attached efflux pump inhibitor (EPI) would enhance bacterial cell phototoxicity and reduce exposure of neighboring host cells to damaging ROS. In this study, we tested the hypothesis by linking NorA EPIs to methylene blue (MB) and examining the photoantimicrobial activity of the EPI–MB hybrids against the human pathogen methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Photochemical/photophysical and <i>in vitro</i> microbiological evaluation of 16 hybrids carrying four different NorA EPIs attached to MB via four linker types identified INF55-(Ac)­en–MB <b>12</b> as a lead. Compound <b>12</b> showed increased uptake into <i>S. aureus</i> cells and enhanced aPDI activity and wound healing effects (relative to MB) in a murine model of an abrasion wound infected by MRSA. The study supports a new approach for treating localized multidrug-resistant MRSA infections and paves the way for wider exploration of the EPI–PS hybrid strategy in aPDI

Irradiation and fluorescence parameters for each photosensitizer.

<p>Irradiation and fluorescence parameters for each photosensitizer.</p

Molecular features that could affect APDI effectiveness.

a<p>As the conjugate has 37 primary amino groups, the number of charges depends of pH and microenviroment.</p>b<p>Average extinction coefficient over range 400–700-nm.</p

Effect of laccase enzyme on photodynamic inactivation of <i>C. neoformans</i> ATCC 208820 (laccase positive strain, black squares), and ATCC 208819 (laccase negative strain, open circles).

<p>(A) methylene blue, (B) rose bengal, (C) EtNBSe, (D) BB6, (E) pL-ce6 were used as photosensitizers at 10 µM in PBS for 30 min followed by a wash and illumination with the wavelengths specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054387#pone-0054387-t002" target="_blank">Table 2</a>. Data are means and bars are the standard deviation. * P<0.05; ** P<0.01; *** P<0.001 for survival of 208820 vs 208820.</p

Chemical structures of the photosensitizers used in this study.

<p>(A) Methylene blue; (B) Rose Bengal; (C) Selenium Nile blue derivatrive (EtNBSe); (D) Tris-cationic fullerene (BB6); poly-L-lysine chlorin (e6) conjugate (pL-ce6).</p

Confocal microscopy image of <i>C. neoformas</i> KN99α.

<p>Cells were treated with APDI mediated by pL-ce6 (10 µM) and then incubated with FITC-annexin V and PI. Green represents fluorescence of externalized phosphatidylserine that is correlated to the initial steps of apoptosis, and red corresponds to fluorescence of PI (advanced apoptosis/necrosis). We present three pictures of the same field: Transmittance in column A, green and red fluorescence in columns B and C respectively. The first line of figures is the stained samples before APDI (0J/cm²), the second line is following an irradiation of 10J/cm² and the last one was irradiated with fluence of 40J/cm². Scale bars 8µm.</p

Confocal fluorescence microscopy images of <i>C. neoformans</i> strains.

<p>Cells were incubated with 10 µM photosensitizers for 30 min as described (methylene blue, rose bengal, EtNBSe, and pL-ce6) and then labeled with MitoTracker Green™ (for methylene blue, EtNBSe, and pL-ce6) or MitoTracker Red™ (for rose bengal). All photosensitizers emitted red fluorescence, except rose bengal that emits green fluorescence. We present one picture of the superimposed images for each strain incubated with each photosensitizer. Scale bars 8µm.</p

Effect of (A) capsule (KN99α and <i>cap59),</i> and (B) laccase enzyme (208820 and 208819) on photosensitize uptake.

<p>Each strain was incubated with photosensitizers at 10 µM for 30 min, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054387#s2" target="_blank">material and methods</a>. Data are means and bars are the standard deviation. * p<0.05 between the positive and negative strains, for each photosensitizer.</p

Effect of capsule on photodynamic inactivation of <i>C. neoformans</i> KN99α (black squares) and <i>cap59</i> (open squares).

<p>(A) methylene blue, (B) rose bengal, (C) EtNBSe, (D) BB6, (E) pL-ce6 were used as photosensitizers at 10 µM in PBS for 30 min followed by a wash and illumination with the wavelengths specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054387#pone-0054387-t002" target="_blank">Table 2</a>. Data are means and bars are the standard deviation. * P<0.05; ** P<0.01; *** P<0.001 for survival of KN99α vs <i>cap59.</i></p