Mutation rate with hydrogen peroxide

Mutation rate of P. aeruginosa WT and mutators before treatment and after treatment with hydrogen peroxide washing and non washing the cells calculated with the MSS maximum likelihood method

H2O2 effect on cell density

Viable cells of P. aeruginosa mutators and WT using BacTiter-Glo™ Reagent (Promega) after treatment with H2O2 relative to viable cells in H2O-treate

Fitness mutators vs WT

Competition experiments between P. aeruginosa PAO1 wild-type and mutators (GmR) in the presence and absence of hydrogen peroxide

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

Mutations in <i>glpT</i> of ten randomly chosen spontaneous mutants of <i>P. aeruginosa</i> PA14 selected from <i>in vitro</i> and <i>in vivo</i> experiments.

<p>Mutants were arranged according to the nucleotide position using the A of the ATG of the ORF as reference.</p

Amplification of fosfomycin resistant mutants in mice treated with the antibiotic.

<p>Fold increase values were calculated as the ratio between the median of the proportion of Fos-R mutants/total bacterial counts (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010193#pone-0010193-g002" target="_blank">figure 2B</a>) of treated and untreated mice.</p

Frequency of mutants resistant to single antibiotics and the combinations of fosfomycin with tobramycin and imipenem.

<p>a: antibiotic concentation in µg/ml.</p

Recovery of bacteria from lungs 48 hours post infection.

<p>Mice were infected with approximately 5×10⁵ cfu/animal and treated with two doses of fosfomycin (200 mg/kg) 24 hours after inoculation or with no antibiotic. PA14 (blue columns) and its mutant derivatives <i>mutS::MAR2xT7</i> (red columns) and <i>glpT::MAR2xT7</i> (gray columns) (A). Ratio of Fos-R mutants/total bacteria in lungs from treated and non-treated mice (B). Values are medians and error bars represent interquartile ranges.</p

Mortality curves.

<p>Mice were infected with two different inocula of <i>P. aeruginosa</i> PA14 (blue lines) and its mutant <i>glpT::MAR2xT7</i> derivative (red lines) strains. The Kaplan-Meier analysis failed to detect any significant difference between curves from the same bacterial inoculum (1×10⁶ cell (crosses) and 5×10⁵ cells (diamonds)) (p = 0.81 and p = 0.21, respectively).</p

Minimal inhibitory concentrations of the antibiotics fosfomycin, tobramycin and imipenem for PA14 and its derivative mutants <i>mutS::MAR2xT7 and glpT::MAR2xT7</i>.

<p>Minimal inhibitory concentrations of the antibiotics fosfomycin, tobramycin and imipenem for PA14 and its derivative mutants <i>mutS::MAR2xT7 and glpT::MAR2xT7</i>.</p