Macrolides show antimicrobial activity against P. aeruginosa in eukaryotic media through increased uptake and reduced efflux. These data may help explain the clinical efficacy of macrolides against pseudomonal infections.

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

The development of novel antimicrobial agents is urgently required to curb the widespread emergence of multidrug-resistant bacteria like colistin-resistant Pseudomonas aeruginosa. We previously synthesized a series of amphiphilic neamine derivatives active against bacterial membranes, among which 3=,6-di-O-[(2؆-naphthyl)propyl]neamine (3=,6-di2NP), 3=,6-di-O-[(2؆-naphthyl)butyl]neamine (3=,6-di2NB), and 3=,6-di-O-nonylneamine (3=,6-diNn) showed high levels of activity and low levels of cytotoxicity (L. Zimmermann et al., J. Med. Chem. 56:7691-7705, 2013). We have now further characterized the activity of these derivatives against colistin-resistant P. aeruginosa and studied their mode of action; specifically, we characterized their ability to interact with lipopolysaccharide (LPS) and to alter the bacterial outer membrane (OM). The three amphiphilic neamine derivatives were active against clinical colistin-resistant strains (MICs, about 2 to 8 g/ml), The most active one (3=,6-diNn) was bactericidal at its MIC and inhibited biofilm formation at 2-fold its MIC. They cooperatively bound to LPSs, increasing the outer membrane permeability. Grafting long and linear alkyl chains (nonyl) optimized binding to LPS and outer membrane permeabilization. The effects of amphiphilic neamine derivatives on LPS micelles suggest changes in the cross-bridging of lipopolysaccharides and disordering in the hydrophobic core of the micelles. The molecular shape of the 3=,6-dialkyl neamine derivatives induced by the nature of the grafted hydrophobic moieties (naphthylalkyl instead of alkyl) and the flexibility of the hydrophobic moiety are critical for their fluidifying effect and their ability to displace cations bridging LPS. Results from this work could be exploited for the development of new amphiphilic neamine derivatives active against colistin-resistant P. aeruginosa.

Pseudomonas aeruginosa invades epithelial and phagocytic cells, which may play an important role in the persistence of infection. We have developed a 24-h model of THP-1 monocyte infection with P. aeruginosa PAO1 in which bacteria are seen multiplying in vacuoles by electron microscopy. The model has been used to quantitatively assess antibiotic activity against intracellular and extracellular bacteria by using a pharmacodynamic approach (concentration-dependent experiments over a wide range of extracellular concentrations to calculate bacteriostatic concentrations [C s ] and maximal relative efficacies [E max ]; Hill-Langmuir equation). Using 16 antipseudomonal antibiotics (three aminoglycosides, nine ␤-lactams, three fluoroquinolones, and colistin), dose-response curves were found to be undistinguishable for antibiotics of the same pharmacological class if data were expressed as a function of the corresponding MICs. Extracellularly, all of the antibiotics reached a bacteriostatic effect at their MIC, and their E max exceeded the limit of detection (؊4.5 log 10 CFU compared to the initial inoculum). Intracellularly, C s values remained unchanged for ␤-lactams, fluoroquinolones, and colistin but were approximately 10 times higher for aminoglycosides, whereas E max values were markedly reduced (less negative), reaching ؊3 log 10 CFU for fluoroquinolones and only ؊1 to ؊1.5 log 10 CFU for all other antibiotics. The decrease in intracellular aminoglycoside potency (higher C s ) can be ascribed to the acid pH to which bacteria are exposed in vacuoles. The decrease in the E max may reflect a reversible alteration of bacterial responsiveness to antibiotics in the intracellular milieu. The model may prove useful for comparison of antipseudomonal antibiotics to reduce the risk of persistence or relapse of pseudomonal infections. P seudomonas aeruginosa is among the leading causes of nosocomial infections in humans, with a particular tropism for the respiratory tract (see references 1 and 2 for reviews). Beside causing acute infections, it also chronically colonizes the lungs of cystic fibrosis patients, causing protracted and relapsing infections that are a primary cause of increased morbidity and mortality (3, 4).P. aeruginosa is difficult to eradicate for at least two non-mutually exclusive reasons. First, P. aeruginosa is not only naturally resistant to many common antibiotics (because of the poor permeability of its outer membrane) but can also express several mechanisms of resistance to most, if not all, of the drugs that have been selected and clinically developed over the years for their antipseudomonal activity (2). Second, P. aeruginosa can adopt specific modes of life that afford protection from host defenses and antibiotic action. The first one consists of the production of biofilm, a physical barrier to the access of antimicrobial agents that contributes to the persistence of infection with P. aeruginosa (5). The second one stems from the capacity of P. aeruginosa to invade and survive within eukaryotic cell...