phenylalanine-arginine-beta-naphthylamide and Pseudomonas-Infections

There is urgent need for new therapeutic strategies to fight the global threat of antibiotic resistance. The focus of this Perspective is on chemical agents that target the most common mechanisms of antibiotic resistance such as enzymatic inactivation of antibiotics, changes in cell permeability, and induction/activation of efflux pumps. Here we assess the current landscape and challenges in the treatment of antibiotic resistance mechanisms at both bacterial cell and community levels. We also discuss the potential clinical application of chemical inhibitors of antibiotic resistance mechanisms as add-on treatments for serious drug-resistant infections. Enzymatic inhibitors, such as the derivatives of the β-lactamase inhibitor avibactam, are closer to the clinic than other molecules. For example, MK-7655, in combination with imipenem, is in clinical development for the treatment of infections caused by carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa, which are difficult to treat. In addition, other molecules targeting multidrug-resistance mechanisms, such as efflux pumps, are under development and hold promise for the treatment of multidrug resistant infections.

Topics: Azabicyclo Compounds; beta-Lactamase Inhibitors; Drug Resistance, Microbial; Enterobacteriaceae Infections; Humans; Imipenem; Pseudomonas Infections

The spread of multidrug-resistant Pseudomonas aeruginosa isolates constitutes a serious clinical challenge. Bacterial efflux machinery is a crucial mechanism of resistance among P. aeruginosa. Efflux inhibitors such as phenylalanine arginyl β-naphthylamide (PAβN) promote the bacterial susceptibility to antimicrobial agents. The pathogenesis of P. aeruginosa is coordinated via quorum sensing (QS). This study aims to find out the impact of efflux pump inhibitor, PAβN, on QS and virulence attributes in clinical isolates of P. aeruginosa. P. aeruginosa isolates were purified from urine and wound samples, and the antimicrobial susceptibility was carried out by disc diffusion method. The multidrug-resistant and the virulent isolates U16, U21, W19 and W23 were selected. PAβN enhanced their susceptibility to most antimicrobial agents. PAβN reduced QS signalling molecules N-3-oxo-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone without affecting bacterial viability. Moreover, PAβN eliminated their virulence factors such as elastase, protease, pyocyanin and bacterial motility. At the transcription level, PAβN significantly (P<0.01) diminished the relative expression of QS cascade (lasI, lasR, rhlI, rhlR, pqsA and pqsR) and QS regulated-type II secretory genes lasB (elastase) and toxA (exotoxin A) compared to the control untreated isolates U16 and U21. In addition, PAβN eliminated the relative expression of pelA (exopolysaccharides) in U16 and U21 isolates. Hence, P. aeruginosa-tested isolates became hypo-virulent upon using PAβN. PAβN significantly blocked the QS circuit and inhibited the virulence factors expressed by clinical isolates of P. aeruginosa. PAβN could be a prime substrate for development of QS inhibitors and prevention of P. aeruginosa pathogenicity.

Topics: Bacterial Proteins; Biofilms; Dipeptides; Humans; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing; Virulence Factors

To assess the prevalence of efflux-driven fluoroquinolone (FQ) resistance in recent clinical isolates of Pseudomonas aeruginosa, a worrisome and often hospital-acquired pathogen, 115 unique strains were collected over a 5-month period, of which 27 and 33 had decreased susceptibility to ciprofloxacin (CIP) and levofloxacin (LVX), respectively. The MIC(50) (minimum inhibitory concentration for 50% of the organisms) was 16 μg/mL for both FQs. The efflux pump inhibitors (EPIs) phenylalanine-arginine-β-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP) were then used to evaluate their efficacy in reducing CIP and LVX MICs. NMP did not significantly modify CIP MICs, whilst PAβN resulted in MIC(50) values of 2 μg/mL and 0.125 μg/mL for CIP and LVX, respectively. With the addition of PAβN, susceptibility to CIP and LVX was recovered in 6 (22.2%) and 31 (93.9%) strains, respectively. The best combination to reverse FQ resistance in this set of strains was LVX with PAβN. The results of this study show that the effect of an EPI is not only dependent on the species on which it is used but also on the molecule associated with it. Therefore, the design of an EPI equally efficient on all resistance-nodulation-cell division (RND) efflux pumps appears to be difficult and, from a practical point of view, if an EPI is developed for clinical use, the efficiency of its combination with a definite molecule should be assessed carefully against a wide range of clinical isolates to evaluate the real benefit of this combination.

Topics: Anti-Bacterial Agents; Ciprofloxacin; Dipeptides; Drug Resistance, Multiple, Bacterial; Fluoroquinolones; Humans; Levofloxacin; Membrane Transport Proteins; Microbial Sensitivity Tests; Ofloxacin; Piperazine; Piperazines; Prevalence; Pseudomonas aeruginosa; Pseudomonas Infections

The objective of this study was to implement a semimechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to describe the effects of ciprofloxacin against Pseudomonas aeruginosa in vitro. Time-kill curves were generated with an initial inoculum close to 5 x 10(6)CFU/ml of P. aeruginosa PAO1 and constant ciprofloxacin concentrations between 0.12 and 4.0 microg/ml (corresponding to 0.5x and 16x MIC). To support the model, phenotypic experiments were conducted with the PAO7H mutant strain, which overexpresses the MexEF OprN efflux pump and phenyl arginine beta-naphthylamide (PAbetaN), a known efflux inhibitor of main Mex multidrug efflux systems. A population approach was used for parameter estimation. At subinhibitory ciprofloxacin concentrations (0.12 and 0.25 microg/ml), an initial CFU decay followed by regrowth was observed, attesting to rapid emergence of bacteria with increased but moderate resistance (8-fold increase of MIC). This phenomenon was mainly due to an overexpression of the Mex protein efflux pumps, as shown by a 16-fold diminution of the MIC in the presence of PAbetaN in these strains with low-level resistance. A PK-PD model with adaptation development was successfully used to describe these data. However, additional experiments are required to validate the robustness of this model after longer exposure periods and multiple dosing regimens, as well as in vivo.

Topics: Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Ciprofloxacin; Colony Count, Microbial; Cross Infection; Dipeptides; Drug Resistance, Bacterial; Genes, Bacterial; Humans; In Vitro Techniques; Microbial Sensitivity Tests; Models, Biological; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Dipeptides; Humans; Microbial Sensitivity Tests; Piperazines; Pseudomonas aeruginosa; Pseudomonas Infections