The Efflux Inhibitor Phenylalanine-Arginine Beta-Naphthylamide (PAβN) Permeabilizes the Outer Membrane of Gram-Negative Bacteria

Abstract

Active efflux of antimicrobial agents is a primary mechanism by which bacterial pathogens can become multidrug resistant. The combined use of efflux pump inhibitors (EPIs) with pump substrates is under exploration to overcome efflux-mediated multidrug resistance. Phenylalanine-arginine β-naphthylamide (PAβN) is a well-studied EPI that is routinely combined with fluoroquinolone antibiotics, but few studies have assessed its utility in combination with β-lactam antibiotics. The initial goal of this study was to assess the efficacy of β-lactams in combination with PAβN against the opportunistic pathogen, Pseudomonas aeruginosa. PAβN reduced the minimal inhibitory concentrations (MICs) of several β-lactam antibiotics against P. aeruginosa; however, the susceptibility changes were not due entirely to efflux inhibition. Upon PAβN treatment, intracellular levels of the chromosomally-encoded AmpC β-lactamase that inactivates β-lactam antibiotics were significantly reduced and AmpC levels in supernatants correspondingly increased, potentially due to permeabilization of the outer membrane. PAβN treatment caused a significant increase in uptake of 8-anilino-1-naphthylenesulfonic acid, a fluorescent hydrophobic probe, and sensitized P. aeruginosa to bulky antibiotics (e.g. vancomycin) that are normally incapable of crossing the outer membrane, as well as to detergent-like bile salts. Supplementation of growth media with magnesium to stabilize the outer membrane increased MICs in the presence of PAβN and restored resistance to vancomycin. Thus, PAβN permeabilizes bacterial membranes in a concentration-dependent manner at levels below those typically used in combination studies, and this additional mode of action should be considered when using PAβN as a control for efflux studies.

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli

“…Highly sensitive fluorescent probes, including 8-anilino-1-naphthalenesulfonic acid (ANS), fluorescein-di-␤-D-galactopyranoside (FDG), and Sytox Green, have been used to gauge PA␤N=s membrane-destabilizing effects in E. coli and P. aeruginosa cells (33,34,35). In these studies, changes in fluorescence, which indicate internalization of these normally impervious fluorescent compounds, were measured 10 to 20 min (33,35) or over an hour (34) after incubation with various amounts of PA␤N.…”

“…In these studies, changes in fluorescence, which indicate internalization of these normally impervious fluorescent compounds, were measured 10 to 20 min (33,35) or over an hour (34) after incubation with various amounts of PA␤N. In contrast, preincubation of cells with PA␤N was avoided in this study to minimize any membrane damage prior to the start of real-time assays.…”

“…The authors showed that in strains expressing RND efflux pumps, PA␤N causes minimum damage to membrane integrity and potential, whereas in strains lacking a major efflux pump, it can impose some toxic effects (20,32). However, the conclusion that PA␤N acts principally as an efflux pump inhibitor was questioned by three recent publications (33,34,35). One of the main objections stems from the observation that PA␤N causes elevated toxicity, by damaging the membrane, in cells lacking major efflux pathways.…”

Possible Role of Curcumin as ection an Efflux Pump Inhibitor in Multi Drug Resistant Clinical Isolates of Pseudomonas aeruginosa

“…permeabilizes bacterial membranes [10]. Among other synthetic compounds, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil has been investigated as potential inhibitor of efflux pump activity in Pseudomonas aeruginosa and Mycobacterium tuberculosis [8,11].…”

Antibacterial and Antibiotic-Potentiation Activities of Levisticum officinale L. Extracts on Pathogenic Bacteria

“…Some compounds, such as phenylalanyl-arginyl-betanaphthylamide (PAßN) are known as efflux pump inhibitors (EPIs) (4). PAßN selectively inhibit the efflux activities of a broad range of efflux pumps such as MexABOprM, MexEF-OprN, MexCD-OprJ and MexXY-OprM in Pseudomonas aeruginosa and AcrAB-TolC in some species of the Enterobacteriaceae (5).…”