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

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 beta-naphthylamide (PA beta N) is a well-studied EPI that is routinely combined with. fluoroquinolone antibiotics, but few studies have assessed its utility in combination with beta-lactam antibiotics. The initial goal of this study was to assess the efficacy of beta-lactams in combination with PA beta N against the opportunistic pathogen pseudomoneas aeruginosa. PA beta N reduced the minimal inhibitory concentrations (MICs) of several beta-lactam antibiotics against P. aeruginosa; however, the susceptibility changes were not due entirely to efflux inhibition. Upon PA beta N treatment, intracellular levels of the chromosomally-encoded AmpC beta-lactamase that inactivates beta-lactam antibiotics were significantly reduced and AmpC levels in supernatants correspondingly increased, potentially due to perrneabilization of the outer membrane. PP PA beta N 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 PA beta N and restored resistance to vancomycin. Thus, PAN 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 PA beta N as a control for efflux studies.