The efflux inhibitor phenylalanine-arginine beta-naphthylamide (PAβN) permeabilizes the outer membrane of gram-negative bacteria

PLoS One. 2013;8(3):e60666. doi: 10.1371/journal.pone.0060666. Epub 2013 Mar 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anilino Naphthalenesulfonates / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / metabolism
  • Cell Membrane Permeability / drug effects*
  • Culture Media
  • Detergents / pharmacology
  • Dipeptides / pharmacology*
  • Drug Resistance, Bacterial / drug effects
  • Gram-Negative Bacteria / cytology*
  • Gram-Negative Bacteria / drug effects*
  • Magnesium / pharmacology
  • Microbial Sensitivity Tests
  • Periplasm / drug effects
  • Periplasm / metabolism
  • Pseudomonas aeruginosa / drug effects
  • beta-Lactamases / metabolism
  • beta-Lactams / pharmacology

Substances

  • Anilino Naphthalenesulfonates
  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Culture Media
  • Detergents
  • Dipeptides
  • beta-Lactams
  • phenylalanine arginine beta-naphthylamide
  • 1-anilino-8-naphthalenesulfonate
  • AmpC beta-lactamases
  • beta-Lactamases
  • Magnesium

Grant support

This study was funded by a Canada-UK Partnership on Antibiotic Resistance-114045 award to LLB. RPL is the recipient of the Michael G. DeGroote Fellowship in Basic Biomedical Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.