dThis study examined the activity of the novel antimicrobial combination ceftazidime-avibactam against Enterobacteriaceae exhibiting different outer membrane permeability profiles, specifically with or without porins and with or without expression of the main efflux pump (AcrAB-TolC). The addition of the outer membrane permeabilizer polymyxin B nonapeptide increased the antibacterial activities of avibactam alone, ceftazidime alone, and ceftazidime-avibactam against the characterized clinical isolates of Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae. This enhancement of activities was mainly due to increased passive penetration of compounds since inhibition of efflux by the addition of phenylalanine-arginine -naphthylamide affected the MICs minimally. OmpF (OmpK35) or OmpC (OmpK36) pores were not the major route by which avibactam crossed the outer membranes of E. coli and K. pneumoniae. In contrast, Omp35 and Omp36 allowed diffusion of avibactam across the outer membrane of E. aerogenes, although other diffusion channels for avibactam were also present in that species. It was clear that outer membrane permeability and outer membrane pore-forming proteins play a key role in the activity of ceftazidime-avibactam. Nevertheless, the MICs of ceftazidime-avibactam (with 4 mg/liter avibactam) against the ceftazidime-resistant clinical isolates of the three species of Enterobacteriaceae studied were <8 mg/liter, regardless of outer membrane permeability changes resulting from an absence of defined porin proteins or upregulation of efflux.T he worldwide dissemination of resistant bacteria has severely reduced the efficacy of our antibiotic arsenal and consequently contributes to increasing frequency of therapeutic failure (1-3). For bacterial pathogens, changing expression of transporters and efflux mechanisms directly alters the intracellular concentrations of antibiotics (4, 5), and mutations that decrease permeability or increase efflux contribute to multidrug resistance (6, 7).These bacterial envelope adaptations act jointly with -lactamase enzymes that inactivate -lactam antibiotics in the periplasm. Consequently, several -lactamase inhibitors are used in combination with -lactams (e.g., piperacillin-tazobactam or amoxicillin-clavulanic acid) to restore -lactam activity by inhibiting -lactamases (8, 9). However, to penetrate the outer membrane, both -lactams and -lactamase inhibitors are understood to diffuse through porin-mediated channels, and a decrease in porin expression potentially impairs the penetration of both (6, 10-12). In addition, just as -lactams are recognized and expelled by efflux pumps (7, 13), physicochemically similar -lactamase inhibitors may also be recognized and pumped by efflux mechanisms (7,14).Avibactam is a first-in-class synthetic, non--lactam -lactamase inhibitor with a novel [3.2.1]-diazabicyclooctane chemical scaffold (15). It inhibits Ambler class A and C -lactamases, as well as some class D enzymes, with a unique covalent and reversible mechanism...