The cell envelope of Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals. Here, we present the results of a genome-wide screen for cell envelope-associated resistance and susceptibility determinants in a Burkholderia cenocepacia clinical isolate. For this purpose, we construct a high-density, randomly-barcoded transposon mutant library and expose it to 19 cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profile over a hundred functional associations and identify mediators of antibiotic susceptibility in the Bcc cell envelope. We reveal connections between β-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism. The synergy of the β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. In comparison with ceftazidime, avibactam more strongly potentiates the activity of aztreonam and meropenem in a panel of Bcc clinical isolates. Finally, we characterize in Bcc the iron and receptor-dependent activity of the siderophore-cephalosporin antibiotic, cefiderocol. Our work has implications for antibiotic target prioritization, and for using additional combinations of β-lactam/β-lactamase inhibitors that can extend the utility of current antibacterial therapies.
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