Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

Abstract

The cell envelope of the Gram-negative 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, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolate B. cenocepacia K56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new functional associations and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We revealed new connections between β-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, which highlight a vulnerability in sharing this lipid intermediate. We then show that the clinically relevant synergy of the β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. Importantly, we found that avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore-cephalosporin antibiotic, cefiderocol. Overall, 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 our current clinical arsenal of antibacterial therapies.

Cell envelope structural and functional contributions to antibiotic resistance inBurkholderia cenocepacia

“…In P. aeruginosa , the disruption of DacB leads to activation of AmpR and the CreBC/BlrAB two-component system, resulting in AmpC overexpression (50). Disruption of the K56-2 CreBC/BlrAB homologues (K562_RS27235-40) did not affect fitness in any condition in the BarSeq experiment (26). Additionally, we did not see a significant change in β-lactamase activity when we knocked down dacB (Figure 4C).…”

“…We leveraged the BarSeq data for a deeper look into cationic antibiotic resistance in Burkholderia . We identified genes important for PMB and CHX fitness associated with O-antigen synthesis, LPS core synthesis, and lipid A synthesis (26). To dissect the importance of the O-antigen versus LPS core, we constructed an unmarked deletion mutant in wbiI , encoding an epimerase/dehydratase required for O-antigen synthesis.…”

“…As expected, this mutant lacked polymeric O-antigen and could be complemented in trans (Figure S1). We also used a previously constructed unmarked deletion mutant in hldD (26), encoding an epimerase required for LPS core synthesis, which lacks both polymeric O-antigen and a complete LPS core. Both the Δ wbiI and Δ hldD mutants were markedly more susceptible than wild-type to PMB, colistin (COL) and CHX; however, the Δ wbiI mutant was generally less susceptible than the Δ hldD mutant, and the increase in CHX susceptibility was only modest for both mutants (Figure 1A).…”

“…Additionally, β-lactamases have previously been implicated as substrates of DsbA in P. aeruginosa (49). As PenB, the dominant β-lactamase in K56-2 (26), was also identified as a putative DSB system substrate (Table S3), we determined β-lactamase activity in targeted CRISPRi mutants using a nitrocefin hydrolysis assay. Knockdown of dsbA and dsbB reduced β-lactamase activity by ∼30% and ∼75%, respectively, while knockdown of dsbC and dsbD showed no difference from the control (Figure 3D).…”

“…To counter the entry of antibiotics, Bcc species have a suite of broad-spectrum efflux pumps (24). Aside from resistance due to the major β-lactamases (25, 26), the contributions of peptidoglycan biosynthesis and recycling factors to antibiotic susceptibility in the Bcc are unclear.…”