The Class A β-Lactamase Produced by Burkholderia Species Compromises the Potency of Tebipenem against a Panel of Isolates from the United States

Abstract

Tebipenem-pivoxil hydrobromide, an orally bioavailable carbapenem, is currently in clinical development for the treatment of extended-spectrum β-lactamase- and AmpC-producing Enterobacterales. Previously, tebipenem was found to possess antimicrobial activity against the biothreat pathogens, Burkholderia pseudomallei and Burkholderia mallei. Thus, herein, tebipenem was evaluated against a panel of 150 curated strains of Burkholderia cepacia complex (Bcc) and Burkholderia gladioli, pathogens that infect people who are immunocompromised or have cystic fibrosis. Using the provisional susceptibility breakpoint of 0.12 mg/L for tebipenem, 100% of the Bcc and B. gladioli tested as being provisionally resistant to tebipenem. Bcc and B. gladioli possess two inducible chromosomal β-lactamases, PenA and AmpC. Using purified PenA1 and AmpC1, model β-lactamases expressed in Burkholderia multivorans ATCC 17616, PenA1 was found to slowly hydrolyze tebipenem, while AmpC1 was inhibited by tebipenem with a k₂/K value of 1.9 ± 0.1 × 10³ M^-1s^-1. In addition, tebipenem was found to be a weak inducer of bla_PenA1 expression. The combination of the slow hydrolysis by PenA1 and weak induction of bla_PenA1 likely compromises the potency of tebipenem against Bcc and B. gladioli.

Keywords: AmpC; Burkholderia; PenA; carbapenem; carbapenemase; tebipenem; β-lactam; β-lactamase.

Figure 1

(A) Bar graph showing the % of isolates that had a specific MIC value. Inset: MIC₅₀ and MIC₉₀ values for antibiotics in next panel [34]. (B) Bar graph comparing the % susceptible, % intermediate, and % resistant of the isolates for tebipenem (TEB), imipenem (IMP), another carbapenem [23], and ceftazidime (CAZ) and trimethoprim-sulfamethoxazole (SXT), two first-line agents [23] used to treat infections caused by Burkholderia species.

Figure 2

Progress curves showing the breakdown of 25 µM tebipenem over time alone (blue), and hydrolysis by the addition of 10 mM sodium hydroxide (NaOH) (orange), 1 µM PenA1 (gray) or 1 µM AmpC1 (yellow).

Figure 3

ESI-MS of PenA1 (red) and AmpC1 (blue) with tebipenem at various time points (1 min, 15 min, 60 min, and 24 h) to reveal the intermediates formed upon incubation, i.e., acyl-enzyme (top left) and acyl-enzyme with loss of R1 hydroxyethyl side chain (top right). Both enzymes are also depicted without tebipenem to show the apo-enzyme’s molecular weight. The ratio of enzyme to tebipenem was chosen based on the t_n at 15 min; see Table 2.

Figure 4

Immunoblotting using anti-PenA1 peptide and anti-AmpC1 polyclonal antibodies as well as an anti-RecA antibody, as an internal standard for the uninduced and induced B. multivorans samples. B. multivorans ATCC 17616 was grown to log phase (OD_{600 nm} ~0.6) in LB, and then cultures were either maintained in LB only (lane 6), or 1 mg/L of imipenem, a known inducer of bla_PenA1 and bla_AmpC1 expression [30], was added (lane 7), or 1 mg/L tebipenem was added (lane 8), and cultures were grown for an additional hour, pelleted, and crude extracts were prepared for immunoblotting. Purified PenA1 (lane 1), purified AmpC (lane 2), E. coli DH10B with pBC SK(+) (lane 3), E. coli DH10B with pBC SK(+) bla_PenA1 (lane 4), and E. coli DH10B with pBC SK(+) bla_AmpC1 (lane 5) were used as controls.