Activity of ETX0462 toward Some Burkholderia spp.

ABSTRACT

Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic human pathogens that are inherently multidrug resistant, limiting treatment options for infections. Here, a novel diazabicyclooctane, ETX0462, was evaluated for activity against Bcc and B. gladioli. Ninety-eight percent of the isolates examined in this study were susceptible. ETX0462 was found to demonstrate in vitro activity superior to that of currently available treatment options (e.g., trimethoprim-sulfamethoxazole and ceftazidime).

TEXT

Among the seven antibiotics recommended by the Clinical and Laboratory Standards Institute (CLSI) to treat Burkholderia cepacia complex (Bcc) infections, three are in the β-lactam family (1). β-Lactams are among the safest and most prescribed antibiotics, comprising 65% of the antibiotic market, with annual sales of close to 15 billion U.S. dollars (2). The most prevalent β-lactam resistance mechanism in Bcc is the expression of β-lactamases (3, 4). Bcc pathogens produce two inducible chromosomally encoded β-lactamases: a PenA class A enzyme and an AmpC enzyme. PenA is the dominant β-lactamase and confers broad resistance to β-lactams (5–8). Therefore, treatment options with β-lactam antibiotics are limited for infections caused by Bcc.

We have previously shown that β-lactams and β-lactam combinations not currently recommended for the treatment of Bcc (e.g., temocillin, sulbactam-durlobactam, piperacillin-avibactam) bypass or inhibit the PenA β-lactamase, resulting in potent antimicrobial activity against Bcc and Burkholderia gladioli (9–11). Here, a novel diazabicyclooctane, ETX0462, which was designed to inhibit penicillin-binding protein 1 (PBP1) and PBP3 in Gram-negative bacteria (12), was evaluated for activity against a panel of 140 multidrug-resistant Bcc strains (representing 14 species) and 10 B. gladioli strains (3). These strains were recovered from respiratory specimens from individuals with cystic fibrosis receiving treatment in 68 cities in 36 different states in the United States (3, 6, 10). All were identified to the species level by using a combination of species-specific PCR assay (i.e., recA gene analysis) (13, 14).

MICs of ETX0462, which was synthesized by Entasis Therapeutics, were determined using an agar dilution assay with cation-adjusted Mueller-Hinton (MH) agar. A Steers Replicator was used to deliver 10 μL containing 10⁴ CFU from overnight cultures grown in MH broth (MHB) at 37°C with shaking. Since CLSI interpretative breakpoints are not available for ETX0462, preliminary interpretative breakpoints of susceptibility at ≤4 μg/mL and resistance at ≥8 μg/mL were selected by Entasis Therapeutics for comparison purposes. These breakpoints were largely derived by projections of achieving clinical exposures with ETX0462 that would safely exceed pharmacokinetics/pharmacodynamics-derived endpoints associated with bactericidal activity of the compound (12). With these interpretative breakpoints, ETX0462 rendered 98% of the examined isolates susceptible (Fig. 1A and B). Three strains, Burkholderia dolosa AU29985, Burkholderia multivorans AU28442, and B. multivorans AU11772 showed MICs of >8 μg/mL. Analysis of the β-lactamases and PBPs present in whole-genome sequences of these three strains revealed that B. dolosa AU29985 has two fewer high-molecular-mass PBPs than B. multivorans ATCC 17616, B. multivorans AU11772 carries a bla_OXA, and B. multivorans AU28442 likely does not produce PBP3 or PBP4, due to fragment mutations in their coding sequences. Considering the binding profile of ETX0462 to PBP1 and PBP3 as well as its inability to be hydrolyzed by β-lactamases, we hypothesize that the increased MICs are related to PBP binding, but we cannot rule out drug impermeability and/or efflux in these strains.

FIG 1.

(A) In vitro susceptibility testing reveals that 147 of 150 Bcc and B. gladioli clinical isolates are provisionally susceptible to ETX0462 with MIC values of ≤4 μg/mL. (B) Percentages of Bcc and B. gladioli clinical isolates testing susceptible, intermediate, or resistant to CLSI-recommended agents, trimethoprim-sulfamethoxazole, ceftazidime, meropenem, minocycline, and levofloxacin, in comparison to ETX0462. (C) Immunoblotting using anti-PenA and anti-RecA antibodies. Lane 1, pure PenA (250 ng); lane 2, B. multivorans ATCC 17616 grown in MHB; lane 3, B. multivorans ATCC 17616 grown in MHB plus 1 mg/L of imipenem for 1 h; lane 4, B. multivorans ATCC 17616 grown in MHB plus 0.125 mg/L of ETX0462 for 1 h.

To assess the impact of ETX0462 on the induction of expression of bla_PenA, B. multivorans ATCC 17616 was grown in MHB to log phase at an optical density at 600 nm between 0.6 and 0.7. The cells were treated with sub-MICs (i.e., concentrations that did not inhibit growth) of imipenem (1 μg/mL) or ETX0462 (0.125 μg/mL) for 1 h. The cells were pelleted and lysed to prepare crude extracts and then subjected to immunoblotting as previously described (9, 15). The bla_PenA gene was not induced when exposed to ETX0462, as PenA protein was not detected using an anti-PenA antibody; imipenem, a strong inducer of bla_PenA expression was used as a control (Fig. 1C). An anti-RecA antibody was used as a control. A limitation is that only one isolate was tested for the induction of expression of bla_PenA. However, given that ETX0462 is not a substrate for PenA, induction of expression of bla_PenA in other strains is not likely to alter the susceptibility to ETX0462.

ETX0462 was found to demonstrate in vitro activity superior to that of currently recommended treatment options (e.g., trimethoprim-sulfamethoxazole and ceftazidime) for Bcc infections (Fig. 1B) (3). Its activity was comparable to that of other novel drug combinations (sulbactam-durlobactam and piperacillin-avibactam) that were previously evaluated by our laboratory against this curated panel of multidrug-resistant bacteria (9, 10). To conclude, 147 of the 150 Bcc and B. gladioli strains tested were provisionally susceptible to ETX0462 according to the presumptive susceptibility breakpoint adopted for this study. Given the high rate of susceptibility, ETX0462 represents a promising addition to our current antibiotic arsenal.