Characterization of GQA as a novel β-lactamase inhibitor of CTX-M-15 and KPC-2 enzymes

Abstract

β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor.In the present study, bla_CTX-M-15 and bla_KPC-2 represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64-128 µg/mL) and cephalosporins (MICs; 128-512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64-512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC₅₀ values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors.

Keywords: Escherichia coli; Klebsiella pneumoniae; Carbapenemase; Extended-spectrum β-lactamases; Galloylquinic acid; β-lactamase inhibitor.

Fig. 1

Effect of different concentrations of GQA on the susceptibility of isogenic strains, namely CTX-M-15-producer (A) to cefotaxime (CTX) disc and KPC-2-producer (B) to imipenem (IPM) disc. A1: CTX disc only showing resistance. A2: CTX disc to which 2 µg/mL of GQA was added showing an inhibition zone with some mutants. A3: CTX disc to which 4 µg/mL of GQA was added showing with an enhanced clear inhibition zone. B1: IPM disc only showing resistance. B2: IPM disc to which 2 µg/mL of GQA was added showing an inhibition zone with a lot of mutants. B3: IPM disc to which 4 µg/mL of GQA was added showing an enhanced clear inhibition zone

Fig. 2

Evaluation of combining 4 µg/mL of GQA with 2 µg/mL of cefotaxime as a cephalosporin (A) or 4 µg/mL of imipenem as a carbapenem (B) against isogenic strains producing the test β-lactamase. Other reference inhibitors, such as clavulanic acid or tazobactam, were also assessed at a fixed concentration of 4 µg/mL. Concentrations of antibiotics represented their breakpoint according to EUCAST guidelines (2024)

Fig. 3

Representation of the half maximal inhibitory concentration (IC₅₀) values for GQAs, clavulanic acid, and tazobactam against the CTX-M-15 (A) and KPC-2 (B) enzymes are shown as percent residual activity of the test enzyme against log concentrations of the compounds and nitrocefin was used as a substrate. IC₅₀ calculated by GraphPad prism software

Fig. 4

Lineweaver–Burk plot showing the mechanism through which GQA hinders CTX-M-15 (A) and KPC-2 (B), via monitoring the hydrolysis of various nitrocefin concentrations using the test enzyme in the presence of 4 µg/mL of GQA

Fig. 5

A Predicted binding mode of LM to the active site of CTX-M-15 (PDB: 5T66). The ligand (GQAs) is shown as CPK-colored thick sticks. The amino acids in the active site are shown as sticks. Electrostatic surface potential (ESP) of the protein active site is shown; Red represents negatively charged (acidic) ESP and Blue is for positive (basic) ESP. Green dashed lines represents the hydrogen bonds between the ligand and the protein MOE Docking score (S Score) of the pose selected = − 7.8 kcal/mol. B 2 D interaction diagram of interaction between GQAs and the active site amino acids of CTX-M-15 (PDB: 5T66). The solvent accessible surface is shown by light blue shades

Fig. 6

A Predicted binding mode of GQA to the active site of KPC-2 (PDB: 6QW9). The ligand (GQAs) is shown as CPK-colored thick sticks. The amino acids in the active site are shown as sticks. Electrostatic surface potential (ESP) of the protein active site is shown; Red represents negatively charged (acidic) ESP and Blue is for positive (basic) ESP. Green dashed lines represents the hydrogen bonds between the ligand and the protein MOE Docking score (S Score) of the pose selected = − 7.6 kcal/mol. B 2 D interaction diagram of interaction between LM and the active site amino acids of KPC-2 (PDB: 6QW9). The solvent accessible surface is shown by light blue shades