Novel PBP3 insertions in MDR Escherichia coli from blood stream infections: A threat for newer β-Lactam combinations

Abstract

Background: Cefepime+zidebactam (FEP-ZID) has recently been reported to be active against multidrug-resistant (MDR) Gram-negatives. Both the compounds target penicillin-binding proteins (PBPs), while zidebactam protects cefepime from β-lactamase hydrolysis. For the first time, this study tested efficiency of FEP-ZID combination on Indian MDR E. coli isolates. Methods and materials: Sixty MDR E. coli isolates from patients with blood stream infections from a tertiary care hospital were selected, and tested against FEP-ZID combination using microbroth dilution with pre-coated antimicrobial panels. Further, two isolates among these were chosen to analyse structural basis behind the reduced binding efficiency of cefepime with PBP3 using molecular docking and dynamics (MD). Docking was performed using AutoDock-Vina v1.1.2 and MM-PBSA analysis was performed using MD simulations to explore the free energy of binding. Results: About 60% (n = 36) of tested isolates harboured four amino acid insertions in PBP3. Notably, 38.3% were INYR and 21.7% were IKYR insertions. Isolates with insertions had higher MICs compared to native PBP3 isolates. In addition, blaOXA-1 was identified in 65%, 69% and 58% among INYR, IKYR and non-mutated isolates, respectively. Docking for VB1 (blaOXA-1-negative) and VB2 (blaOXA-1-positive) revealed that native-cefipime-PBP3 complex exhibited higher binding energy (-8.6) when compared to mutant complexes (<-6.9). The native complex possessed a crucial interaction with conserved serine residue S307 which was not observed in mutants. Native-complex exhibited stronger binding strength as shown by higher number of hydrogen bonds (LYS499, THR 497, SER 307, THR495) than mutants (VB1-GLN540, THR501, GLN361; VB2-ASP413). MD simulation of native complex showed ∼0.2 to ∼0.25 nm of RMSD, whereas mutants exhibited ∼0.3 to ∼0.4 nm of RMSD denoting stability of native-complex. MD simulation deciphered that mutations alter active-site conformation, thereby reducing binding potential and causing resistance against cefepime. Conclusion: INYR and IKYR insertions were observed for the first time in E. coli with high MICs against FEP-ZID combination. In silico MD approach confirmed that resistance was due to insertion of amino acids causing alteration in active binding pockets of PBP3. Such resistance mechanism in addition to presence of blaOXA-1 enzyme is a great threat for both existing and novel/unique drug combinations.