A mechanistic approach to prove the efficacy of combination therapy against New Delhi metallo-β-lactamases producing bacterial strain: A molecular and biochemical approach

Abstract

Background: NDM-1 is a novel broad-spectrum metallo-β-lactamase with the capability to grant resistance to almost all β-lactam antibiotics. Its widespread dissemination made treatment options a major challenge to combat, causing threat to public health worldwide. Due to antibiotic resistance problems, development of effective therapeutics for infections caused by NDM-1 producing strains is urgently required. Since combination therapies are proved to be effective in many cases, this study was initiated to put forward novel effective antibiotics combinations for fighting infections caused by NDM-1 producing strains. Methods: Streptomycin and amikacin combination and streptomycin and ciprofloxacin combination were tested by checkerboard assay. NDM-1 protein/enzyme was then expressed and purified to carry out enzyme kinetics study, CD and fluorescence spectroscopic studies. Results: Streptomycin and amikacin combination and streptomycin and ciprofloxacin combination showed synergistic effect towards NDM-1 producing bacterial strains as shown by FICI results. NDM-1 producing bacterial cells were expressed and purified to obtain protein as the source of enzyme. When NDM-1 enzyme was treated with streptomycin along with amikacin, the efficiency of enzyme was decreased by 49.37% and when the enzyme was treated with streptomycin along with ciprofloxacin, the efficiency of enzyme was decreased by 29.66% as revealed by enzyme kinetic studies. Due to binding of streptomycin and amikacin in combination and streptomycin and ciprofloxacin in combination, conformational changes in the secondary structure of NDM-1 enzyme were observed by CD spectroscopic studies. Antibiotics streptomycin and ciprofloxacin bind with NDM-1 through exothermic processes, whereas amikacin binds through an endothermic process. All three antibiotics bind spontaneously with an association constant of the order of 104 M-1 as revealed by fluorescence spectroscopic studies. Conclusions: The therapeutic combination of streptomycin with amikacin and ciprofloxacin plays an important role in inhibiting NDM-1 producing bacterial strains. Therefore, these combinations can be used as effective future therapeutic candidates against NDM-1 producing bacterial cells.