Synergistic Effects of a Novel combination therapy Active Against Hospital-Acquired Methicillin-Resistant Staphylococcus aureus (HA- MRSA)

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major cause of nosocomial infections, demonstrating significant resistance to both β-lactam and non-β-lactam antibiotics. This resistance is largely attributable to the transpeptidase penicillin-binding protein-2a (PBP2a), which is encoded by the mecA gene, in addition to numerus other auxiliary factors. The rising minimum inhibitory concentration of vancomycin among MRSA isolates underscores the urgent need for novel therapeutic strategies to combat MRSA infections. Combination therapy has emerged as a promising approach to this challenge. In a previous screening of 1200-FDA approved drug library, the antioxidant Compound AMC18 has been found to potentiate the activity of Amoxicillin against the community acquired MRSA USA300. This study aimed to assess the efficacy of AMC18 in combination with amoxicillin against several hospital-acquired MRSA (HA-MRSA) strains in vitro and in vivo using Galleria mellonella larvae model. The synergistic effects of the AMC18 and AMX combination were evaluated using a checkerboard assay, while further analyses included time- kill assays to examine bactericidal kinetics and resistance assays to assess the combination's ability to prevent resistance development in the N315 MRSA strain. The chequerboard assay revealed a synergistic activity of the AMX/AMC18 combination against all tested HA-MRSA strains, with fractional inhibitory concentration index (FICI) values below 0.5, achieving a 16- fold reduction in AMX MIC value in significant strain such as N315. Moreover, the combination significantly enhanced bactericidal activity against the N315 strain in time-kill assays compared to either agent alone. Although the N315 strain displayed a two- fold increase in AMX MIC after 14 days of sublethal AMX exposure, this resistance was effectively counteracted by the AMX/AMC18 combination. Furthermore, biofilm eradication assays indicated that the AMX/AMC18 combination significantly reduced biofilm biomass in the HA-MRSA strain N315 relative to monotherapies. In the Galleria mellonella larvae model, the combination therapy significantly improved survival rate of infected larvae up to 80 %. Collectively, these findings suggest that AMC18, when used in conjunction with AMX, can synergistically enhance the in vitro killing of HA-MRSA, highlighting its potential as an effective β-lactam adjuvant in MRSA infection treatment.z