Comprehensive biological evaluation of infuzide as a potent antimicrobial, alone and in combination with gentamicin, linezolid, and minocycline targeting MDR Staphylococcus aureus and Enterococcus sp.

Antimicrobial resistance is the greatest threat to modern healthcare systems. The unrelentingly increasing drug resistance manifests in ESKAPE pathogens, thus necessitating urgent novel drug discovery. Whole cell-screening assays were used to identify Infuzide, expressing potent antimicrobial activity against gram-positive pathogens Staphylococcus aureus and Enterococcus. To probe if Infuzide's inactivity against gram-negative pathogens was due to entry issues, its activity was determined in the presence of Polymyxin B nonapeptide, a known membrane permeabilizer. Furthermore, its activity against clinical drug-resistant strains was evaluated. As the next step, the compound's time-kill kinetics against S. aureus was determined, along with its ability to synergize with approved drugs via checkerboard assays; this was further validated via combination time-kill kinetics assays. Infuzide's in vitro post-antibiotic effect, ability to eradicate S. aureus biofilms, and ability to isolate resistant mutants were also ascertained. Eventually, Infuzide's in vivo efficacy alone and in combination was assessed in murine neutropenic thigh and skin infection model against drug-susceptible and drug-resistant S. aureus. Infuzide exhibits highly potent activity against S. aureus and Enterococcus, which is comparable with standard of care. In addition, infuzide expresses concentration-dependent bactericidal activity with ~5.9 log10cfu/mL reduction within 6 h of treatment and synergizes with gentamicin, linezolid, and minocycline, even against MDR strains. Infuzide also inhibits bacterial growth in both biofilm and intracellular phases and exhibits robust in vivo efficacy in both neutropenic thigh and murine skin infection models without inducing resistance. Taken together, Infuzide exhibits all characteristics required for pre-clinical development as an anti-staphylococcal compound.IMPORTANCEThis study identifies Infuzide as a novel antimicrobial against S. aureus.