Comprehensive biological evaluation of infuzide as a potent antimicrobial, alone and in combination with gentamicin, linezolid, and minocycline targeting MDR <i>Staphylococcus aureus</i> and <i>Enterococcus</i> 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 <i>Staphylococcus aureus</i> and <i>Enterococcus</i>. 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 <i>S. aureus</i> 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 <i>in vitro</i> post-antibiotic effect, ability to eradicate <i>S. aureus</i> biofilms, and ability to isolate resistant mutants were also ascertained. Eventually, Infuzide's <i>in vivo</i> efficacy alone and in combination was assessed in murine neutropenic thigh and skin infection model against drug-susceptible and drug-resistant <i>S. aureus</i>. Infuzide exhibits highly potent activity against <i>S. aureus</i> and <i>Enterococcus</i>, 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 <i>in vivo</i> 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 <i>S. aureus.</i>