Comparative analysis of various infection models to provide methodological insights for the characterization of new antivirulence drugs to fight antibioresistance

ABSTRACT Bacterial infections remain a major global health burden, amplified by the rapid emergence of antimicrobial resistance (AMR). Among priority pathogens, the ESKAPEE group represents a critical threat due to their high prevalence in healthcare-associated infections, and their capacity to acquire multidrug resistance. Antivirulence strategies, aiming at disarming pathogens rather than inhibiting their growth, constitute a promising alternative to conventional antibiotics. Their development, however, is hindered by the lack of suitable experimental models to evaluate virulence and antivirulence efficacy. Here, we establish complementary in vitro and in vivo infection models to characterize pathogenicity and assess antivirulence activity against clinically relevant ESKAPEE bacteria ( Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli ). We developed a robust nitric oxide (NO)-based in vitro assay that mimics host immune nitrosative stress and enables quantitative evaluation of bacterial resistance to innate immune defenses. In parallel, we established comparative insect infection models using Galleria mellonella and Bombyx species, allowing reproducible assessment of bacterial virulence across multiple hosts and pathogens. These models highlighted marked differences in pathogenic potential depending on both bacterial species and host background. As a proof of concept, we applied these systems to characterize NM102, a first-in-class antivirulence compound targeting the DNA repair protein Mfd. NM102 significantly reduced bacterial survival under NO stress in vitro and decreased bacterial burden in vivo in insect infection models. Together, our results demonstrate that combining immune-mimicking in vitro assays with complementary insect models provides a powerful and ethically acceptable platform to study bacterial pathogenicity and to accelerate the development of innovative antivirulence therapeutics to combat AMR. IMPORTANCE Bacterial infections remain a major public health challenge, worsened by the rapid rise of antimicrobial resistance. Among the most concerning pathogens are the ESKAPEE bacteria, which frequently cause hospital-acquired infections and are often resistant to multiple antibiotics. Antivirulence strategies offer promising alternatives to traditional treatments by disarming bacteria rather than killing them, potentially reducing the risk of resistance. However, progress in this field has been limited by the lack of reliable models to measure bacterial virulence and evaluate antivirulence compounds. In this study, we developed complementary models to better assess bacterial pathogenicity and treatment efficacy. We created an in vitro assay that mimics immune defenses and established insect infection models to compare virulence across pathogens and hosts. Using these systems, we tested NM102, a first-in-class antivirulence compound targeting bacterial DNA repair. NM102 reduced bacterial survival and infection severity, highlighting the potential of antivirulence approaches as innovative therapies against drug-resistant infections.