Comparative Combinatorial Effects of Endolysins LNT103 with Ten Conventional Antibiotics against Gram-Negative...

Bacteriophage-derived endolysins have emerged as promising antibacterial agents; however, their combinatorial interactions with conventional antibiotics remain insufficiently characterized across Gram-negative bacterial species. Here, we systematically evaluated synergistic, additive, or indifferent interactions between the Gram-negative-targeting endolysin LNT103 and ten clinically relevant antibiotics across 15 Escherichia coli, 10 Pseudomonas aeruginosa, and 5 Acinetobacter baumannii strains. Minimum inhibitory concentrations (MICs) and fractional inhibitory concentration indices (FICIs) were determined using checkerboard assays. Among 300 combinations tested, synergistic interactions were observed with chloramphenicol (three cases), sulfamethoxazole (three cases), colistin (two cases), and trimethoprim (one case), indicating pronounced strain dependence. When synergistic and additive interactions were considered together, positive effects were most frequent in P. aeruginosa (76.0%), followed by E. coli (54.6%) and A. baumannii (40.0%). Notably, these trends were inversely correlated with median endolysin MIC values (72, 16, and 4 μg/mL, respectively). In contrast, no correlation was observed between intrinsic antibiotic MICs and combination outcomes, and no antagonism was detected across all combinations. Time-kill assays performed on representative strains further demonstrated that endolysin concentration exerted a greater influence on bactericidal activity than antibiotic concentration. Collectively, these results indicate that intrinsic endolysin susceptibility is a primary determinant of combination efficacy and provide a rational framework for optimizing endolysin-antibiotic adjunctive therapies against multidrug-resistant Gram-negative pathogens.