Investigation of the Inoculum Effect of Antimicrobial Peptides Utilizing a Microfluidic Chip Platform

In infections with high bacterial loads, such as peritonitis, antibiotic efficacy is often compromised by the inoculum effect. Antimicrobial peptides, despite their broad-spectrum activity, are similarly affected. To elucidate the mechanisms underlying the inoculum effect of antimicrobial peptides, this study employed microfluidic chips to isolate and analyze single bacterial cells. Comparison of population-level minimum inhibitory concentration obtained from 96-well assays with single-cell minimum inhibitory concentration revealed that minimum inhibitory concentration increased with inoculum density, whereas single-cell minimum inhibitory concentration remained unchanged. Further investigation revealed that intracellular components released from lysed cells and OmpT-mediated peptide degradation substantially elevated population resistance, effects that were absent under single-cell isolation. These results demonstrate that the inoculum effect of antimicrobial peptides stems from population-dependent interactions, intracellular material release, and enzymatic degradation rather than intrinsic single-cell resistance. This work provides mechanistic insight into the inoculum effect of antimicrobial peptides and informs the optimization of their clinical application.