A Cell-Penetrating Peptide-Derived Fluorescent Probe for Selective Isolation of Bacterial Spores

In this study, we identify a peptide discovered through fluorescence-based screening of a cell-penetrating peptide (CPP) library that preferentially associates with bacterial spores and evaluate its utility as a fluorescence-guided tool for spore isolation. Specifically, an eight-ornithine peptide conjugated with 5(6)-carboxyfluorescein (Orn₈-K-FAM) generates a distinct and spatially confined fluorescence signal in spores. Across multiple spore-forming bacterial species, more than 90% of spores were labelled by Orn₈-K-FAM, whereas only a minor fraction of vegetative cells from non-spore-forming bacteria exhibited detectable fluorescence, indicating a high degree of preference at the cellular level under the conditions tested. To assess practical applicability, Orn₈-K-FAM labeling was combined with flow-cytometric sorting, enabling fluorescence-guided isolation of spores from mixed microbial populations, including both a defined synthetic community and an environmentally derived bacterial fraction. Compared with conventional spore isolation approaches based on selective cultivation or heat-mediated inactivation of vegetative cells, this workflow provides a cultivation-independent means to separate spore-associated cellular states from heterogeneous microbial samples. This work highlights the potential of CPP-derived probes as analytical tools for spore-focused studies in applied and environmental microbiology, while providing a foundation for further investigation into the scope and mechanistic basis of CPP-spore interactions.