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 (Orn 8 ‐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 Orn 8 ‐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, Orn 8 ‐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.