Metabolic Gadolinium Labeling of Clostridium novyi -Nontoxic for Magnetic Resonance Imaging Visualization of Spores and Germinated Bacteria

Bacterial cancer therapy using Clostridium novyi- nontoxic ( C. novyi- NT) has emerged as a promising strategy to selectively eradicate hypoxic and necrotic tumor regions. However, noninvasive visualization of injected spores and germinated bacteria remains a major barrier to clinical translation. Here, we developed metabolically responsive gadolinium-labeled C. novyi- NT (Gd- C. novyi- NT) to enable magnetic resonance imaging (MRI)-based tracking of spore localization and bacterial germination. Gd ions were electrostatically condensed onto the negatively charged spore surface, forming Gd clusters that produced strong T2-weighted contrast without affecting spore viability or germination efficiency. Under anaerobic conditions, these Gd aggregates were progressively diluted and redistributed along the bacterial cell wall through metal-chelating metabolites, converting the MRI signal from T2-dominant to T1-weighted contrast. In an orthotopic liver tumor model, intra-arterial infusion of Gd-spores led to selective intratumoral accumulation, with early T2 contrast followed by sustained T1 enhancement persisting for up to 9 days post-administration. This metabolically driven T2-to-T1 contrast transition provides a dynamic, noninvasive imaging signature that correlates with bacterial germination and proliferation. The Gd- C. novyi- NT platform therefore offers a powerful tool for real-time monitoring of bacterial therapeutic behavior in vivo, advancing precision imaging and therapeutic control of bacterial-based oncologic therapies.