The utilization of a magneto-fluorescence sequential labeling strategy for the visualization and tracking of intestinal translocation of oral microbiota

The microbiota of the oral-gut axis influences systemic physical health, but the exact mechanisms remain unknown. In this study, magnetic supraparticles modified with polyacrylic acid and 3-azido-D-alanine hydrochloride (MSP@PAA-ADA) were constructed, and a magnetic-fluorescence sequential labeling strategy was established for microbiota viability labeling in combination with a cyanine 5-labeled D-amino acid (Cy5ADA) fluorescent probe. MSP@PAA-ADA, with a particle size of approximately 160 nm, exhibited good hydrophilicity, high magnetic responsiveness, and low bacterial toxicity. MSP@PAA-ADA achieved efficient labeling of oral bacteria through interaction with peptidoglycan in bacterial cell walls. The experimental results indicated that MSP@PAA-ADA can be able to widely bind salivary microbiota, and the labeled salivary bacteria translocated along the oral-gut axis. A small amount of fluorescent signal could still be detected in the mice 36 hours after gavage. MSP@PAA-ADA was employed as a magnetic probe for the in vitro labeling of oral bacteria, and sequential labeling was conducted using the fluorescent metabolic probe Cy5ADA. The oral bacteria labeled with MSP@PAA-ADA can be effectively magnetically separated after intestinal translocation through the gastrointestinal tract. The magnetically responsive and fluorescently imaged oral bacteria demonstrate their activity after intestinal translocation. The magneto-fluorescence dual-mode strategy that enables controllable spatiotemporal tracing and simultaneous viability assessment of intestinal oral bacteria in the gut, providing a new technical approach for dissecting biological mechanisms along the oral–gut microbiota axis. • MSP@PAA-ADA enables efficient labeling of salivary bacteria without affecting viability. • Co-labeling of MSP@PAA-ADA and Cy5ADA reveals salivary microbiota intestinal translocation. • Magneto-fluorescence tracking reveals transient bioactivity of oral bacteria in gut. • The strategy enables real-time longitudinal tracking for oral-gut axis microbiota research.