In vivo tracking and survival analysis of lactobacillus strains using fluorescent d-amino acids

The viability of probiotics is essential for their health-promoting effects, yet in vivo tracking of their survival and behavior remains technically challenging due to limitations in conventional methods. A fluorescent d-amino acid (FDAA)-based labeling strategy was established to trace Lactobacillus rhamnosus GG and Lactobacillus plantarum Lpc-115 in the mouse gastrointestinal tract. Both strains were efficiently labeled (efficiency > 95%) without affecting viability, with L. rhamnosus displaying higher fluorescence intensity. Real-time distribution and intestinal transit were visualized using small animal imaging, confirming the utility of a "2 + 4-h time model" for optimized survival assessment. Flow cytometry and confocal microscopy validated the in vivo persistence of labeled bacteria in both cecal contents and feces, showing consistent trends and higher survival rates for L. rhamnosus. This genome-independent method enables accurate, non-invasive tracking of probiotics and offers a scalable approach for evaluating their viability, functionality, and host interactions.