Anaerobic Fluorescent Reporters Enable In Vivo Tracking of VRE Dynamics in Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated bloodstream infection and a dominant colonizer of the antibiotic-perturbed gut. Experimental interrogation of VREfm colonization dynamics has been constrained by three major barriers: widespread resistance to conventional selection markers in contemporary clinical isolates, instability of plasmid-based constructs, and failure of oxygen-dependent fluorescent reporters in the anaerobic intestinal environment. To address these limitations, we develop genetic tools optimized for VREfm that enable stable anaerobic fluorescent labeling and longitudinal tracking of colonization dynamics. We show that bile pigment-binding fluorescent proteins, including eUnaG2 and smURFP, generate robust signal under anaerobic conditions across diverse clinical backgrounds. Systematic analysis of resistance profiles revealed that conventional selection markers are frequently incompatible with modern isolates; accordingly, we identify puromycin as a broadly effective entry-point strategy and demonstrate that a puromycin-selectable plasmid enables reporter expression for otherwise genetically intractable extensively drug-resistant (XDR) strains. To support long-term studies without continuous selection, we establish an enhanced pheS** counterselection system for chromosomal integration at neutral loci and engineer a tandem 2xsmURFP construct to optimize single-copy fluorescence. Using these tools, we directly track competition between fluorescently labeled VREfm strains in a murine model via flow cytometric analysis of stool samples, enabling longitudinal strain-level quantification without antibiotic selection or selective plating. This work provides a unified framework for genetic manipulation and anaerobic strain tracking in XDR E. faecium, establishing a practical platform for dissecting ecological processes that govern persistence and competition in the gut. ImportanceVancomycin-resistant Enterococcus faecium (VREfm) is a dangerous and difficult-to-treat hospital pathogen. How VREfm establishes itself in the gastrointestinal tract and competes with other strains remains poorly understood, yet this knowledge is essential for preventing infection. Studying these processes has been impeded by widespread antibiotic resistance and by the failure of conventional fluorescent proteins in the oxygen-free gut environment. We describe tools that overcome these barriers and provide a practical foundation for mechanistic studies of VREfm gut colonization and persistence.