Abstract
Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a major nosocomial pathogen, but studying its host and microbiome interactions remains challenging due to limited genetic tools. Current obstacles include extensive intrinsic and acquired resistance that precludes use of conventional selection markers, poor plasmid maintenance, and oxygen-dependent reporters unsuitable for gut environments. Here we present an integrated toolkit that enables robust genetic manipulation and in vivo tracking of VREfm. We developed a universal puromycin selection system effective across diverse clinical isolates and an enhanced pheS** counterselection marker for stable genomic integration. We identified two neutral genomic loci that support reporter genes insertion without fitness cost. Using this system, we demonstrate long-term tracking of VREfm colonization, strain competition and host-microbiome interactions in mouse models via chromosomally-integrated anaerobic fluorescent proteins (eUnaG2 and smURFP). This toolkit advances the study of VREfm pathogenesis by enabling stable genetic manipulation of even the most resistant clinical isolates and direct visualization of host-microbe interactions in ecologically and immunologically relevant in vivo environments.