Heterogeneity and Metabolic Diversity among Enterococcus Species during Long-term Colonization

Urinary tract infections (UTIs), traditionally dominated by Gram-negative pathogens, are increasingly complicated by antimicrobial-resistant Enterococcus spp. in hospital settings. This study screened urine samples from 210 ICU patients at Uppsala University Hospital (June 2020 - September 2021), identifying 39 unique PhenePlate-RF types across E. faecium, E. faecalis, and E. durans. E. faecium isolates showed considerable genetic diversity, primarily within clonal complex 17 (CC17), known for its virulence and antibiotic resistance. We identified multiple lineages and sequence types (STs), such as in patient HWP143, who had isolates from both ST80 and ST22 (an ancestral CC17 lineage). Notably, metabolic adaptations, such as increased L-arabinose metabolism, and shifts in antibiotic resistance were observed. Variations and similarities in plasmid content between individual linages suggest horizontal gene transfer. E. faecalis isolates exhibited less genetic diversity but significant metabolic variability across patients and mixed infections, as seen in patient HWP051, colonized by both ST16 (CC58) and ST287. E. durans, though less common, shared important metabolic traits with E. faecium and displayed polyclonal characteristics, highlighting its potential role in UTIs and the complexity of enterococcal infections. E. durans was sometimes misidentified, underscoring the need for accurate identification methods. This research underscores the importance of understanding genetic and metabolic diversity, plasmid variations, and horizontal gene transfer in Enterococcus spp., which influence antibiotic resistance, virulence, and ultimately, treatment outcomes. IMPORTANCE STATEMENTOur study uncovers novel insights into the genetic and metabolic diversity of Enterococcus species within individual patients, focusing on E. faecium, E. faecalis, and E. durans. Unlike prior studies, which often focused on single lineages, we reveal multiple clones and lineages across individual patients, including clones from clonal complex 17 and the emerging sequence type (ST) 192, highlighting notable metabolic adaptations and shifts in antibiotic resistance. The detection of mixed colonization with varied ST-types, and E. durans misidentification by MALDI-TOF, later corrected by sequencing, further emphasizes the challenges in Enterococcus species identification. For the first time, we demonstrate likely horizontal gene transfer among E. faecium, E. faecalis, and E. durans within the same patient, underscoring the dynamic nature of these infections. Our findings have significant implications for understanding the complexity of Enterococcus infections, stressing the need to consider genetic and metabolic diversity to improve disease management and treatment outcomes.