Clinical, Genomic, and Functional Characterization of Vancomycin-Resistant Enterococci from Immunocompromised Patients: Insights into Epithelial Dysfunction and Bloodstream Infections

Giuseppe Sangiorgio¹Ilenia Martina Pia Filannino¹Giuseppe Migliorisi²Dafne Bongiorno¹Nicolò Musso³Grete Francesca Privitera⁴Gabriella Santuccio⁵Dario Leotta⁵MADDALENA CALVO^6*Stefania Stefani¹

• ¹Universita degli Studi di Catania Dipartimento di Scienze biomediche e biotecnologiche, Catania, Italy
• ²Ospedale Ingrassia, Palermo, Italy
• ³Universita degli Studi di Enna 'Kore', Enna, Italy
• ⁴Universita degli Studi di Catania Dipartimento di Medicina Clinica e Sperimentale, Catania, Italy
• ⁵Azienda Ospedaliero Universitaria Policlinico G Rodolico San Marco, Catania, Italy
• ⁶U.O.C. Laboratory Analysis Unit, A.O.U. “Policlinico-San Marco”, Via S. Sofia 78, Catania, 95123, Italy, Catania, Italy

Vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE) are recognized as major opportunistic pathogens in immunocompromised patients, where they may cause bloodstream infections (BSIs). The present study aimed to characterize a cohort of immunocompromised patients colonized or infected with VRE, performing genomic analysis of these isolates. We investigated the impact of bacterial culture supernatants on Caco-2 epithelial cells, focusing on adhesion and cytotoxicity to elucidate mechanisms related to epithelial dysfunction and bacterial translocation. We conducted a retrospective study including 46 VRE from two Italian hospitals. Clinical and epidemiological data were collected, and isolates were characterized by antimicrobial susceptibility testing and whole-genome sequencing. Four representative isolates (E. faecium ST80, E. faecium ST117, E. faecalis ST28, and E. faecalis ST179) and two reference strains (ATCC 29212™ and ATCC 51299™) were selected for in vitro analyses. Adhesion to Caco-2 monolayers was quantified, while cytotoxicity was assessed using MTT assays with bacterial cell-free supernatants (CS). Hydrogen peroxide (H₂O₂) production was measured using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. The majority of isolates were E. faecium (78.3%), predominantly ST80 and ST117, possessed multiple resistance determinants. E. faecalis isolates displayed greater sequence type diversity with a ST28 predominance, carrying virulence genes as ebp, gelE, and elrA. In vitro, bloodstream-derived isolates (E. faecium 51, E. faecalis 52) and reference strain ATCC 29212™ adhered more strongly to Caco-2 cells than other isolates. CS from invasive isolates and ATCC 51299™ significantly reduced epithelial cell viability at 24 h (p < 0.01). In these isolates, H₂O₂ higher quantification was documented in a cellular model. Discussion Our findings highlighted the convergence of antimicrobial resistance and virulence traits in VRE, alongside functional evidence of strain-dependent adhesion and secretion of cytotoxic metabolites. Elevated H₂O₂ production provides a possible path between enterococcal secretomes and epithelial injury, suggesting oxidative stress as a contributor to epithelial dysfunction and potential translocation. These insights expand current understanding of VRE pathogenesis and point to novel therapeutic approaches aimed at preserving epithelial integrity and mitigating oxidative damage in high-risk patients.