Mechanisms underlying Phage-Antibiotic Synergy and Resistance in Daptomycin-Resistant Enterococcus faecium

Summary Enterococcus faecium (E.fcm) is a formidable multidrug-resistant (MDR) pathogen associated with high morbidity and mortality . Daptomycin (DAP) is a preferred treatment modality for serious MDR E.fcm infections and has demonstrated rapid bactericidal activity against this pathogen. Unfortunately, DAP resistance is increasing in part due to mutations in the LiaFSR pathway, a three-component regulatory system involved in cellular membrane stress response. Notably, strains harboring LiaFSR mutations have demonstrated nonresponsiveness to DAP (8-14 mg/kg/day) and beta-lactam combinations (DAP+BL) that previously displayed synergistic and bactericidal activity against other nonresponsive MDR E.fcm strains. Bacteriophages (“phages”) are an emerging anti-infective therapy for MDR infections refractory to conventional antibiotics. Given their ability to target specific pathogens, replicate within the cell, and penetrate high burden inoculum infection sites, phage therapy is increasingly sought after as an adjunct to antibiotics for patients with deep- seated MDR infections, such as infective endocarditis, that are refractory to antibiotics alone. However, little is known regarding the mechanisms for synergy observed with phages and antibiotics, which is critical to the optimizing phage-antibiotic combinations (PACs). Our central hypothesis is that we can enhance the efficacy of PACs against MDR E.fcm by analyzing the mechanisms for phage-antibiotic synergy. Our long-term goals are to: i) optimize therapy against MDR E.fcm infections by defining phage-antibiotic regimens that maximize bactericidal killing while preventing treatment-emergent resistance, and ii) determine frequency of administration, duration of therapy, and underlying synergy mechanisms for further optimization PACs. Our short-term goal is to determine the mechanisms for effective PAC synergistic bactericidal activity and prevention of antibiotic and phage resistant MDR E.fcm by conducting high inoculum modified checkerboard (CB) assays, time kill analyses (TKA), and ex-vivo SEV models to measure the impact of adjunctive therapy on E.fcm eradication. The proposed research is innovative as we will employ novel mechanistic fingerprinting techniques developed by our group and a validated ex-vivo simulated endocardial vegetation model that mimics human PK parameters to determine effective phage-antibiotic combinations against MDR E.fcm pathogens. The proposed research is expected to lay the groundwork for effective in-vivo phage-antibiotic experiments investigating the eradication of high bacterial burden MDR E.fcm while preventing treatment- emergent resistance. Attainment of this data is crucial to help guide effective clinical management of refractory MDR E.fcm due to antibiotic resistance and/or suboptimal exposure at the site of infection. This will positively impact public health by prolonging the useful life of conventional antibiotics used to treat refractory MDR E.fcm and alleviate devastating consequences endured by impacted patients. Project Number: 1R21AI196281-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Michael Rybak | Institution: WAYNE STATE UNIVERSITY, DETROIT, MI | Award Amount: $455,719 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 DCAI-K (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19628101