Related Experiment Video
Updated: Aug 28, 2025
Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings
Published on: August 19, 2021
Phage Cocktails with Daptomycin and Ampicillin Eradicates Biofilm-Embedded Multidrug-Resistant Enterococcus faecium
Ashlan J Kunz Coyne1, Kyle Stamper1, Razieh Kebriaei1
1Anti-Infective Research Laboratory, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA.
Abstract:
Multidrug-resistant (MDR) Enterococcus faecium is a challenging nosocomial pathogen known to colonize medical device surfaces and form biofilms. Bacterio (phages) may constitute an emerging anti-infective option for refractory, biofilm-mediated infections. This study evaluates eight MDR E. faecium strains for biofilm production and phage susceptibility against nine phages. Two E. faecium strains isolated from patients with bacteremia and identified to be biofilm producers, R497 (daptomycin (DAP)-resistant) and HOU503 (DAP-susceptible dose-dependent (SDD), in addition to four phages with the broadest host ranges (ATCC 113, NV-497, NV-503-01, NV-503-02) were selected for further experiments. Preliminary phage-antibiotic screening was performed with modified checkerboard minimum biofilm inhibitory concentration (MBIC) assays to efficiently screen for bacterial killing and phage-antibiotic synergy (PAS). Data were compared by one-way ANOVA and Tukey (HSD) tests. Time kill analyses (TKA) were performed against R497 and HOU503 with DAP at 0.5× MBIC, ampicillin (AMP) at free peak = 72 µg/mL, and phage at a multiplicity of infection (MOI) of 0.01. In 24 h TKA against R497, phage-antibiotic combinations (PAC) with DAP, AMP, or DAP + AMP combined with 3- or 4-phage cocktails demonstrated significant killing compared to the most effective double combination (ANOVA range of mean differences 2.998 to 3.102 log10 colony forming units (CFU)/mL; p = 0.011, 2.548 to 2.868 log10 colony forming units (CFU)/mL; p = 0.023, and 2.006 to 2.329 log10 colony forming units (CFU)/mL; p = 0.039, respectively), with preserved phage susceptibility identified in regimens with 3-phage cocktails containing NV-497 and the 4-phage cocktail. Against HOU503, AMP combined with any 3- or 4-phage cocktail and DAP + AMP combined with the 3-phage cocktail ATCC 113 + NV-497 + NV-503-01 demonstrated significant PAS and bactericidal activity (ANOVA range of mean differences 2.251 to 2.466 log10 colony forming units (CFU)/mL; p = 0.044 and 2.119 to 2.350 log10 colony forming units (CFU)/mL; p = 0.028, respectively), however, only PAC with DAP + AMP maintained phage susceptibility at the end of 24 h TKA. R497 and HOU503 exposure to DAP, AMP, or DAP + AMP in the presence of single phage or phage cocktail resulted in antibiotic resistance stabilization (i.e., no antibiotic MBIC elevation compared to baseline) without identified antibiotic MBIC reversion (i.e., lowering of antibiotic MBIC compared to baseline in DAP-resistant and DAP-SDD isolates) at the end of 24 h TKA. In conclusion, against DAP-resistant R497 and DAP-SDD HOU503 E. faecium clinical blood isolates, the use of DAP + AMP combined with 3- and 4-phage cocktails effectively eradicated biofilm-embedded MDR E. faecium without altering antibiotic MBIC or phage susceptibility compared to baseline.
Insights
Phage-antibiotic combinations effectively eradicated multidrug-resistant Enterococcus faecium biofilms. This approach prevented antibiotic resistance development and maintained phage susceptibility in clinical isolates.
Area of Science:
- Microbiology
- Infectious Diseases
- Bacteriology
Background:
- Multidrug-resistant (MDR) Enterococcus faecium is a significant nosocomial pathogen, often forming biofilms on medical devices.
- Bacteriophages (phages) present a potential therapeutic strategy for challenging, biofilm-mediated infections.
Purpose of the Study:
- To evaluate the efficacy of phage-antibiotic combinations against MDR Enterococcus faecium biofilms.
- To assess phage-antibiotic synergy (PAS) and the impact on antibiotic resistance and phage susceptibility.
Main Methods:
- Screening of eight MDR E. faecium strains for biofilm production and susceptibility to nine phages.
- Modified checkerboard minimum biofilm inhibitory concentration (MBIC) assays for preliminary screening.
- Time kill analyses (TKA) using daptomycin (DAP), ampicillin (AMP), and phage cocktails against biofilm-producing strains R497 (DAP-resistant) and HOU503 (DAP-susceptible dose-dependent).
Main Results:
- Phage-antibiotic combinations (PAC) with DAP, AMP, or DAP + AMP and 3- or 4-phage cocktails demonstrated significant bacterial killing against R497.
- Significant PAS and bactericidal activity were observed for AMP with 3- or 4-phage cocktails, and DAP + AMP with a 3-phage cocktail against HOU503.
- Antibiotic resistance stabilization, without reversion, was observed in both strains when exposed to antibiotics and phages.
Conclusions:
- DAP + AMP combined with 3- and 4-phage cocktails effectively eradicated biofilm-embedded MDR E. faecium clinical isolates.
- This combination therapy maintained antibiotic and phage susceptibility without inducing resistance.
- Phage-antibiotic combinations offer a promising strategy for treating refractory E. faecium biofilm infections.
