Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia

Abstract

Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient's gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient's antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections.

Importance: Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Keywords: bacteriophage therapy; phage-neutralizing antibodies; vancomycin-resistant Enterococcus faecium.

Fig 1

Clinical timeline and GI tract enterococcal populations. (A) The timeline represents the observation period (June 2020 through July 2021). The three bars above the timeline depict the type and duration of treatments used throughout the observation period: blue (Φ9184) and red (ΦHi3) for phages, gray for daptomycin exposure, and the multicolored bar highlights other systemic antibiotics given (vancomycin, purple; ceftaroline, pink; oritavancin, yellow; tigecycline, green; tedizolid, dark teal; doxycycline, light teal). PO indicates that the antibiotic was given orally, and all other antibiotics were given intravenously. Triangles below the timeline indicate positive blood cultures that grew vancomycin-sensitive E. faecium (VSE, open triangles) and vancomycin-resistant E. faecium (VRE, closed triangles). Gray, open diamonds indicate blood cultures that were obtained after starting phage therapy but remained negative. Red dashes indicated time points when serum was collected to test for phage neutralization. Below the timeline, BSI isolates that underwent WGS are numbered 1 through 20 and are shaded white for VSE and black for VRE. (B) GI enterococcal population metagenomics of pooled colonies from stool samples plated on bile esculin azide agar. The observation day indicates when each stool sample was collected. The relation to BSI events is listed below and corresponds to BSI events described in panel A. The bar graph shows the relative abundance of reads mapping to E. faecalis (gray bars), E. faecium (pink bars), and the VanA operon (black dotted line). Colored squares above each bar indicate which phage (top row), systemic intravenous antibiotics (rows 2–5), and oral antibiotics (rows 6 and 7) the patient was being treated with at the time each stool sample was collected.

Fig 2

Development of a phage-specific neutralizing immune response. (A) Efficiency of plating (EOP) of Φ9184 (blue circles), ΦHi3 (red circles), and a 50:50 cocktail of both phages (purple squares) on clinical E. faecium BSI isolates compared to the phage host strain. Isolates 13 and 14 were the last isolates collected prior to phage initiation (Before Φ), and isolates 15–20 represent breakthrough BSI events occurring after phage therapy was started (After Φ). Black boxes indicate VRE, and white boxes indicate VSE isolates. (B) Average EOP of Φ9184 and ΦHi3 on the host strain after incubation with patient serum versus incubation with buffer alone, across three replicates. (C) ELISA IgG-binding curves from host sera against Φ9184 (blue) and ΦHi3 (red). “Before Φ” indicates the serum sample that was collected prior to initiation of phage therapy (observation day 181), and "Day 337” indicates the serum sample collected on observation day 337, which was 34 days after starting the Φ9184 and ΦHi3 cocktail. (D) Half-maximal IgG titers (-log₁₀ EC50) of each ELISA replicate from the conditions shown in panel C. Error bars indicate the standard error mean of replicates. *P < 0.05 and **P < 0.01.