In vitro resensitization of multidrug-resistant clinical isolates of Enterococcus faecium and E. faecalis through phage-antibiotic synergy

Abstract

Bacteriophages are an increasingly attractive option for the treatment of antibiotic-resistant infections, but their efficacy is difficult to discern due to the confounding effects of antibiotics. Phages are generally delivered in conjunction with antibiotics, and thus, when patients improve, it is unclear whether the phages, antibiotics, or both are responsible. This question is particularly relevant for enterococcus infections, as limited data suggest phages might restore antibiotic efficacy against resistant strains. Enterococci can develop high-level resistance to vancomycin, a primary treatment. We assessed clinical and laboratory isolates of Enterococcus faecium and Enterococcus faecalis to determine whether we could observe synergistic interactions between phages and antibiotics. We identified synergy between multiple phages and antibiotics including linezolid, ampicillin, and vancomycin. Notably, antibiotic susceptibility did not predict synergistic interactions with phages. Vancomycin-resistant isolates (n = 6) were eradicated by the vancomycin-phage combination as effectively as vancomycin-susceptible isolates (n = 2). Transcriptome analysis revealed significant gene expression changes under antibiotic-phage conditions, especially for linezolid and vancomycin, with upregulated genes involved in nucleotide and protein biosynthesis and downregulated stress response and prophage-related genes. While our results do not conclusively determine the mechanism of the observed synergistic interactions between antibiotics and phages, they do confirm and build upon previous research that observed these synergistic interactions. Our work highlights how using phages can restore the effectiveness of vancomycin against resistant isolates. This finding provides a promising, although unexpected, strategy for moving forward with phage treatments for vancomycin-resistant Enterococcus infections.

Keywords: antibiotics; bacteriophages; cooperativity; resensitization; synergy.

Fig 1

Morphological and genomic characterization of E. faecium and E. faecalis phages. Myovirus morphologies were observed for phages Ben, Bob, and Bop (A–C). Siphovirus morphologies were observed for phages PL and ReUmp (D and E). Genome maps of phage PL (F) and ReUmp (G). The outermost circle shows open reading frames (ORF) of predicted proteins. Phage structural proteins are highlighted in cyan; DNA-associated proteins are shown in yellow; and hypothetical proteins are shown in purple.

Fig 2

Synograms and growth curves showing the effect of various treatments (antibiotics, phages, antibiotic + phages) on growth dynamics of Enterococcus faecium. The color gradient in synograms represents the percentage of growth reduction. Growth reduction was calculated using following the formula: Percentage reduction = [(OD growth control – OD treatment)/OD growth control] * 100. The average of three biological replicates is shown. Antibiotics vancomycin, ampicillin, and linezolid were tested in all strains. (A) E. faecium EF98PII in the presence of phage Ben. (B) E. faecium NYU in the presence of phage Ben. (C) E. faecium Tx13301 in the presence of phage Bop. (D) E. faecium EF208PII in the presence of phage Bop. Conditions from wells marked with red squares were selected for RNAseq experiments.

Fig 3

Synograms and growth curves showing the effect of various treatments (antibiotics, phages, antibiotic + phages) on growth dynamics of Enterococcus faecalis. The color gradient in synograms represents the percentage of growth reduction. Growth reduction was calculated using following the formula: Percentage reduction = [(OD growth control – OD treatment)/OD growth control] * 100. The average of three biological replicates is shown. Antibiotics vancomycin, ampicillin, and linezolid were tested in all strains. (A) E. faecalis EF116PII in the presence of phage PL. (B) E. faecalis DP11 in the presence of phage ReUmp. (C) E. faecalis EF140PII in the presence of phage Bob. (D) E. faecalis V587 in the presence of phage Ben. Conditions from wells marked with red squares were selected for RNAseq experiments.

Fig 4

PCA shows clustering of samples at different time points and pathway analysis of PCA rotation. (A) Scatter plots of the first two principal components of the normalized gene expression profile of all the samples. The ellipse encircles the three biological replicates for individual experimental conditions and was drawn at a tolerance cutoff of 0.01.

Fig 5

Heatmap shows the top 50 differentially expressed genes. in E. faecium EF98PII (A) and E. faecalis EF116PII (B) for various comparisons between “Antibiotic +Phage” versus “Antibiotic” only treatment samples at 18 hours post co-culture. The data were centered by subtracting the average expression level for each gene and samples were normalized by dividing with SD. Pearson’s correlation coefficient and average linkage were used to calculate the distance matrix. Each sample group includes three biological replicates and is highlighted by colored bars atop the heatmaps. The color key legend next to each heatmap represents the degree of variation in the expression of genes between samples.

Fig 6

Pathway analysis of significantly upregulated/downregulated genes during “Phage + Antibiotic” co-culture as compared to “Antibiotic” only culture at 18 hours. Network of pathways involving differentially expressed genes isolated from E. faecium EF98PII (A, B) and E. faecalis EF116PII (C, D) treated with “Antibiotic + Phage” versus “Antibiotics” only at 18 hours. Genes were filtered as background and “all available gene sets” were used for enrichment analysis of differentially expressed genes. Two pathways (nodes) are connected if they share 30% or more genes. Red represents upregulated and green represents downregulated. The node sizes correspond to their respective gene set sizes. Darker-colored nodes correlate to more significantly enriched gene sets. Thicker edges represent more overlapped genes.