Phage Cocktails Constrain the Growth of <i>Enterococcus</i>

Abstract

Phages that infect pathogenic bacteria present a valuable resource for treating antibiotic-resistant infections. We isolated and developed a collection of 19 <i>Enterococcus</i> phages, including myoviruses, siphoviruses, and a podovirus, that can infect both Enterococcus faecalis and Enterococcus faecium. Several of the <i>Myoviridae</i> phages that we found in southern California wastewater were from the <i>Brockvirinae</i> subfamily (formerly <i>Spounavirinae</i>) and had a broad host range across both E. faecium and E. faecalis. By searching the NCBI Sequence Read Archive, we showed that these phages are prevalent globally in human and animal microbiomes. <i>Enterococcus</i> is a regular member of healthy human gut microbial communities; however, it is also an opportunistic pathogen responsible for an increasing number of antibiotic-resistant infections. We tested the ability of each phage to clear <i>Enterococcus</i> host cultures and delay the emergence of phage-resistant <i>Enterococcus</i>. We found that some phages were ineffective at clearing <i>Enterococcus</i> cultures individually but were effective when combined into cocktails. Quantitative PCR was used to track phage abundance in cocultures and revealed dynamics ranging from one dominant phage to an even distribution of phage growth. Genomic characterization showed that mutations in <i>Enterococcus</i> exopolysaccharide synthesis genes were consistently found in the presence of phage infection. This work will help to inform cocktail design for <i>Enterococcus</i>, which is an important target for phage therapy applications. <b>IMPORTANCE</b> Due to the rise in antibiotic resistance, <i>Enterococcus</i> infections are a major health crisis that requires the development of alternative therapies. Phage therapy offers an alternative to antibiotics and has shown promise in both <i>in vitro</i> and early clinical studies. Here, we established a collection of 19 <i>Enterococcus</i> phages and tested whether combining phages into cocktails could delay growth and the emergence of resistant mutants in comparison with individual phages. We showed that cocktails of two or three phages often prevented the growth of phage-resistant mutants, and we identified which phages were replicating the most in each cocktail. When resistant mutants emerged to single phages, they showed consistent accumulation of mutations in exopolysaccharide synthesis genes. These data serve to demonstrate that a cocktail approach can inform efforts to improve efficacy against <i>Enterococcus</i> isolates and reduce the emergence of resistance.