Abstract
Enterococcus is a genus of Gram-positive bacteria that are commensal to the gastrointestinal tracts of humans but some species have been increasingly implicated as agents of nosocomial infections. The increase in infections and the spread of antibiotic-resistant strains have contributed to renewed interest in the discovery of Enterococcus phages. The aims of this study were (1) the isolation, characterization, and genome sequencing of a phage capable of infecting an antibiotic-resistant E. faecalis strain, and (2) the comparative genomic analysis of publicly-available Enterococcus phages. For this purpose, multiple phages were isolated from wastewater treatment plant (WWTP) influent using a high-level aminoglycoside-resistant (HLAR) E. faecalis strain as the host. One phage, phiNASRA1, demonstrated a high lytic efficiency (∼97.52%). Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) showed that phiNASRA1 belongs to the Siphoviridae family of double-stranded DNA viruses. The phage was approximately 250 nm in length and its complete genome (40,139 bp, 34.7% GC) contained 62 open reading frames (ORFs). Phylogenetic comparisons of phiNASRA1 and 31 publicly-available Enterococcus phages, based on the large subunit terminase and portal proteins, grouped phage by provenance, size, and GC content. In particular, both phylogenies grouped phages larger than 100 kbp into distinct clades. A phylogeny based on a pangenome analysis of the same 32 phages also grouped phages by provenance, size, and GC content although agreement between the two single-locus phylogenies was higher. Per the pangenome phylogeny, phiNASRA1 was most closely related to phage LY0322 that was similar in size, GC content, and number of ORFs (40,139 and 40,934 bp, 34.77 and 34.80%, and 60 and 64 ORFs, respectively). The pangenome analysis did illustrate the high degree of sequence diversity and genome plasticity as no coding sequence was homologous across all 32 phages, and even ‘conserved’ structural proteins (e.g., the large subunit terminase and portal proteins) were homologous in no more than half of the 32 phage genomes. These findings contribute to a growing body of literature devoted to understanding phage biology and diversity. We propose that this high degree of diversity limited the value of the single-locus and pangenome phylogenies. By contrast, the high degree of homology between phages larger than 100 kbp suggests that pangenome analyses of more similar phages is a viable method for assessing subclade diversity. Future work is focused on validating phiNASRA1 as a potential therapeutic agent to eradicate antibiotic-resistant E. faecalis infections in an animal model.
Highlights
Enterococcus is a diverse genus of Gram-positive bacteria and a component of the human gastrointestinal microflora (Murray, 1990)
Following the guidelines established by the International Committee of Taxonomy of Viruses (ICTV), phiNASRA1 was classified as a double-stranded DNA virus of the Siphoviridae family in the order Caudovirales
The absence of genes associated with drug resistance were encouraging but further testing is needed to determine the phage’s ability to clear an antibiotic-resistant infection in an animal model
Summary
Enterococcus is a diverse genus of Gram-positive bacteria and a component of the human gastrointestinal microflora (Murray, 1990). Most species and strains are commensal but a minority are human pathogens (Fisher & Phillips, 2009). In the past 30 years, a growing number of nosocomial infections have been attributed to E. faecalis and E. faecium (Moellering, 1992; Guzman Prieto et al, 2016). Antibiotic-resistant strains have been increasingly associated with nosocomial bacteremia, surgical wound infections, and urinary tract infections (Gilmore, Lebreton & Van Schaik, 2013; Lebreton et al, 2013). Enterococcus infections are routinely treated with aminoglycosides (e.g., gentamicin and streptomycin) in combination with a cell wall inhibitor like ampicillin (Moellering, 1971). Clinical E. faecalis and E. faecium isolates are frequently resistant to both aminoglycosides and cell wall inhibitors (Lebreton et al, 2013; Guzman Prieto et al, 2016). Mechanisms of resistance to aminoglycosides include mutation of the 30S ribosomal subunit and the acquisition of aminoglycoside modifying enzymes (AMEs) via horizontal gene transfer (HGT), while mechanisms of resistance to cell wall inhibitors include the mutation of penicillin-binding proteins and the HGT-mediated acquisition of B-lactamases (Hollenbeck & Rice, 2012)
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