Related Experiment Video
Updated: Dec 19, 2025
Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing
Published on: December 23, 2022
Isolation, characterization, and comparative genomic analysis of a phage infecting high-level
Danial Nasr Azadani1, Daiyuan Zhang2, J Robert Hatherill2
1Life Sciences, Texas A&M University-Corpus Christi, Corpus Christi, TX, United States of America.
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.
Insights
Researchers isolated phiNASRA1, a phage effective against antibiotic-resistant Enterococcus faecalis. Comparative genomic analysis revealed significant phage diversity, highlighting potential for novel therapeutic agents.
Area of Science:
- Microbiology
- Virology
- Genomics
Background:
- Enterococcus species are Gram-positive bacteria, commensal in the human gut but increasingly causing nosocomial infections.
- Rising antibiotic resistance in Enterococcus strains necessitates the discovery of novel therapeutic strategies, such as bacteriophages.
- Bacteriophages (phages) are viruses that infect bacteria and show promise in combating resistant infections.
Purpose of the Study:
- To isolate, characterize, and sequence the genome of a phage targeting an antibiotic-resistant Enterococcus faecalis strain.
- To perform a comparative genomic analysis of Enterococcus phages available in public databases.
- To assess the phylogenetic relationships and genomic diversity among Enterococcus phages.
Main Methods:
- Isolation of phages from wastewater treatment plant influent using a high-level aminoglycoside-resistant Enterococcus faecalis strain.
- Characterization of isolated phage phiNASRA1 using transmission electron microscopy (TEM) and whole-genome sequencing (WGS).
- Comparative genomic analysis involving phylogenetic comparisons based on specific proteins and pangenome analysis of 32 Enterococcus phages.
Main Results:
- Phage phiNASRA1, belonging to the Siphoviridae family, was isolated with high lytic efficiency (∼97.52%).
- phiNASRA1 has a double-stranded DNA genome of 40,139 bp with 62 open reading frames (ORFs).
- Phylogenetic analyses grouped phages by provenance, size, and GC content, revealing significant genomic diversity and plasticity among Enterococcus phages.
Conclusions:
- The study successfully isolated and characterized phiNASRA1, a potential candidate for combating antibiotic-resistant Enterococcus faecalis.
- Comparative genomics highlights extensive diversity within Enterococcus phages, complicating single-locus phylogenetic interpretations.
- Pangenome analysis is effective for assessing diversity within closely related phage groups, and phiNASRA1 shows promise for future therapeutic applications.
