A novel thermostable lytic phage vB_EF_Enf3_CCASU-2024-3 against clinical Enterococcus faecium and Enterococcus faecalis.

Enterococci, Gram-positive bacteria, have become a major concern in healthcare settings due to their significant virulence and antibiotic resistance. This research focuses on isolating, phenotypic, and genotypic analysis of enterococci-specific lytic phages to be used as potential candidates in combating multidrug-resistant (MDR) Enterococcus clinical isolates. The virulence of Enterococcus isolates was analyzed by testing for gelatinase and biofilm formation. The phage(s) was isolated from a sewage sample, then purified, propagated, and physiochemically analyzed. The phage was examined using transmission electron microscopy, and the whole genome sequence (WGS) was performed. Sixety-five clinical enterococci including, 27 (41.5%), 33 (50.7%) 3 (4.6%), and 2 (3%) E. faecalis, E. faecium, E. avium, and E. durans, respectively were isolated. Linezolid, teicoplanin, chloramphenicol, and vancomycin exhibited the lowest resistance. Twenty-five (38.5%) isolates were both gelatinase- and biofilm-producers. A novel lytic vB_EF_Enf3 phage belonging to Caudoviricetes class, characterized by an icosahedral head with a diameter of 100 ± 5 nm and a tail measuring 70 ± 5 nm in length was isolated. The phage demonstrated good thermal stability, and viability across various pH levels and exhibited a broad- spectrum of activity against E. faecium and E. faecalis. The vB_EF_Enf3 phage (36,202 bp length) harbored 36 open reading frames (ORFs) with a GC content of 34.4% (GenBank accession, PP747318). In conclusion, a novel thermostable lytic bacteriophage vB_EF_Enf3, belonging to class Caudoviricetes, was isolated from sewage showing broad-spectrum potent lytic activity against E. faecium and E. faecalis and maintained stability under various extreme conditions, including temperature, and pH fluctuations.