Genomic, Functional, and Evolutionary Insights into a Novel T7-like Phage B1 Infecting Multidrug-Resistant Enterobacter cloacae

Multidrug-resistant (MDR) <i>Enterobacter cloacae</i> is a growing public health issue worldwide, highlighting the urgent need for alternative antimicrobial strategies. This study reports on a lytic phage, designated B1, isolated from sewage, which exhibits specificity and lytic efficiency against MDR <i>E. cloacae</i>. Morphological observation revealed that B1 possesses an icosahedral head (~54 nm) and a short tail (~13 nm). Phage B1 showed a narrow host range, demonstrated stability within a temperature range of 4-37 °C, tolerance to pH values between 5 and 11, and showed an excellent bacteriolytic capacity with a short latent period of less than 10 min and a burst size of approximately 150 PFU/initially infected cell, indicating a rapid lytic cycle and efficient replication capability. Whole-genome sequencing revealed that the phage genome consists of 40,163 base pairs of double-stranded DNA containing 52 open reading frames (ORFs) with a GC content of 52%. Comparative genome-wide analysis using VIRIDIC revealed that B1 shares 75% to 92% similarity with <i>Escherichia</i> phage IMM-002 (accession: NC_048071), <i>Citrobacter</i> phage SH4, and <i>Cronobacter</i> phage Dev2 (accession: NC_023558), but shares less than 70% similarity with other <i>Enterobacter</i> phages. According to ICTV criteria, B1 represents a new species within the same genus as T7-like phages belonging to <i>Autographiviridae</i>, subfamily <i>Studiervirinae</i>, genus <i>Kayfunavirus</i>. In addition, B1 lacks lysogeny-associated or virulence genes and exhibits potent lytic activity against multidrug-resistant <i>E. cloacae</i>, making it a promising candidate for phage therapy. These findings opened up our understanding of the diversity of T7-like phages and provided insights into their evolutionary adaptability and therapeutic potential.