Genomic, Morphological and Functional Characterization of Virulent Bacteriophage IME-JL8 Targeting Citrobacter freundii

Abstract

<i>Citrobacter freundii</i> refers to a fish pathogen extensively reported to be able to cause injury and high mortality. Phage therapy is considered a process to alternatively control bacterial infections and contaminations. In the present study, the isolation of a virulent bacteriophage IME-JL8 isolated from sewage was presented, and such bacteriophage was characterized to be able to infect <i>Citrobacter freundii</i> specifically. Phage IME-JL8 has been classified as the member of the <i>Siphoviridae</i> family, which exhibits the latent period of 30-40 min. The pH and thermal stability of phage IME-JL8 demonstrated that this bacteriophage achieved a pH range of 4-10 as well as a temperature range of 4, 25, and 37°C. As revealed from the results of whole genomic sequence analysis, IME-JL8 covers a double-stranded genome of 49,838 bp (exhibiting 47.96% G+C content), with 80 putative coding sequences contained. No bacterial virulence- or lysogenesis-related ORF was identified in the IME-JL8 genome, so it could be applicable to phage therapy. As indicated by the <i>in vitro</i> experiments, phage IME-JL8 is capable of effectively removing bacteria (the colony count decreased by 6.8 log units at 20 min), and biofilm can be formed in 24 h. According to the <i>in vivo</i> experiments, administrating IME-JL8 (1 × 10⁷ PFU) was demonstrated to effectively protect the fish exhibiting a double median lethal dose (2 × 10⁹ CFU/carp). Moreover, the phage treatment led to the decline of pro-inflammatory cytokines in carp with lethal infections. IME-JL8 was reported to induce efficient lysis of <i>Citrobacter freundii</i> both <i>in vitro</i> and <i>in vivo</i>, thereby demonstrating its potential as an alternative treatment strategy for infections attributed to <i>Citrobacter freundii.</i>