Genomic characterization of a novel Pseudomonas aeruginosa bacteriophage representing the newly proposed genus Angoravirus: in vitro antimicrobial and antibiofilm activity.

Abstract

Multi-drug-resistant (MDR) Pseudomonas aeruginosa is an important pathogen that poses a critical threat due to its metallo-beta-lactamase (MBL)-mediated carbapenem resistance and biofilm-forming ability, making bacterial treatment very complicated and requiring alternative strategies. Bacteriophages are promising alternatives; however, the discovery of novel phages targeting MDR strains remains urgent. In this study, Pseudomonas phage Baskent_P4_1, a novel virulent siphovirus that infects clinical MDR P. aeruginosa isolates, was isolated from wastewater and characterized comprehensively. Its efficacy was tested against biofilm-forming, MDR isolates with MBL activity by spot test and efficiency of plating (EOP). Biological characterization showed that phage Baskent_P4_1 is stable at pH 4-10 and temperatures up to 50 °C, while its stability decreases  >60 °C temperature. It has a short latent period of 10 min and a high burst size of 253 phages per cell. The phage lysed 40% of the MDR P. aeruginosa isolates tested, including strong biofilm producers. In vitro assays showed significant biofilm inhibition (48.8% reduction at 10⁹ PFU/mL) and degradation of pre-formed biofilms. Transmission electron microscopy (TEM) revealed an icosahedral head (70 nm) and a long non-contractile tail (150 nm). Whole genome sequencing by Illumina demonstrated a linear dsDNA genome of 41.947 bp (62.8% GC content) with 53 predicted coding sequences. No virulence factors, antibiotic resistance genes, or tRNAs were detected, thus ensuring therapeutic safety. Along with phylogenetic and vConTACT2 analysis, these results suggested that phage Baskent_P4_1 belongs to a new genus, which was proposed here as the genus Angoravirus with three other species. Genomic analysis identified hydrolases (ORF 13/14) and 7-deazaguanine modification enzymes (ORF 46/47) that may contribute to host lysis and evasion of bacterial defenses. These findings highlight Baskent_P4_1's potential as a therapeutic candidate against MDR P. aeruginosa infections. The study underscores the importance of expanding phage diversity libraries and provides a framework for characterizing novel phages to combat antimicrobial resistance.