Isolation and genomic characterization of a lytic bacteriophage against multidrug-resistant Proteus mirabilis

Multidrug-resistant (MDR) Proteus mirabilis is a significant opportunistic pathogen, largely due to its ability to form robust biofilms that are recalcitrant to conventional antibiotic therapy. Bacteriophages represent a promising alternative, yet their therapeutic potential is contingent on a thorough characterization of their biological functions and genomic architecture. This study details the isolation and comprehensive analysis of a novel bacteriophage targeting MDR P. mirabilis. A novel lytic bacteriophage, designated Proteus phage ram_arti_1324, was isolated from sewage. Transmission electron microscopy classified it as a member of the family Podoviridae. Functional assays demonstrated its potent efficacy against established P. mirabilis biofilms, achieving a 55% reduction in total biomass in vitro conditions. Whole-genome sequencing revealed a linear, double-stranded DNA genome of 91,735 bp with a GC content of 34.7%. Genomic annotation identified 146 putative coding sequences (CDS), including key lytic genes encoding two distinct endolysins, and four tRNA genes. Crucially, the genome lacks any identifiable virulence factors, antibiotic resistance genes, or markers indicative of a lysogenic life cycle. Comparative genomic analysis against 51 known Proteus phages confirmed its novelty. Proteus phage ram_arti_1324 shares less than 95% average nucleotide identity with its closest relatives, suggesting it represents a novel viral species. This uniqueness was further supported by whole-genome alignment, which revealed a distinct arrangement of locally co-linear blocks not observed in other related phages. The genomic and functional data establish that Proteus phage ram_arti_1324 is a novel, strictly lytic bacteriophage with significant therapeutic potential against both planktonic and biofilm-associated MDR P. mirabilis. Its potent anti-biofilm activity, combined with a genome free of deleterious genes, makes it a strong candidate for further development in phage-based clinical applications. The complete genome sequence provides a valuable resource for future research into phage-host dynamics and the bioengineering of next-generation antimicrobial agents. KEY POINTS: • Novel lytic bacteriophage Proteus phage ram_arti_1324 was isolated with genome size 91735 bp. • Proteus phage ram_arti_1324 displayed absence of virulence factors, antibiotic resistance genes (ARGs) and deleterious genes. • Proteus phage ram_arti_1324 displayed antibiofilm activity against MDR P. mirabilis.