Phage-Derived Endolysin PlyPM1 as a Rapid Enzybiotic for Detection and Lysis of Proteus mirabilis in Clinical Urine Samples.

Abstract

Proteus mirabilis is a formidable nosocomial pathogen notorious for causing catheter-associated urinary tract infections (CAUTIs), catheter encrustation, and urolithiasis due to its potent urease activity and biofilm formation. The rise of multidrug-resistant (MDR) P. mirabilis strains, including those resistant to broad-spectrum cephalosporins and carbapenems, underscores the critical need for novel, rapid diagnostic and therapeutic strategies. While whole bacteriophages have shown therapeutic promise, their use in diagnostics can be limited by host range specificity and regulatory complexities. Bacteriophage endolysins—highly evolved enzymes that degrade the bacterial peptidoglycan cell wall from within—offer a superior alternative as precision “enzybiotics.” This study describes the identification, recombinant production, and functional characterization of a novel endolysin, PlyPM1, derived from a Myoviridae phage specific to P. mirabilis, and evaluates its dual utility for rapid pathogen detection and direct bacteriolysis in clinical urine matrices.The gene encoding PlyPM1 was identified from the genome of the lytic phage φPM-M1, isolated from wastewater. Bioinformatic analysis revealed a modular structure featuring a conserved N-terminal catalytic domain (predicted CHAP [cysteine, histidine-dependent amidohydrolase/peptidase] domain) and a C-terminal cell wall binding domain (CBD) with high specificity for P. mirabilis surface epitopes. The recombinant 28-kDa enzyme was overexpressed in E. coli BL21(DE3), purified via nickel-affinity chromatography, and demonstrated potent, rapid lytic activity against all 25 clinical P. mirabilis isolates tested, including 18 MDR strains. PlyPM1 exhibited remarkable specificity, showing no activity against other common uropathogens (E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) or commensal urinary flora (Lactobacillus spp.), thereby minimizing dysbiosis risk. In a standardized turbidity reduction assay, 50 µg/mL of PlyPM1 achieved a >3-log reduction in P. mirabilis planktonic cell counts in phosphate-buffered saline within 30 minutes at 37°C.Critically, PlyPM1 retained significant lytic activity in synthetic and human urine, overcoming the inhibitory effects of urea and ionic strength that often hinder antimicrobial agents. Furthermore, PlyPM1 efficiently disrupted pre-formed P. mirabilis biofilms on silicone catheter surfaces, reducing viable biofilm-embedded cells by 99.8% after a 2-hour treatment, as quantified by confocal laser scanning microscopy and colony-forming unit counts.Leveraging its specificity and rapid activity, we developed a proof-of-concept PlyPM1-based detection assay. The assay utilizes PlyPM1 conjugated to fluorescent microspheres. Upon binding to P. mirabilis cells in spiked urine samples, the complex catalyzes the release of a fluorescent signal from a quenched peptidoglycan substrate. This method allowed for the sensitive detection of P. mirabilis at concentrations as low as 10³ CFU/mL directly in urine within 25 minutes, outperforming standard culture-based identification (requiring 24-48 hours) and showing comparable sensitivity to PCR but without the need for nucleic acid extraction.

In conclusion, PlyPM1 represents a first-in-class, phage-derived enzybiotic with dual diagnostic and therapeutic potential against P. mirabilis uropathogens. Its rapid, specific bacteriolytic activity in urine and against biofilms positions it as an excellent candidate for development into (1) a novel therapeutic adjunct for MDR CAUTIs, and (2) a rapid point-of-care diagnostic tool to guide targeted antibiotic therapy, thereby addressing both treatment and antimicrobial stewardship challenges in clinical urology.