Kayvirus-Based Sensing of Lower Respiratory Tract Infection and Taxonomic Dissection of Prophages in Clinical Strains of Staphylococcus aureus

Staphylococcus aureus is one of the major pathogenic species affiliated to ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) group, frequently encountered in the lower respiratory tract infections (LRTI). Here, we investigated the applicability of Staphylococcus phage RuSa1 as a bioreporter for S. aureus infections in human respiratory tract and assessed the prophage landscape of clinical strains of S. aureus with reference to P. aeruginosa for safe phage therapeutics. While BOX-A1R-based repetitive extragenic palindromic-polymerase chain reaction (BOX-PCR) displayed discrete signatures corresponding to Staphylococcus in sputum samples, RuSa1 specifically identified S. aureus in LRTI. Pangenome analysis revealed the prevalence of holin and major capsid proteins as prophages in clinical strains of S. aureus (n = 50). Peeveelvirus, Biseptimavirus, Dubowvirus and Triavirus are the major lysogenic taxa in S. aureus, whereas genetic signatures corresponding to Kayvirus-like prophages were absent. The abundance and evenness of prophages were high in S. aureus, whereas the diversity and richness peaked in P. aeruginosa. Interestingly, holins exhibited high diversity, abundance, richness and evenness specifically in S. aureus. In contrast, tail proteins showed high diversity, abundance, and richness in P. aeruginosa, while their distribution was more even in S. aureus. We identified a tail fibre protein in RuSa1, which exhibited strong binding with wall teichoic acids of S. aureus (binding energy −8.1 kcal/mol), followed by S. aureus ST395 (binding energy −7.2 kcal/mol). This study demonstrated the potential ability of RuSa1 to sense and lyse respiratory S. aureus, and decoded taxonomic landscape of prophages, which encourages safe applicability of Kayvirus in diagnostics and therapy targeted to S. aureus in LRTI.