Isolation and identification of Klebsiella pneumoniae phage ΦK2046: optimizing its antibacterial potential in combination with chlorhexidine.

Background: Hospital-acquired infections (HAIs) significantly increase morbidity and mortality worldwide, with Klebsiella pneumoniae (K. pneumoniae) being a leading HAI pathogen requiring targeted eradication in healthcare settings. The growing bacterial tolerance to chemical disinfectants, like chlorhexidine, highlights an urgent need for novel disinfection strategies. Bacteriophages, which employ unique mechanisms to lyse bacteria, offer a potential solution. Combining phages with disinfectants could reduce the use of chemical agents and delay the development of bacterial resistance. However, the use of phages for contamination control in clinical environments remains underexplored.

Methods: ΦK2046 was isolated from hospital wastewater and characterized by transmission electron microscopy, one-step growth curve, optimal multiplicity of infection, and stability analysis. Whole-genome sequencing was performed to identify the genomic characteristics of ΦK2046. The antibacterial and antibiofilm effects of ΦK2046 combined with chlorhexidine were assessed through growth curves, time-kill assays, crystal violet staining, and scanning electron microscopy. A contaminated medical device model was established to assess the ΦK2046-chlorhexidine combination's biofilm reduction efficacy, and different dosing sequences and timing intervals were evaluated for their impact on biofilms formed on urinary catheters.

Results: ΦK2046, characterized by a short latency period, strong environmental stability, safety, and tolerance to chlorhexidine, significantly enhanced the antibacterial and antibiofilm effects of chlorhexidine against FK2046, and reduce the emergence of resistant strains. In contaminated medical device models, the combination of ΦK2046 and chlorhexidine diminished bacterial load and biofilm formation on surfaces. A "phage-first" dosing sequence, particularly with a 90-min interval before chlorhexidine treatment, showed superior efficacy in biofilm reduction.

Conclusions: This study, using ΦK2046 as an example, demonstrates the potential of phages to enhance the antibacterial and antibiofilm effects of chlorhexidine and their feasibility in medical device disinfection. This innovative approach not only improves chlorhexidine's disinfecting power but also effectively tackles the issue of reduced susceptibility of K. pneumoniae to chlorhexidine. The research advances the development and application of phage-based disinfectants and lays a foundation for establishing a phage library with adjuvant properties for disinfectants.