Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization

Abstract

Background: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections.

Methods: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10.

Results: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family.

Conclusion: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

Keywords: K. pneumoniae; And Antibiotic resistance; Bacteriophage KZag1; Biofilm reduction; Genomic analysis; Phage Therapy.

Fig. 1

Morphological Characteristics of K. pneumoniae Phage KZag1 (A) Phage Zag1 Plaque Morphology NA double-layer agar plate showing the plaque morphology of phage Zag1. Clear zones indicate areas where phage KZag1 has lysed the host K. pneumoniae cells. B Transmission Electron Microscopy (TEM) Image of Phage Zag1 shows three individual phage Zag1 particles, with a scale bar of 0.5 µm. C Transmission Electron Microscopy (TEM) Image of Phage Zag1 High-resolution TEM image revealing the detailed structure of phage Zag1. Scale represents 100 nm. D Phage Zag1 Adsorption on K. pneumoniae Cell Visual depiction of phage Zag1 attached to the surface of a K. pneumoniae bacterial cell

Fig. 2

Single-step growth curve for K. pneumoniae KZag1 phage. The plaque forming units (PFUs) per infected cell in cultures of K. pneumoniae K9 at different time post infection are shown. Samples were taken at intervals every 10 min

Fig. 3

Effect of temperature and pH on the stability of K. pneumoniae KZag1 phage. A The stability of phage of K. pneumoniae Kzag1 at different temperatures. B The stability of KZag1 phage at different pH values. The number of phage was estimated by plaque assay using K. pneumoniae. Results are shown as means ± standard error

Fig. 4

Phage Treatment of K. pneumoniae K9 Biofilm. The figure demonstrates the impact of Kzag1 phage treatment on K. pneumoniae bacterial biofilms using different multiplicities of infection (MOIs) of 0.1, 1 and10. Each data point on the graph represents the mean of three independent experiments. The results indicate the efficacy of phage treatment in reducing K. pneumoniae biofilm formation at varying MOI values

Fig. 5

Whole genome map of phage Zag1. The figure displays the whole genome map of phage Zag1. The circular representation showcases the phage's genomic sequence, indicating the positions of various ORFs and functional elements. The map highlights key features and regions of interest within the phage's genetic structure

Fig. 6

Phylogenetic tree depicting the evolutionary relationships among various bacteriophages, including Klebsiella phage cp16, cp21, P01, KP6, Menlow, PWKp5, vB_KpnM-20, vB_KpnM_KpS110, vB_KpnS_MDA2066, UPM 2146, vB_KqM-Westerburg, vB_KqM-LilBean, and vB_KqM-Bilbo, as well as Escherichia phage vB_EcoM_KWBSE43-6, Klebsiella phage T751, Klebsiella phage 0507-KN2-1 DNA, and KZag1. The scale bar represents a genetic distance corresponding to 0.01 substitutions per site, indicating minimal evolutionary divergence among the depicted phages