Isolation, Characterization, and Anti-Biofilm Activity of a Novel Kaypoctavirus Against K24 Capsular Type, Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates

Abstract

Background/Objectives: The significant outbreak of multidrug-resistant Klebsiella pneumoniae has emerged as a primary global concern associated with high morbidity and mortality rates. Certain strains of K. pneumoniae are highly resistant to most antibiotics available in clinical practice, exacerbating the challenge of bacterial infections. Methods: Phage vB_KpnP_PW7 (vKPPW7) was isolated and characterized. Its morphology, stability, adsorption rate, one-step growth curve, lytic activity, whole-genome sequence analysis, and antibacterial and antibiofilm activities were evaluated. Results: The virulent phage has a 73,658 bp linear dsDNA genome and was classified as a new species of the genus Kaypoctavirus, subfamily Enquatrovirinae, and family Schitoviridae. Phage vKPPW7 has a high adsorption rate, a short latent period, and a large burst size. The phage showed activity against 18 K. pneumoniae isolates with the K24 capsular type but was unable to lyse K. pneumoniae isolates whose capsular type was not classified as K24. Additionally, phage vKPPW7 demonstrated strong stability across various temperatures and pH values. The phage exhibited antibacterial activity, and scanning electron microscopy (SEM) confirmed its ability to lyse MDR K. pneumoniae with the K24 capsular type. Furthermore, phage vKPPW7 effectively removed preformed biofilm and prevented biofilm formation, resulting in reduced biofilm biomass and biofilm viability compared to controls. The architecture of phage-treated biofilms was confirmed under SEM. Conclusions: These findings suggest that phage vKPPW7 holds promise for development as a therapeutic or biocontrol agent.

Keywords: K24 capsular type; Klebsiella pneumoniae; antibacterial activity; bacteriophage; biofilms; phage therapy.

Figure 1

Plaque, virion morphology and stability of phage vKPPW7. (a) Representative photographs of phage vKPPW7 plaques formed on a double-layer agar plate; (b) Transmission electron micrograph showing the virion morphology of phage vKPPW7; (c) Stability of phage vKPPW7 after 2 h incubation at temperatures ranging from 4 to 80 °C; (d) Stability of phage vKPPW7 after 2 h incubation at pH levels ranging from 1 to 14. Data are expressed as the mean ± standard error of the mean (SEM), and the * symbol indicates significance at p ≤ 0.05.

Figure 2

Infective properties of phage vKPPW7. (a) Adsorption curve of phage vKPPW7 to the MDR K. pneumoniae clinical isolate KPPW67, which served as the host cells; (b) One-step growth curve showing latent period and burst size of phage vKPPW7 on its host bacteria; (c) The lytic activity of phage vKPPW7 against its host bacteria at different multiplicities of infection. The bars represent the SEM, and asterisks denote statistically significant differences (* p ≤ 0.05).

Figure 3

Structural changes in MDR K. pneumoniae following treatment of phage vKPPW7. (a) MDR K. pneumoniae bacterial cells; (b) MDR K. pneumoniae cells treated with phage vKPPW7.

Figure 4

Genome map of phage vKPPW7. The innermost circles depicted the GC skew and GC content, respectively. Arrows represented the predicted coding sequences (CDSs), with their colors indicating the grouping of CDSs based on the protein functions.

Figure 5

Phage vKPPW7 genomic analysis. (a) A phylogenetic tree of phage vKPPW7 and related phages was generated using ViPTree, a whole-genome-based phylogenomic tool. The query sequence is indicated by an asterisk.; (b) A genome-wide comparison of phage vKPPW7 and Klebsiella phage KP8 was performed using ViPTree. Homologous regions identified by tBLASTX search are connected by segments, which are colored-coded based on amino acid identity.

Figure 6

Evolutionary relationship between phage vKPPW7 and other phages. The phylogenetic trees for phage terminase large subunit (a) and capsid proteins (b) were constructed using the maximum likelihood method based on the alignment of amino acid sequences. The JTT matrix-based model was utilized with 1000 bootstrap replicates. The sequence of phage vKPPW7, used as the query, is indicated by an asterisk (*).

Figure 7

Ability of phage vKPPW7 to reduce biofilm formation. (a) The effect of phage vKPPW7 on the biomass of biofilm formation in 1-day-old biofilms; (b) The effect of phage vKPPW7 on the biomass of biofilm formation at 5-day-old biofilms; (c) The effect of phage vKPPW7 on the biofilm cell viability in 1-day-old biofilms; (d) Effect of phage vKPPW7 on the biofilm cell viability in 5-day-old biofilms. The bars represent the SEM, and asterisks denote statistically significant differences (* p ≤ 0.05).

Figure 8

Ability of phage vKPPW7 to remove preformed biofilms. (a) The effect of phage vKPPW7 on the biomass of 1-day-old preformed biofilms; (b) The effect of phage vKPPW7 on the biomass of 5-day-old preformed biofilms; (c) The effect of phage vKPPW7 on the biofilm cell viability within 1-day-old preformed biofilms; (d) Effect of phage vKPPW7 on the biofilm cell viability within 5-day-old preformed biofilms. The bars represent the SEM, and asterisks denote statistically significant differences (* p ≤ 0.05).

Figure 9

Observation of biofilm structures using SEM. (a–c) Five-day-old biofilms of MDR K. pneumoniae; (d–f) Five-day-old biofilms of MDR K. pneumoniae treated with phage vKPPW7. Figures (a) and (d) were captured at 2000× magnification, while figures (b) and (e) were captured at 10,000× magnification, and figures c and f were captured at 20,000× magnification.