南京农大张炜/聊城大学任庆海团队揭示K1 型特异性噬菌体可高效防控高毒力肺炎克雷伯菌感染 | VS推荐

Fig. 1. Morphology of phage vB_Kp_Z1. (A) Plaque morphology of phage vB_Kp_Z1 observed at 12, 24, 36, and 48 h post-infection. (B) Transmission electron micrograph of phage vB_Kp_Z1 following negative staining with 2% (w/v) phosphotungstic acid. Scale bar, 100 nm.

Fig. 2. Biological characterization of phage vB_Kp_Z1. (A) Determination of the optimal multiplicity of infection (MOI). Progeny phage titers were measured at different MOIs, and the optimal MOI was determined to be 1. (B) One-step growth curve. The latent period was approximately 10 min, and the phage titer reached a plateau at 100 min post-infection. (C) Thermal stability. Phage vB_Kp_Z1 was incubated at temperatures ranging from 4 °C to 80 °C for 1 h, and the remaining titers were determined by the double-layer agar method. (D) pH stability. Phage vB_Kp_Z1 was incubated at 25 °C for 1 h in buffers ranging from pH 1 to 12, and the remaining titers were determined by the double-layer agar method.

Fig. 3. Genome map and comparative genomic analysis of phage vB_Kp_Z1. (A) Circular genome map of phage vB_Kp_Z1 generated using Pharokka, showing GC content, GC skew, annotated functional proteins, and putative proteins. A total of 61 coding sequences (CDSs) were identified, including 34 with assigned functions and 27 hypothetical proteins. (B) Whole-genome comparison between phage vB_Kp_Z1 and the related phage RCIP0053 performed using Clinker, illustrating genome synteny.

Fig. 4. Intergenomic similarity analysis of phage vB_Kp_Z1 using VIRIDIC. Heatmap showing pairwise intergenomic similarities between phage vB_Kp_Z1 and 12 closely related K. pneumoniae phages. Matrix values represent percentage similarities. Phage vB_Kp_Z1 is highlighted with a red star, and the color scale indicating similarity is shown on the right.

Fig.5. Growth inhibition and antibiofilm activity of phage vB_Kp_Z1. (A) Growth curves of KP1897 and KP177 under SM buffer (control) and phage vB_Kp_Z1 treatment at a multiplicity of infection (MOI) of 1. Bacterial growth was monitored by measuring OD600 over time. (B) OD595 values of biofilms formed at 24 h, 48 h, and 72 h following treatment with phage vB_Kp_Z1 at multiplicities of infection (MOI) of 0.1, 1, and 10. Phage treatment reduced biofilm biomass compared with the positive control. Statistical significance was assessed by one-way ANOVA, and asterisks indicate significant differences (*P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Fig. 6. Therapeutic efficacy of phage vB_Kp_Z1 in a mouse model of Klebsiella pneumoniae infection, with pathological evaluation of liver and lung injury. (A) Experimental design. Mice were intraperitoneally challenged with K. pneumoniae (KP1897 or KP177) at 0 h, followed by administration of phage vB_Kp_Z1 or PBS at 1 h. Blood, lung, and liver samples (n = 5) were collected at 10 h, and the remaining mice (n = 8) were monitored for survival up to 168 h. (B) Survival of mice infected with KP1897 and treated with phage vB_Kp_Z1 or control. Survival of mice infected with KP177 and treated with phage vB_Kp_Z1 or control. (C) Bacterial loads in blood, liver, and lung tissues of mice infected with KP1897 (green symbols) or KP177 (blue symbols), comparing phage-treated and control groups. Statistical significance was assessed by unpaired t-test (****P < 0.0001). (D) Representative H&E-stained liver and lung sections from the four experimental groups. Scale bar, 100 μm. (E) Semi-quantitative pathological scores of liver and lung tissues. Statistical significance was assessed by unpaired t-test (****P < 0.0001).

Supplementary Fig. S1. TPositive string test of Klebsiella pneumoniae KP177. KP177 colonies formed a viscous string exceeding 5 mm in length, indicating a hypermucoviscous phenotype.

 Supplementary Fig. S2. Phylogenetic analysis of phage vB_Kp_Z1. (A) Proteomic tree constructed using VIPTree based on complete phage genomes. The inner ring indicates viral families, and the outer ring shows host bacterial groups. The position of phage vB_Kp_Z1 is highlighted by a red star. (B) Phylogenetic tree inferred from the amino acid sequences of the large terminase subunit using the neighbor-joining method with 1,000 bootstrap replicates. Bootstrap values are shown at the nodes. Phage vB_Kp_Z1 is indicated.

Supplementary Fig. S3. Representative crystal violet staining images of biofilm formation. Biofilms were formed at 24 h (A), 48 h (B), and 72 h (C) following treatment with phage vB_Kp_Z1 at MOI of 0.1, 1, and 10. Images show the remaining adherent cells after staining with 0.1% crystal violet, with staining intensity reflecting the relative biofilm biomass. Positive control: untreated biofilm; negative control: culture medium only.